Mapping microscopic order in plant and mammalian cells and tissues: novel differential polarization attachment for new generation confocal microscopes (DP-LSM)

NASA Astrophysics Data System (ADS)

Steinbach, G.; Pawlak, K.; Pomozi, I.; Tóth, E. A.; Molnár, A.; Matkó, J.; Garab, G.

2014-03-01

Elucidation of the molecular architecture of complex, highly organized molecular macro-assemblies is an important, basic task for biology. Differential polarization (DP) measurements, such as linear (LD) and circular dichroism (CD) or the anisotropy of the fluorescence emission (r), which can be carried out in a dichrograph or spectrofluorimeter, respectively, carry unique, spatially averaged information about the molecular organization of the sample. For inhomogeneous samples—e.g. cells and tissues—measurements on macroscopic scale are not satisfactory, and in some cases not feasible, thus microscopic techniques must be applied. The microscopic DP-imaging technique, when based on confocal laser scanning microscope (LSM), allows the pixel by pixel mapping of anisotropy of a sample in 2D and 3D. The first DP-LSM configuration, which, in fluorescence mode, allowed confocal imaging of different DP quantities in real-time, without interfering with the ‘conventional’ imaging, was built on a Zeiss LSM410. It was demonstrated to be capable of determining non-confocally the linear birefringence (LB) or LD of a sample and, confocally, its FDLD (fluorescence detected LD), the degree of polarization (P) and the anisotropy of the fluorescence emission (r), following polarized and non-polarized excitation, respectively (Steinbach et al 2009 Acta Histochem.111 316-25). This DP-LSM configuration, however, cannot simply be adopted to new generation microscopes with considerably more compact structures. As shown here, for an Olympus FV500, we designed an easy-to-install DP attachment to determine LB, LD, FDLD and r, in new-generation confocal microscopes, which, in principle, can be complemented with a P-imaging unit, but specifically to the brand and type of LSM.

Spectro-microscopy of living plant cells.

PubMed

Harter, Klaus; Meixner, Alfred J; Schleifenbaum, Frank

2012-01-01

Spectro-microscopy, a combination of fluorescence microscopy with spatially resolved spectroscopic techniques, provides new and exciting tools for functional cell biology in living organisms. This review focuses on recent developments in spectro-microscopic applications for the investigation of living plant cells in their native tissue context. The application of spectro-microscopic methods led to the recent discovery of a fast signal response pathway for the brassinosteroide receptor BRI1 in the plasma membrane of living plant cells. Moreover, the competence of different plant cell types to respond to environmental or endogenous stimuli was determined in vivo by correlation analysis of different optical and spectroscopic readouts such as fluorescence lifetime (FLT). Furthermore, a new spectro-microscopic technique, fluorescence intensity decay shape analysis microscopy (FIDSAM), has been developed. FIDSAM is capable of imaging low-expressed fluorophore-tagged proteins at high spatial resolution and precludes the misinterpretation of autofluorescence artifacts. In addition, FIDSAM provides a very effective and sensitive tool on the basis of Förster resonance energy transfer (FRET) for the qualitative and quantitative determination of protein-protein interaction. Finally, we report on the quantitative analysis of the photosystem I and II (PSI/PSII) ratio in the chloroplasts of living Arabidopsis plants at room temperature, using high-resolution, spatially resolved fluorescence spectroscopy. With this technique, it was not only possible to measure PSI/PSII ratios, but also to demonstrate the differential competence of wild-type and carbohydrate-deficient plants to adapt the PSI/PSII ratio to different light conditions. In summary, the information content of standard microscopic images is extended by several dimensions by the use of spectro-microscopic approaches. Therefore, novel cell physiological and molecular topics can be addressed and valuable insights into molecular and subcellular processes can be obtained in living plants.

The design of a microscopic system for typical fluorescent in-situ hybridization applications

NASA Astrophysics Data System (ADS)

Yi, Dingrong; Xie, Shaochuan

2013-12-01

Fluorescence in situ hybridization (FISH) is a modern molecular biology technique used for the detection of genetic abnormalities in terms of the number and structure of chromosomes and genes. The FISH technique is typically employed for prenatal diagnosis of congenital dementia in the Obstetrics and Genecology department. It is also routinely used to pick up qualifying breast cancer patients that are known to be highly curable by the prescription of Her2 targeted therapy. During the microscopic observation phase, the technician needs to count typically green probe dots and red probe dots contained in a single nucleus and calculate their ratio. This procedure need to be done to over hundreds of nuclei. Successful implementation of FISH tests critically depends on a suitable fluorescent microscope which is primarily imported from overseas due to the complexity of such a system beyond the maturity of the domestic optoelectrical industry. In this paper, the typical requirements of a fluorescent microscope that is suitable for FISH applications are first reviewed. The focus of this paper is on the system design and computational methods of an automatic florescent microscopy with high magnification APO objectives, a fast spinning automatic filter wheel, an automatic shutter, a cooled CCD camera used as a photo-detector, and a software platform for image acquisition, registration, pseudo-color generation, multi-channel fusing and multi-focus fusion. Preliminary results from FISH experiments indicate that this system satisfies routine FISH microscopic observation tasks.

Near-field microscopy and fluorescence spectroscopy: application to chromosomes labelled with different fluorophores.

PubMed

Mahieu-Williame, L; Falgayrettes, P; Nativel, L; Gall-Borrut, P; Costa, L; Salehzada, T; Bisbal, C

2010-04-01

We have coupled a spectrophotometer with a scanning near-field optical microscope to obtain, with a single scan, simultaneously scanning near-field optical microscope fluorescence images at different wavelengths as well as topography and transmission images. Extraction of the fluorescence spectra enabled us to decompose the different wavelengths of the fluorescence signals which normally overlap. We thus obtained images of the different fluorescence emissions of acridine orange bound to single or double stranded nucleic acids in human metaphase chromosomes before and after DNAse I or RNAse A treatment. The analysis of these images allowed us to visualize some specific chromatin areas where RNA is associated with DNA showing that such a technique could be used to identify multiple components within a cell.

Quantitative high dynamic range beam profiling for fluorescence microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mitchell, T. J., E-mail: t.j.mitchell@dur.ac.uk; Saunter, C. D.; O’Nions, W.

2014-10-15

Modern developmental biology relies on optically sectioning fluorescence microscope techniques to produce non-destructive in vivo images of developing specimens at high resolution in three dimensions. As optimal performance of these techniques is reliant on the three-dimensional (3D) intensity profile of the illumination employed, the ability to directly record and analyze these profiles is of great use to the fluorescence microscopist or instrument builder. Though excitation beam profiles can be measured indirectly using a sample of fluorescent beads and recording the emission along the microscope detection path, we demonstrate an alternative approach where a miniature camera sensor is used directly withinmore » the illumination beam. Measurements taken using our approach are solely concerned with the illumination optics as the detection optics are not involved. We present a miniature beam profiling device and high dynamic range flux reconstruction algorithm that together are capable of accurately reproducing quantitative 3D flux maps over a large focal volume. Performance of this beam profiling system is verified within an optical test bench and demonstrated for fluorescence microscopy by profiling the low NA illumination beam of a single plane illumination microscope. The generality and success of this approach showcases a widely flexible beam amplitude diagnostic tool for use within the life sciences.« less

High-Speed Fluorescence Microscopy: Lifetime Imaging in the Biomedical Sciences

NASA Astrophysics Data System (ADS)

Periasamy, Ammasi; Wang, Xue F.; Wodnick, Pawel; Gordon, Gerald W.; Kwon, Seongwook; Diliberto, Pamela A.; Herman, Brian

1995-02-01

The ability to observe the behavior of living cells and tissues provides unparalleled access to information regarding the organization and dynamics of complex cellular structures. While great strides have been made over the past 30 to 40 years in the design and application of a variety of novel optical microscopic techniques, until recently, it has not been possible to image biological phenomena that occur over very short time periods (nanosecond to millisecond) or over short distances (10 to 1000 [Angstrom capital A, ring]). However, the recent combination of (1) very rapidly gated and sensitive image intensifiers and (2) the ability to deliver fluorescence excitation energy to intact living biological specimens in a pulsed or sinusoidally modulated fashion has allowed such measurements to become a reality through the imaging of the lifetimes of fluorescent molecules. This capability has resulted in the ability to observe the dynamic organization and interaction of cellular components on a spatial and temporal scale previously not possible using other microscopic techniques. This paper discusses the implementation of a fluorescence lifetime imaging microscope (FLIM) and provides a review of some of the applications of such an instrument. These include measurements of receptor topography and subunit interactions using fluorescence resonance energy transfer (FRET), fluorescence anisotropy of phospholipids in cell membranes, cytosolic free calcium (Ca2+)i and the detection of human papillomavirus (HPV) infection in clinical cervicovaginal smears.

An optical clearing technique for plant tissues allowing deep imaging and compatible with fluorescence microscopy.

PubMed

Warner, Cherish A; Biedrzycki, Meredith L; Jacobs, Samuel S; Wisser, Randall J; Caplan, Jeffrey L; Sherrier, D Janine

2014-12-01

We report on a nondestructive clearing technique that enhances transmission of light through specimens from diverse plant species, opening unique opportunities for microscope-enabled plant research. After clearing, plant organs and thick tissue sections are amenable to deep imaging. The clearing method is compatible with immunocytochemistry techniques and can be used in concert with common fluorescent probes, including widely adopted protein tags such as GFP, which has fluorescence that is preserved during the clearing process. © 2014 American Society of Plant Biologists. All Rights Reserved.

Comparison of Fluorescence Microscopy and Different Growth Media Culture Methods for Acanthamoeba Keratitis Diagnosis.

PubMed

Peretz, Avi; Geffen, Yuval; Socea, Soergiu D; Pastukh, Nina; Graffi, Shmuel

2015-08-01

Acanthamoeba keratitis (AK), a potentially blinding infection of the cornea, is caused by a free-living protozoan. Culture and microscopic examination of corneal scraping tissue material is the conventional method for identifying Acanthamoeba. In this article, we compared several methods for AK diagnosis of 32 patients: microscopic examination using fluorescent dye, specific culture on growth media-non-nutrient agar (NNA), culture on liquid growth media-peptone yeast glucose (PYG), and TYI-S-33. AK was found in 14 patients. Thirteen of the specimens were found AK positive by fluorescence microscopic examination, 11 specimens were found AK positive on PYG growth media, and 9 specimens were found AK positive on TYI-S-33 growth media. Only five specimens were found AK positive on NNA growth media. Therefore, we recommend using fluorescence microscopy technique and culture method, especially PYG liquid media. © The American Society of Tropical Medicine and Hygiene.

Augmented microscopy: real-time overlay of bright-field and near-infrared fluorescence images

PubMed Central

Watson, Jeffrey R.; Gainer, Christian F.; Martirosyan, Nikolay; Skoch, Jesse; Lemole, G. Michael; Anton, Rein; Romanowski, Marek

2015-01-01

Abstract. Intraoperative applications of near-infrared (NIR) fluorescent contrast agents can be aided by instrumentation capable of merging the view of surgical field with that of NIR fluorescence. We demonstrate augmented microscopy, an intraoperative imaging technique in which bright-field (real) and electronically processed NIR fluorescence (synthetic) images are merged within the optical path of a stereomicroscope. Under luminance of 100,000 lx, representing typical illumination of the surgical field, the augmented microscope detects 189 nM concentration of indocyanine green and produces a composite of the real and synthetic images within the eyepiece of the microscope at 20 fps. Augmentation described here can be implemented as an add-on module to visualize NIR contrast agents, laser beams, or various types of electronic data within the surgical microscopes commonly used in neurosurgical, cerebrovascular, otolaryngological, and ophthalmic procedures. PMID:26440760

Any Way You Slice It—A Comparison of Confocal Microscopy Techniques

PubMed Central

Jonkman, James

2015-01-01

The confocal fluorescence microscope has become a popular tool for life sciences researchers, primarily because of its ability to remove blur from outside of the focal plane of the image. Several different kinds of confocal microscopes have been developed, each with advantages and disadvantages. This article will cover the grid confocal, classic confocal laser-scanning microscope (CLSM), the resonant scanning-CLSM, and the spinning-disk confocal microscope. The way each microscope technique works, the best applications the technique is suited for, the limitations of the technique, and new developments for each technology will be presented. Researchers who have access to a range of different confocal microscopes (e.g., through a local core facility) should find this paper helpful for choosing the best confocal technology for specific imaging applications. Others with funding to purchase an instrument should find the article helpful in deciding which technology is ideal for their area of research. PMID:25802490

Use of a night vision intensifier for direct visualization by eye of far-red and near-infrared fluorescence through an optical microscope.

PubMed

Siddiqi, M A; Kilduff, G M; Gearhart, J D

2003-11-01

We describe the design, construction and testing of a prototype device that allows the direct visualization by eye of far-red and near-infrared (NIR) fluorescence through an optical microscope. The device incorporates a gallium arsenide (GaAs) image intensifier, typically utilized in low-light or 'night vision' applications. The intensifier converts far-red and NIR light into electrons and then into green light, which is visible to the human eye. The prototype makes possible the direct, real-time viewing by eye of normally invisible far-red and NIR fluorescence from a wide variety of fluorophores, using the full field of view of the microscope to which it is applied. The high sensitivity of the image intensifier facilitates the viewing of a wide variety of photosensitive specimens, including live cells and embryos, at vastly reduced illumination levels in both fluorescence and bright-field microscopy. Modifications to the microscope are not required in order to use the prototype, which is fully compatible with all current fluorescence techniques. Refined versions of the prototype device will have broad research and clinical applications.

Physics and engineering aspects of cell and tissue imaging systems: microscopic devices and computer assisted diagnosis.

PubMed

Chen, Xiaodong; Ren, Liqiang; Zheng, Bin; Liu, Hong

2013-01-01

The conventional optical microscopes have been used widely in scientific research and in clinical practice. The modern digital microscopic devices combine the power of optical imaging and computerized analysis, archiving and communication techniques. It has a great potential in pathological examinations for improving the efficiency and accuracy of clinical diagnosis. This chapter reviews the basic optical principles of conventional microscopes, fluorescence microscopes and electron microscopes. The recent developments and future clinical applications of advanced digital microscopic imaging methods and computer assisted diagnosis schemes are also discussed.

Miniaturized integration of a fluorescence microscope

PubMed Central

Ghosh, Kunal K.; Burns, Laurie D.; Cocker, Eric D.; Nimmerjahn, Axel; Ziv, Yaniv; Gamal, Abbas El; Schnitzer, Mark J.

2013-01-01

The light microscope is traditionally an instrument of substantial size and expense. Its miniaturized integration would enable many new applications based on mass-producible, tiny microscopes. Key prospective usages include brain imaging in behaving animals towards relating cellular dynamics to animal behavior. Here we introduce a miniature (1.9 g) integrated fluorescence microscope made from mass-producible parts, including semiconductor light source and sensor. This device enables high-speed cellular-level imaging across ∼0.5 mm2 areas in active mice. This capability allowed concurrent tracking of Ca2+ spiking in >200 Purkinje neurons across nine cerebellar microzones. During mouse locomotion, individual microzones exhibited large-scale, synchronized Ca2+ spiking. This is a mesoscopic neural dynamic missed by prior techniques for studying the brain at other length scales. Overall, the integrated microscope is a potentially transformative technology that permits distribution to many animals and enables diverse usages, such as portable diagnostics or microscope arrays for large-scale screens. PMID:21909102

Miniaturized integration of a fluorescence microscope.

PubMed

Ghosh, Kunal K; Burns, Laurie D; Cocker, Eric D; Nimmerjahn, Axel; Ziv, Yaniv; Gamal, Abbas El; Schnitzer, Mark J

2011-09-11

The light microscope is traditionally an instrument of substantial size and expense. Its miniaturized integration would enable many new applications based on mass-producible, tiny microscopes. Key prospective usages include brain imaging in behaving animals for relating cellular dynamics to animal behavior. Here we introduce a miniature (1.9 g) integrated fluorescence microscope made from mass-producible parts, including a semiconductor light source and sensor. This device enables high-speed cellular imaging across ∼0.5 mm2 areas in active mice. This capability allowed concurrent tracking of Ca2+ spiking in >200 Purkinje neurons across nine cerebellar microzones. During mouse locomotion, individual microzones exhibited large-scale, synchronized Ca2+ spiking. This is a mesoscopic neural dynamic missed by prior techniques for studying the brain at other length scales. Overall, the integrated microscope is a potentially transformative technology that permits distribution to many animals and enables diverse usages, such as portable diagnostics or microscope arrays for large-scale screens.

Confocal Fluorescence Microscopy of Mung Beanleaves

NASA Astrophysics Data System (ADS)

Chen, Zhiwei; Liu, Dongwu

Recently, confocal microscope has become a routine technique and indispensable tool for cell biological studies and molecular investigations. The light emitted from the point out-of-focus is blocked by the pinhole and can not reach the detector, which is one of the critical features of the confocal microscope. In present studies, the probes acridine orange (AO) and rhodamine-123 were used to research stoma and mitochondria of mung bean leaves, respectively. The results indicated that the stomatal guard cells and mitochondria were clearly seen in epidermic tissue of mung bean leaves. Taken together, it is a good method to research plant cells with confocal microscope and fluorescence probes.

Single-shot optical sectioning using two-color probes in HiLo fluorescence microscopy.

PubMed

Muro, Eleonora; Vermeulen, Pierre; Ioannou, Andriani; Skourides, Paris; Dubertret, Benoit; Fragola, Alexandra; Loriette, Vincent

2011-06-08

We describe a wide-field fluorescence microscope setup which combines HiLo microscopy technique with the use of a two-color fluorescent probe. It allows one-shot fluorescence optical sectioning of thick biological moving sample which is illuminated simultaneously with a flat and a structured pattern at two different wavelengths. Both homogenous and structured fluorescence images are spectrally separated at detection and combined similarly with the HiLo microscopy technique. We present optically sectioned full-field images of Xenopus laevis embryos acquired at 25 images/s frame rate. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Measurement of resistance to solute transport across surfactant-laden interfaces using a Fluorescence Recovery After Photobleaching (FRAP) technique

NASA Technical Reports Server (NTRS)

Browne, Edward P.; Nivaggioli, Thierry; Hatton, T. Alan

1994-01-01

A noninvasive fluorescence recovery after photobleaching (FRAP) technique is under development to measure interfacial transport in two phase systems without disturbing the interface. The concentration profiles of a probe solute are measured in both sides of the interface by argon-ion laser, and the system relaxation is then monitored by a microscope-mounted CCD camera.

Fluorescence lifetime imaging microscopy using near-infrared contrast agents.

PubMed

Nothdurft, R; Sarder, P; Bloch, S; Culver, J; Achilefu, S

2012-08-01

Although single-photon fluorescence lifetime imaging microscopy (FLIM) is widely used to image molecular processes using a wide range of excitation wavelengths, the captured emission of this technique is confined to the visible spectrum. Here, we explore the feasibility of utilizing near-infrared (NIR) fluorescent molecular probes with emission >700 nm for FLIM of live cells. The confocal microscope is equipped with a 785 nm laser diode, a red-enhanced photomultiplier tube, and a time-correlated single photon counting card. We demonstrate that our system reports the lifetime distributions of NIR fluorescent dyes, cypate and DTTCI, in cells. In cells labelled separately or jointly with these dyes, NIR FLIM successfully distinguishes their lifetimes, providing a method to sort different cell populations. In addition, lifetime distributions of cells co-incubated with these dyes allow estimate of the dyes' relative concentrations in complex cellular microenvironments. With the heightened interest in fluorescence lifetime-based small animal imaging using NIR fluorophores, this technique further serves as a bridge between in vitro spectroscopic characterization of new fluorophore lifetimes and in vivo tissue imaging. © 2012 The Author Journal of Microscopy © 2012 Royal Microscopical Society.

Closed loop adaptive optics for microscopy without a wavefront sensor.

PubMed

Kner, Peter; Winoto, Lukman; Agard, David A; Sedat, John W

2010-02-24

A three-dimensional wide-field image of a small fluorescent bead contains more than enough information to accurately calculate the wavefront in the microscope objective back pupil plane using the phase retrieval technique. The phase-retrieved wavefront can then be used to set a deformable mirror to correct the point-spread function (PSF) of the microscope without the use of a wavefront sensor. This technique will be useful for aligning the deformable mirror in a widefield microscope with adaptive optics and could potentially be used to correct aberrations in samples where small fluorescent beads or other point sources are used as reference beacons. Another advantage is the high resolution of the retrieved wavefont as compared with current Shack-Hartmann wavefront sensors. Here we demonstrate effective correction of the PSF in 3 iterations. Starting from a severely aberrated system, we achieve a Strehl ratio of 0.78 and a greater than 10-fold increase in maximum intensity.

Two-Photon Excitation, Fluorescence Microscopy, and Quantitative Measurement of Two-Photon Absorption Cross Sections

NASA Astrophysics Data System (ADS)

DeArmond, Fredrick Michael

As optical microscopy techniques continue to improve, most notably the development of super-resolution optical microscopy which garnered the Nobel Prize in Chemistry in 2014, renewed emphasis has been placed on the development and use of fluorescence microscopy techniques. Of particular note is a renewed interest in multiphoton excitation due to a number of inherent properties of the technique including simplified optical filtering, increased sample penetration, and inherently confocal operation. With this renewed interest in multiphoton fluorescence microscopy, comes an increased demand for robust non-linear fluorescent markers, and characterization of the associated tool set. These factors have led to an experimental setup to allow a systematized approach for identifying and characterizing properties of fluorescent probes in the hopes that the tool set will provide researchers with additional information to guide their efforts in developing novel fluorophores suitable for use in advanced optical microscopy techniques as well as identifying trends for their synthesis. Hardware was setup around a software control system previously developed. Three experimental tool sets were set up, characterized, and applied over the course of this work. These tools include scanning multiphoton fluorescence microscope with single molecule sensitivity, an interferometric autocorrelator for precise determination of the bandwidth and pulse width of the ultrafast Titanium Sapphire excitation source, and a simplified fluorescence microscope for the measurement of two-photon absorption cross sections. Resulting values for two-photon absorption cross sections and two-photon absorption action cross sections for two standardized fluorophores, four commercially available fluorophores, and ten novel fluorophores are presented as well as absorption and emission spectra.

Immunofluorescence Staining — EDRN Public Portal

Cancer.gov

Direct immunofluorescence method is used to detect the deposit of immunoglobulins, complement components, fibrinogen, etc. in tissues. This technique is usually performed on frozen sections. The primary antibody is conjugated to fluorescein binds directly with the antigen and can be detected by the fluorescent tag using a fluorescent microscope.

Biological applications of confocal fluorescence polarization microscopy

NASA Astrophysics Data System (ADS)

Bigelow, Chad E.

Fluorescence polarization microscopy is a powerful modality capable of sensing changes in the physical properties and local environment of fluorophores. In this thesis we present new applications for the technique in cancer diagnosis and treatment and explore the limits of the modality in scattering media. We describe modifications to our custom-built confocal fluorescence microscope that enable dual-color imaging, optical fiber-based confocal spectroscopy and fluorescence polarization imaging. Experiments are presented that indicate the performance of the instrument for all three modalities. The limits of confocal fluorescence polarization imaging in scattering media are explored and the microscope parameters necessary for accurate polarization images in this regime are determined. A Monte Carlo routine is developed to model the effect of scattering on images. Included in it are routines to track the polarization state of light using the Mueller-Stokes formalism and a model for fluorescence generation that includes sampling the excitation light polarization ellipse, Brownian motion of excited-state fluorophores in solution, and dipole fluorophore emission. Results from this model are compared to experiments performed on a fluorophore-embedded polymer rod in a turbid medium consisting of polystyrene microspheres in aqueous suspension. We demonstrate the utility of the fluorescence polarization imaging technique for removal of contaminating autofluorescence and for imaging photodynamic therapy drugs in cell monolayers. Images of cells expressing green fluorescent protein are extracted from contaminating fluorescein emission. The distribution of meta-tetrahydroxypheny1chlorin in an EMT6 cell monolayer is also presented. A new technique for imaging enzyme activity is presented that is based on observing changes in the anisotropy of fluorescently-labeled substrates. Proof-of-principle studies are performed in a model system consisting of fluorescently labeled bovine serum albumin attached to sepharose beads. The action of trypsin and proteinase K on the albumin is monitored to demonstrate validity of the technique. Images of the processing of the albumin in J774 murine macrophages are also presented indicating large intercellular differences in enzyme activity. Future directions for the technique are also presented, including the design of enzyme probes specific for prostate specific antigen based on fluorescently-labeled dendrimers. A technique for enzyme imaging based on extracellular autofluorescence is also proposed.

Real-time imaging of nitric oxide production in living cells with 1,3,5,7-tetramethyl-2,6-dicarbethoxy-8-(3',4'-diaminophenyl)-difluoroboradiaza-s-indacence by invert fluorescence microscope.

PubMed

Huang, Ke-Jing; Wang, Hong; Ma, Ming; Zhang, Xian; Zhang, Hua-Shan

2007-02-01

Although the importance of nitric oxide (NO) as a signalling molecule in many biological processes is becoming increasingly evident, many proposed and potential biological functions of NO still remain unclear. Bioimaging is a good technique to visualize observation of nitric oxide in biological samples. In this report, a fluorescent probe, 1,3,5,7-tetramethyl-2,6-dicarbethoxy-8-(3',4'-diaminophenyl)-difluoroboradiaza-s-indacence (TMDCDABODIPY), has been first applied to real-time image NO produced in PC12 cells, Sf9 cells and human vascular endothelial cells at the presence of l-arginine with inverted fluorescence microscope. NO production in the cells is successfully captured and imaged with fine temporal and spatial resolution. The results prove that the probe combined with inverted fluorescence microscope can be developed into a sensitive and selective method for further study of NO release from cells.

Two-photon imaging in living brain slices.

PubMed

Mainen, Z F; Maletic-Savatic, M; Shi, S H; Hayashi, Y; Malinow, R; Svoboda, K

1999-06-01

Two-photon excitation laser scanning microscopy (TPLSM) has become the tool of choice for high-resolution fluorescence imaging in intact neural tissues. Compared with other optical techniques, TPLSM allows high-resolution imaging and efficient detection of fluorescence signal with minimal photobleaching and phototoxicity. The advantages of TPLSM are especially pronounced in highly scattering environments such as the brain slice. Here we describe our approaches to imaging various aspects of synaptic function in living brain slices. To combine several imaging modes together with patch-clamp electrophysiological recordings we found it advantageous to custom-build an upright microscope. Our design goals were primarily experimental convenience and efficient collection of fluorescence. We describe our TPLSM imaging system and its performance in detail. We present dynamic measurements of neuronal morphology of neurons expressing green fluorescent protein (GFP) and GFP fusion proteins as well as functional imaging of calcium dynamics in individual dendritic spines. Although our microscope is a custom instrument, its key advantages can be easily implemented as a modification of commercial laser scanning microscopes. Copyright 1999 Academic Press.

Fluorescent speckle microscopy of microtubules: how low can you go?

PubMed

Waterman-Storer, C M; Salmon, E D

1999-12-01

Fluorescent speckle microscopy (FSM) is a new technique for visualizing the movement, assembly, and turnover of macromolecular assemblies like the cytoskeleton in living cells. In this method, contrast is created by coassembly of a small fraction of fluorescent subunits in a pool of unlabeled subunits. Random variation in association creates a nonuniform "fluorescent speckle" pattern. Fluorescent speckle movements in time-lapse recordings stand out to the eye and can be measured. Because fluorescent speckles represent fiduciary marks on the polymer lattice, FSM provides the opportunity for the first time to see the 2- and 3-dimensional trajectories of lattice movements within large arrays of polymers as well as identifying sites of assembly and disassembly of individual polymers. The technique works with either microinjection of fluorescently labeled subunits or expression of subunits ligated to green fluorescent protein (GFP). We have found for microtubules assembled in vitro that speckles containing one fluorophore can be detected and recorded using a conventional wide-field epi-fluorescence light microscope and digital imaging with a low noise cooled CCD camera. In living cells, optimal speckle contrast occurs at fractions of labeled tubulin of approximately 0.1-0.5% where the fluorescence of each speckle corresponds to one to seven fluorophores per resolvable unit (approximately 0.27 microm) in the microscope. This small fraction of labeled subunits significantly reduces out-of-focus fluorescence and greatly improves visibility of fluorescently labeled structures and their dynamics in thick regions of living cells.

Dual-modality single particle orientation and rotational tracking of intracellular transport of nanocargos.

PubMed

Sun, Wei; Gu, Yan; Wang, Gufeng; Fang, Ning

2012-01-17

The single particle orientation and rotational tracking (SPORT) technique was introduced recently to follow the rotational motion of plasmonic gold nanorod under a differential interference contrast (DIC) microscope. In biological studies, however, cellular activities usually involve a multiplicity of molecules; thus, tracking the motion of a single molecule/object is insufficient. Fluorescence-based techniques have long been used to follow the spatial and temporal distributions of biomolecules of interest thanks to the availability of multiplexing fluorescent probes. To know the type and number of molecules and the timing of their involvement in a biological process under investigation by SPORT, we constructed a dual-modality DIC/fluorescence microscope to simultaneously image fluorescently tagged biomolecules and plasmonic nanoprobes in living cells. With the dual-modality SPORT technique, the microtubule-based intracellular transport can be unambiguously identified while the dynamic orientation of nanometer-sized cargos can be monitored at video rate. Furthermore, the active transport on the microtubule can be easily separated from the diffusion before the nanocargo docks on the microtubule or after it undocks from the microtubule. The potential of dual-modality SPORT is demonstrated for shedding new light on unresolved questions in intracellular transport.

Micro axial tomography: A miniaturized, versatile stage device to overcome resolution anisotropy in fluorescence light microscopy

NASA Astrophysics Data System (ADS)

Staier, Florian; Eipel, Heinz; Matula, Petr; Evsikov, Alexei V.; Kozubek, Michal; Cremer, Christoph; Hausmann, Michael

2011-09-01

With the development of novel fluorescence techniques, high resolution light microscopy has become a challenging technique for investigations of the three-dimensional (3D) micro-cosmos in cells and sub-cellular components. So far, all fluorescence microscopes applied for 3D imaging in biosciences show a spatially anisotropic point spread function resulting in an anisotropic optical resolution or point localization precision. To overcome this shortcoming, micro axial tomography was suggested which allows object tilting on the microscopic stage and leads to an improvement in localization precision and spatial resolution. Here, we present a miniaturized device which can be implemented in a motor driven microscope stage. The footprint of this device corresponds to a standard microscope slide. A special glass fiber can manually be adjusted in the object space of the microscope lens. A stepwise fiber rotation can be controlled by a miniaturized stepping motor incorporated into the device. By means of a special mounting device, test particles were fixed onto glass fibers, optically localized with high precision, and automatically rotated to obtain views from different perspective angles under which distances of corresponding pairs of objects were determined. From these angle dependent distance values, the real 3D distance was calculated with a precision in the ten nanometer range (corresponding here to an optical resolution of 10-30 nm) using standard microscopic equipment. As a proof of concept, the spindle apparatus of a mature mouse oocyte was imaged during metaphase II meiotic arrest under different perspectives. Only very few images registered under different rotation angles are sufficient for full 3D reconstruction. The results indicate the principal advantage of the micro axial tomography approach for many microscopic setups therein and also those of improved resolutions as obtained by high precision localization determination.

Chemical reactivation of quenched fluorescent protein molecules enables resin-embedded fluorescence microimaging

PubMed Central

Xiong, Hanqing; Zhou, Zhenqiao; Zhu, Mingqiang; Lv, Xiaohua; Li, Anan; Li, Shiwei; Li, Longhui; Yang, Tao; Wang, Siming; Yang, Zhongqin; Xu, Tonghui; Luo, Qingming; Gong, Hui; Zeng, Shaoqun

2014-01-01

Resin embedding is a well-established technique to prepare biological specimens for microscopic imaging. However, it is not compatible with modern green-fluorescent protein (GFP) fluorescent-labelling technique because it significantly quenches the fluorescence of GFP and its variants. Previous empirical optimization efforts are good for thin tissue but not successful on macroscopic tissue blocks as the quenching mechanism remains uncertain. Here we show most of the quenched GFP molecules are structurally preserved and not denatured after routine embedding in resin, and can be chemically reactivated to a fluorescent state by alkaline buffer during imaging. We observe up to 98% preservation in yellow-fluorescent protein case, and improve the fluorescence intensity 11.8-fold compared with unprocessed samples. We demonstrate fluorescence microimaging of resin-embedded EGFP/EYFP-labelled tissue block without noticeable loss of labelled structures. This work provides a turning point for the imaging of fluorescent protein-labelled specimens after resin embedding. PMID:24886825

Hyperspectral Imaging and Spectroscopy of Fluorescently Coupled Acyl-CoA: Cholesterol Acyltransferase in Insect Cells

NASA Technical Reports Server (NTRS)

Malak, H.; Mahtani, H.; Herman, P.; Vecer, J.; Lu, X.; Chang, T. Y.; Richmond, Robert C.; Whitaker, Ann F. (Technical Monitor)

2001-01-01

A high-performance hyperspectral imaging module with high throughput of light suitable for low-intensity fluorescence microscopic imaging and subsequent analysis, including single-pixel-defined emission spectroscopy, was tested on Sf21 insect cells expressing green fluorescence associated with recombinant green fluorescent protein linked or not with the membrane protein acyl-CoA:cholesterol acyltransferase. The imager utilized the phenomenon of optical activity as a new technique providing information over a spectral range of 220-1400 nm, and was inserted between the microscope and an 8-bit CCD video-rate camera. The resulting fluorescence image did not introduce observable image aberrations. The images provided parallel acquisition of well resolved concurrent spatial and spectral information such that fluorescence associated with green fluorescent protein alone was demonstrated to be diffuse within the Sf21 insect cell, and that green fluorescence associated with the membrane protein was shown to be specifically concentrated within regions of the cell cytoplasm. Emission spectra analyzed from different regions of the fluorescence image showed blue shift specific for the regions of concentration associated with the membrane protein.

Measurement of drug-target engagement in live cells by two-photon fluorescence anisotropy imaging.

PubMed

Vinegoni, Claudio; Fumene Feruglio, Paolo; Brand, Christian; Lee, Sungon; Nibbs, Antoinette E; Stapleton, Shawn; Shah, Sunil; Gryczynski, Ignacy; Reiner, Thomas; Mazitschek, Ralph; Weissleder, Ralph

2017-07-01

The ability to directly image and quantify drug-target engagement and drug distribution with subcellular resolution in live cells and whole organisms is a prerequisite to establishing accurate models of the kinetics and dynamics of drug action. Such methods would thus have far-reaching applications in drug development and molecular pharmacology. We recently presented one such technique based on fluorescence anisotropy, a spectroscopic method based on polarization light analysis and capable of measuring the binding interaction between molecules. Our technique allows the direct characterization of target engagement of fluorescently labeled drugs, using fluorophores with a fluorescence lifetime larger than the rotational correlation of the bound complex. Here we describe an optimized protocol for simultaneous dual-channel two-photon fluorescence anisotropy microscopy acquisition to perform drug-target measurements. We also provide the necessary software to implement stream processing to visualize images and to calculate quantitative parameters. The assembly and characterization part of the protocol can be implemented in 1 d. Sample preparation, characterization and imaging of drug binding can be completed in 2 d. Although currently adapted to an Olympus FV1000MPE microscope, the protocol can be extended to other commercial or custom-built microscopes.

Adaptive optics plug-and-play setup for high-resolution microscopes with multi-actuator adaptive lens

NASA Astrophysics Data System (ADS)

Quintavalla, M.; Pozzi, P.; Verhaegen, Michelle; Bijlsma, Hielke; Verstraete, Hans; Bonora, S.

2018-02-01

Adaptive Optics (AO) has revealed as a very promising technique for high-resolution microscopy, where the presence of optical aberrations can easily compromise the image quality. Typical AO systems however, are almost impossible to implement on commercial microscopes. We propose a simple approach by using a Multi-actuator Adaptive Lens (MAL) that can be inserted right after the objective and works in conjunction with an image optimization software allowing for a wavefront sensorless correction. We presented the results obtained on several commercial microscopes among which a confocal microscope, a fluorescence microscope, a light sheet microscope and a multiphoton microscope.

Flash lamp-excited time-resolved fluorescence microscope suppresses autofluorescence in water concentrates to deliver an 11-fold increase in signal-to-noise ratio.

PubMed

Connally, Russell; Veal, Duncan; Piper, James

2004-01-01

The ubiquity of naturally fluorescing components (autofluorophores) encountered in most biological samples hinders the detection and identification of labeled targets through fluorescence-based techniques. Time-resolved fluorescence (TRF) is a technique by which the effects of autofluorescence are reduced by using specific fluorescent labels with long fluorescence lifetimes (compared with autofluorophores) in conjunction with time-gated detection. A time-resolved fluorescence microscope (TRFM) is described that is based on a standard epifluorescence microscope modified by the addition of a pulsed excitation source and an image-intensified time-gateable CCD camera. The choice of pulsed excitation source for TRFM has a large impact on the price and performance of the instrument. A flash lamp with rapid discharge characteristics was selected for our instrument because of the high spectral energy in the UV region and short pulse length. However, the flash output decayed with an approximate lifetime of 18 micros and the TRFM required a long-lived lanthanide chelate label to ensure that probe fluorescence was visible after decay of the flash plasma. We synthesized a recently reported fluorescent chelate (BHHCT) and conjugated it to a monoclonal antibody directed against the waterborne parasite Giardia lamblia. For a 600-nm bandpass filter set and a gate delay of 60 micros, the TRFM provided an 11.3-fold improvement in the signal-to-noise ratio (S/N) of labeled Giardia over background. A smaller gain in an SNR of 9.69-fold was achieved with a 420-nm longpass filter set; however, the final contrast ratio between labeled cyst and background was higher (11.3 versus 8.5). Despite the decay characteristics of the light pulse, flash lamps have many practical advantages compared with optical chopper wheels and modulated lasers for applications in TRFM.

Deep two-photon microscopic imaging through brain tissue using the second singlet state from fluorescent agent chlorophyll α in spinach leaf

NASA Astrophysics Data System (ADS)

Shi, Lingyan; Rodríguez-Contreras, Adrián; Budansky, Yury; Pu, Yang; An Nguyen, Thien; Alfano, Robert R.

2014-06-01

Two-photon (2P) excitation of the second singlet (S) state was studied to achieve deep optical microscopic imaging in brain tissue when both the excitation (800 nm) and emission (685 nm) wavelengths lie in the "tissue optical window" (650 to 950 nm). S2 state technique was used to investigate chlorophyll α (Chl α) fluorescence inside a spinach leaf under a thick layer of freshly sliced rat brain tissue in combination with 2P microscopic imaging. Strong emission at the peak wavelength of 685 nm under the 2P S state of Chl α enabled the imaging depth up to 450 μm through rat brain tissue.

Deep two-photon microscopic imaging through brain tissue using the second singlet state from fluorescent agent chlorophyll α in spinach leaf.

PubMed

Shi, Lingyan; Rodríguez-Contreras, Adrián; Budansky, Yury; Pu, Yang; Nguyen, Thien An; Alfano, Robert R

2014-06-01

Two-photon (2P) excitation of the second singlet (S₂) state was studied to achieve deep optical microscopic imaging in brain tissue when both the excitation (800 nm) and emission (685 nm) wavelengths lie in the "tissue optical window" (650 to 950 nm). S₂ state technique was used to investigate chlorophyll α (Chl α) fluorescence inside a spinach leaf under a thick layer of freshly sliced rat brain tissue in combination with 2P microscopic imaging. Strong emission at the peak wavelength of 685 nm under the 2P S₂ state of Chl α enabled the imaging depth up to 450 μm through rat brain tissue.

Closed loop adaptive optics for microscopy without a wavefront sensor

PubMed Central

Kner, Peter; Winoto, Lukman; Agard, David A.; Sedat, John W.

2013-01-01

A three-dimensional wide-field image of a small fluorescent bead contains more than enough information to accurately calculate the wavefront in the microscope objective back pupil plane using the phase retrieval technique. The phase-retrieved wavefront can then be used to set a deformable mirror to correct the point-spread function (PSF) of the microscope without the use of a wavefront sensor. This technique will be useful for aligning the deformable mirror in a widefield microscope with adaptive optics and could potentially be used to correct aberrations in samples where small fluorescent beads or other point sources are used as reference beacons. Another advantage is the high resolution of the retrieved wavefont as compared with current Shack-Hartmann wavefront sensors. Here we demonstrate effective correction of the PSF in 3 iterations. Starting from a severely aberrated system, we achieve a Strehl ratio of 0.78 and a greater than 10-fold increase in maximum intensity. PMID:24392198

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miranda, Adelaide; De Beule, Pieter A. A., E-mail: pieter.de-beule@inl.int; Martins, Marco

Combined microscopy techniques offer the life science research community a powerful tool to investigate complex biological systems and their interactions. Here, we present a new combined microscopy platform based on fluorescence optical sectioning microscopy through aperture correlation microscopy with a Differential Spinning Disk (DSD) and nanomechanical mapping with an Atomic Force Microscope (AFM). The illumination scheme of the DSD microscope unit, contrary to standard single or multi-point confocal microscopes, provides a time-independent illumination of the AFM cantilever. This enables a distortion-free simultaneous operation of fluorescence optical sectioning microscopy and atomic force microscopy with standard probes. In this context, we discussmore » sample heating due to AFM cantilever illumination with fluorescence excitation light. Integration of a DSD fluorescence optical sectioning unit with an AFM platform requires mitigation of mechanical noise transfer of the spinning disk. We identify and present two solutions to almost annul this noise in the AFM measurement process. The new combined microscopy platform is applied to the characterization of a DOPC/DOPS (4:1) lipid structures labelled with a lipophilic cationic indocarbocyanine dye deposited on a mica substrate.« less

Optimal model-based sensorless adaptive optics for epifluorescence microscopy.

PubMed

Pozzi, Paolo; Soloviev, Oleg; Wilding, Dean; Vdovin, Gleb; Verhaegen, Michel

2018-01-01

We report on a universal sample-independent sensorless adaptive optics method, based on modal optimization of the second moment of the fluorescence emission from a point-like excitation. Our method employs a sample-independent precalibration, performed only once for the particular system, to establish the direct relation between the image quality and the aberration. The method is potentially applicable to any form of microscopy with epifluorescence detection, including the practically important case of incoherent fluorescence emission from a three dimensional object, through minor hardware modifications. We have applied the technique successfully to a widefield epifluorescence microscope and to a multiaperture confocal microscope.

Hyper-spectral imaging in scanning-confocal-fluorescence microscopy using a novel broadband diffractive optic

NASA Astrophysics Data System (ADS)

Wang, Peng; Ebeling, Carl G.; Gerton, Jordan; Menon, Rajesh

In this paper, we demonstrate hyper-spectral imaging of fluorescent microspheres in a scanning-confocal-fluorescence microscope by spatially dispersing the spectra using a novel broadband diffractive optic, and applying a nonlinear optimization technique to extract the full-incident spectra. This broadband diffractive optic has a designed optical efficiency of over 90% across the entire visible spectrum. We used this technique to create two-color images of two fluorophores and also extracted their emission spectra with good fidelity. This method can be extended to image both spatially and spectrally overlapping fluorescent samples. Full control in the number of emission spectra and the feasibility of enhanced imaging speed are demonstrated as well.

A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging

PubMed Central

Poland, Simon P.; Krstajić, Nikola; Monypenny, James; Coelho, Simao; Tyndall, David; Walker, Richard J.; Devauges, Viviane; Richardson, Justin; Dutton, Neale; Barber, Paul; Li, David Day-Uei; Suhling, Klaus; Ng, Tony; Henderson, Robert K.; Ameer-Beg, Simon M.

2015-01-01

We demonstrate diffraction limited multiphoton imaging in a massively parallel, fully addressable time-resolved multi-beam multiphoton microscope capable of producing fluorescence lifetime images with sub-50ps temporal resolution. This imaging platform offers a significant improvement in acquisition speed over single-beam laser scanning FLIM by a factor of 64 without compromising in either the temporal or spatial resolutions of the system. We demonstrate FLIM acquisition at 500 ms with live cells expressing green fluorescent protein. The applicability of the technique to imaging protein-protein interactions in live cells is exemplified by observation of time-dependent FRET between the epidermal growth factor receptor (EGFR) and the adapter protein Grb2 following stimulation with the receptor ligand. Furthermore, ligand-dependent association of HER2-HER3 receptor tyrosine kinases was observed on a similar timescale and involved the internalisation and accumulation or receptor heterodimers within endosomes. These data demonstrate the broad applicability of this novel FLIM technique to the spatio-temporal dynamics of protein-protein interaction. PMID:25780724

Ultrafast superresolution fluorescence imaging with spinning disk confocal microscope optics.

PubMed

Hayashi, Shinichi; Okada, Yasushi

2015-05-01

Most current superresolution (SR) microscope techniques surpass the diffraction limit at the expense of temporal resolution, compromising their applications to live-cell imaging. Here we describe a new SR fluorescence microscope based on confocal microscope optics, which we name the spinning disk superresolution microscope (SDSRM). Theoretically, the SDSRM is equivalent to a structured illumination microscope (SIM) and achieves a spatial resolution of 120 nm, double that of the diffraction limit of wide-field fluorescence microscopy. However, the SDSRM is 10 times faster than a conventional SIM because SR signals are recovered by optical demodulation through the stripe pattern of the disk. Therefore a single SR image requires only a single averaged image through the rotating disk. On the basis of this theory, we modified a commercial spinning disk confocal microscope. The improved resolution around 120 nm was confirmed with biological samples. The rapid dynamics of micro-tubules, mitochondria, lysosomes, and endosomes were observed with temporal resolutions of 30-100 frames/s. Because our method requires only small optical modifications, it will enable an easy upgrade from an existing spinning disk confocal to a SR microscope for live-cell imaging. © 2015 Hayashi and Okada. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Capillary array scanner for time-resolved detection and identification of fluorescently labelled DNA fragments.

PubMed

Neumann, M; Herten, D P; Dietrich, A; Wolfrum, J; Sauer, M

2000-02-25

The first capillary array scanner for time-resolved fluorescence detection in parallel capillary electrophoresis based on semiconductor technology is described. The system consists essentially of a confocal fluorescence microscope and a x,y-microscope scanning stage. Fluorescence of the labelled probe molecules was excited using a short-pulse diode laser emitting at 640 nm with a repetition rate of 50 MHz. Using a single filter system the fluorescence decays of different labels were detected by an avalanche photodiode in combination with a PC plug-in card for time-correlated single-photon counting (TCSPC). The time-resolved fluorescence signals were analyzed and identified by a maximum likelihood estimator (MLE). The x,y-microscope scanning stage allows for discontinuous, bidirectional scanning of up to 16 capillaries in an array, resulting in longer fluorescence collection times per capillary compared to scanners working in a continuous mode. Synchronization of the alignment and measurement process were developed to allow for data acquisition without overhead. Detection limits in the subzeptomol range for different dye molecules separated in parallel capillaries have been achieved. In addition, we report on parallel time-resolved detection and separation of more than 400 bases of single base extension DNA fragments in capillary array electrophoresis. Using only semiconductor technology the presented technique represents a low-cost alternative for high throughput DNA sequencing in parallel capillaries.

A combined confocal and magnetic resonance microscope for biological studies

NASA Astrophysics Data System (ADS)

Majors, Paul D.; Minard, Kevin R.; Ackerman, Eric J.; Holtom, Gary R.; Hopkins, Derek F.; Parkinson, Christopher I.; Weber, Thomas J.; Wind, Robert A.

2002-12-01

Complementary data acquired with different microscopy techniques provide a basis for establishing a more comprehensive understanding of cell function in health and disease, particularly when results acquired with different methodologies can be correlated in time and space. In this article, a novel microscope is described for studying live cells simultaneously with both confocal scanning laser fluorescence optical microscopy and magnetic resonance microscopy. The various design considerations necessary for integrating these two complementary techniques are discussed, the layout and specifications of the instrument are given, and examples of confocal and magnetic resonance images of large frog cells and model tumor spheroids obtained with the compound microscope are presented.

Dual-labeling with 5-aminolevulinic acid and fluorescein for fluorescence-guided resection of high-grade gliomas: technical note.

PubMed

Suero Molina, Eric; Wölfer, Johannes; Ewelt, Christian; Ehrhardt, André; Brokinkel, Benjamin; Stummer, Walter

2018-02-01

OBJECTIVE Fluorescence guidance with 5-aminolevulinic acid (5-ALA) helps improve resections of malignant gliomas. However, one limitation is the low intensity of blue light for background illumination. Fluorescein has recently been reintroduced into neurosurgery, and novel microscope systems are available for visualizing this fluorochrome, which highlights all perfused tissues but has limited selectivity for tumor detection. Here, the authors investigate a combination of both fluorochromes: 5-ALA for distinguishing tumor and fluorescein for providing tissue fluorescence of adjacent brain tissue. METHODS The authors evaluated 6 patients who harbored cerebral lesions suggestive of high-grade glioma. Patients received 5-ALA (20 mg/kg) orally 4 hours before induction of anesthesia. Low-dose fluorescein (3 mg/kg intravenous) was injected immediately after anesthesia induction. Pentero microscopes (equipped either with Yellow 560 or Blue 400 filters) were used to visualize fluorescence. To simultaneously visualize both fluorochromes, the Yellow 560 module was combined with external blue light illumination (D-light C System). RESULTS Fluorescein-induced fluorescence created a useful background for protoporphyrin IX (PPIX) fluorescence, which appeared orange to red, surrounded by greenly fluorescent normal brain and edematous tissue. Green brain-tissue fluorescence was helpful in augmenting background. Levels of blue illumination that were too strong obscured PPIX fluorescence. Unspecific extravasation of fluorescein was noted at resection margins, which did not interfere with PPIX fluorescence detection. CONCLUSIONS Dual labeling with both PPIX and fluorescein fluorescence is feasible and gives superior background information during fluorescence-guided resections. The authors believe that this technique carries potential as a next step in fluorescence-guided resections if it is completely integrated into the surgical microscope.

Pulp tissue in sex determination: A fluorescent microscopic study

PubMed Central

Nayar, Amit; Singh, Harkanwal Preet; Leekha, Swati

2014-01-01

Aims: To determine and compare the reliability of pulp tissue in determination of sex and to analyze whether caries have any effect on fluorescent body test. Materials and Methods: This study was carried on 50 maxillary and mandibular teeth (25 male teeth and 25 female teeth), which were indicated for extraction. The teeth are categorized into 5 groups, 10 each (5 from males and 5 from females) on the basis of caries progression. The pulp cells are stained with quinacrine hydrochloride and observed with fluorescent microscope for fluorescent body. Gender is determined by identification of Y chromosome fluorescence in dental pulp. Results: Fluorescent bodies were found to be more in sound teeth in males as the caries increase the mean percentage of fluorescent bodies observed decreases in males. We also observed the fluorescent spots in females, and the value of the spot increases in female as the caries progresses, thereby giving false positive results in females. Conclusion: Sex determination by fluorescent staining of the Y chromosome is a reliable technique in teeth with healthy pulps or caries with enamel or up to half way of dentin. Teeth with caries involving pulp cannot be used for sex determination. PMID:25125912

Novel snapshot hyperspectral imager for fluorescence imaging

NASA Astrophysics Data System (ADS)

Chandler, Lynn; Chandler, Andrea; Periasamy, Ammasi

2018-02-01

Hyperspectral imaging has emerged as a new technique for the identification and classification of biological tissue1. Benefitting recent developments in sensor technology, the new class of hyperspectral imagers can capture entire hypercubes with single shot operation and it shows great potential for real-time imaging in biomedical sciences. This paper explores the use of a SnapShot imager in fluorescence imaging via microscope for the very first time. Utilizing the latest imaging sensor, the Snapshot imager is both compact and attachable via C-mount to any commercially available light microscope. Using this setup, fluorescence hypercubes of several cells were generated, containing both spatial and spectral information. The fluorescence images were acquired with one shot operation for all the emission range from visible to near infrared (VIS-IR). The paper will present the hypercubes obtained images from example tissues (475-630nm). This study demonstrates the potential of application in cell biology or biomedical applications for real time monitoring.

Correlative Fluorescence and Electron Microscopy in 3D-Scanning Electron Microscope Perspective.

PubMed

Franks, Jonathan; Wallace, Callen T; Shibata, Masateru; Suga, Mitsuo; Erdman, Natasha; Stolz, Donna B; Watkins, Simon C

2017-04-03

The ability to correlate fluorescence microscopy (FM) and electron microscopy (EM) data obtained on biological (cell and tissue) specimens is essential to bridge the resolution gap between the data obtained by these different imaging techniques. In the past such correlations were limited to either EM navigation in two dimensions to the locations previously highlighted by fluorescence markers, or subsequent high-resolution acquisition of tomographic information using a TEM. We present a novel approach whereby a sample previously investigated by FM is embedded and subjected to sequential mechanical polishing and backscatter imaging by scanning electron microscope. The resulting three dimensional EM tomogram of the sample can be directly correlated to the FM data. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Pump-probe optical microscopy for imaging nonfluorescent chromophores.

PubMed

Wei, Lu; Min, Wei

2012-06-01

Many chromophores absorb light intensely but have undetectable fluorescence. Hence microscopy techniques other than fluorescence are highly desirable for imaging these chromophores inside live cells, tissues, and organisms. The recently developed pump-probe optical microscopy techniques provide fluorescence-free contrast mechanisms by employing several fundamental light-molecule interactions including excited state absorption, stimulated emission, ground state depletion, and the photothermal effect. By using the pump pulse to excite molecules and the subsequent probe pulse to interrogate the created transient states on a laser scanning microscope, pump-probe microscopy offers imaging capability with high sensitivity and specificity toward nonfluorescent chromophores. Single-molecule sensitivity has even been demonstrated. Here we review and summarize the underlying principles of this emerging class of molecular imaging techniques.

Laser-Induced Fluorescence Detection in High-Throughput Screening of Heterogeneous Catalysts and Single Cells Analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Su, Hui

2001-01-01

Laser-induced fluorescence detection is one of the most sensitive detection techniques and it has found enormous applications in various areas. The purpose of this research was to develop detection approaches based on laser-induced fluorescence detection in two different areas, heterogeneous catalysts screening and single cell study. First, the author introduced laser-induced imaging (LIFI) as a high-throughput screening technique for heterogeneous catalysts to explore the use of this high-throughput screening technique in discovery and study of various heterogeneous catalyst systems. This scheme is based on the fact that the creation or the destruction of chemical bonds alters the fluorescence properties ofmore » suitably designed molecules. By irradiating the region immediately above the catalytic surface with a laser, the fluorescence intensity of a selected product or reactant can be imaged by a charge-coupled device (CCD) camera to follow the catalytic activity as a function of time and space. By screening the catalytic activity of vanadium pentoxide catalysts in oxidation of naphthalene, they demonstrated LIFI has good detection performance and the spatial and temporal resolution needed for high-throughput screening of heterogeneous catalysts. The sample packing density can reach up to 250 x 250 subunits/cm 2 for 40-μm wells. This experimental set-up also can screen solid catalysts via near infrared thermography detection. In the second part of this dissertation, the author used laser-induced native fluorescence coupled with capillary electrophoresis (LINF-CE) and microscope imaging to study the single cell degranulation. On the basis of good temporal correlation with events observed through an optical microscope, they have identified individual peaks in the fluorescence electropherograms as serotonin released from the granular core on contact with the surrounding fluid.« less

Detection of Infectious Bovine Rhinotracheitis and Bovine Viral Diarrhea Viruses in the Nasal Epithelial Cells by the Direct Immunofluorescence Technique

PubMed Central

Silim, A.; Elazhary, M.A.S.Y.

1983-01-01

Nasal epithelial cells were collected by cotton swabs for the diagnosis in experimental and field cases of infectious bovine rhinotracheitis and field cases of bovine viral diarrhea in calves. A portion of the cells was washed twice in phosphate buffered saline and a 25 µL drop was placed on microscope slides. The cells were dried, fixed and stained according to the direct fluorescent antibody technique. Another portion of the same specimen was inoculated onto primary bovine skin cell cultures for virus isolation. In the experimental studies for infectious bovine rhinotracheitis, 29/35 specimens were positive by fluorescent antibody technique and 32/35 by cell culture and in the field cases, 22/119 were positive by fluorescent antibody technique and 19/119 by cell culture. In the field cases of bovine viral diarrhea, 28/69 samples were positive by fluorescent antibody technique and 14/69 by cell culture. When fluorescent antibody technique was performed on inoculated cell cultures a total of 24/69 specimens were positive for bovine viral diarrhea. The sensitivity of fluorescent antibody technique was thus comparable to that of cell culture method for infectious bovine rhinotracheitis and bovine viral diarrhea. ImagesFig. 1.Fig. 2.Fig. 3. PMID:6299484

Automated detection of fluorescent cells in in-resin fluorescence sections for integrated light and electron microscopy.

PubMed

Delpiano, J; Pizarro, L; Peddie, C J; Jones, M L; Griffin, L D; Collinson, L M

2018-04-26

Integrated array tomography combines fluorescence and electron imaging of ultrathin sections in one microscope, and enables accurate high-resolution correlation of fluorescent proteins to cell organelles and membranes. Large numbers of serial sections can be imaged sequentially to produce aligned volumes from both imaging modalities, thus producing enormous amounts of data that must be handled and processed using novel techniques. Here, we present a scheme for automated detection of fluorescent cells within thin resin sections, which could then be used to drive automated electron image acquisition from target regions via 'smart tracking'. The aim of this work is to aid in optimization of the data acquisition process through automation, freeing the operator to work on other tasks and speeding up the process, while reducing data rates by only acquiring images from regions of interest. This new method is shown to be robust against noise and able to deal with regions of low fluorescence. © 2018 The Authors. Journal of Microscopy published by JohnWiley & Sons Ltd on behalf of Royal Microscopical Society.

Structured illumination microscopy for dual-modality 3D sub-diffraction resolution fluorescence and refractive-index reconstruction

PubMed Central

Chowdhury, Shwetadwip; Eldridge, Will J.; Wax, Adam; Izatt, Joseph A.

2017-01-01

Though structured illumination (SI) microscopy is a popular imaging technique conventionally associated with fluorescent super-resolution, recent works have suggested its applicability towards sub-diffraction resolution coherent imaging with quantitative endogenous biological contrast. Here, we demonstrate that SI can efficiently integrate together the principles of fluorescent super-resolution and coherent synthetic aperture to achieve 3D dual-modality sub-diffraction resolution, fluorescence and refractive-index (RI) visualizations of biological samples. We experimentally demonstrate this framework by introducing a SI microscope capable of 3D sub-diffraction resolution fluorescence and RI imaging, and verify its biological visualization capabilities by experimentally reconstructing 3D RI/fluorescence visualizations of fluorescent calibration microspheres as well as alveolar basal epithelial adenocarcinoma (A549) and human colorectal adenocarcinmoa (HT-29) cells, fluorescently stained for F-actin. This demonstration may suggest SI as an especially promising imaging technique to enable future biological studies that explore synergistically operating biophysical/biochemical and molecular mechanisms at sub-diffraction resolutions. PMID:29296504

Combination of hand-held probe and microscopy for fluorescence guided surgery in the brain tumor marginal zone.

PubMed

Richter, Johan C O; Haj-Hosseini, Neda; Hallbeck, Martin; Wårdell, Karin

2017-06-01

Visualization of the tumor is crucial for differentiating malignant tissue from healthy brain during surgery, especially in the tumor marginal zone. The aim of the study was to introduce a fluorescence spectroscopy-based hand-held probe (HHF-probe) for tumor identification in combination with the fluorescence guided resection surgical microscope (FGR-microscope), and evaluate them in terms of diagnostic performance and practical aspects of fluorescence detection. Eighteen operations were performed on 16 patients with suspected high-grade glioma. The HHF-probe and the FGR-microscope were used for detection of protoporphyrin (PpIX) fluorescence induced by 5-aminolevulinic acid (5-ALA) and evaluated against histopathological analysis and visual grading done through the FGR-microscope by the surgeon. A ratio of PpIX fluorescence intensity to the autofluorescence intensity (fluorescence ratio) was used to quantify the spectra detected by the probe. Fluorescence ratio medians (range 0 - 40) measured by the probe were related to the intensity of the fluorescence in the FGR-microscope, categorized as "none" (0.3, n=131), "weak" (1.6, n=34) and "strong" (5.4, n=28). Of 131 "none" points in the FGR-microscope, 88 (67%) exhibited fluorescence with the HHF-probe. For the tumor marginal zone, the area under the receiver operator characteristics (ROC) curve was 0.49 for the FGR-microscope and 0.65 for the HHF-probe. The probe was integrated in the established routine of tumor resection using the FGR-microscope. The HHF-probe was superior to the FGR-microscope in sensitivity; it detected tumor remnants after debulking under the FGR-microscope. The combination of the HHF-probe and the FGR-microscope was beneficial especially in the tumor marginal zone. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Nanoroughened plasmonic films for enhanced biosensing detection

NASA Astrophysics Data System (ADS)

LeMoal, Eric; Lévêque-Fort, Sandrine; Potier, Marie-Claude; Fort, Emmanuel

2009-06-01

Although fluorescence is the prevailing labeling technique in biosensing applications, sensitivity improvement is still a striving challenge. We show that coating standard microscope slides with nanoroughened silver films provides a high fluorescence signal enhancement due to plasmonic interactions. As a proof of concept, we applied these films with tailored plasmonic properties to DNA microarrays. Using common optical scanning devices, we achieved signal amplifications of more than 40-fold.

Laser-Stimulated Fluorescence in Paleontology

PubMed Central

Kaye, Thomas G.; Falk, Amanda R.; Pittman, Michael; Sereno, Paul C.; Burnham, David A.; Gong, Enpu; Xu, Xing; Wang, Yinan

2015-01-01

Fluorescence using ultraviolet (UV) light has seen increased use as a tool in paleontology over the last decade. Laser-stimulated fluorescence (LSF) is a next generation technique that is emerging as a way to fluoresce paleontological specimens that remain dark under typical UV. A laser’s ability to concentrate very high flux rates both at the macroscopic and microscopic levels results in specimens fluorescing in ways a standard UV bulb cannot induce. Presented here are five paleontological case histories that illustrate the technique across a broad range of specimens and scales. Novel uses such as back-lighting opaque specimens to reveal detail and detection of specimens completely obscured by matrix are highlighted in these examples. The recent cost reductions in medium-power short wavelength lasers and use of standard photographic filters has now made this technique widely accessible to researchers. This technology has the potential to automate multiple aspects of paleontology, including preparation and sorting of microfossils. This represents a highly cost-effective way to address paleontology's preparatory bottleneck. PMID:26016843

Eukaryotic cell flattening

NASA Astrophysics Data System (ADS)

Bae, Albert; Westendorf, Christian; Erlenkamper, Christoph; Galland, Edouard; Franck, Carl; Bodenschatz, Eberhard; Beta, Carsten

2010-03-01

Eukaryotic cell flattening is valuable for improving microscopic observations, ranging from bright field to total internal reflection fluorescence microscopy. In this talk, we will discuss traditional overlay techniques, and more modern, microfluidic based flattening, which provides a greater level of control. We demonstrate these techniques on the social amoebae Dictyostelium discoideum, comparing the advantages and disadvantages of each method.

Histochemical study of trans-polyisoprene accumulation by spectral confocal laser scanning microscopy and a specific dye showing fluorescence solvatochromism in the rubber-producing plant, Eucommia ulmoides Oliver.

PubMed

Nakazawa, Yoshihisa; Takeda, Tsuyoshi; Suzuki, Nobuaki; Hayashi, Tatsushi; Harada, Yoko; Bamba, Takeshi; Kobayashi, Akio

2013-09-01

A microscopic technique combining spectral confocal laser scanning microscopy with a lipophilic fluorescent dye, Nile red, which can emit trans-polyisoprene specific fluorescence, was developed, and unmixed images of synthesized trans-polyisoprene in situ in Eucommia ulmoides were successfully obtained. The images showed that trans-polyisoprene was initially synthesized as granules in non-articulated laticifers that changed shape to fibers during laticifer maturation. Non-articulated laticifers are developed from single laticiferous cells, which are differentiated from surrounding parenchyma cells in the cambium. Therefore, these observations suggested that trans-polyisoprene biosynthesis first started in laticifer cells as granules and then the granules accumulated and fused in the inner space of the laticifers over time. Finally, laticifers were filled with the synthesized trans-polyisoprene, which formed a fibrous structure fitting the laticifers shape. Both trans- and cis-polyisoprene are among the most important polymers naturally produced by plants, and this microscopic technique combined with histological study should provide useful information in the fields of plant histology, bioindustry and phytochemistry.

Design and Construction of a Multi-wavelength, Micromirror Total Internal Reflectance Fluorescence Microscope

PubMed Central

Larson, Joshua; Kirk, Matt; Drier, Eric A.; O’Brien, William; MacKay, James F.; Friedman, Larry; Hoskins, Aaron

2015-01-01

Colocalization Single Molecule Spectroscopy (CoSMoS) has proven to be a useful method for studying the composition, kinetics, and mechanisms of complex cellular machines. Key to the technique is the ability to simultaneously monitor multiple proteins and/or nucleic acids as they interact with one another. Here we describe a protocol for constructing a CoSMoS micromirror Total Internal Reflection Fluorescence Microscope (mmTIRFM). Design and construction of a scientific microscope often requires a number of custom components and a significant time commitment. In our protocol, we have streamlined this process by implementation of a commercially available microscopy platform designed to accommodate the optical components necessary for a mmTIRFM. The mmTIRF system eliminates the need for machining custom parts by the end-user and facilitates optical alignment. Depending on the experience-level of the microscope builder, these time-savings and the following protocol can enable mmTIRF construction to be completed within two months. PMID:25188633

Design and construction of a multiwavelength, micromirror total internal reflectance fluorescence microscope.

PubMed

Larson, Joshua; Kirk, Matt; Drier, Eric A; O'Brien, William; MacKay, James F; Friedman, Larry J; Hoskins, Aaron A

2014-10-01

Colocalization single-molecule spectroscopy (CoSMoS) has proven to be a useful method for studying the composition, kinetics and mechanisms of complex cellular machines. Key to the technique is the ability to simultaneously monitor multiple proteins and/or nucleic acids as they interact with one another. Here we describe a protocol for constructing a CoSMoS micromirror total internal reflection fluorescence microscope (mmTIRFM). Design and construction of a scientific microscope often requires a number of custom components and a substantial time commitment. In our protocol, we have streamlined this process by implementation of a commercially available microscopy platform designed to accommodate the optical components necessary for an mmTIRFM. The mmTIRF system eliminates the need for machining custom parts by the end user and facilitates optical alignment. Depending on the experience level of the microscope builder, these time savings and the following protocol can enable mmTIRF construction to be completed within 2 months.

Evanescent field microscopy techniques for studying dynamics at the surface of living cells

NASA Astrophysics Data System (ADS)

Sund, Susan E.

This thesis presents two distinct optical microscopy techniques for applications in cell biophysics: (a)the extension to living cells of an established technique, total internal reflection/fluorescence recovery after photobleaching (TIR/FRAP) for the first time in imaging mode; and (b)the novel development of polarized total internal reflection fluorescence (p- TIRF) to study membrane orientation in living cells. Although reversible chemistry is crucial to dynamical processes in living cells, relatively little is known about the relevant chemical kinetic rates in vivo. TIR/FRAP, an established technique which can measure reversible biomolecular kinetic rates at surfaces, is extended here to measure kinetic parameters of microinjected rhodamine actin at the cytofacial surface of the plasma membrane of living cultured smooth muscle cells. For the first time, spatial imaging (with a CCD camera) is used in conjunction with TIR/FRAP. TIR/FRAP imaging allows production of spatially resolved images of kinetic data, and calculation of correlation distances, cell-wide gradients, and kinetic parameter dependence on initial fluorescence intensity. In living cells, membrane curvature occurs both in easily imaged large scale morphological features, and also in less visualizable submicroscopic regions of activity such as endocytosis, exocytosis, and cell surface ruffling. A fluorescence microscopic method, p-TIRF, is introduced here to visualize such regions. The method is based on fluorescence of the oriented membrane probe diI- C18-(3) (diI) excited by evanescent field light polarized either perpendicular or parallel to the plane of the substrate coverslip. The excitation efficiency from each polarization depends on the membrane orientation, and thus the ratio of the observed fluorescence excited by these two polarizations vividly shows regions of microscopic and submicroscopic curvature of the membrane. A theoretical background of the technique and experimental verifications are presented in samples of protein solutions, model lipid bilayers, and living cells. Sequential digital images of the polarized TIR fluorescence ratios show spatially-resolved time- course maps of membrane orientations on diI labeled macrophages from which low visibility membrane structures can be identified and quantified. The TIR images are sharpened and contrast-enhanced by deconvoluting them with an experimentally-measured point spread function.

Probing plasmodesmata function with biochemical inhibitors.

PubMed

White, Rosemary G

2015-01-01

To investigate plasmodesmata (PD) function, a useful technique is to monitor the effect on cell-to-cell transport of applying an inhibitor of a physiological process, protein, or other cell component of interest. Changes in PD transport can then be monitored in one of several ways, most commonly by measuring the cell-to-cell movement of fluorescent tracer dyes or of free fluorescent proteins. Effects on PD structure can be detected in thin sections of embedded tissue observed using an electron microscope, most commonly a Transmission Electron Microscope (TEM). This chapter outlines commonly used inhibitors, methods for treating different tissues, how to detect altered cell-to-cell transport and PD structure, and important caveats.

Quantitative comparison between full-spectrum and filter-based imaging in hyperspectral fluorescence microscopy

PubMed Central

GAO, L.; HAGEN, N.; TKACZYK, T.S.

2012-01-01

Summary We implement a filterless illumination scheme on a hyperspectral fluorescence microscope to achieve full-range spectral imaging. The microscope employs polarisation filtering, spatial filtering and spectral unmixing filtering to replace the role of traditional filters. Quantitative comparisons between full-spectrum and filter-based microscopy are provided in the context of signal dynamic range and accuracy of measured fluorophores’ emission spectra. To show potential applications, a five-colour cell immunofluorescence imaging experiment is theoretically simulated. Simulation results indicate that the use of proposed full-spectrum imaging technique may result in three times improvement in signal dynamic range compared to that can be achieved in the filter-based imaging. PMID:22356127

A microscopic evaluation of collagen-bilirubin interactions: in vitro surface phenomenon.

PubMed

Usharani, N; Jayakumar, G C; Rao, J R; Chandrasekaran, B; Nair, B U

2014-02-01

This study is carried out to understand the morphology variations of collagen I matrices influenced by bilirubin. The characteristics of bilirubin interaction with collagen ascertained using various techniques like XRD, CLSM, fluorescence, SEM and AFM. These techniques are used to understand the distribution, expression and colocalization patterns of collagen-bilirubin complexes. The present investigation mimic the in vivo mechanisms created during the disorder condition like jaundice. Fluorescence technique elucidates the crucial role played by bilirubin deposition and interaction during collagen organization. Influence of bilirubin during collagen fibrillogenesis and banding patterns are clearly visualize using SEM. As a result, collagen-bilirubin complex provides different reconstructed patterns because of the influence of bilirubin concentration. Selectivity, specificity and spatial organization of collagen-bilirubin are determined through AFM imaging. Consequently, it is observed that the morphology and quantity of the bilirubin binding to collagen varied by the concentrations and the adsorption rate in protein solutions. Microscopic studies of collagen-bilirubin interaction confirms that bilirubin influence the fibrillogenesis and alter the rate of collagen organization depending on the bilirubin concentration. This knowledge helps to develop a novel drug to inhibit the interface point of interaction between collagen and bilirubin. © 2013 The Authors Journal of Microscopy © 2013 Royal Microscopical Society.

Combined multi-plane phase retrieval and super-resolution optical fluctuation imaging for 4D cell microscopy

NASA Astrophysics Data System (ADS)

Descloux, A.; Grußmayer, K. S.; Bostan, E.; Lukes, T.; Bouwens, A.; Sharipov, A.; Geissbuehler, S.; Mahul-Mellier, A.-L.; Lashuel, H. A.; Leutenegger, M.; Lasser, T.

2018-03-01

Super-resolution fluorescence microscopy provides unprecedented insight into cellular and subcellular structures. However, going `beyond the diffraction barrier' comes at a price, since most far-field super-resolution imaging techniques trade temporal for spatial super-resolution. We propose the combination of a novel label-free white light quantitative phase imaging with fluorescence to provide high-speed imaging and spatial super-resolution. The non-iterative phase retrieval relies on the acquisition of single images at each z-location and thus enables straightforward 3D phase imaging using a classical microscope. We realized multi-plane imaging using a customized prism for the simultaneous acquisition of eight planes. This allowed us to not only image live cells in 3D at up to 200 Hz, but also to integrate fluorescence super-resolution optical fluctuation imaging within the same optical instrument. The 4D microscope platform unifies the sensitivity and high temporal resolution of phase imaging with the specificity and high spatial resolution of fluorescence microscopy.

Denaturing of single electrospun fibrinogen fibers studied by deep ultraviolet fluorescence microscopy.

PubMed

Kim, Jeongyong; Song, Hugeun; Park, Inho; Carlisle, Christine R; Bonin, Keith; Guthold, Martin

2011-03-01

Deep ultraviolet (DUV) microscopy is a fluorescence microscopy technique to image unlabeled proteins via the native fluorescence of some of their amino acids. We constructed a DUV fluorescence microscope, capable of 280 nm wavelength excitation by modifying an inverted optical microscope. Moreover, we integrated a nanomanipulator-controlled micropipette into this instrument for precise delivery of picoliter amounts of fluid to selected regions of the sample. In proof-of-principle experiments, we used this instrument to study, in situ, the effect of a denaturing agent on the autofluorescence intensity of single, unlabeled, electrospun fibrinogen nanofibers. Autofluorescence emission from the nanofibers was excited at 280 nm and detected at ∼350 nm. A denaturant solution was discretely applied to small, select sections of the nanofibers and a clear local reduction in autofluorescence intensity was observed. This reduction is attributed to the dissolution of the fibers and the unfolding of proteins in the fibers. Copyright © 2010 Wiley-Liss, Inc.

Mesoscopic Fluorescence Molecular Tomography for Evaluating Engineered Tissues.

PubMed

Ozturk, Mehmet S; Chen, Chao-Wei; Ji, Robin; Zhao, Lingling; Nguyen, Bao-Ngoc B; Fisher, John P; Chen, Yu; Intes, Xavier

2016-03-01

Optimization of regenerative medicine strategies includes the design of biomaterials, development of cell-seeding methods, and control of cell-biomaterial interactions within the engineered tissues. Among these steps, one paramount challenge is to non-destructively image the engineered tissues in their entirety to assess structure, function, and molecular expression. It is especially important to be able to enable cell phenotyping and monitor the distribution and migration of cells throughout the bulk scaffold. Advanced fluorescence microscopic techniques are commonly employed to perform such tasks; however, they are limited to superficial examination of tissue constructs. Therefore, the field of tissue engineering and regenerative medicine would greatly benefit from the development of molecular imaging techniques which are capable of non-destructive imaging of three-dimensional cellular distribution and maturation within a tissue-engineered scaffold beyond the limited depth of current microscopic techniques. In this review, we focus on an emerging depth-resolved optical mesoscopic imaging technique, termed laminar optical tomography (LOT) or mesoscopic fluorescence molecular tomography (MFMT), which enables longitudinal imaging of cellular distribution in thick tissue engineering constructs at depths of a few millimeters and with relatively high resolution. The physical principle, image formation, and instrumentation of LOT/MFMT systems are introduced. Representative applications in tissue engineering include imaging the distribution of human mesenchymal stem cells embedded in hydrogels, imaging of bio-printed tissues, and in vivo applications.

AUTOMATED CELL SEGMENTATION WITH 3D FLUORESCENCE MICROSCOPY IMAGES.

PubMed

Kong, Jun; Wang, Fusheng; Teodoro, George; Liang, Yanhui; Zhu, Yangyang; Tucker-Burden, Carol; Brat, Daniel J

2015-04-01

A large number of cell-oriented cancer investigations require an effective and reliable cell segmentation method on three dimensional (3D) fluorescence microscopic images for quantitative analysis of cell biological properties. In this paper, we present a fully automated cell segmentation method that can detect cells from 3D fluorescence microscopic images. Enlightened by fluorescence imaging techniques, we regulated the image gradient field by gradient vector flow (GVF) with interpolated and smoothed data volume, and grouped voxels based on gradient modes identified by tracking GVF field. Adaptive thresholding was then applied to voxels associated with the same gradient mode where voxel intensities were enhanced by a multiscale cell filter. We applied the method to a large volume of 3D fluorescence imaging data of human brain tumor cells with (1) small cell false detection and missing rates for individual cells; and (2) trivial over and under segmentation incidences for clustered cells. Additionally, the concordance of cell morphometry structure between automated and manual segmentation was encouraging. These results suggest a promising 3D cell segmentation method applicable to cancer studies.

An automated image processing routine for segmentation of cell cytoplasms in high-resolution autofluorescence images

NASA Astrophysics Data System (ADS)

Walsh, Alex J.; Skala, Melissa C.

2014-02-01

The heterogeneity of genotypes and phenotypes within cancers is correlated with disease progression and drug-resistant cellular sub-populations. Therefore, robust techniques capable of probing majority and minority cell populations are important both for cancer diagnostics and therapy monitoring. Herein, we present a modified CellProfiler routine to isolate cytoplasmic fluorescence signal on a single cell level from high resolution auto-fluorescence microscopic images.

A simple approach to spectrally resolved fluorescence and bright field microscopy over select regions of interest

NASA Astrophysics Data System (ADS)

Dahlberg, Peter D.; Boughter, Christopher T.; Faruk, Nabil F.; Hong, Lu; Koh, Young Hoon; Reyer, Matthew A.; Shaiber, Alon; Sherani, Aiman; Zhang, Jiacheng; Jureller, Justin E.; Hammond, Adam T.

2016-11-01

A standard wide field inverted microscope was converted to a spatially selective spectrally resolved microscope through the addition of a polarizing beam splitter, a pair of polarizers, an amplitude-mode liquid crystal-spatial light modulator, and a USB spectrometer. The instrument is capable of simultaneously imaging and acquiring spectra over user defined regions of interest. The microscope can also be operated in a bright-field mode to acquire absorption spectra of micron scale objects. The utility of the instrument is demonstrated on three different samples. First, the instrument is used to resolve three differently labeled fluorescent beads in vitro. Second, the instrument is used to recover time dependent bleaching dynamics that have distinct spectral changes in the cyanobacteria, Synechococcus leopoliensis UTEX 625. Lastly, the technique is used to acquire the absorption spectra of CH3NH3PbBr3 perovskites and measure differences between nanocrystal films and micron scale crystals.

A simple approach to spectrally resolved fluorescence and bright field microscopy over select regions of interest.

PubMed

Dahlberg, Peter D; Boughter, Christopher T; Faruk, Nabil F; Hong, Lu; Koh, Young Hoon; Reyer, Matthew A; Shaiber, Alon; Sherani, Aiman; Zhang, Jiacheng; Jureller, Justin E; Hammond, Adam T

2016-11-01

A standard wide field inverted microscope was converted to a spatially selective spectrally resolved microscope through the addition of a polarizing beam splitter, a pair of polarizers, an amplitude-mode liquid crystal-spatial light modulator, and a USB spectrometer. The instrument is capable of simultaneously imaging and acquiring spectra over user defined regions of interest. The microscope can also be operated in a bright-field mode to acquire absorption spectra of micron scale objects. The utility of the instrument is demonstrated on three different samples. First, the instrument is used to resolve three differently labeled fluorescent beads in vitro. Second, the instrument is used to recover time dependent bleaching dynamics that have distinct spectral changes in the cyanobacteria, Synechococcus leopoliensis UTEX 625. Lastly, the technique is used to acquire the absorption spectra of CH 3 NH 3 PbBr 3 perovskites and measure differences between nanocrystal films and micron scale crystals.

ultraLM and miniLM: Locator tools for smart tracking of fluorescent cells in correlative light and electron microscopy.

PubMed

Brama, Elisabeth; Peddie, Christopher J; Wilkes, Gary; Gu, Yan; Collinson, Lucy M; Jones, Martin L

2016-12-13

In-resin fluorescence (IRF) protocols preserve fluorescent proteins in resin-embedded cells and tissues for correlative light and electron microscopy, aiding interpretation of macromolecular function within the complex cellular landscape. Dual-contrast IRF samples can be imaged in separate fluorescence and electron microscopes, or in dual-modality integrated microscopes for high resolution correlation of fluorophore to organelle. IRF samples also offer a unique opportunity to automate correlative imaging workflows. Here we present two new locator tools for finding and following fluorescent cells in IRF blocks, enabling future automation of correlative imaging. The ultraLM is a fluorescence microscope that integrates with an ultramicrotome, which enables 'smart collection' of ultrathin sections containing fluorescent cells or tissues for subsequent transmission electron microscopy or array tomography. The miniLM is a fluorescence microscope that integrates with serial block face scanning electron microscopes, which enables 'smart tracking' of fluorescent structures during automated serial electron image acquisition from large cell and tissue volumes.

Aberration correction in wide-field fluorescence microscopy by segmented-pupil image interferometry.

PubMed

Scrimgeour, Jan; Curtis, Jennifer E

2012-06-18

We present a new technique for the correction of optical aberrations in wide-field fluorescence microscopy. Segmented-Pupil Image Interferometry (SPII) uses a liquid crystal spatial light modulator placed in the microscope's pupil plane to split the wavefront originating from a fluorescent object into an array of individual beams. Distortion of the wavefront arising from either system or sample aberrations results in displacement of the images formed from the individual pupil segments. Analysis of image registration allows for the local tilt in the wavefront at each segment to be corrected with respect to a central reference. A second correction step optimizes the image intensity by adjusting the relative phase of each pupil segment through image interferometry. This ensures that constructive interference between all segments is achieved at the image plane. Improvements in image quality are observed when Segmented-Pupil Image Interferometry is applied to correct aberrations arising from the microscope's optical path.

Identification of powdered Chinese herbal medicines by fluorescence microscopy, Part 1: Fluorescent characteristics of mechanical tissues, conducting tissues, and ergastic substances.

PubMed

Wang, Ya-Qiong; Liang, Zhi-Tao; Li, Qin; Yang, Hua; Chen, Hu-Biao; Zhao, Zhong-Zhen; Li, Ping

2011-03-01

The light microscope has been successfully used in identification of Chinese herbal medicines (CHMs) for more than a century. However, positive identification is not always possible. Given the popularity of fluorescence microscopy in bioanalysis, researchers dedicated to finding new ways to identify CHMs more effectively are now turning to fluorescence microscopy for authentication purposes. Some studies on distinguishing confused species from the same genus and on exploring distributions of chemicals in tissues of CHMs by fluorescence microscopy have been reported; however, no systematic investigations on fluorescent characteristics of powdered CHMs have been reported. Here, 46 samples of 16 CHMs were investigated. Specifically, the mechanical tissues including stone cells and fibers, the conducting tissues including three types of vessels, and ergastic substances including crystals of calcium oxalate and secretions, in various powdered CHMs were investigated by both light microscope and fluorescence microscope. The results showed many microscopic features emit fluorescence that makes them easily observed, even against complex backgrounds. Under the fluorescence microscope, different microscopic features from the same powdered CHM or some same features from different powdered CHMs emitted the different fluorescence, making this information very helpful for the authentication of CHMs in powder form. Moreover, secretions with unique chemical profiles from different powdered CHMs showed different fluorescent characteristics. Hence, fluorescence microscopy could be a useful additional method for the authentication of powdered CHMs if the fluorescent characteristics of specific CHMs are known. Copyright © 2010 Wiley-Liss, Inc.

A Simple low-cost device enables four epi-illumination techniques on standard light microscopes.

PubMed

Ishmukhametov, Robert R; Russell, Aidan N; Wheeler, Richard J; Nord, Ashley L; Berry, Richard M

2016-02-08

Back-scattering darkfield (BSDF), epi-fluorescence (EF), interference reflection contrast (IRC), and darkfield surface reflection (DFSR) are advanced but expensive light microscopy techniques with limited availability. Here we show a simple optical design that combines these four techniques in a simple low-cost miniature epi-illuminator, which inserts into the differential interference-contrast (DIC) slider bay of a commercial microscope, without further additions required. We demonstrate with this device: 1) BSDF-based detection of Malarial parasites inside unstained human erythrocytes; 2) EF imaging with and without dichroic components, including detection of DAPI-stained Leishmania parasite without using excitation or emission filters; 3) RIC of black lipid membranes and other thin films, and 4) DFSR of patterned opaque and transparent surfaces. We believe that our design can expand the functionality of commercial bright field microscopes, provide easy field detection of parasites and be of interest to many users of light microscopy.

A Simple low-cost device enables four epi-illumination techniques on standard light microscopes

NASA Astrophysics Data System (ADS)

Ishmukhametov, Robert R.; Russell, Aidan N.; Wheeler, Richard J.; Nord, Ashley L.; Berry, Richard M.

2016-02-01

Back-scattering darkfield (BSDF), epi-fluorescence (EF), interference reflection contrast (IRC), and darkfield surface reflection (DFSR) are advanced but expensive light microscopy techniques with limited availability. Here we show a simple optical design that combines these four techniques in a simple low-cost miniature epi-illuminator, which inserts into the differential interference-contrast (DIC) slider bay of a commercial microscope, without further additions required. We demonstrate with this device: 1) BSDF-based detection of Malarial parasites inside unstained human erythrocytes; 2) EF imaging with and without dichroic components, including detection of DAPI-stained Leishmania parasite without using excitation or emission filters; 3) RIC of black lipid membranes and other thin films, and 4) DFSR of patterned opaque and transparent surfaces. We believe that our design can expand the functionality of commercial bright field microscopes, provide easy field detection of parasites and be of interest to many users of light microscopy.

Resolution improvement by nonconfocal theta microscopy.

PubMed

Lindek, S; Stelzer, E H

1999-11-01

We present a novel scanning fluorescence microscopy technique, nonconfocal theta microscopy (NCTM), that provides almost isotropic resolution. In NCTM, multiphoton absorption from two orthogonal illumination directions is used to induce fluorescence emission. Therefore the point-spread function of the microscope is described by the product of illumination point-spread functions with reduced spatial overlap, which provides the resolution improvement and the more isotropic observation volume. We discuss the technical details of this new method.

Optical filters for wavelength selection in fluorescence instrumentation.

PubMed

Erdogan, Turan

2011-04-01

Fluorescence imaging and analysis techniques have become ubiquitous in life science research, and they are poised to play an equally vital role in in vitro diagnostics (IVD) in the future. Optical filters are crucial for nearly all fluorescence microscopes and instruments, not only to provide the obvious function of spectral control, but also to ensure the highest possible detection sensitivity and imaging resolution. Filters make it possible for the sample to "see" light within only the absorption band, and the detector to "see" light within only the emission band. Without filters, the detector would not be able to distinguish the desired fluorescence from scattered excitation light and autofluorescence from the sample, substrate, and other optics in the system. Today the vast majority of fluorescence instruments, including the widely popular fluorescence microscope, use thin-film interference filters to control the spectra of the excitation and emission light. Hence, this unit emphasizes thin-film filters. After briefly introducing different types of thin-film filters and how they are made, the unit describes in detail different optical filter configurations in fluorescence instruments, including both single-color and multicolor imaging systems. Several key properties of thin-film filters, which can significantly affect optical system performance, are then described. In the final section, tunable optical filters are also addressed in a relative comparison.

Fluorescence of fungi in superficial and deep fungal infections

PubMed Central

Elston, Dirk M

2001-01-01

Background Fluorescence of many fungi is noted when H&E stained sections are examined under a fluorescent microscope. In theory, this phenomenon could aid in the diagnosis of cutaneous and disseminated fungal infections without the delay associated with special stains. Seventy-six cases of superficial and deep fungal infections and 3 cases of protothecosis were studied to determine the clinical usefulness of this technique. Results In most cases, fluorescence was noted, but was not intense. Fluorescence of fungi did not correlate with the age of the specimen. In most cases, organisms in H&E stained sections were more easily identified with routine light microscopy than with fluorescent microscopy. Conclusion This report suggests that in H&E stained skin specimens, fluorescent microscopy is of little benefit in the identification of fungal organisms. PMID:11602016

5-Aminolevulinic Acid-Protoporphyrin IX Fluorescence-Guided Surgery of High-Grade Gliomas: A Systematic Review.

PubMed

Guyotat, Jacques; Pallud, Johan; Armoiry, Xavier; Pavlov, Vladislav; Metellus, Philippe

2016-01-01

The current first-line treatment of malignant gliomas consists in surgical resection (if possible) as large as possible. The existing tools don't permit to identify the limits of tumor infiltration, which goes beyond the zone of contrast enhancement on MRI. The fluorescence-guided malignant gliomas surgery was started 15 years ago and had become a standard of care in many countries. The technique is based on fluorescent molecule revelation using the filters, positioned within the surgical microscope. The fluorophore, protoporphyrin IX (PpIX), is converted in tumoral cells from 5-aminolevulinic acid (5-ALA), given orally before surgery. Many studies have shown that the ratio of gross total resections was higher if the fluorescence technique was used. The fluorescence signal intensity is correlated to the cell density and the PpIX concentration. The current method has a very high specificity but still lower sensibility, particularly regarding the zones with poor tumoral infiltration. This book reviews the principles of the technique and the results (extent of resection and survival).

Nano-fEM: protein localization using photo-activated localization microscopy and electron microscopy.

PubMed

Watanabe, Shigeki; Richards, Jackson; Hollopeter, Gunther; Hobson, Robert J; Davis, Wayne M; Jorgensen, Erik M

2012-12-03

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for protein localization, but subcellular context is often absent in fluorescence images. Immuno-electron microscopy, on the other hand, can localize proteins, but the technique is limited by a lack of compatible antibodies, poor preservation of morphology and because most antigens are not exposed to the specimen surface. Correlative approaches can acquire the fluorescence image from a whole cell first, either from immuno-fluorescence or genetically tagged proteins. The sample is then fixed and embedded for electron microscopy, and the images are correlated (1-3). However, the low-resolution fluorescence image and the lack of fiducial markers preclude the precise localization of proteins. Alternatively, fluorescence imaging can be done after preserving the specimen in plastic. In this approach, the block is sectioned, and fluorescence images and electron micrographs of the same section are correlated (4-7). However, the diffraction limit of light in the correlated image obscures the locations of individual molecules, and the fluorescence often extends beyond the boundary of the cell. Nano-resolution fluorescence electron microscopy (nano-fEM) is designed to localize proteins at nano-scale by imaging the same sections using photo-activated localization microscopy (PALM) and electron microscopy. PALM overcomes the diffraction limit by imaging individual fluorescent proteins and subsequently mapping the centroid of each fluorescent spot (8-10). We outline the nano-fEM technique in five steps. First, the sample is fixed and embedded using conditions that preserve the fluorescence of tagged proteins. Second, the resin blocks are sectioned into ultrathin segments (70-80 nm) that are mounted on a cover glass. Third, fluorescence is imaged in these sections using the Zeiss PALM microscope. Fourth, electron dense structures are imaged in these same sections using a scanning electron microscope. Fifth, the fluorescence and electron micrographs are aligned using gold particles as fiducial markers. In summary, the subcellular localization of fluorescently tagged proteins can be determined at nanometer resolution in approximately one week.

Sodium Fluorescein-Guided Resection under the YELLOW 560 nm Surgical Microscope Filter in Malignant Gliomas: Our First 38 Cases Experience.

PubMed

Zhang, Ningning; Tian, Hailong; Huang, Dezhang; Meng, Xianbing; Guo, Wenqiang; Wang, Chaochao; Yin, Xin; Zhang, Hongying; Jiang, Bin; He, Zheng; Wang, Zhigang

2017-01-01

Sodium fluorescein (FL) had been safely used in fluorescence-guided microsurgery for imaging various brain tumors. Under the YELLOW 560 nm surgical microscope filter, low-dose FL as a fluorescent dye helps in visualization. Our study investigated the safety and efficacy of this innovative technique in malignant glioma (MG) patients. 38 patients suffering from MGs confirmed by pathology underwent FL-guided resection under YELLOW 560 nm surgical microscope filter. We retrospectively analyzed the clinical characters, microsurgery procedure, extent of resection, pathology of MGs, progression-free survival (PFS), and overall survival (OS). Thirty-eight patients had MGs (10 WHO grade III, 28 WHO grade IV). With YELLOW 560 nm surgical microscope filter combined with neuronavigation, sodium fluorescein-guided gross total resection (GTR) was achieved in 35 (92.1%) patients and subtotal resection in 3 (7.69%). The sensitivity and specificity of FL were 94.4% and 88.6% regardless of radiographic localization. Intraoperatively, 10 biopsies (10/28 FL[+]) showed "low" or "high" fluorescence in non-contrast-enhancement region and are also confirmed by pathology. Our data showed 6-month PFS of 92.3% and median survival of 11 months. FL-guided resection of MGs under the YELLOW 560 nm surgical microscope filter combined with neuronavigation was safe and effective, especially in non-contrast-MRI regions. It is feasible for improving the extent of resection in MGs especially during emergency cases.

Eco-friendly intracellular biosynthesis of CdS quantum dots without changing Escherichia coli's antibiotic resistance.

PubMed

Yan, Zheng-Yu; Du, Qing-Qing; Qian, Jing; Wan, Dong-Yu; Wu, Sheng-Mei

2017-01-01

In the paper, a green and efficient biosynthetical technique was reported for preparing cadmium sulfide (CdS) quantum dots, in which Escherichia coli (E. coli) was chosen as a biomatrix. Fluorescence emission spectra and fluorescent microscopic photographs revealed that as-produced CdS quantum dots had an optimum fluorescence emission peak located at 470nm and emitted a blue-green fluorescence under ultraviolet excitation. After extracted from bacterial cells and located the nanocrystals' foci in vivo, the CdS quantum dots showed a uniform size distribution by transmission electron microscope. Through the systematical investigation of the biosynthetic conditions, including culture medium replacement, input time point of cadmium source, working concentrations of raw inorganic ions, and co-cultured time spans of bacteria and metal ions in the bio-manufacture, the results revealed that CdS quantum dots with the strongest fluorescence emission were successfully prepared when E. coli cells were in stationary phase, with the replacement of culture medium and following the incubation with 1.0×10 -3 mol/L cadmium source for 2 days. Results of antimicrobial susceptibility testing indicated that the sensitivities to eight types of antibiotics of E. coli were barely changed before and after CdS quantum dots were prepared in the mild temperature environment, though a slight fall of antibiotic resistance could be observed, suggesting hinted the proposed technique of producing quantum dots is a promising environmentally low-risk protocol. Copyright © 2016 Elsevier Inc. All rights reserved.

Optically Sectioned Imaging of Microvasculature of In-Vivo and Ex-Vivo Thick Tissue Models with Speckle-illumination HiLo Microscopy and HiLo Image Processing Implementation in MATLAB Architecture

NASA Astrophysics Data System (ADS)

Suen, Ricky Wai

The work described in this thesis covers the conversion of HiLo image processing into MATLAB architecture and the use of speckle-illumination HiLo microscopy for use of ex-vivo and in-vivo imaging of thick tissue models. HiLo microscopy is a wide-field fluorescence imaging technique and has been demonstrated to produce optically sectioned images comparable to confocal in thin samples. The imaging technique was developed by Jerome Mertz and the Boston University Biomicroscopy Lab and has been implemented in our lab as a stand-alone optical setup and a modification to a conventional fluorescence microscope. Speckle-illumination HiLo microscopy combines two images taken under speckle-illumination and standard uniform-illumination to generate an optically sectioned image that reject out-of-focus fluorescence. The evaluated speckle contrast in the images is used as a weighting function where elements that move out-of-focus have a speckle contrast that decays to zero. The experiments shown here demonstrate the capability of our HiLo microscopes to produce optically-sectioned images of the microvasculature of ex-vivo and in-vivo thick tissue models. The HiLo microscope were used to image the microvasculature of ex-vivo mouse heart sections prepared for optical histology and the microvasculature of in-vivo rodent dorsal window chamber models. Studies in label-free surface profiling with HiLo microscopy is also presented.

Development of HiLo Microscope and its use in In-Vivo Applications

NASA Astrophysics Data System (ADS)

Patel, Shreyas J.

The functionality of achieving optical sectioning in biomedical research is invaluable as it allows for visualization of a biological sample at different depths while being free of background scattering. Most current microscopy techniques that offer optical sectioning, unfortunately, require complex instrumentation and thus are generally costly. HiLo microscopy, on the other hand, offers the same functionality and advantage at a relatively low cost. Hence, the work described in this thesis involves the design, build, and application of a HiLo microscope. More specifically, a standalone HiLo microscope was built in addition to implementing HiLo microscopy on a standard fluorescence microscope. In HiLo microscopy, optical sectioning is achieved by acquiring two different types of images per focal plane. One image is acquired under uniform illumination and the other is acquired under speckle illumination. These images are processed using an algorithm that extracts in-focus information and removes features and glare that occur as a result of background fluorescence. To show the benefits of the HiLo microscopy, several imaging experiments on various samples were performed under a HiLo microscope and compared against a traditional fluorescence microscope and a confocal microscope, which is considered the gold standard in optical imaging. In-vitro and ex-vivo imaging was performed on a set of pollen grains, and optically cleared mouse brain and heart slices. Each of these experiments showed great reduction in background scattering at different depths under HiLo microscopy. More importantly, HiLo imaging of optically cleared heart slice demonstrated emergence of different vasculature at different depths. Reduction of out-of-focus light increased the spatial resolution and allowed better visualization of capillary vessels. Furthermore, HiLo imaging was tested in an in-vivo model of a rodent dorsal window chamber model. When imaging the same sample under confocal microscope, the results were comparable between the two modalities. Additionally, a method of achieving blood flow maps at different depth using a combination of HiLo and LSI imaging is also discussed. The significance of this combined technique could help categorize blood flow to particular depths; this can help improve outcomes of medical treatments such pulse dye laser and photodynamic therapy treatments.

Planetary Surface Analysis Using Fast Laser Spectroscopic Techniques: Combined Microscopic Raman, LIBS, and Fluorescence Spectroscopy

NASA Astrophysics Data System (ADS)

Blacksberg, J.; Rossman, G. R.; Maruyama, Y.; Charbon, E.

2011-12-01

In situ exploration of planetary surfaces has to date required multiple techniques that, when used together, yield important information about their formation histories and evolution. We present a time-resolved laser spectroscopic technique that could potentially collect complementary sets of data providing information on mineral structure, composition, and hydration state. Using a picosecond-scale pulsed laser and a fast time-resolved detector we can simultaneously collect spectra from Raman, Laser Induced Breakdown Spectroscopy (LIBS), and fluorescence emissions that are separated in time due to the unique decay times of each process. The use of a laser with high rep rate (40 KHz) and low pulse energy (1 μJ/pulse) allows us to rapidly collect high signal to noise Raman spectra while minimizing sample damage. Increasing the pulse energy by about an order of magnitude creates a microscopic plasma near the surface and enables the collection of LIBS spectra at an unusually high rep rate and low pulse energy. Simultaneously, broader fluorescence peaks can be detected with lifetimes varying from nanosecond to microsecond. We will present Raman, LIBS, and fluorescence spectra obtained on natural mineral samples such as sulfates, clays, pyroxenes and carbonates that are of interest for Mars mineralogy. We demonstrate this technique using a photocathode-based streak camera detector as well as a newly-developed solid state Single Photon Avalanche Diode (SPAD) sensor array based on Complementary Metal-Oxide Semiconductor (CMOS) technology. We will discuss the impact of system design and detector choice on science return of a potential planetary surface mission, with a specific focus on size, weight, power, and complexity. The research described here was carried out at the Jet Propulsion Laboratory, California Institute of Technology, under a contract with the National Aeronautics and Space Administration (NASA).

Generation of Micropatterned Substrates Using Micro Photopatterning

PubMed Central

Doyle, Andrew D.

2010-01-01

Micro photopatterning (µPP) has been developed to rapidly test and generate different patterns for extracellular matrix adsorption without being hindered with the process of making physical stamps through nanolithography techniques. It uses two-photon excitation guided through a point-scanning confocal microscope to locally photoablate poly(vinyl) alcohol (PVA) thin films in user-defined computer-controlled patterns. PVA thin films are ideal for surface blocking, being hydrophilic substrates that deter protein adsorption and cell attachment. Because gold substrates are not used during µPP, all live-cell fluorescent imaging techniques including total internal reflection fluorescence microscopy of GFP–linked proteins can be performed with minimal loss of fluorescence signal. Furthermore, because µPP does not require physical stamps for pattern generation, multiple ECMs or other proteins can be localized within microns of each other. This unit details the setup of µPP as well as giving troubleshooting techniques. PMID:20013752

Agglutination Assays of the Plasmodium falciparum-Infected Erythrocyte.

PubMed

Tan, Joshua; Bull, Peter C

2015-01-01

The agglutination assay is used to determine the ability of antibodies to recognize parasite variant antigens on the surface of Plasmodium falciparum-infected erythrocytes. In this technique, infected erythrocytes are selectively labelled with a DNA-binding fluorescent dye and mixed with antibodies of interest to allow antibody-surface antigen binding. Recognition of surface antigens by the antibodies can result in the formation of agglutinates containing multiple parasite-infected erythrocytes. These can be viewed and quantified using a fluorescence microscope.

Dual-channel (green and red) fluorescence microendoscope with subcellular resolution

NASA Astrophysics Data System (ADS)

de Paula D'Almeida, Camila; Fortunato, Thereza Cury; Teixeira Rosa, Ramon Gabriel; Romano, Renan Arnon; Moriyama, Lilian Tan; Pratavieira, Sebastião.

2018-02-01

Usually, tissue images at cellular level need biopsies to be done. Considering this, diagnostic devices, such as microendoscopes, have been developed with the purpose of do not be invasive. This study goal is the development of a dual-channel microendoscope, using two fluorescent labels: proflavine and protoporphyrin IX (PpIX), both approved by Food and Drug Administration. This system, with the potential to perform a microscopic diagnosis and to monitor a photodynamic therapy (PDT) session, uses a halogen lamp and an image fiber bundle to perform subcellular image. Proflavine fluorescence indicates the nuclei of the cell, which is the reference for PpIX localization on image tissue. Preliminary results indicate the efficacy of this optical technique to detect abnormal tissues and to improve the PDT dosimetry. This was the first time, up to our knowledge, that PpIX fluorescence was microscopically observed in vivo, in real time, combined to other fluorescent marker (Proflavine), which allowed to simultaneously observe the spatial localization of the PpIX in the mucosal tissue. We believe this system is very promising tool to monitor PDT in mucosa as it happens. Further experiments have to be performed in order to validate the system for PDT monitoring.

Global analysis of microscopic fluorescence lifetime images using spectral segmentation and a digital micromirror spatial illuminator.

PubMed

Bednarkiewicz, Artur; Whelan, Maurice P

2008-01-01

Fluorescence lifetime imaging (FLIM) is very demanding from a technical and computational perspective, and the output is usually a compromise between acquisition/processing time and data accuracy and precision. We present a new approach to acquisition, analysis, and reconstruction of microscopic FLIM images by employing a digital micromirror device (DMD) as a spatial illuminator. In the first step, the whole field fluorescence image is collected by a color charge-coupled device (CCD) camera. Further qualitative spectral analysis and sample segmentation are performed to spatially distinguish between spectrally different regions on the sample. Next, the fluorescence of the sample is excited segment by segment, and fluorescence lifetimes are acquired with a photon counting technique. FLIM image reconstruction is performed by either raster scanning the sample or by directly accessing specific regions of interest. The unique features of the DMD illuminator allow the rapid on-line measurement of global good initial parameters (GIP), which are supplied to the first iteration of the fitting algorithm. As a consequence, a decrease of the computation time required to obtain a satisfactory quality-of-fit is achieved without compromising the accuracy and precision of the lifetime measurements.

Multistage morphological segmentation of bright-field and fluorescent microscopy images

NASA Astrophysics Data System (ADS)

Korzyńska, A.; Iwanowski, M.

2012-06-01

This paper describes the multistage morphological segmentation method (MSMA) for microscopic cell images. The proposed method enables us to study the cell behaviour by using a sequence of two types of microscopic images: bright field images and/or fluorescent images. The proposed method is based on two types of information: the cell texture coming from the bright field images and intensity of light emission, done by fluorescent markers. The method is dedicated to the image sequences segmentation and it is based on mathematical morphology methods supported by other image processing techniques. The method allows for detecting cells in image independently from a degree of their flattening and from presenting structures which produce the texture. It makes use of some synergic information from the fluorescent light emission image as the support information. The MSMA method has been applied to images acquired during the experiments on neural stem cells as well as to artificial images. In order to validate the method, two types of errors have been considered: the error of cell area detection and the error of cell position using artificial images as the "gold standard".

Improved axial point spread function in a two-frequency laser scanning confocal fluorescence microscope

NASA Astrophysics Data System (ADS)

Wu, Jheng-Syong; Chung, Yung-Chin; Chien, Jun-Jei; Chou, Chien

2018-01-01

A two-frequency laser scanning confocal fluorescence microscope (TF-LSCFM) based on intensity modulated fluorescence signal detection was proposed. The specimen-induced spherical aberration and scattering effect were suppressed intrinsically, and high image contrast was presented due to heterodyne interference. An improved axial point spread function in a TF-LSCFM compared with a conventional laser scanning confocal fluorescence microscope was demonstrated and discussed.

Comparison of LED and Conventional Fluorescence Microscopy for Detection of Acid Fast Bacilli in a Low-Incidence Setting

PubMed Central

Minion, Jessica; Pai, Madhukar; Ramsay, Andrew; Menzies, Dick; Greenaway, Christina

2011-01-01

Introduction Light emitting diode fluorescence microscopes have many practical advantages over conventional mercury vapour fluorescence microscopes, which would make them the preferred choice for laboratories in both low- and high-resource settings, provided performance is equivalent. Methods In a nested case-control study, we compared diagnostic accuracy and time required to read slides with the Zeiss PrimoStar iLED, LW Scientific Lumin, and a conventional fluorescence microscope (Leica DMLS). Mycobacterial culture was used as the reference standard, and subgroup analysis by specimen source and organism isolated were performed. Results There was no difference in sensitivity or specificity between the three microscopes, and agreement was high for all comparisons and subgroups. The Lumin and the conventional fluorescence microscope were equivalent with respect to time required to read smears, but the Zeiss iLED was significantly time saving compared to both. Conclusions Light emitting diode microscopy should be considered by all tuberculosis diagnostic laboratories, including those in high income countries, as a replacement for conventional fluorescence microscopes. Our findings provide support to the recent World Health Organization policy recommending that conventional fluorescence microscopy be replaced by light emitting diode microscopy using auramine staining in all settings where fluorescence microscopy is currently used. PMID:21811622

Optimized optical clearing method for imaging central nervous system

NASA Astrophysics Data System (ADS)

Yu, Tingting; Qi, Yisong; Gong, Hui; Luo, Qingming; Zhu, Dan

2015-03-01

The development of various optical clearing methods provides a great potential for imaging entire central nervous system by combining with multiple-labelling and microscopic imaging techniques. These methods had made certain clearing contributions with respective weaknesses, including tissue deformation, fluorescence quenching, execution complexity and antibody penetration limitation that makes immunostaining of tissue blocks difficult. The passive clarity technique (PACT) bypasses those problems and clears the samples with simple implementation, excellent transparency with fine fluorescence retention, but the passive tissue clearing method needs too long time. In this study, we not only accelerate the clearing speed of brain blocks but also preserve GFP fluorescence well by screening an optimal clearing temperature. The selection of proper temperature will make PACT more applicable, which evidently broaden the application range of this method.

In Vivo Mitochondrial Oxygen Tension Measured by a Delayed Fluorescence Lifetime Technique

PubMed Central

Mik, Egbert G.; Johannes, Tanja; Zuurbier, Coert J.; Heinen, Andre; Houben-Weerts, Judith H. P. M.; Balestra, Gianmarco M.; Stap, Jan; Beek, Johan F.; Ince, Can

2008-01-01

Mitochondrial oxygen tension (mitoPO2) is a key parameter for cellular function, which is considered to be affected under various pathophysiological circumstances. Although many techniques for assessing in vivo oxygenation are available, no technique for measuring mitoPO2 in vivo exists. Here we report in vivo measurement of mitoPO2 and the recovery of mitoPO2 histograms in rat liver by a novel optical technique under normal and pathological circumstances. The technique is based on oxygen-dependent quenching of the delayed fluorescence lifetime of protoporphyrin IX. Application of 5-aminolevulinic acid enhanced mitochondrial protoporphyrin IX levels and induced oxygen-dependent delayed fluorescence in various tissues, without affecting mitochondrial respiration. Using fluorescence microscopy, we demonstrate in isolated hepatocytes that the signal is of mitochondrial origin. The delayed fluorescence lifetime was calibrated in isolated hepatocytes and isolated perfused livers. Ultimately, the technique was applied to measure mitoPO2 in rat liver in vivo. The results demonstrate mitoPO2 values of ∼30–40 mmHg. mitoPO2 was highly sensitive to small changes in inspired oxygen concentration around atmospheric oxygen level. Ischemia-reperfusion interventions showed altered mitoPO2 distribution, which flattened overall compared to baseline conditions. The reported technology is scalable from microscopic to macroscopic applications, and its reliance on an endogenous compound greatly enhances its potential field of applications. PMID:18641065

Plasmonics Enhanced Smartphone Fluorescence Microscopy.

PubMed

Wei, Qingshan; Acuna, Guillermo; Kim, Seungkyeum; Vietz, Carolin; Tseng, Derek; Chae, Jongjae; Shir, Daniel; Luo, Wei; Tinnefeld, Philip; Ozcan, Aydogan

2017-05-18

Smartphone fluorescence microscopy has various applications in point-of-care (POC) testing and diagnostics, ranging from e.g., quantification of immunoassays, detection of microorganisms, to sensing of viruses. An important need in smartphone-based microscopy and sensing techniques is to improve the detection sensitivity to enable quantification of extremely low concentrations of target molecules. Here, we demonstrate a general strategy to enhance the detection sensitivity of a smartphone-based fluorescence microscope by using surface-enhanced fluorescence (SEF) created by a thin metal-film. In this plasmonic design, the samples are placed on a silver-coated glass slide with a thin spacer, and excited by a laser-diode from the backside through a glass hemisphere, generating surface plasmon polaritons. We optimized this mobile SEF system by tuning the metal-film thickness, spacer distance, excitation angle and polarization, and achieved ~10-fold enhancement in fluorescence intensity compared to a bare glass substrate, which enabled us to image single fluorescent particles as small as 50 nm in diameter and single quantum-dots. Furthermore, we quantified the detection limit of this platform by using DNA origami-based brightness standards, demonstrating that ~80 fluorophores per diffraction-limited spot can be readily detected by our mobile microscope, which opens up new opportunities for POC diagnostics and sensing applications in resource-limited-settings.

A Fluorescence Recovery After Photobleaching (FRAP) Technique for the Measurement of Solute Transport Across Surfactant-Laden Interfaces

NASA Technical Reports Server (NTRS)

Browne, Edward P.; Hatton, T. Alan

1996-01-01

The technique of Fluorescence Recovery After Photobleaching (FRAP) has been applied to the measurement of interfacial transport in two-phase systems. FRAP exploits the loss of fluorescence exhibited by certain fluorophores when over-stimulated (photobleached), so that a two-phase system, originally at equilibrium, can be perturbed without disturbing the interface by strong light from an argon-ion laser and its recovery monitored by a microscope-mounted CCD camera as it relaxes to a new equilibrium. During this relaxation, the concentration profiles of the probe solute are measured on both sides of the interface as a function of time, yielding information about the transport characteristics of the system. To minimize the size of the meniscus between the two phases, a photolithography technique is used to selectively treat the glass walls of the cell in which the phases are contained. This allows concentration measurements to be made very close to the interface and increases the sensitivity of the FRAP technique.

Laser excited confocal microscope fluorescence scanner and method

DOEpatents

Mathies, Richard A.; Peck, Konan

1992-01-01

A fluorescent scanner for scanning the fluorescence from a fluorescence labeled separated sample on a sample carrier including a confocal microscope for illuminating a predetermined volume of the sample carrier and/or receiving and processing fluorescence emissions from said volume to provide a display of the separated sample.

An Efficient Referencing And Sample Positioning System To Investigate Heterogeneous Substances With Combined Microfocused Synchrotron X-ray Techniques

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spangenberg, Thomas; Goettlicher, Joerg; Steininger, Ralph

2009-01-29

A referencing and sample positioning system has been developed to transfer object positions measured with an offline microscope to a synchrotron experimental station. The accuracy should be sufficient to deal with heterogeneous samples on micrometer scale. Together with an online fluorescence mapping visualisation the optical alignment helps to optimize measuring procedures for combined microfocused X-ray techniques.

Correlative super-resolution fluorescence microscopy combined with optical coherence microscopy

NASA Astrophysics Data System (ADS)

Kim, Sungho; Kim, Gyeong Tae; Jang, Soohyun; Shim, Sang-Hee; Bae, Sung Chul

2015-03-01

Recent development of super-resolution fluorescence imaging technique such as stochastic optical reconstruction microscopy (STORM) and photoactived localization microscope (PALM) has brought us beyond the diffraction limits. It allows numerous opportunities in biology because vast amount of formerly obscured molecular structures, due to lack of spatial resolution, now can be directly observed. A drawback of fluorescence imaging, however, is that it lacks complete structural information. For this reason, we have developed a super-resolution multimodal imaging system based on STORM and full-field optical coherence microscopy (FF-OCM). FF-OCM is a type of interferometry systems based on a broadband light source and a bulk Michelson interferometer, which provides label-free and non-invasive visualization of biological samples. The integration between the two systems is simple because both systems use a wide-field illumination scheme and a conventional microscope. This combined imaging system gives us both functional information at a molecular level (~20nm) and structural information at the sub-cellular level (~1μm). For thick samples such as tissue slices, while FF-OCM is readily capable of imaging the 3D architecture, STORM suffer from aberrations and high background fluorescence that substantially degrade the resolution. In order to correct the aberrations in thick tissues, we employed an adaptive optics system in the detection path of the STORM microscope. We used our multimodal system to obtain images on brain tissue samples with structural and functional information.

Moxifloxacin: Clinically compatible contrast agent for multiphoton imaging

NASA Astrophysics Data System (ADS)

Wang, Taejun; Jang, Won Hyuk; Lee, Seunghun; Yoon, Calvin J.; Lee, Jun Ho; Kim, Bumju; Hwang, Sekyu; Hong, Chun-Pyo; Yoon, Yeoreum; Lee, Gilgu; Le, Viet-Hoan; Bok, Seoyeon; Ahn, G.-One; Lee, Jaewook; Gho, Yong Song; Chung, Euiheon; Kim, Sungjee; Jang, Myoung Ho; Myung, Seung-Jae; Kim, Myoung Joon; So, Peter T. C.; Kim, Ki Hean

2016-06-01

Multiphoton microscopy (MPM) is a nonlinear fluorescence microscopic technique widely used for cellular imaging of thick tissues and live animals in biological studies. However, MPM application to human tissues is limited by weak endogenous fluorescence in tissue and cytotoxicity of exogenous probes. Herein, we describe the applications of moxifloxacin, an FDA-approved antibiotic, as a cell-labeling agent for MPM. Moxifloxacin has bright intrinsic multiphoton fluorescence, good tissue penetration and high intracellular concentration. MPM with moxifloxacin was demonstrated in various cell lines, and animal tissues of cornea, skin, small intestine and bladder. Clinical application is promising since imaging based on moxifloxacin labeling could be 10 times faster than imaging based on endogenous fluorescence.

A simple approach to spectrally resolved fluorescence and bright field microscopy over select regions of interest

PubMed Central

Dahlberg, Peter D.; Boughter, Christopher T.; Faruk, Nabil F.; Hong, Lu; Koh, Young Hoon; Reyer, Matthew A.; Sherani, Aiman; Hammond, Adam T.

2016-01-01

A standard wide field inverted microscope was converted to a spatially selective spectrally resolved microscope through the addition of a polarizing beam splitter, a pair of polarizers, an amplitude-mode liquid crystal-spatial light modulator, and a USB spectrometer. The instrument is capable of simultaneously imaging and acquiring spectra over user defined regions of interest. The microscope can also be operated in a bright-field mode to acquire absorption spectra of micron scale objects. The utility of the instrument is demonstrated on three different samples. First, the instrument is used to resolve three differently labeled fluorescent beads in vitro. Second, the instrument is used to recover time dependent bleaching dynamics that have distinct spectral changes in the cyanobacteria, Synechococcus leopoliensis UTEX 625. Lastly, the technique is used to acquire the absorption spectra of CH3NH3PbBr3 perovskites and measure differences between nanocrystal films and micron scale crystals. PMID:27910631

Polymer-and glass-based fluorescence standards for the near infrared (NIR) spectral region.

PubMed

Würth, Christian; Hoffmann, Katrin; Behnke, Thomas; Ohnesorge, Marius; Resch-Genger, Ute

2011-05-01

The widespread use and acceptance of fluorescence techniques especially in regulated areas like medical diagnostics is closely linked to standardization concepts that guarantee and improve the comparability and reliability of fluorescence measurements. At the core of such concepts are dependable fluorescence standards that are preferably certified. The ever rising interest in fluorescence measurements in the near-infrared (NIR) spectral region renders the availability of spectral and intensity standards for this wavelength region increasingly important. This encouraged us to develop approaches to solid NIR standards based upon dye-doped polymers and assess their application-relevant properties in comparison to metal ion-doped glasses. The overall goal is here to provide inexpensive, easily fabricated, and robust internal and external calibration tools for a broad variety of fluorescence instruments ranging e.g. from spectrofluorometers over fluorescence microscopes to miniaturized fluorescence sensors. © Springer Science+Business Media, LLC 2010

Intestine pH measurements using fluorescence imaging: an in-vivo preliminary study

NASA Astrophysics Data System (ADS)

Marechal, Xavier-Marie; Mordon, Serge R.; Devoisselle, Jean-Marie; Begu, Sylvie; Mathieu, D.; Buys, Bruno; Dhelin, Guy; Lesage, Jean C.; Neviere, Remi; Chopin, Claude

1999-02-01

Measurement of gastrointestinal intramucosal pH has been recognized as an important factor in the detection of hypoxia-induced dysfunctions. However, current pH measurement techniques are limited in terms of time and spatial resolution. A major advance in accurate pH measurement was the development of the ratiometric fluorescent indicator dye, 2',7'-bis(carboxyethyl)-4,5- carboxyfluorescein (BCECF). This study aimed to demonstrate the feasibility of fluorescence imaging technique to measure in vivo the pH of intestine. The intestine was inserted in an optical chamber placed under a microscope. Animals were injected i.v. with the pH-sensitive fluorescent dye BCECF. Fluorescence was visualized by illuminating the intestine alternately at 490 and 470 nm. The emitted fluorescence was directed to an intensified camera. The ratio of emitted fluorescence at excitation wavelengths of 490 and 470 nm was measured, corrected and converted to pH by constructing a calibration curve. The pH controls were performed with a pH microelectrode correlated with venous blood gas sampling. We concluded that accurate pH measurements of rat intestine can be obtained by fluorescence imaging using BCECF. This technology could be easily adapted for endoscopic pH measurement.

Laser excited confocal microscope fluorescence scanner and method

DOEpatents

Mathies, R.A.; Peck, K.

1992-02-25

A fluorescent scanner is designed for scanning the fluorescence from a fluorescence labeled separated sample on a sample carrier. The scanner includes a confocal microscope for illuminating a predetermined volume of the sample carrier and/or receiving and processing fluorescence emissions from the volume to provide a display of the separated sample. 8 figs.

All-plastic, miniature, digital fluorescence microscope for three part white blood cell differential measurements at the point of care

PubMed Central

Forcucci, Alessandra; Pawlowski, Michal E.; Majors, Catherine; Richards-Kortum, Rebecca; Tkaczyk, Tomasz S.

2015-01-01

Three-part differential white blood cell counts are used for disease diagnosis and monitoring at the point-of-care. A low-cost, miniature achromatic microscope was fabricated for identification of lymphocytes, monocytes, and granulocytes in samples of whole blood stained with acridine orange. The microscope was manufactured using rapid prototyping techniques of diamond turning and 3D printing and is intended for use at the point-of-care in low-resource settings. The custom-designed microscope requires no manual adjustment between samples and was successfully able to classify three white blood cell types (lymphocytes, granulocytes, and monocytes) using samples of peripheral whole blood stained with acridine orange. PMID:26601006

Computational efficient segmentation of cell nuclei in 2D and 3D fluorescent micrographs

NASA Astrophysics Data System (ADS)

De Vylder, Jonas; Philips, Wilfried

2011-02-01

This paper proposes a new segmentation technique developed for the segmentation of cell nuclei in both 2D and 3D fluorescent micrographs. The proposed method can deal with both blurred edges as with touching nuclei. Using a dual scan line algorithm its both memory as computational efficient, making it interesting for the analysis of images coming from high throughput systems or the analysis of 3D microscopic images. Experiments show good results, i.e. recall of over 0.98.

Enhanced emission of Nile Red on plasmonic platforms

NASA Astrophysics Data System (ADS)

Synak, Anna; Bojarski, Piotr; Grobelna, Beata; Gryczyński, Ignacy; Fudala, Rafał; Mońka, Michal

2018-04-01

Strongly enhanced fluorescence of Nile Red deposited in the vicinity of silver nanoparticles and gold semitransparent mirror was observed. The properties of three different plasmonic platforms based on TiO2, TiO2-GLYMO (1:1) and SiO2 matrices were studied with spectroscopic and microscopic techniques. Significant differences of Nile Red spectroscopic properties in both matrices were observed. In particular, the sensitivity of Nile Red fluorescence enhancement and its peak location to the polarity of local surrounding was found.

High-Throughput Accurate Single-Cell Screening of Euglena gracilis with Fluorescence-Assisted Optofluidic Time-Stretch Microscopy.

PubMed

Guo, Baoshan; Lei, Cheng; Ito, Takuro; Jiang, Yiyue; Ozeki, Yasuyuki; Goda, Keisuke

2016-01-01

The development of reliable, sustainable, and economical sources of alternative fuels is an important, but challenging goal for the world. As an alternative to liquid fossil fuels, algal biofuel is expected to play a key role in alleviating global warming since algae absorb atmospheric CO2 via photosynthesis. Among various algae for fuel production, Euglena gracilis is an attractive microalgal species as it is known to produce wax ester (good for biodiesel and aviation fuel) within lipid droplets. To date, while there exist many techniques for inducing microalgal cells to produce and accumulate lipid with high efficiency, few analytical methods are available for characterizing a population of such lipid-accumulated microalgae including E. gracilis with high throughout, high accuracy, and single-cell resolution simultaneously. Here we demonstrate high-throughput, high-accuracy, single-cell screening of E. gracilis with fluorescence-assisted optofluidic time-stretch microscopy-a method that combines the strengths of microfluidic cell focusing, optical time-stretch microscopy, and fluorescence detection used in conventional flow cytometry. Specifically, our fluorescence-assisted optofluidic time-stretch microscope consists of an optical time-stretch microscope and a fluorescence analyzer on top of a hydrodynamically focusing microfluidic device and can detect fluorescence from every E. gracilis cell in a population and simultaneously obtain its image with a high throughput of 10,000 cells/s. With the multi-dimensional information acquired by the system, we classify nitrogen-sufficient (ordinary) and nitrogen-deficient (lipid-accumulated) E. gracilis cells with a low false positive rate of 1.0%. This method holds promise for evaluating cultivation techniques and selective breeding for microalgae-based biofuel production.

Chip-based wide field-of-view nanoscopy

NASA Astrophysics Data System (ADS)

Diekmann, Robin; Helle, Øystein I.; Øie, Cristina I.; McCourt, Peter; Huser, Thomas R.; Schüttpelz, Mark; Ahluwalia, Balpreet S.

2017-04-01

Present optical nanoscopy techniques use a complex microscope for imaging and a simple glass slide to hold the sample. Here, we demonstrate the inverse: the use of a complex, but mass-producible optical chip, which hosts the sample and provides a waveguide for the illumination source, and a standard low-cost microscope to acquire super-resolved images via two different approaches. Waveguides composed of a material with high refractive-index contrast provide a strong evanescent field that is used for single-molecule switching and fluorescence excitation, thus enabling chip-based single-molecule localization microscopy. Additionally, multimode interference patterns induce spatial fluorescence intensity variations that enable fluctuation-based super-resolution imaging. As chip-based nanoscopy separates the illumination and detection light paths, total-internal-reflection fluorescence excitation is possible over a large field of view, with up to 0.5 mm × 0.5 mm being demonstrated. Using multicolour chip-based nanoscopy, we visualize fenestrations in liver sinusoidal endothelial cells.

Sodium Fluorescein-Guided Resection under the YELLOW 560 nm Surgical Microscope Filter in Malignant Gliomas: Our First 38 Cases Experience

PubMed Central

Tian, Hailong; Huang, Dezhang; Meng, Xianbing; Guo, Wenqiang; Wang, Chaochao; Yin, Xin; Zhang, Hongying; Jiang, Bin; He, Zheng

2017-01-01

Objective Sodium fluorescein (FL) had been safely used in fluorescence-guided microsurgery for imaging various brain tumors. Under the YELLOW 560 nm surgical microscope filter, low-dose FL as a fluorescent dye helps in visualization. Our study investigated the safety and efficacy of this innovative technique in malignant glioma (MG) patients. Patients and Method 38 patients suffering from MGs confirmed by pathology underwent FL-guided resection under YELLOW 560 nm surgical microscope filter. We retrospectively analyzed the clinical characters, microsurgery procedure, extent of resection, pathology of MGs, progression-free survival (PFS), and overall survival (OS). Results Thirty-eight patients had MGs (10 WHO grade III, 28 WHO grade IV). With YELLOW 560 nm surgical microscope filter combined with neuronavigation, sodium fluorescein-guided gross total resection (GTR) was achieved in 35 (92.1%) patients and subtotal resection in 3 (7.69%). The sensitivity and specificity of FL were 94.4% and 88.6% regardless of radiographic localization. Intraoperatively, 10 biopsies (10/28 FL[+]) showed “low” or “high” fluorescence in non-contrast-enhancement region and are also confirmed by pathology. Our data showed 6-month PFS of 92.3% and median survival of 11 months. Conclusion FL-guided resection of MGs under the YELLOW 560 nm surgical microscope filter combined with neuronavigation was safe and effective, especially in non-contrast-MRI regions. It is feasible for improving the extent of resection in MGs especially during emergency cases. PMID:29124069

Towards native-state imaging in biological context in the electron microscope

PubMed Central

Weston, Anne E.; Armer, Hannah E. J.

2009-01-01

Modern cell biology is reliant on light and fluorescence microscopy for analysis of cells, tissues and protein localisation. However, these powerful techniques are ultimately limited in resolution by the wavelength of light. Electron microscopes offer much greater resolution due to the shorter effective wavelength of electrons, allowing direct imaging of sub-cellular architecture. The harsh environment of the electron microscope chamber and the properties of the electron beam have led to complex chemical and mechanical preparation techniques, which distance biological samples from their native state and complicate data interpretation. Here we describe recent advances in sample preparation and instrumentation, which push the boundaries of high-resolution imaging. Cryopreparation, cryoelectron microscopy and environmental scanning electron microscopy strive to image samples in near native state. Advances in correlative microscopy and markers enable high-resolution localisation of proteins. Innovation in microscope design has pushed the boundaries of resolution to atomic scale, whilst automatic acquisition of high-resolution electron microscopy data through large volumes is finally able to place ultrastructure in biological context. PMID:19916039

Blood-Brain Barrier Disruption, Sodium Fluorescein, And Fluorescence-Guided Surgery Of Gliomas.

PubMed

Xiang, Yan; Zhu, Xiao-Peng; Zhao, Jian-Nong; Huang, Guo-Hao; Tang, Jun-Hai; Chen, Huan-Ran; Du, Lei; Zhang, Dong; Tang, Xue-Feng; Yang, Hui; Lv, Sheng-Qing

2018-01-22

Sodium fluorescein (SF) is an ideal dye for intraoperative guided-resection of high-grade gliomas (HGGs). However, it is not well understood whether the SF-guided technique is suitable for different grades of gliomas, and the correlation between fluorescence and pathology is also not yet clear. In this study, we investigated 28 patients, including 23 patients with HGG and 5 patients with low-grade glioma (LGG). All patients were treated using the SF-guided technique on a Pentero 900 microscope (Carl Zeiss, Oberkochen, Germany). Claudin-5 immunohistochemical (IHC) staining for the tumours and peritumour tissues was analyzed. Intraoperative yellow fluorescence was noted in all the HGGs but not in the LGGs. Claudin-5 expression in the blood brain barrier endothelial cells was downregulated and disconnected in the HGGs (p < 0.05), but had no difference or slightly decreased in the LGGs (p > 0.05). The SF-guided technique is suitable for HGG surgery but not for LGG surgery. Downregulation of claudin-5 expression may contribute to the presence of yellow fluorescence in the glioma in SF-guided surgery.

Time-Correlated Single-Photon Counting Fluorescence Imaging of Lipid Domains In Raft-Mimicking Giant Unilamellar Vesicles

NASA Astrophysics Data System (ADS)

Clarke, James; Cheng, Kwan; Shindell, Orrin; Wang, Exing

We have designed and constructed a high-throughput electrofusion chamber and an incubator to fabricate Giant Unilamellar Vesicles (GUVs) consisting of high-melting lipids, low-melting lipids, cholesterol and both ordered and disordered phase sensitive fluorescent probes (DiIC12, dehydroergosterol and BODIPY-Cholesterol). GUVs were formed in a 3 stage pulse sequence electrofusion process with voltages ranging from 50mVpp to 2.2Vpp and frequencies from 5Hz to 10Hz. Steady state and time-correlated single-photon counting (TCSPC) fluorescence lifetime (FLIM) based confocal and/or multi-photon microscopic techniques were used to characterize phase separated lipid domains in GUVs. Confocal imaging measures the probe concentration and the chemical environment of the system. TCSPC techniques determine the chemical environment through the perturbation of fluorescent lifetimes of the probes in the system. The above techniques will be applied to investigate the protein-lipid interactions involving domain formation. Specifically, the mechanisms governing lipid domain formations in the above systems that mimic the lipid rafts in cells will be explored. Murchison Fellowship at Trinity University.

In vivo optical detection of pH in microscopic tissue samples of Arabidopsis thaliana.

PubMed

Kašík, Ivan; Podrazký, Ondřej; Mrázek, Jan; Martan, Tomáš; Matějec, Vlastimil; Hoyerová, Klára; Kamínek, Miroslav

2013-12-01

Minimally invasive in vivo measurement of pH in microscopic biological samples of μm or μl size, e.g. plant cells, tissues and saps, may help to explain complex biological processes. Consequently, techniques to achieve such measurements are a focus of interest for botanists. This paper describes a technique for the in vivo measurement of pH in the range pH5.0 to pH7.8 in microscopic plant tissue samples of Arabidopsis thaliana based on a ratiometric fluorescence method using low-loss robust tapered fiber probes. For this purpose tapered fiber probes were prepared and coated with a detection layer containing ion-paired fluorescent pH-transducer 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (c-HPTS). A fluorescence ratiometric approach was employed based on excitation at 415 nm and 450 nm and on the comparison of the fluorescence response at 515 nm. The suitability of tapered fiber probes for local detection of pH between 5.0 and 7.8 was demonstrated. A pH sensitivity of 0.15 pH units was achieved within the pH ranges 5.0-5.9 and 7.1-7.8, and this was improved to 0.04 pH units within the pH range 5.9-7.1. Spatial resolution of the probes was better than 20 μm and a time response within 15-20s was achieved. Despite the minute dimensions of the tapered fiber probes the setup developed was relatively robust and compact in construction and performed reliably. It has been successfully employed for the in vivo local determination of pH of mechanically resistant plant tissues of A. thaliana of microscopic scale. The detection of momentary pH gradients across the intact plant seems to be a good tool for the determination of changes in pH in response to experimental treatments affecting for example enzyme activities, availability of mineral nutrients, hormonal control of plant development and plant responses to environmental cues. © 2013.

Fluorescence microscopy: A tool to study autophagy

NASA Astrophysics Data System (ADS)

Rai, Shashank; Manjithaya, Ravi

2015-08-01

Autophagy is a cellular recycling process through which a cell degrades old and damaged cellular components such as organelles and proteins and the degradation products are reused to provide energy and building blocks. Dysfunctional autophagy is reported in several pathological situations. Hence, autophagy plays an important role in both cellular homeostasis and diseased conditions. Autophagy can be studied through various techniques including fluorescence based microscopy. With the advancements of newer technologies in fluorescence microscopy, several novel processes of autophagy have been discovered which makes it an essential tool for autophagy research. Moreover, ability to tag fluorescent proteins with sub cellular targets has enabled us to evaluate autophagy processes in real time under fluorescent microscope. In this article, we demonstrate different aspects of autophagy in two different model organisms i.e. yeast and mammalian cells, with the help of fluorescence microscopy.

Laser scanning confocal microscopy: history, applications, and related optical sectioning techniques.

PubMed

Paddock, Stephen W; Eliceiri, Kevin W

2014-01-01

Confocal microscopy is an established light microscopical technique for imaging fluorescently labeled specimens with significant three-dimensional structure. Applications of confocal microscopy in the biomedical sciences include the imaging of the spatial distribution of macromolecules in either fixed or living cells, the automated collection of 3D data, the imaging of multiple labeled specimens and the measurement of physiological events in living cells. The laser scanning confocal microscope continues to be chosen for most routine work although a number of instruments have been developed for more specific applications. Significant improvements have been made to all areas of the confocal approach, not only to the instruments themselves, but also to the protocols of specimen preparation, to the analysis, the display, the reproduction, sharing and management of confocal images using bioinformatics techniques.

Micro-scale temperature measurement method using fluorescence polarization

NASA Astrophysics Data System (ADS)

Tatsumi, K.; Hsu, C.-H.; Suzuki, A.; Nakabe, K.

2016-09-01

A novel method that can measure the fluid temperature in microscopic scale by measuring the fluorescence polarization is described in this paper. The measurement technique is not influenced by the quenching effects which appears in conventional LIF methods and is believed to show a higher reliability in temperature measurements. Experiment was performed using a microchannel flow and fluorescent molecule probes, and the effects of the fluid temperature, fluid viscosity, measurement time, and pH of the solution on the measured fluorescence polarization degree are discussed to understand the basic characteristics of the present method. The results showed that fluorescence polarization is considerably less sensible to these quenching factors. A good correlation with the fluid temperature, on the other hand, was obtained and agreed well with the theoretical values confirming the feasibility of the method.

Simultaneous optical and electrical recording of a single ion-channel.

PubMed

Ide, Toru; Takeuchi, Yuko; Aoki, Takaaki; Yanagida, Toshio

2002-10-01

In recent years, the single-molecule imaging technique has proven to be a valuable tool in solving many basic problems in biophysics. The technique used to measure single-molecule functions was initially developed to study electrophysiological properties of channel proteins. However, the technology to visualize single channels at work has not received as much attention. In this study, we have for the first time, simultaneously measured the optical and electrical properties of single-channel proteins. The large conductance calcium-activated potassium channel (BK-channel) labeled with fluorescent dye molecules was incorporated into a planar bilayer membrane and the fluorescent image captured with a total internal reflection fluorescence microscope simultaneously with single-channel current recording. This innovative technology will greatly advance the study of channel proteins as well as signal transduction processes that involve ion permeation processes.

Cryo X-ray microscope with flat sample geometry for correlative fluorescence and nanoscale tomographic imaging.

PubMed

Schneider, Gerd; Guttmann, Peter; Rehbein, Stefan; Werner, Stephan; Follath, Rolf

2012-02-01

X-ray imaging offers a new 3-D view into cells. With its ability to penetrate whole hydrated cells it is ideally suited for pairing fluorescence light microscopy and nanoscale X-ray tomography. In this paper, we describe the X-ray optical set-up and the design of the cryo full-field transmission X-ray microscope (TXM) at the electron storage ring BESSY II. Compared to previous TXM set-ups with zone plate condenser monochromator, the new X-ray optical layout employs an undulator source, a spherical grating monochromator and an elliptically shaped glass capillary mirror as condenser. This set-up improves the spectral resolution by an order of magnitude. Furthermore, the partially coherent object illumination improves the contrast transfer of the microscope compared to incoherent conditions. With the new TXM, cells grown on flat support grids can be tilted perpendicular to the optical axis without any geometrical restrictions by the previously required pinhole for the zone plate monochromator close to the sample plane. We also developed an incorporated fluorescence light microscope which permits to record fluorescence, bright field and DIC images of cryogenic cells inside the TXM. For TXM tomography, imaging with multi-keV X-rays is a straightforward approach to increase the depth of focus. Under these conditions phase contrast imaging is necessary. For soft X-rays with shrinking depth of focus towards 10nm spatial resolution, thin optical sections through a thick specimen might be obtained by deconvolution X-ray microscopy. As alternative 3-D X-ray imaging techniques, the confocal cryo-STXM and the dual beam cryo-FIB/STXM with photoelectron detection are proposed. Copyright © 2012 Elsevier Inc. All rights reserved.

Upgrade of a Scanning Confocal Microscope to a Single-Beam Path STED Microscope

PubMed Central

Klauss, André; König, Marcelle; Hille, Carsten

2015-01-01

By overcoming the diffraction limit in light microscopy, super-resolution techniques, such as stimulated emission depletion (STED) microscopy, are experiencing an increasing impact on life sciences. High costs and technically demanding setups, however, may still hinder a wider distribution of this innovation in biomedical research laboratories. As far-field microscopy is the most widely employed microscopy modality in the life sciences, upgrading already existing systems seems to be an attractive option for achieving diffraction-unlimited fluorescence microscopy in a cost-effective manner. Here, we demonstrate the successful upgrade of a commercial time-resolved confocal fluorescence microscope to an easy-to-align STED microscope in the single-beam path layout, previously proposed as “easy-STED”, achieving lateral resolution < λ/10 corresponding to a five-fold improvement over a confocal modality. For this purpose, both the excitation and depletion laser beams pass through a commercially available segmented phase plate that creates the STED-doughnut light distribution in the focal plane, while leaving the excitation beam unaltered when implemented into the joint beam path. Diffraction-unlimited imaging of 20 nm-sized fluorescent beads as reference were achieved with the wavelength combination of 635 nm excitation and 766 nm depletion. To evaluate the STED performance in biological systems, we compared the popular phalloidin-coupled fluorescent dyes Atto647N and Abberior STAR635 by labeling F-actin filaments in vitro as well as through immunofluorescence recordings of microtubules in a complex epithelial tissue. Here, we applied a recently proposed deconvolution approach and showed that images obtained from time-gated pulsed STED microscopy may benefit concerning the signal-to-background ratio, from the joint deconvolution of sub-images with different spatial information which were extracted from offline time gating. PMID:26091552

Trace Element Mapping of a Biological Specimen by a Full-Field X-ray Fluorescence Imaging Microscope with a Wolter Mirror

NASA Astrophysics Data System (ADS)

Hoshino, Masato; Yamada, Norimitsu; Ishino, Toyoaki; Namiki, Takashi; Watanabe, Norio; Aoki, Sadao

2007-01-01

A full-field X-ray fluorescence imaging microscope with a Wolter mirror was applied to the element mapping of alfalfa seeds. The X-ray fluorescence microscope was built at the Photon Factory BL3C2 (KEK). X-ray fluorescence images of several growing stages of the alfalfa seeds were obtained. X-ray fluorescence energy spectra were measured with either a solid state detector or a CCD photon counting method. The element distributions of iron and zinc which were included in the seeds were obtained using a photon counting method.

Color image analysis of contaminants and bacteria transport in porous media

NASA Astrophysics Data System (ADS)

Rashidi, Mehdi; Dehmeshki, Jamshid; Daemi, Mohammad F.; Cole, Larry; Dickenson, Eric

1997-10-01

Transport of contaminants and bacteria in aqueous heterogeneous saturated porous systems have been studied experimentally using a novel fluorescent microscopic imaging technique. The approach involves color visualization and quantification of bacterium and contaminant distributions within a transparent porous column. By introducing stained bacteria and an organic dye as a contaminant into the column and illuminating the porous regions with a planar sheet of laser beam, contaminant and bacterial transport processes through the porous medium can be observed and measured microscopically. A computer controlled color CCD camera is used to record the fluorescent images as a function of time. These images are recorded by a frame accurate high resolution VCR and are then analyzed using a color image analysis code written in our laboratories. The color images are digitized this way and simultaneous concentration and velocity distributions of both contaminant and bacterium are evaluated as a function of time and pore characteristics. The approach provides a unique dynamic probe to observe these transport processes microscopically. These results are extremely valuable in in-situ bioremediation problems since microscopic particle-contaminant- bacterium interactions are the key to understanding and optimization of these processes.

A Fresh Cadaver Study on Indocyanine Green Fluorescence Lymphography: A New Whole-Body Imaging Technique for Investigating the Superficial Lymphatics.

PubMed

Shinaoka, Akira; Koshimune, Seijiro; Yamada, Kiyoshi; Kumagishi, Kanae; Suami, Hiroo; Kimata, Yoshihiro; Ohtsuka, Aiji

2018-05-01

Identification of the lymphatic system in cadavers is painstaking because lymphatic vessels have very thin walls and are transparent. Selection of appropriate contrast agents is a key factor for successfully visualizing the lymphatics. In this study, the authors introduce a new imaging technique of lymphatic mapping in the whole bodies of fresh cadavers. Ten fresh human cadavers were used for this study. The authors injected 0.1 ml of indocyanine green fluorescence solution subcutaneously at multiple spots along the watershed lines between lymphatic territories and hand and foot regions. After the body was scanned by the near-infrared camera system, fluorescent tissues were harvested and histologic examination was performed under the microscope equipped with the infrared camera system to confirm that they were the lymphatics. Subcutaneously injected indocyanine green was immediately transported into the lymphatic vessels after gentle massage on the injection points. Sweeping massage along the lymphatic vessels facilitated indocyanine green transport inside the lymphatic vessel to move toward the lymph nodes. The lymphatic system was visualized well in the whole body. Histologic examinations confirmed that indocyanine green was detected in the lymphatic lumens specifically, even when located far from the injected points. The lymphatic system could be visualized in whole-body fresh cadavers, as in living bodies, using indocyanine green fluorescence lymphography. Compatibility of indocyanine green lymphography would facilitate the use of cadaveric specimens for macroscopic and microscopic analyses.

Planetary Surface Exploration Using Time-Resolved Laser Spectroscopy on Rovers and Landers

NASA Astrophysics Data System (ADS)

Blacksberg, Jordana; Alerstam, Erik; Maruyama, Yuki; Charbon, Edoardo; Rossman, George

2013-04-01

Planetary surface exploration using laser spectroscopy has become increasingly relevant as these techniques become a reality on Mars surface missions. The ChemCam instrument onboard the Curiosity rover is currently using laser induced breakdown spectroscopy (LIBS) on a mast-mounted platform to measure elemental composition of target rocks. The RLS Raman Spectrometer is included on the payload for the ExoMars mission to be launched in 2018 and will identify minerals and organics on the Martian surface. We present a next-generation instrument that builds on these widely used techniques to provide a means for performing both Raman spectroscopy and LIBS in conjunction with microscopic imaging. Microscopic Raman spectroscopy with a laser spot size smaller than the grains of interest can provide surface mapping of mineralogy while preserving morphology. A very small laser spot size (~ 1 µm) is often necessary to identify minor phases that are often of greater interest than the matrix phases. In addition to the difficulties that can be posed by fine-grained material, fluorescence interference from the very same material is often problematic. This is particularly true for many of the minerals of interest that form in environments of aqueous alteration and can be highly fluorescent. We use time-resolved laser spectroscopy to eliminate fluorescence interference that can often make it difficult or impossible to obtain Raman spectra. As an added benefit, we have found that with small changes in operating parameters we can include microscopic LIBS using the same hardware. This new technique relies on sub-ns, high rep-rate lasers with relatively low pulse energy and compact solid state detectors with sub-ns time resolution. The detector technology that makes this instrument possible is a newly developed Single-Photon Avalanche Diode (SPAD) sensor array based on Complementary Metal-Oxide Semiconductor (CMOS) technology. The use of this solid state time-resolved detector offers a significant reduction in size, weight, power, and overall complexity - making time resolved detection feasible for planetary applications. We will discuss significant advances leading to the feasibility of a compact time-resolved spectrometer. We will present results on planetary analog minerals to demonstrate the instrument performance including fluorescence rejection and combined Raman-LIBS capability.

Observing secretory granules with a multiangle evanescent wave microscope.

PubMed Central

Rohrbach, A

2000-01-01

In total internal reflection fluorescence microscopy (TIRFM), fluorophores near a surface can be excited with evanescent waves, which decay exponentially with distance from the interface. Penetration depths of evanescent waves from 60 nm to 300 nm were generated by varying the angle of incidence of a laser beam. With a novel telecentric multiangle evanescent wave microscope, we monitored and investigated both single secretory granules and pools of granules in bovine chromaffin cells. By measuring the fluorescence intensity as a function of penetration depth, it is possible through a Laplace transform to obtain the fluorophore distribution as a function of axial position. We discuss the extent to which it is possible to determine distances and diameters of granules with this microscopy technique by modeling the fluorescent volumes of spheres in evanescent fields. The anisotropic near-field detection of fluorophores and the influence of the detection point-spread function are considered. The diameters of isolated granules between 70 nm and 300 nm have been reconstructed, which is clearly beyond the resolution limit of a confocal microscope. Furthermore, the paper demonstrates how evanescent waves propagate along surfaces and scatter at objects with a higher refractive index. TIRFM will have a limited applicability for quantitative measurements when the parameters used to define evanescent waves are not optimally selected. PMID:10777760

A STED-FLIM microscope applied to imaging the natural killer cell immune synapse

NASA Astrophysics Data System (ADS)

Lenz, M. O.; Brown, A. C. N.; Auksorius, E.; Davis, D. M.; Dunsby, C.; Neil, M. A. A.; French, P. M. W.

2011-03-01

We present a stimulated emission depletion (STED) fluorescence lifetime imaging (FLIM) microscope, excited by a microstructured optical fibre supercontinuum source that is pumped by a femtosecond Ti:Sapphire-laser, which is also used for depletion. Implemented using a piezo-scanning stage on a laser scanning confocal fluorescence microscope system with FLIM realised using time correlated single photon counting (TCSPC), this provides convenient switching between confocal and STED-FLIM with spatial resolution down to below 60 nm. We will present our design considerations to make a robust instrument for biological applications including a comparison between fixed phase plate and spatial light modulator (SLM) approaches to shape the STED beam and the correlation of STED and confocal FLIM microscopy. Following our previous application of FLIM-FRET to study intercellular signalling at the immunological synapse (IS), we are employing STED microscopy to characterize the spatial distribution of cellular molecules with subdiffraction resolution at the IS. In particular, we are imaging cytoskeletal structure at the Natural Killer cell activated immune synapse. We will also present our progress towards multilabel STED microscopy to determine how relative spatial molecular organization, previously undetectable by conventional microscopy techniques, is important for NK cell cytotoxic function. Keywords: STED, Stimulated Emission Depletion Microscopy, Natural Killer (NK) cell, Fluorescence lifetime imaging, FLIM, Super resolution microscopy.

Measuring protein dynamics in live cells: protocols and practical considerations for fluorescence fluctuation microscopy

PubMed Central

Youker, Robert T.; Teng, Haibing

2014-01-01

Abstract. Quantitative analysis of protein complex stoichiometries and mobilities are critical for elucidating the mechanisms that regulate cellular pathways. Fluorescence fluctuation spectroscopy (FFS) techniques can measure protein dynamics, such as diffusion coefficients and formation of complexes, with extraordinary precision and sensitivity. Complete calibration and characterization of the microscope instrument is necessary in order to avoid artifacts during data acquisition and to capitalize on the full capabilities of FFS techniques. We provide an overview of the theory behind FFS techniques, discuss calibration procedures, provide protocols, and give practical considerations for performing FFS experiments. One important parameter recovered from FFS measurements is the relative molecular brightness that can correlate with oligomerization. Three methods for measuring molecular brightness (fluorescence correlation spectroscopy, photon-counting histogram, and number and brightness analysis) recover similar values when measuring samples under ideal conditions in vitro. However, examples are given illustrating that these different methods used for calculating molecular brightness of fluorescent molecules in cells are not always equivalent. Methods relying on spot measurements are more prone to bleaching and movement artifacts that can lead to underestimation of brightness values. We advocate for the use of multiple FFS techniques to study molecular brightnesses to overcome and compliment limitations of individual techniques. PMID:25260867

Optimal resolution in Fresnel incoherent correlation holographic fluorescence microscopy

PubMed Central

Brooker, Gary; Siegel, Nisan; Wang, Victor; Rosen, Joseph

2011-01-01

Fresnel Incoherent Correlation Holography (FINCH) enables holograms and 3D images to be created from incoherent light with just a camera and spatial light modulator (SLM). We previously described its application to microscopic incoherent fluorescence wherein one complex hologram contains all the 3D information in the microscope field, obviating the need for scanning or serial sectioning. We now report experiments which have led to the optimal optical, electro-optic, and computational conditions necessary to produce holograms which yield high quality 3D images from fluorescent microscopic specimens. An important improvement from our previous FINCH configurations capitalizes on the polarization sensitivity of the SLM so that the same SLM pixels which create the spherical wave simulating the microscope tube lens, also pass the plane waves from the infinity corrected microscope objective, so that interference between the two wave types at the camera creates a hologram. This advance dramatically improves the resolution of the FINCH system. Results from imaging a fluorescent USAF pattern and a pollen grain slide reveal resolution which approaches the Rayleigh limit by this simple method for 3D fluorescent microscopic imaging. PMID:21445140

Freezing cytorrhysis and critical temperature thresholds for photosystem II in the peat moss Sphagnum capillifolium.

PubMed

Buchner, Othmar; Neuner, Gilbert

2010-07-01

Leaflets of Sphagnum capillifolium were exposed to temperatures from -5 degrees C to +60 degrees C under controlled conditions while mounted on a microscope stage. The resultant cytological response to these temperature treatments was successfully monitored using a light and fluorescence microscope. In addition to the observable cytological changes during freezing cytorrhysis and heat exposure on the leaflets, the concomitant critical temperature thresholds for inactivation of photosystem II (PS II) were studied using a micro fibre optic and a chlorophyll fluorometer mounted to the microscope stage. Chlorophyllous cells of S. capillifolium showed extended freezing cytorrhysis immediately after ice nucleation at -1.1 degrees C in the water in which the leaflets were submersed during the measurement. The occurrence of freezing cytorrhysis, which was visually manifested by cell shrinkage, was highly dynamic and was completed within 2 s. A total reduction of the mean projected diameter of the chloroplast containing area during freezing cytorrhysis from 8.9 to 3.8 microm indicates a cell volume reduction of approximately -82%. Simultaneous measurement of chlorophyll fluorescence of PS II was possible even through the frozen water in which the leaf samples were submersed. Freezing cytorrhysis was accompanied by a sudden rise of basic chlorophyll fluorescence. The critical freezing temperature threshold of PS II was identical to the ice nucleation temperature (-1.1 degrees C). This is significantly above the temperature threshold at which frost damage to S. capillifolium leaflets occurs (-16.1 degrees C; LT(50)) which is higher than observed in most higher plants from the European Alps during summer. High temperature thresholds of PS II were 44.5 degrees C which is significantly below the heat tolerance of chlorophyllous cells (49.9 degrees C; LT(50)). It is demonstrated that light and fluorescence microscopic techniques combined with simultaneous chlorophyll fluorescence measurements may act as a useful tool to study heat, low temperature, and ice-encasement effects on the cellular structure and primary photosynthetic processes of intact leaf tissues.

Electrically tunable lens speeds up 3D orbital tracking

PubMed Central

Annibale, Paolo; Dvornikov, Alexander; Gratton, Enrico

2015-01-01

3D orbital particle tracking is a versatile and effective microscopy technique that allows following fast moving fluorescent objects within living cells and reconstructing complex 3D shapes using laser scanning microscopes. We demonstrated notable improvements in the range, speed and accuracy of 3D orbital particle tracking by replacing commonly used piezoelectric stages with Electrically Tunable Lens (ETL) that eliminates mechanical movement of objective lenses. This allowed tracking and reconstructing shape of structures extending 500 microns in the axial direction. Using the ETL, we tracked at high speed fluorescently labeled genomic loci within the nucleus of living cells with unprecedented temporal resolution of 8ms using a 1.42NA oil-immersion objective. The presented technology is cost effective and allows easy upgrade of scanning microscopes for fast 3D orbital tracking. PMID:26114037

Confocal endomicroscopy for in vivo microscopic analysis of upper gastrointestinal tract premalignant and malignant lesions.

PubMed

Gheorghe, Cristian; Iacob, Razvan; Becheanu, Gabriel; Dumbrav Abreve, Mona

2008-03-01

Confocal LASER endomicroscopy (CLE) is a new endoscopic technique which allows subsurface in vivo microscopic analysis during ongoing endoscopy, using systemically or topically administered fluorescent agents. It allows targeted biopsies to be taken, potentially improving the diagnostic rate in certain gastrointestinal diseases. Worldwide experience with CLE for upper gastrointestinal malignant and premalignant lesions is still reduced. Potential clinical applications are presented, including diagnosis of NERD, Barrett's esophagus, atrophic gatritis, gastric intestinal metaplasia and dysplasia, gastric adenomatous or hyperplastic polyps, gastric cancer.

Visualizing Fluorescence: Using a Homemade Fluorescence "Microscope" to View Latent Fingerprints on Paper

ERIC Educational Resources Information Center

LaFratta, Christopher N.; Huh, Sun Phill; Mallillin, Allistair C.; Riviello, Peter J.; Walt, David R.

2010-01-01

We describe an inexpensive hand-held fluorescence imager (low-magnification microscope), constructed from poly(vinyl chloride) pipe and other inexpensive components for use as a teaching tool to understand the principles of fluorescence detection. Optical filters are used to select the excitation and emission wavelengths and can be easily…

Microscopic fluorescence spectral analysis of basal cell carcinomas

NASA Astrophysics Data System (ADS)

He, Qingli; Lui, Harvey; Zloty, David; Cowan, Bryce; Warshawski, Larry; McLean, David I.; Zeng, Haishan

2007-05-01

Background and Objectives. Laser-induced autofluorescence (LIAF) is a promising tool for cancer diagnosis. This method is based on the differences in autofluorescence spectra between normal and cancerous tissues, but the underlined mechanisms are not well understood. The objective of this research is to study the microscopic origins and intrinsic fluorescence properties of basal cell carcinoma (BCC) for better understanding of the mechanism of in vivo fluorescence detection and margin delineation of BCCs on skin patients. A home-made micro- spectrophotometer (MSP) system was used to image the fluorophore distribution and to measure the fluorescence spectra of various microscopic structures and regions on frozen tissue sections. Materials and Methods. BCC tissue samples were obtained from 14 patients undergoing surgical resections. After surgical removal, each tissue sample was immediately embedded in OCT medium and snap-frozen in liquid nitrogen. The frozen tissue block was then cut into 16-μm thickness sections using a cryostat microtome and placed on microscopic glass slides. The sections for fluorescence study were kept unstained and unfixed, and then analyzed by the MSP system. The adjacent tissue sections were H&E stained for histopathological examination and also served to help identify various microstructures on the adjacent unstained sections. The MSP system has all the functions of a conventional microscope, plus the ability of performing spectral analysis on selected micro-areas of a microscopic sample. For tissue fluorescence analysis, 442nm He-Cd laser light is used to illuminate and excite the unstained tissue sections. A 473-nm long pass filter was inserted behind the microscope objective to block the transmitted laser light while passing longer wavelength fluorescence signal. The fluorescence image of the sample can be viewed through the eyepieces and also recorded by a CCD camera. An optical fiber is mounted onto the image plane of the photograph port of the microscope to collect light from a specific micro area of the sample. The collected light is transmitted via the fiber to a disperserve type CCD spectrometer for spectral analysis. Results. The measurement results showed significant spectral differences between normal and cancerous tissues. For normal tissue regions, the spectral results agreed with our previous findings on autofluorescence of normal skin sections. For the cancerous regions, the epidermis showed very weak fluorescence signal, while the stratum corneum exhibited fluorescence emissions peaking at about 510 nm. In the dermis, the basal cell island and a band of surrounding areas showed very weak fluorescence signal, while distal dermis above and below the basal cell island showed greater fluorescence signal but with different spectral shapes. The very weak autofluorescence from the basal cell island and its surrounding area may be attributed to their degenerative properties that limited the production of collagens. Conclusions. The obtained microscopic results very well explain the in vivo fluorescence properties of BCC lesions in that they have decreased fluorescence intensity compared to the surrounding normal skin. The intrinsic spectra of various microstructures and the microscopic fluorescence images (corresponding fluorophore distribution in tissue) obtained in this study will be used for further theoretical modeling of in vivo fluorescence spectroscopy and imaging of skin cancers.

Extracellular oxygen concentration mapping with a confocal multiphoton laser scanning microscope and TCSPC card

NASA Astrophysics Data System (ADS)

Hosny, Neveen A.; Lee, David A.; Knight, Martin M.

2010-02-01

Extracellular oxygen concentrations influence cell metabolism and tissue function. Fluorescence Lifetime Imaging Microscopy (FLIM) offers a non-invasive method for quantifying local oxygen concentrations. However, existing methods show limited spatial resolution and/or require custom made systems. This study describes a new optimised approach for quantitative extracellular oxygen detection, providing an off-the-shelf system with high spatial resolution and an improved lifetime determination over previous techniques, while avoiding systematic photon pile-up. Fluorescence lifetime detection of an oxygen sensitive fluorescent dye, tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate [Ru(bipy)3]2+, was measured using a Becker&Hickl time-correlated single photon counting (TCSPC) card with excitation provided by a multi-photon laser. This technique was able to identify a subpopulation of isolated chondrocyte cells, seeded in three-dimensional agarose gel, displaying a significant spatial oxygen gradient. Thus this technique provides a powerful tool for quantifying spatial oxygen gradients within three-dimensional cellular models.

Toward quantitative fluorescence microscopy with DNA origami nanorulers.

PubMed

Beater, Susanne; Raab, Mario; Tinnefeld, Philip

2014-01-01

The dynamic development of fluorescence microscopy has created a large number of new techniques, many of which are able to overcome the diffraction limit. This chapter describes the use of DNA origami nanostructures as scaffold for quantifying microscope properties such as sensitivity and resolution. The DNA origami technique enables placing of a defined number of fluorescent dyes in programmed geometries. We present a variety of DNA origami nanorulers that include nanorulers with defined labeling density and defined distances between marks. The chapter summarizes the advantages such as practically free choice of dyes and labeling density and presents examples of nanorulers in use. New triangular DNA origami nanorulers that do not require photoinduced switching by imaging transient binding to DNA nanostructures are also reported. Finally, we simulate fluorescence images of DNA origami nanorulers and reveal that the optimal DNA nanoruler for a specific application has an intermark distance that is roughly 1.3-fold the expected optical resolution. © 2014 Elsevier Inc. All rights reserved.

Correlative cryogenic tomography of cells using light and soft x-rays

PubMed Central

Smith, Elizabeth A.; Cinquin, Bertrand P.; Do, Myan; McDermott, Gerry; Le Gros, Mark A.; Larabell, Carolyn A.

2013-01-01

Correlated imaging is the process of imaging a specimen with two complementary modalities, and then combining the two data sets to create a highly informative, composite view. A recent implementation of this concept has been the combination of soft x-ray tomography (SXT) with fluorescence cryogenic microscopy (FCM). SXT-FCM is used to visualize cells that are held in a near-native, cryo-preserved state. The resultant images are, therefore, highly representative of both the cellular architecture and molecular organization in vivo. SXT quantitatively visualizes the cell and sub-cellular structures; FCM images the spatial distribution of fluorescently labeled molecules. Here, we review the characteristics of SXT-FCM, and briefly discuss how this method compares with existing correlative imaging techniques. We also describe how the incorporation of a cryo-rotation stage into a cryogenic fluorescence microscope allows acquisition of fluorescence cryogenic tomography (FCT) data. FCT is optimally suited to correlation with SXT, since both techniques image the specimen in 3-D, potentially with similar, isotropic spatial resolution. PMID:24355261

Enhanced weak-signal sensitivity in two-photon microscopy by adaptive illumination.

PubMed

Chu, Kengyeh K; Lim, Daryl; Mertz, Jerome

2007-10-01

We describe a technique to enhance both the weak-signal relative sensitivity and the dynamic range of a laser scanning optical microscope. The technique is based on maintaining a fixed detection power by fast feedback control of the illumination power, thereby transferring high measurement resolution to weak signals while virtually eliminating the possibility of image saturation. We analyze and demonstrate the benefits of adaptive illumination in two-photon fluorescence microscopy.

Fluorescence Lifetime Imaging Microscopy Using Near-Infrared Contrast Agents

PubMed Central

Nothdurft, Ralph; Sarder, Pinaki; Bloch, Sharon; Culver, Joseph; Achilefu, Samuel

2013-01-01

Although single-photon fluorescence lifetime imaging microscopy (FLIM) is widely used to image molecular processes using a wide range of excitation wavelengths, the captured emission of this technique is confined to the visible spectrum. Here, we explore the feasibility of utilizing near-infrared (NIR) fluorescent molecular probes with emission >700 nm for FLIM of live cells. The confocal microscope is equipped with a 785 nm laser diode, a red-enhanced photomultiplier tube, and a time-correlated single photon counting card. We demonstrate that our system reports the lifetime distributions of NIR fluorescent dyes, cypate and DTTCI, in cells. In cells labeled separately or jointly with these dyes, NIR FLIM successfully distinguishes their lifetimes, providing a method to sort different cell populations. In addition, lifetime distributions of cells co-incubated with these dyes allow estimate of the dyes’ relative concentrations in complex cellular microenvironments. With the heightened interest in fluorescence lifetime-based small animal imaging using NIR fluorophores, this technique further serves as a bridge between in vitro spectroscopic characterization of new fluorophore lifetimes and in vivo tissue imaging. PMID:22788550

Reliable measurement of E. coli single cell fluorescence distribution using a standard microscope set-up.

PubMed

Cortesi, Marilisa; Bandiera, Lucia; Pasini, Alice; Bevilacqua, Alessandro; Gherardi, Alessandro; Furini, Simone; Giordano, Emanuele

2017-01-01

Quantifying gene expression at single cell level is fundamental for the complete characterization of synthetic gene circuits, due to the significant impact of noise and inter-cellular variability on the system's functionality. Commercial set-ups that allow the acquisition of fluorescent signal at single cell level (flow cytometers or quantitative microscopes) are expensive apparatuses that are hardly affordable by small laboratories. A protocol that makes a standard optical microscope able to acquire quantitative, single cell, fluorescent data from a bacterial population transformed with synthetic gene circuitry is presented. Single cell fluorescence values, acquired with a microscope set-up and processed with custom-made software, are compared with results that were obtained with a flow cytometer in a bacterial population transformed with the same gene circuitry. The high correlation between data from the two experimental set-ups, with a correlation coefficient computed over the tested dynamic range > 0.99, proves that a standard optical microscope- when coupled with appropriate software for image processing- might be used for quantitative single-cell fluorescence measurements. The calibration of the set-up, together with its validation, is described. The experimental protocol described in this paper makes quantitative measurement of single cell fluorescence accessible to laboratories equipped with standard optical microscope set-ups. Our method allows for an affordable measurement/quantification of intercellular variability, whose better understanding of this phenomenon will improve our comprehension of cellular behaviors and the design of synthetic gene circuits. All the required software is freely available to the synthetic biology community (MUSIQ Microscope flUorescence SIngle cell Quantification).

Fluorescence microscope (Cyscope) for malaria diagnosis in pregnant women in Medani Hospital, Sudan.

PubMed

Hassan, Saad El-Din H; Haggaz, Abd Elrahium D; Mohammed-Elhassan, Ehab B; Malik, Elfatih M; Adam, Ishag

2011-09-24

Accuracy of diagnosis is the core for malaria control. Although microscopy is the gold standard in malaria diagnosis, its reliability is largely dependent on user skill. We compared performance of Cyscope fluorescence microscope with the Giemsa stained light microscopy for the diagnosis of malaria among pregnant women at Medani Hospital in Central Sudan. The area is characterized by unstable malaria transmission. Socio-demographic characteristics and obstetrics history were gathered using pre-tested questionnaires. Blood samples were collected from febrile pregnant women who were referred as malaria case following initial diagnosis by general microscopist. During the study period 128 febrile pregnant women presented at the hospital. Among them, Plasmodium falciparum malaria was detected in 82 (64.1%) and 80 (62.5%) by the Giemsa-stained light microscopy and the Cyscope fluorescence microscope, respectively. The sensitivity of the Cyscope fluorescence microscope was 97.6% (95% CI: 92.2%-99.6%). Out of 46 which were negative by Giemsa-stained light microscopy, 5 were positive by the Cyscope fluorescence microscope. This is translated in specificity of 89.1% (95% CI: 77.5%-95.9%). The positive and negative predictive value of Cyscope fluorescence microscope was 94.1% (95% CI: 87.4% -97.8%) and 95.3% (95% CI: 85.4% - 99.2%), respectively. This study has shown that Cyscope fluorescence microscope is a reliable diagnostic, sensitive and specific in diagnosing P. falciparum malaria among pregnant women in this setting. Further studies are needed to determine effectiveness in diagnosing other Plasmodium species and to compare it with other diagnostic tools e.g. rapid diagnostic tests and PCR.

Discerning the Chemistry in Individual Organelles with Small-Molecule Fluorescent Probes.

PubMed

Xu, Wang; Zeng, Zebing; Jiang, Jian-Hui; Chang, Young-Tae; Yuan, Lin

2016-10-24

Principle has it that even the most advanced super-resolution microscope would be futile in providing biological insight into subcellular matrices without well-designed fluorescent tags/probes. Developments in biology have increasingly been boosted by advances of chemistry, with one prominent example being small-molecule fluorescent probes that not only allow cellular-level imaging, but also subcellular imaging. A majority, if not all, of the chemical/biological events take place inside cellular organelles, and researchers have been shifting their attention towards these substructures with the help of fluorescence techniques. This Review summarizes the existing fluorescent probes that target chemical/biological events within a single organelle. More importantly, organelle-anchoring strategies are described and emphasized to inspire the design of new generations of fluorescent probes, before concluding with future prospects on the possible further development of chemical biology. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Measurement of two-photon-absorption spectra through nonlinear fluorescence produced by a line-shaped excitation beam.

PubMed

Hasani, E; Parravicini, J; Tartara, L; Tomaselli, A; Tomassini, D

2018-05-01

We propose an innovative experimental approach to estimate the two-photon absorption (TPA) spectrum of a fluorescent material. Our method develops the standard indirect fluorescence-based method for the TPA measurement by employing a line-shaped excitation beam, generating a line-shaped fluorescence emission. Such a configuration, which requires a relatively high amount of optical power, permits to have a greatly increased fluorescence signal, thus avoiding the photon counterdetection devices usually used in these measurements, and allowing to employ detectors such as charge-coupled device (CCD) cameras. The method is finally tested on a fluorescent isothiocyanate sample, whose TPA spectrum, which is measured with the proposed technique, is compared with the TPA spectra reported in the literature, confirming the validity of our experimental approach. © 2018 The Authors Journal of Microscopy © 2018 Royal Microscopical Society.

Multistage Spatial Property Based Segmentation for Quantification of Fluorescence Distribution in Cells

NASA Astrophysics Data System (ADS)

Zhang, Guangyun; Jia, Xiuping; Pham, Tuan D.; Crane, Denis I.

2010-01-01

The interpretation of the distribution of fluorescence in cells is often by simple visualization of microscope-derived images for qualitative studies. In other cases, however, it is desirable to be able to quantify the distribution of fluorescence using digital image processing techniques. In this paper, the challenges of fluorescence segmentation due to the noise present in the data are addressed. We report that intensity measurements alone do not allow separation of overlapping data between target and background. Consequently, spatial properties derived from neighborhood profile were included. Mathematical Morphological operations were implemented for cell boundary extraction and a window based contrast measure was developed for fluorescence puncta identification. All of these operations were applied in the proposed multistage processing scheme. The testing results show that the spatial measures effectively enhance the target separability.

Combining total internal reflection sum frequency spectroscopy spectral imaging and confocal fluorescence microscopy.

PubMed

Allgeyer, Edward S; Sterling, Sarah M; Gunewardene, Mudalige S; Hess, Samuel T; Neivandt, David J; Mason, Michael D

2015-01-27

Understanding surface and interfacial lateral organization in material and biological systems is critical in nearly every field of science. The continued development of tools and techniques viable for elucidation of interfacial and surface information is therefore necessary to address new questions and further current investigations. Sum frequency spectroscopy (SFS) is a label-free, nonlinear optical technique with inherent surface specificity that can yield critical organizational information on interfacial species. Unfortunately, SFS provides no spatial information on a surface; small scale heterogeneities that may exist are averaged over the large areas typically probed. Over the past decade, this has begun to be addressed with the advent of SFS microscopy. Here we detail the construction and function of a total internal reflection (TIR) SFS spectral and confocal fluorescence imaging microscope directly amenable to surface investigations. This instrument combines, for the first time, sample scanning TIR-SFS imaging with confocal fluorescence microscopy.

Assembly and microscopic characterization of DNA origami structures.

PubMed

Scheible, Max; Jungmann, Ralf; Simmel, Friedrich C

2012-01-01

DNA origami is a revolutionary method for the assembly of molecular nanostructures from DNA with precisely defined dimensions and with an unprecedented yield. This can be utilized to arrange nanoscale components such as proteins or nanoparticles into pre-defined patterns. For applications it will now be of interest to arrange such components into functional complexes and study their geometry-dependent interactions. While commonly DNA nanostructures are characterized by atomic force microscopy or electron microscopy, these techniques often lack the time-resolution to study dynamic processes. It is therefore of considerable interest to also apply fluorescence microscopic techniques to DNA nanostructures. Of particular importance here is the utilization of novel super-resolved microscopy methods that enable imaging beyond the classical diffraction limit.

New solutions for standardization, monitoring and quality management of fluorescence-based imaging systems (Conference Presentation)

NASA Astrophysics Data System (ADS)

Royon, Arnaud; Papon, Gautier

2016-03-01

Fluorescence microscopes have become ubiquitous in life sciences laboratories, including those focused on pharmaceuticals, diagnosis, and forensics. For the past few years, the need for both performance guarantees and quantifiable results has driven development in this area. However, the lack of appropriate standards and reference materials makes it difficult or impossible to compare the results of two fluorescence microscopes, or to measure performance fluctuations of one microscope over time. Therefore, the operation of fluorescence microscopes is not monitored as often as their use warrants - an issue that is recognized by both systems manufacturers and national metrology institutes. We have developed a new process that enables the etching of long-term stable fluorescent patterns with sub-micrometer sizes in three dimensions inside glass. In this paper, we present, based on this new process, a fluorescent multi-dimensional ruler and a dedicated software that are suitable for monitoring and quality management of fluorescence-based imaging systems (wide-field, confocal, multiphoton, high content machines). In addition to fluorescence, the same patterns exhibit bright- and dark-field contrast, DIC, and phase contrast, which make them also relevant to monitor these types of microscopes. Non-exhaustively, this new solution enables the measurement of: The stage repositioning accuracy; The illumination and detection homogeneities; The field flatness; The detectors' characteristics; The lateral and axial spatial resolutions; The spectral response (spectrum, intensity and lifetime) of the system. Thanks to the stability of the patterns, microscope performance assessment can be carried out as well in a daily basis as in the long term.

Intraoperative Fluorescence Cerebral Angiography by Laser Surgical Microscopy: Comparison With Xenon Microscopy and Simultaneous Observation of Cerebral Blood Flow and Surrounding Structures.

PubMed

Ito, Yuhei; Suzuki, Kyouichi; Ichikawa, Tsuyoshi; Watanabe, Yoichi; Sato, Taku; Sakuma, Jun; Saito, Kiyoshi

2018-06-12

Laser surgical microscopes should enable uniform illumination of the operative field, and require less luminous energy compared with existing xenon surgical microscopes. To examine the utility of laser illumination in fluorescence cerebral angiography. Fluorescein sodium (fluorescein) was used as a fluorescent dye. We first compared the clarity of cerebral blood flow images collected by fluorescence angiography between the laser illumination and xenon illumination methods. We then assessed use of the laser illuminator for simultaneous observation of blood flow and surrounding structures during fluorescence angiography. Furthermore, the study was designed to evaluate usefulness of the thus determined excitation light in clinical cases. Fluorescence angiography using blue light laser for excitation provided higher clarity and contrast blood flow images compared with using blue light generated from a xenon lamp. Further, illumination with excitation light consisting of a combination of 3 types of laser (higher level of blue light, no green light, and lower level of red light) enabled both blood flow and surrounding structures to be observed through the microscope directly by the surgeon. Laser-illuminated fluorescence angiography provides high clarity and contrast images of cerebral blood flow. Further, a laser providing strong blue light and weak red light for excitation light enables simultaneous visual observation of fluorescent blood flow and surrounding structures by the surgeon using a surgical microscope. Overall, these data suggest that laser surgical microscopes are useful for both ordinary operative manipulations and fluorescence angiography.

FluoroSim: A Visual Problem-Solving Environment for Fluorescence Microscopy

PubMed Central

Quammen, Cory W.; Richardson, Alvin C.; Haase, Julian; Harrison, Benjamin D.; Taylor, Russell M.; Bloom, Kerry S.

2010-01-01

Fluorescence microscopy provides a powerful method for localization of structures in biological specimens. However, aspects of the image formation process such as noise and blur from the microscope's point-spread function combine to produce an unintuitive image transformation on the true structure of the fluorescing molecules in the specimen, hindering qualitative and quantitative analysis of even simple structures in unprocessed images. We introduce FluoroSim, an interactive fluorescence microscope simulator that can be used to train scientists who use fluorescence microscopy to understand the artifacts that arise from the image formation process, to determine the appropriateness of fluorescence microscopy as an imaging modality in an experiment, and to test and refine hypotheses of model specimens by comparing the output of the simulator to experimental data. FluoroSim renders synthetic fluorescence images from arbitrary geometric models represented as triangle meshes. We describe three rendering algorithms on graphics processing units for computing the convolution of the specimen model with a microscope's point-spread function and report on their performance. We also discuss several cases where the microscope simulator has been used to solve real problems in biology. PMID:20431698

Temporal and spatial binning of TCSPC data to improve signal-to-noise ratio and imaging speed

NASA Astrophysics Data System (ADS)

Walsh, Alex J.; Beier, Hope T.

2016-03-01

Time-correlated single photon counting (TCSPC) is the most robust method for fluorescence lifetime imaging using laser scanning microscopes. However, TCSPC is inherently slow making it ineffective to capture rapid events due to the single photon product per laser pulse causing extensive acquisition time limitations and the requirement of low fluorescence emission efficiency to avoid bias of measurement towards short lifetimes. Furthermore, thousands of photons per pixel are required for traditional instrument response deconvolution and fluorescence lifetime exponential decay estimation. Instrument response deconvolution and fluorescence exponential decay estimation can be performed in several ways including iterative least squares minimization and Laguerre deconvolution. This paper compares the limitations and accuracy of these fluorescence decay analysis techniques to accurately estimate double exponential decays across many data characteristics including various lifetime values, lifetime component weights, signal-to-noise ratios, and number of photons detected. Furthermore, techniques to improve data fitting, including binning data temporally and spatially, are evaluated as methods to improve decay fits and reduce image acquisition time. Simulation results demonstrate that binning temporally to 36 or 42 time bins, improves accuracy of fits for low photon count data. Such a technique reduces the required number of photons for accurate component estimation if lifetime values are known, such as for commercial fluorescent dyes and FRET experiments, and improve imaging speed 10-fold.

Parallel excitation-emission multiplexed fluorescence lifetime confocal microscopy for live cell imaging.

PubMed

Zhao, Ming; Li, Yu; Peng, Leilei

2014-05-05

We present a novel excitation-emission multiplexed fluorescence lifetime microscopy (FLIM) method that surpasses current FLIM techniques in multiplexing capability. The method employs Fourier multiplexing to simultaneously acquire confocal fluorescence lifetime images of multiple excitation wavelength and emission color combinations at 44,000 pixels/sec. The system is built with low-cost CW laser sources and standard PMTs with versatile spectral configuration, which can be implemented as an add-on to commercial confocal microscopes. The Fourier lifetime confocal method allows fast multiplexed FLIM imaging, which makes it possible to monitor multiple biological processes in live cells. The low cost and compatibility with commercial systems could also make multiplexed FLIM more accessible to biological research community.

Enhanced fluorescence microscope and its application

NASA Astrophysics Data System (ADS)

Wang, Susheng; Li, Qin; Yu, Xin

1997-12-01

A high gain fluorescence microscope is developed to meet the needs in medical and biological research. By the help of an image intensifier with luminance gain of 4 by 104 the sensitivity of the system can achieve 10-6 1x level and be 104 times higher than ordinary fluorescence microscope. Ultra-weak fluorescence image can be detected by it. The concentration of fluorescent label and emitting light intensity of the system are decreased as much as possible, therefore, the natural environment of the detected call can be kept. The CCD image acquisition set-up controlled by computer obtains the quantitative data of each point according to the gray scale. The relation between luminous intensity and output of CCD is obtained by using a wide range weak photometry. So the system not only shows the image of ultra-weak fluorescence distribution but also gives the intensity of fluorescence of each point. Using this system, we obtained the images of distribution of hypocrellin A (HA) in Hela cell, the images of Hela cell being protected by antioxidant reagent Vit. E, SF and BHT. The images show that the digitized ultra-sensitive fluorescence microscope is a useful tool for medical and biological research.

Fast widefield techniques for fluorescence and phase endomicroscopy

NASA Astrophysics Data System (ADS)

Ford, Tim N.

Endomicroscopy is a recent development in biomedical optics which gives researchers and physicians microscope-resolution views of intact tissue to complement macroscopic visualization during endoscopy screening. This thesis presents HiLo endomicroscopy and oblique back-illumination endomicroscopy, fast wide-field imaging techniques with fluorescence and phase contrast, respectively. Fluorescence imaging in thick tissue is often hampered by strong out-of-focus background signal. Laser scanning confocal endomicroscopy has been developed for optically-sectioned imaging free from background, but reliance on mechanical scanning fundamentally limits the frame rate and represents significant complexity and expense. HiLo is a fast, simple, widefield fluorescence imaging technique which rejects out-of-focus background signal without the need for scanning. It works by acquiring two images of the sample under uniform and structured illumination and synthesizing an optically sectioned result with real-time image processing. Oblique back-illumination microscopy (OBM) is a label-free technique which allows, for the first time, phase gradient imaging of sub-surface morphology in thick scattering tissue with a reflection geometry. OBM works by back-illuminating the sample with the oblique diffuse reflectance from light delivered via off-axis optical fibers. The use of two diametrically opposed illumination fibers allows simultaneous and independent measurement of phase gradients and absorption contrast. Video-rate single-exposure operation using wavelength multiplexing is demonstrated.

Nonlinear Structured Illumination Using a Fluorescent Protein Activating at the Readout Wavelength

PubMed Central

Hou, Wenya; Kielhorn, Martin; Arai, Yoshiyuki; Nagai, Takeharu; Kessels, Michael M.; Qualmann, Britta; Heintzmann, Rainer

2016-01-01

Structured illumination microscopy (SIM) is a wide-field technique in fluorescence microscopy that provides fast data acquisition and two-fold resolution improvement beyond the Abbe limit. We observed a further resolution improvement using the nonlinear emission response of a fluorescent protein. We demonstrated a two-beam nonlinear structured illumination microscope by introducing only a minor change into the system used for linear SIM (LSIM). To achieve the required nonlinear dependence in nonlinear SIM (NL-SIM) we exploited the photoswitching of the recently introduced fluorophore Kohinoor. It is particularly suitable due to its positive contrast photoswitching characteristics. Contrary to other reversibly photoswitchable fluorescent proteins which only have high photostability in living cells, Kohinoor additionally showed little degradation in fixed cells over many switching cycles. PMID:27783656

Compression of multispectral fluorescence microscopic images based on a modified set partitioning in hierarchal trees

NASA Astrophysics Data System (ADS)

Mansoor, Awais; Robinson, J. Paul; Rajwa, Bartek

2009-02-01

Modern automated microscopic imaging techniques such as high-content screening (HCS), high-throughput screening, 4D imaging, and multispectral imaging are capable of producing hundreds to thousands of images per experiment. For quick retrieval, fast transmission, and storage economy, these images should be saved in a compressed format. A considerable number of techniques based on interband and intraband redundancies of multispectral images have been proposed in the literature for the compression of multispectral and 3D temporal data. However, these works have been carried out mostly in the elds of remote sensing and video processing. Compression for multispectral optical microscopy imaging, with its own set of specialized requirements, has remained under-investigated. Digital photography{oriented 2D compression techniques like JPEG (ISO/IEC IS 10918-1) and JPEG2000 (ISO/IEC 15444-1) are generally adopted for multispectral images which optimize visual quality but do not necessarily preserve the integrity of scientic data, not to mention the suboptimal performance of 2D compression techniques in compressing 3D images. Herein we report our work on a new low bit-rate wavelet-based compression scheme for multispectral fluorescence biological imaging. The sparsity of signicant coefficients in high-frequency subbands of multispectral microscopic images is found to be much greater than in natural images; therefore a quad-tree concept such as Said et al.'s SPIHT1 along with correlation of insignicant wavelet coefficients has been proposed to further exploit redundancy at high-frequency subbands. Our work propose a 3D extension to SPIHT, incorporating a new hierarchal inter- and intra-spectral relationship amongst the coefficients of 3D wavelet-decomposed image. The new relationship, apart from adopting the parent-child relationship of classical SPIHT, also brought forth the conditional "sibling" relationship by relating only the insignicant wavelet coefficients of subbands at the same level of decomposition. The insignicant quadtrees in dierent subbands in the high-frequency subband class are coded by a combined function to reduce redundancy. A number of experiments conducted on microscopic multispectral images have shown promising results for the proposed method over current state-of-the-art image-compression techniques.

Fourier transform Raman spectroscopy and archaeology: a preliminary study of human teeth

NASA Astrophysics Data System (ADS)

Edwards, Howell G.; Farwell, Dennis W.; Roberts, Charlotte A.; Williams, Adrian C.

1994-01-01

The FT-Raman spectra of human bones and teeth in archaeological specimens dating to the 4th and 10th centuries AD from Romano-British and Anglo-Saxon burial sites in the U.K. have been recorded successfully using microscopic and remote sensing techniques. The samples exhibit fluorescence ascribed to mineral absorption from the grave soils but, nevertheless, good quality spectra are obtained. The versatility of the technique for non destructive sampling is demonstrated.

Two-photon excitation based photochemistry and neural imaging

NASA Astrophysics Data System (ADS)

Hatch, Kevin Andrew

Two-photon microscopy is a fluorescence imaging technique which provides distinct advantages in three-dimensional cellular and molecular imaging. The benefits of this technology may extend beyond imaging capabilities through exploitation of the quantum processes responsible for fluorescent events. This study utilized a two-photon microscope to investigate a synthetic photoreactive collagen peptidomimetic, which may serve as a potential material for tissue engineering using the techniques of two-photon photolysis and two-photon polymerization. The combination of these techniques could potentially be used to produce a scaffold for the vascularization of engineered three-dimensional tissues in vitro to address the current limitations of tissue engineering. Additionally, two-photon microscopy was used to observe the effects of the application of the neurotransmitter dopamine to the mushroom body neural structures of Drosophila melanogaster to investigate dopamine's connection to cognitive degeneration.

Spatial and temporal single-cell volume estimation by a fluorescence imaging technique with application to astrocytes in primary culture

NASA Astrophysics Data System (ADS)

Khatibi, Siamak; Allansson, Louise; Gustavsson, Tomas; Blomstrand, Fredrik; Hansson, Elisabeth; Olsson, Torsten

1999-05-01

Cell volume changes are often associated with important physiological and pathological processes in the cell. These changes may be the means by which the cell interacts with its surrounding. Astroglial cells change their volume and shape under several circumstances that affect the central nervous system. Following an incidence of brain damage, such as a stroke or a traumatic brain injury, one of the first events seen is swelling of the astroglial cells. In order to study this and other similar phenomena, it is desirable to develop technical instrumentation and analysis methods capable of detecting and characterizing dynamic cell shape changes in a quantitative and robust way. We have developed a technique to monitor and to quantify the spatial and temporal volume changes in a single cell in primary culture. The technique is based on two- and three-dimensional fluorescence imaging. The temporal information is obtained from a sequence of microscope images, which are analyzed in real time. The spatial data is collected in a sequence of images from the microscope, which is automatically focused up and down through the specimen. The analysis of spatial data is performed off-line and consists of photobleaching compensation, focus restoration, filtering, segmentation and spatial volume estimation.

A dark-field microscope for background-free detection of resonance fluorescence from single semiconductor quantum dots operating in a set-and-forget mode

NASA Astrophysics Data System (ADS)

Kuhlmann, Andreas V.; Houel, Julien; Brunner, Daniel; Ludwig, Arne; Reuter, Dirk; Wieck, Andreas D.; Warburton, Richard J.

2013-07-01

Optically active quantum dots, for instance self-assembled InGaAs quantum dots, are potentially excellent single photon sources. The fidelity of the single photons is much improved using resonant rather than non-resonant excitation. With resonant excitation, the challenge is to distinguish between resonance fluorescence and scattered laser light. We have met this challenge by creating a polarization-based dark-field microscope to measure the resonance fluorescence from a single quantum dot at low temperature. We achieve a suppression of the scattered laser exceeding a factor of 107 and background-free detection of resonance fluorescence. The same optical setup operates over the entire quantum dot emission range (920-980 nm) and also in high magnetic fields. The major development is the outstanding long-term stability: once the dark-field point has been established, the microscope operates for days without alignment. The mechanical and optical designs of the microscope are presented, as well as exemplary resonance fluorescence spectroscopy results on individual quantum dots to underline the microscope's excellent performance.

Deep UV laser-induced fluorescence detection of unlabeled drugs and proteins in microchip electrophoresis.

PubMed

Schulze, Philipp; Ludwig, Martin; Kohler, Frank; Belder, Detlev

2005-03-01

Deep UV fluorescence detection at 266-nm excitation wavelength has been realized for sensitive detection in microchip electrophoresis. For this purpose, an epifluorescence setup was developed enabling the coupling of a deep UV laser into a commercial fluorescence microscope. Deep UV laser excitation utilizing a frequency quadrupled pulsed laser operating at 266 nm shows an impressive performance for native fluorescence detection of various compounds in fused-silica microfluidic devices. Aromatic low molecular weight compounds such as serotonin, propranolol, a diol, and tryptophan could be detected at low-micromolar concentrations. Deep UV fluorescence detection was also successfully employed for the detection of unlabeled basic proteins. For this purpose, fused-silica chips dynamically coated with hydroxypropylmethyl cellulose were employed to suppress analyte adsorption. Utilizing fused-silica chips permanently coated with poly(vinyl alcohol), it was also possible to separate and detect egg white chicken proteins. These data show that deep UV fluorescence detection significantly widens the application range of fluorescence detection in chip-based analysis techniques.

Multimodal full-field optical coherence tomography on biological tissue: toward all optical digital pathology

NASA Astrophysics Data System (ADS)

Harms, F.; Dalimier, E.; Vermeulen, P.; Fragola, A.; Boccara, A. C.

2012-03-01

Optical Coherence Tomography (OCT) is an efficient technique for in-depth optical biopsy of biological tissues, relying on interferometric selection of ballistic photons. Full-Field Optical Coherence Tomography (FF-OCT) is an alternative approach to Fourier-domain OCT (spectral or swept-source), allowing parallel acquisition of en-face optical sections. Using medium numerical aperture objective, it is possible to reach an isotropic resolution of about 1x1x1 ìm. After stitching a grid of acquired images, FF-OCT gives access to the architecture of the tissue, for both macroscopic and microscopic structures, in a non-invasive process, which makes the technique particularly suitable for applications in pathology. Here we report a multimodal approach to FF-OCT, combining two Full-Field techniques for collecting a backscattered endogeneous OCT image and a fluorescence exogeneous image in parallel. Considering pathological diagnosis of cancer, visualization of cell nuclei is of paramount importance. OCT images, even for the highest resolution, usually fail to identify individual nuclei due to the nature of the optical contrast used. We have built a multimodal optical microscope based on the combination of FF-OCT and Structured Illumination Microscopy (SIM). We used x30 immersion objectives, with a numerical aperture of 1.05, allowing for sub-micron transverse resolution. Fluorescent staining of nuclei was obtained using specific fluorescent dyes such as acridine orange. We present multimodal images of healthy and pathological skin tissue at various scales. This instrumental development paves the way for improvements of standard pathology procedures, as a faster, non sacrificial, operator independent digital optical method compared to frozen sections.

Fluorescence Behavior and Dural Infiltration of Meningioma Analyzed by 5-Aminolevulinic Acid-Based Fluorescence: Operating Microscope Versus Mini-Spectrometer.

PubMed

Knipps, Johannes; Beseoglu, Kerim; Kamp, Marcel; Fischer, Igor; Felsberg, Joerg; Neumann, Lisa M; Steiger, Hans-Jakob; Cornelius, Jan F

2017-12-01

To compare fluorescence intensity of tumor specimens, as measured by a fluorescence-guided surgery microscope and a spectrometer, to evaluate tumor infiltration of dura mater around meningiomas with help of these 2 different 5-aminolevulinic acid (5-ALA)-based fluorescence tools, and to correlate fluorescence intensity with histopathologic data. In a clinical series, meningiomas were resected by 5-ALA fluorescence-guided surgery. Fluorescence intensity was semiquantitatively rated by the surgeon at predefined points. Biopsies were harvested and fluorescence intensity measured by a spectrometer and histopathologically analyzed. Sampling was realized at the level of the dura in a centrifugal direction. A total of 104 biopsies (n = 13 tumors) were analyzed. Specificity and sensitivity of the microscope were 0.96 and 0.53 and of the spectrometer 0.95 and 0.93, respectively. Fluorescence intensity as measured by the spectrometer was correlated to histologically confirmed tumor burden. In a centrifugal direction, tumor burden and fluorescence intensity continuously decreased (along the dural tail). Below a threshold value of 639 arbitrary units no tumor was histologically detectable. At the level of the dura the spectrometer was highly sensitive for detection of meningioma cells. The surgical microscope showed false negative results and missed residual tumor cells in more than one half of the cases. The complementary use of both fluorescence tools may improve resection quality. Copyright © 2017 Elsevier Inc. All rights reserved.

Time-gated FLIM microscope for corneal metabolic imaging

NASA Astrophysics Data System (ADS)

Silva, Susana F.; Batista, Ana; Domingues, José Paulo; Quadrado, Maria João.; Morgado, António Miguel

2016-03-01

Detecting corneal cells metabolic alterations may prove a valuable tool in the early diagnosis of corneal diseases. Nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are autofluorescent metabolic co-factors that allow the assessment of metabolic changes through non-invasive optical methods. These co-factors exhibit double-exponential fluorescence decays, with well-separated short and lifetime components, which are related to their protein-bound and free-states. Corneal metabolism can be assessed by measuring the relative contributions of these two components. For that purpose, we have developed a wide-field time-gated fluorescence lifetime microscope based on structured illumination and one-photon excitation to record FAD lifetime images from corneas. NADH imaging was not considered as its UV excitation peak is regarded as not safe for in vivo measurements. The microscope relies on a pulsed blue diode laser (λ=443 nm) as excitation source, an ultra-high speed gated image intensifier coupled to a CCD camera to acquire fluorescence signals and a Digital Micromirror Device (DMD) to implement the Structured Illumination technique. The system has a lateral resolution better than 2.4 μm, a field of view of 160 per 120 μm and an optical sectioning of 6.91 +/- 0.45 μm when used with a 40x, 0.75 NA, Water Immersion Objective. With this setup we were able to measure FAD contributions from ex-vivo chicken corneas collected from a local slaughterhouse..

Multifocal Fluorescence Microscope for Fast Optical Recordings of Neuronal Action Potentials

PubMed Central

Shtrahman, Matthew; Aharoni, Daniel B.; Hardy, Nicholas F.; Buonomano, Dean V.; Arisaka, Katsushi; Otis, Thomas S.

2015-01-01

In recent years, optical sensors for tracking neural activity have been developed and offer great utility. However, developing microscopy techniques that have several kHz bandwidth necessary to reliably capture optically reported action potentials (APs) at multiple locations in parallel remains a significant challenge. To our knowledge, we describe a novel microscope optimized to measure spatially distributed optical signals with submillisecond and near diffraction-limit resolution. Our design uses a spatial light modulator to generate patterned illumination to simultaneously excite multiple user-defined targets. A galvanometer driven mirror in the emission path streaks the fluorescence emanating from each excitation point during the camera exposure, using unused camera pixels to capture time varying fluorescence at rates that are ∼1000 times faster than the camera’s native frame rate. We demonstrate that this approach is capable of recording Ca2+ transients resulting from APs in neurons labeled with the Ca2+ sensor Oregon Green Bapta-1 (OGB-1), and can localize the timing of these events with millisecond resolution. Furthermore, optically reported APs can be detected with the voltage sensitive dye DiO-DPA in multiple locations within a neuron with a signal/noise ratio up to ∼40, resolving delays in arrival time along dendrites. Thus, the microscope provides a powerful tool for photometric measurements of dynamics requiring submillisecond sampling at multiple locations. PMID:25650920

A compact light-sheet microscope for the study of the mammalian central nervous system

PubMed Central

Yang, Zhengyi; Haslehurst, Peter; Scott, Suzanne; Emptage, Nigel; Dholakia, Kishan

2016-01-01

Investigation of the transient processes integral to neuronal function demands rapid and high-resolution imaging techniques over a large field of view, which cannot be achieved with conventional scanning microscopes. Here we describe a compact light sheet fluorescence microscope, featuring a 45° inverted geometry and an integrated photolysis laser, that is optimized for applications in neuroscience, in particular fast imaging of sub-neuronal structures in mammalian brain slices. We demonstrate the utility of this design for three-dimensional morphological reconstruction, activation of a single synapse with localized photolysis, and fast imaging of neuronal Ca2+ signalling across a large field of view. The developed system opens up a host of novel applications for the neuroscience community. PMID:27215692

Multimodality hard-x-ray imaging of a chromosome with nanoscale spatial resolution

DOE PAGES

Yan, Hanfei; Nazaretski, Evgeny; Lauer, Kenneth R.; ...

2016-02-05

Here, we developed a scanning hard x-ray microscope using a new class of x-ray nano-focusing optic called a multilayer Laue lens and imaged a chromosome with nanoscale spatial resolution. The combination of the hard x-ray's superior penetration power, high sensitivity to elemental composition, high spatial-resolution and quantitative analysis creates a unique tool with capabilities that other microscopy techniques cannot provide. Using this microscope, we simultaneously obtained absorption-, phase-, and fluorescence-contrast images of Pt-stained human chromosome samples. The high spatial-resolution of the microscope and its multi-modality imaging capabilities enabled us to observe the internal ultra-structures of a thick chromosome without sectioningmore » it.« less

Multimodality hard-x-ray imaging of a chromosome with nanoscale spatial resolution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yan, Hanfei; Nazaretski, Evgeny; Lauer, Kenneth R.

Here, we developed a scanning hard x-ray microscope using a new class of x-ray nano-focusing optic called a multilayer Laue lens and imaged a chromosome with nanoscale spatial resolution. The combination of the hard x-ray's superior penetration power, high sensitivity to elemental composition, high spatial-resolution and quantitative analysis creates a unique tool with capabilities that other microscopy techniques cannot provide. Using this microscope, we simultaneously obtained absorption-, phase-, and fluorescence-contrast images of Pt-stained human chromosome samples. The high spatial-resolution of the microscope and its multi-modality imaging capabilities enabled us to observe the internal ultra-structures of a thick chromosome without sectioningmore » it.« less

Multiphoton microscopic imaging of human normal and cancerous oesophagus tissue.

PubMed

Chen, W S; Wang, Y; Liu, N R; Zhang, J X; Chen, R

2014-01-01

In this paper, microstructures of human oesophageal submucosa are evaluated using multiphoton microscopy, based on two-photon excited fluorescence and second harmonic generation. The content and distribution of collagen, elastic fibers and cancer cells in normal and cancerous submucosa layer have been distinctly obtained and briefly discussed. The variation of these components is very relevant to the pathology in oesophagus, especially in early oesophageal cancer. Our results further indicate that the multiphoton microscopy technique has the potential application in vivo in clinical diagnosis and monitoring of early oesophageal cancer. © 2013 The Authors Journal of Microscopy © 2013 Royal Microscopical Society.

Examining the contents of isolated Xenopus germinal vesicles.

PubMed

Gall, Joseph G; Wu, Zheng'an

2010-05-01

One can manually isolate the giant oocyte nucleus or germinal vesicle (GV) of Xenopus from a living oocyte with nothing more complicated than jewelers' forceps and a dissecting microscope. Similarly, one can remove the nuclear envelope by hand and allow the lampbrush chromosomes and other nuclear organelles to spread on a microscope slide. After centrifugation, the nuclear contents adhere tightly to the slide, where they can be subjected to immunostaining or fluorescent in situ hybridization for visualization by conventional or confocal microscopy. Preparations of isolated GV contents reveal details of nuclear structure that are almost impossible to attain by more conventional techniques.

SU-E-T-98: Towards Cell Nucleus Microdosimetry: Construction of a Confocal Laser-Scanning Fluorescence Microscope to Readout Fluorescence Nuclear Track Detectors (FNTDs).

PubMed

McFadden, C; Bartz, J; Akselrod, M; Sawakuchi, G

2012-06-01

To construct a custom confocal laser scanning microscope (CLSM) capable of resolving individual proton tracks in the volume of an Al 2 O 3 :C,Mg fluorescent nuclear track detector (FNTD). The spatial resolution of the FNTD technique is at the sub-micrometer scale. Therefore the FNTD technique has the potential to perform radiation measurements at the cell nucleus scale. The crystal volume of an FNTD contains defects which become fluorescent F 2 + centers after trapping delta electrons from ionizing radiation. These centers have an absorption band centered at 620 nm and an emission band in the near infrared. Events of energy deposition in the crystal are read-out using a CLSM with sub-micrometer spatial resolution. Excitation light from a 635 nm laser is focused in the crystal volume by an objective lens. Fluorescence is collected back through the same path, filtered through a dichroic mirror, and focused through a small pinhole onto an avalanche photodiode. Lateral scanning of the focal point is performed with a scanning mirror galvanometer, and axial scanning is performed using a stepper-motor stage. Control of electronics and image acquisition was performed using a custom built LabVIEW VI and further image processing was done using Java. The system was used to scan FNTDs exposed to a 6 MV x-ray beam and an unexposed FNTD. Fluorescence images above the unexposed background were obtained at scan depths ranging from 5 - 10 micrometer below the crystal surface using a 100 micrometer pinhole size. Further work needs to be done to increase the resolution and the signal to noise ratio of the images so that energy deposition events may be identified more easily. Natural Sciences and Engineering Research Council of Canada. © 2012 American Association of Physicists in Medicine.

Time-resolved fluorescence microscopy to study biologically related applications using sol-gel derived and cellular media

NASA Astrophysics Data System (ADS)

Toury, Marion; Chandler, Lin; Allison, Archie; Campbell, David; McLoskey, David; Holmes-Smith, A. Sheila; Hungerford, Graham

2011-03-01

Fluorescence microscopy provides a non-invasive means for visualising dynamic protein interactions. As well as allowing the calculation of kinetic processes via the use of time-resolved fluorescence, localisation of the protein within cells or model systems can be monitored. These fluorescence lifetime images (FLIM) have become the preferred technique for elucidating protein dynamics due to the fact that the fluorescence lifetime is an absolute measure, in the main independent of fluorophore concentration and intensity fluctuations caused by factors such as photobleaching. In this work we demonstrate the use of a time-resolved fluorescence microscopy, employing a high repetition rate laser excitation source applied to study the influence of a metal surface on fluorescence tagged protein and to elucidate viscosity using the fluorescence lifetime probe DASPMI. These were studied in a cellular environment (yeast) and in a model system based on a sol-gel derived material, in which silver nanostructures were formed in situ using irradiation from a semiconductor laser in CW mode incorporated on a compact time-resolved fluorescence microscope (HORIBA Scientific DeltaDiode and DynaMyc).

A nano grating tunable MEMS optical filter for high-speed on-chip multispectral fluorescent detection.

PubMed

Truxal, Steven C; Huang, Nien-Tsu; Kurabayashi, Katsuo

2009-01-01

We report a microelectromechanical (MEMS) tunable optical filter and its integration in a fluorescence microscope for high speed on-chip spectral measurements. This integration allows for measurements of any fluorescence sample placed onto the microscope stage. We demonstrate the system capabilities by taking spectral measurements of multicolor fluorescent beads and fluorescently labeled cells passing through a microfluidic cytometer. The system has applications in biological studies where the measurement of multiple fluorescent peaks is restricted by the detection method's speed and sensitivity.

A Generalization of Theory for Two-Dimensional Fluorescence Recovery after Photobleaching Applicable to Confocal Laser Scanning Microscopes

PubMed Central

Kang, Minchul; Day, Charles A.; Drake, Kimberly; Kenworthy, Anne K.; DiBenedetto, Emmanuele

2009-01-01

Abstract Fluorescence recovery after photobleaching (FRAP) using confocal laser scanning microscopes (confocal FRAP) has become a valuable technique for studying the diffusion of biomolecules in cells. However, two-dimensional confocal FRAP sometimes yields results that vary with experimental setups, such as different bleaching protocols and bleaching spot sizes. In addition, when confocal FRAP is used to measure diffusion coefficients (D) for fast diffusing molecules, it often yields D-values that are one or two orders-of-magnitude smaller than that predicted theoretically or measured by alternative methods such as fluorescence correlation spectroscopy. Recently, it was demonstrated that this underestimation of D can be corrected by taking diffusion during photobleaching into consideration. However, there is currently no consensus on confocal FRAP theory, and no efforts have been made to unify theories on conventional and confocal FRAP. To this end, we generalized conventional FRAP theory to incorporate diffusion during photobleaching so that analysis by conventional FRAP theory for a circular region of interest is easily applicable to confocal FRAP. Finally, we demonstrate the accuracy of these new (to our knowledge) formulae by measuring D for soluble enhanced green fluorescent protein in aqueous glycerol solution and in the cytoplasm and nucleus of COS7 cells. PMID:19720039

Long-working-distance fluorescence microscope with high-numerical-aperture objectives for variable-magnification imaging in live mice from macro- to subcellular

NASA Astrophysics Data System (ADS)

Kimura, Hiroaki; Momiyama, Masashi; Tomita, Katsuro; Tsuchiya, Hiroyuki; Hoffman, Robert M.

2010-11-01

We demonstrate the development of a long-working-distance fluorescence microscope with high-numerical-aperture objectives for variable-magnification imaging in live mice from macro- to subcellular. To observe cytoplasmic and nuclear dynamics of cancer cells in the living mouse, 143B human osteosarcoma cells are labeled with green fluorescent protein in the nucleus and red fluorescent protein in the cytoplasm. These dual-color cells are injected by a vascular route in an abdominal skin flap in nude mice. The mice are then imaged with the Olympus MVX10 macroview fluorescence microscope. With the MVX10, the nuclear and cytoplasmic behavior of cancer cells trafficking in blood vessels of live mice is observed. We also image lung metastases in live mice from the macro- to the subcellular level by opening the chest wall and imaging the exposed lung in live mice. Injected splenocytes, expressing cyan fluorescent protein, could also be imaged on the lung of live mice. We demonstrate that the MVX10 microscope offers the possibility of full-range in vivo fluorescence imaging from macro- to subcellular and should enable widespread use of powerful imaging technologies enabled by genetic reporters and other fluorophores.

Protein immobilization onto electrochemically synthesized CoFe nanowires

PubMed Central

Torati, Sri Ramulu; Reddy, Venu; Yoon, Seok Soo; Kim, CheolGi

2015-01-01

CoFe nanowires have been synthesized by the electrodeposition technique into the pores of a polycarbonate membrane with a nominal pore diameter of 50 nm, and the composition of CoFe nanowires varying by changing the source concentration of iron. The synthesized nanowire surfaces were functionalized with amine groups by treatment with aminopropyltriethoxysilane (APTES) linker, and then conjugated with streptavidin-Cy3 protein via ethyl (dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide coupling chemistry. The oxide surface of CoFe nanowire is easily modified with aminopropyltriethoxysilane to form an amine terminating group, which is covalently bonded to streptavidin-Cy3 protein. The physicochemical properties of the nanowires were analyzed through different characterization techniques such as scanning electron microscope, energy dispersive spectroscopy, and vibrating sample magnetometer. Fluorescence microscopic studies and Fourier transform infrared studies confirmed the immobilization of protein on the nanowire surface. In addition, the transmission electron microscope analysis reveals the thin protein layer which is around 12–15 nm on the nanowire surfaces. PMID:25609966

Dental enamel defect diagnosis through different technology-based devices.

PubMed

Kobayashi, Tatiana Yuriko; Vitor, Luciana Lourenço Ribeiro; Carrara, Cleide Felício Carvalho; Silva, Thiago Cruvinel; Rios, Daniela; Machado, Maria Aparecida Andrade Moreira; Oliveira, Thais Marchini

2018-06-01

Dental enamel defects (DEDs) are faulty or deficient enamel formations of primary and permanent teeth. Changes during tooth development result in hypoplasia (a quantitative defect) and/or hypomineralisation (a qualitative defect). To compare technology-based diagnostic methods for detecting DEDs. Two-hundred and nine dental surfaces of anterior permanent teeth were selected in patients, 6-11 years of age, with cleft lip with/without cleft palate. First, a conventional clinical examination was conducted according to the modified Developmental Defects of Enamel Index (DDE Index). Dental surfaces were evaluated using an operating microscope and a fluorescence-based device. Interexaminer reproducibility was determined using the kappa test. To compare groups, McNemar's test was used. Cramer's V test was used for comparing the distribution of index codes obtained after classification of all dental surfaces. Cramer's V test revealed statistically significant differences (P < .0001) in the distribution of index codes obtained using the different methods; the coefficients were 0.365 for conventional clinical examination versus fluorescence, 0.961 for conventional clinical examination versus operating microscope and 0.358 for operating microscope versus fluorescence. The sensitivity of the operating microscope and fluorescence method was statistically significant (P = .008 and P < .0001, respectively). Otherwise, the results did not show statistically significant differences in accuracy and specificity for either the operating microscope or the fluorescence methods. This study suggests that the operating microscope performed better than the fluorescence-based device and could be an auxiliary method for the detection of DEDs. © 2017 FDI World Dental Federation.

O5.05FLUORESCEIN-GUIDED REMOVAL OF HIGH-GRADE GLIOMAS WITH A DEDICATED FILTER ON THE SURGICAL MICROSCOPE: PRELIMINARY RESULTS OF THE FLUOGLIO STUDY

PubMed Central

Acerbi, F.; Broggi, M.; Cavallo, C.; Anghileri, E.; Eoli, M.; Schiariti, M.; Corte, E. La; Pollo, B.; Boffano, C.; Ferroli, P.

2014-01-01

BACKGROUND: Fluorescein is a fluorescent tracer that can be used for many applications. It is able to accumulate in brain areas with blood-brain barrier disruption, and thus it can be considered an ideal dye for intraoperative visualization of high-grade gliomas (HGG). We report the preliminary results of a phase II trail (FLUOGLIO) on a new fluorescein-guided technique to remove HHG with a dedicated filter on the surgical microscope. METHODS: In September 2011 we started a prospective phase II-trial (FLUOGLIO) to evaluated safety and obtain initial indications about efficacy of fluorescein-guided surgery for HGG. Patients with suspected HGG amenable to complete resection of contrast-enhancing area were eligible to participate in this study. This report is based on the analysis of the short- and long-term results in 28 consecutive patients with HGG (age range 45-74 years), enrolled since September 2011. Fluorescein was intravenous (i.v.) injected after intubation (5-10 mg/Kg). Tumor was removed with microsurgical technique and fluorescence visualization by BLU400 or YELLOW560 filters on Pentero microscope (Carl Zeiss, Germany). The study was approved by our Ethical Committee and registered on the European Regulatory Authorities website (EudraCT No. 2011-002527-18). RESULTS: Median pre-operative tumor volume was 33.1 cm3 (2.4-87.8 cm3). We found no adverse reaction to fluorescein administration. Tumor was completely removed in 80% of the patients. Median follow-up was 10 months. 6 months Progression-free Survival (PFS) and median survival were respectively 71.4 % and 11 months. CONCLUSION: Our analysis suggested that fluorescein-guided technique with a dedicated filter on the surgical microscope is safe and allows high-rate of complete resection of contrast-enhanced tumor at the early post-operative MRI.

Detection of polycyclic aromatic hydrocarbons (PAHs) in Medicago sativa L. by fluorescence microscopy.

PubMed

Alves, Wilber S; Manoel, Evelin A; Santos, Noemi S; Nunes, Rosane O; Domiciano, Giselli C; Soares, Marcia R

2017-04-01

Green technologies, such as phytoremediation, are effective for removing organic pollutants derived from oil and oil products, including polycyclic aromatic hydrocarbons (PAHs). Given the increasing popularity of these sustainable remediation techniques, methods based on fluorescence microscopy and multiphoton microscopy for the environmental monitoring of such pollutants have emerged in recent decades as effective tools for phytoremediation studies aimed at understanding the fate of these contaminants in plants. However, little is known about the cellular and molecular mechanisms involved in PAH uptake, responses and degradation by plants. Thus, the present study aimed to detect the location of pyrene, anthracene and phenanthrene using fluorescence microscopy techniques in shoots and roots of Medicago sativa L. (alfalfa) plants grown in artificially contaminated soil (150ppm PAHs) for 40days. Leaflet and root samples were then collected and observed under a fluorescence microscope to detect the presence of PAHs in various tissues. One important finding of the present study was intense fluorescence in the glandular secreting trichomes (GSTs) of plants grown in contaminated soil. These trichomes, with a previously unknown function, may be sites of PAH conjugation and degradation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Assessment of incomplete clipping of aneurysms intraoperatively by a near-infrared indocyanine green-video angiography (Niicg-Va) integrated microscope.

PubMed

Imizu, S; Kato, Y; Sangli, A; Oguri, D; Sano, H

2008-08-01

The objective of this article was to assess the clinical use and the completeness of clipping with total occlusion of the aneurysmal lumen, real-time assessment of vascular patency in the parent, branching and perforating vessels, intraoperative assessment of blood flow, image quality, spatial resolution and clinical value in difficult aneurysms using near infrared indocyanine green video angiography integrated on to an operative Pentero neurosurgical microscope (Carl Zeiss, Oberkochen Germany). Thirteen patients with aneurysms were operated upon. An infrared camera with near infrared technology was adapted on to the OPMI Pentero microscope with a special filter and infrared excitation light to illuminate the operating field which was designed to allow passage of the near infrared light required for excitation of indocyanine green (ICG) which was used as the intravascular marker. The intravascular fluorescence was imaged with a video camera attached to the microscope. ICG fluorescence (700-850 nm) from a modified microscope light source on to the surgical field and passage of ICG fluorescence (780-950 nm) from the surgical field, back into the optical path of the microscope was used to detect the completeness of aneurysmal clipping Incomplete clipping in three patients (1 female and 2 males) with unruptured complicated aneurysms was detected using indocyanine green video angiography. There were no adverse effects after injection of indocyanine green. The completeness of clipping was inadequately detected by Doppler ultrasound miniprobe and rigid endoscopy and was thus complemented by indocyanine green video angiography. The operative microscope-integrated ICG video angiography as a new intraoperative method for detecting vascular flow, was found to be quick, reliable, cost-effective and possibly a substitute or adjunct for Doppler ultrasonography or intraoperative DSA, which is presently the gold standard. The simplicity of the method, the speed with which the investigation can be performed, the quality of the images, and the outcome of surgical procedures have all reduced the need for angiography. This technique may be useful during routine aneurysm surgery as an independent form of angiography and/or as an adjunct to intraoperative or postoperative DSA.

Confocal laser scanning microscopic photoconversion: a new method to stabilize fluorescently labeled cellular elements for electron microscopic analysis.

PubMed

Colello, Raymond J; Tozer, Jordan; Henderson, Scott C

2012-01-01

Photoconversion, the method by which a fluorescent dye is transformed into a stable, osmiophilic product that can be visualized by electron microscopy, is the most widely used method to enable the ultrastructural analysis of fluorescently labeled cellular structures. Nevertheless, the conventional method of photoconversion using widefield fluorescence microscopy requires long reaction times and results in low-resolution cell targeting. Accordingly, we have developed a photoconversion method that ameliorates these limitations by adapting confocal laser scanning microscopy to the procedure. We have found that this method greatly reduces photoconversion times, as compared to conventional wide field microscopy. Moreover, region-of-interest scanning capabilities of a confocal microscope facilitate the targeting of the photoconversion process to individual cellular or subcellular elements within a fluorescent field. This reduces the area of the cell exposed to light energy, thereby reducing the ultrastructural damage common to this process when widefield microscopes are employed. © 2012 by John Wiley & Sons, Inc.

Scanning Fiber Endoscope Improves Detection of 5-Aminolevulinic Acid-Induced Protoporphyrin IX Fluorescence at the Boundary of Infiltrative Glioma.

PubMed

Belykh, Evgenii; Miller, Eric J; Hu, Danying; Martirosyan, Nikolay L; Woolf, Eric C; Scheck, Adrienne C; Byvaltsev, Vadim A; Nakaji, Peter; Nelson, Leonard Y; Seibel, Eric J; Preul, Mark C

2018-05-01

Fluorescence-guided surgery with protoporphyrin IX (PpIX) as a photodiagnostic marker is gaining acceptance for resection of malignant gliomas. Current wide-field imaging technologies do not have sufficient sensitivity to detect low PpIX concentrations. We evaluated a scanning fiber endoscope (SFE) for detection of PpIX fluorescence in gliomas and compared it to an operating microscope (OPMI) equipped with a fluorescence module and to a benchtop confocal laser scanning microscope (CLSM). 5-Aminolevulinic acid-induced PpIX fluorescence was assessed in GL261-Luc2 cells in vitro and in vivo after implantation in mouse brains, at an invading glioma growth stage, simulating residual tumor. Intraoperative fluorescence of high and low PpIX concentrations in normal brain and tumor regions with SFE, OPMI, CLSM, and histopathology were compared. SFE imaging of PpIX correlated to CLSM at the cellular level. PpIX accumulated in normal brain cells but significantly less than in glioma cells. SFE was more sensitive to accumulated PpIX in fluorescent brain areas than OPMI (P < 0.01) and dramatically increased imaging time (>6×) before tumor-to-background contrast was diminished because of photobleaching. SFE provides new endoscopic capabilities to view PpIX-fluorescing tumor regions at cellular resolution. SFE may allow accurate imaging of 5-aminolevulinic acid labeling of gliomas and other tumor types when current detection techniques have failed to provide reliable visualization. SFE was significantly more sensitive than OPMI to low PpIX concentrations, which is relevant to identifying the leading edge or metastasizing cells of malignant glioma or to treating low-grade gliomas. This new application has the potential to benefit surgical outcomes. Copyright © 2018 Elsevier Inc. All rights reserved.

Automated four color CD4/CD8 analysis of leukocytes by scanning fluorescence microscopy using Quantum dots

NASA Astrophysics Data System (ADS)

Bocsi, Jozsef; Mittag, Anja; Varga, Viktor S.; Molnar, Bela; Tulassay, Zsolt; Sack, Ulrich; Lenz, Dominik; Tarnok, Attila

2006-02-01

Scanning Fluorescence Microscope (SFM) is a new technique for automated motorized microscopes to measure multiple fluorochrome labeled cells (Bocsi et al. Cytometry 2004, 61A:1). The ratio of CD4+/CD8+ cells is an important in immune diagnostics in immunodeficiency and HIV. Therefor a four-color staining protocol (DNA, CD3, CD4 and CD8) for automated SFM analysis of lymphocytes was developed. EDTA uncoagulated blood was stained with organic and inorganic (Quantum dots) fluorochromes in different combinations. Aliquots of samples were measured by Flow Cytometry (FCM) and SFM. By SFM specimens were scanned and digitized using four fluorescence filter sets. Automated cell detection (based on Hoechst 33342 fluorescence), CD3, CD4 and CD8 detection were performed, CD4/CD8 ratio was calculated. Fluorescence signals were well separable on SFM and FCM. Passing and Bablok regression of all CD4/CD8 ratios obtained by FCM and SFM (F(X)=0.0577+0.9378x) are in the 95% confidence interval. Cusum test did not show significant deviation from linearity (P>0.10). This comparison indicates that there is no systemic bias between the two different methods. In SFM analyses the inorganic Quantum dot staining was very stable in PBS in contrast to the organic fluorescent dyes, but bleached shortly after mounting with antioxidant and free radical scavenger mounting media. This shows the difficulty of combinations of organic dyes and Quantum dots. Slide based multi-fluorescence labeling system and automated SFM are applicable tools for the CD4/CD8 ratio determination in peripheral blood samples. Quantum Dots are stable inorganic fluorescence labels that may be used as reliable high resolution dyes for cell labeling.

Correlative cryogenic tomography of cells using light and soft x-rays.

PubMed

Smith, Elizabeth A; Cinquin, Bertrand P; Do, Myan; McDermott, Gerry; Le Gros, Mark A; Larabell, Carolyn A

2014-08-01

Correlated imaging is the process of imaging a specimen with two complementary modalities, and then combining the two data sets to create a highly informative, composite view. A recent implementation of this concept has been the combination of soft x-ray tomography (SXT) with fluorescence cryogenic microscopy (FCM). SXT-FCM is used to visualize cells that are held in a near-native, cryopreserved. The resultant images are, therefore, highly representative of both the cellular architecture and molecular organization in vivo. SXT quantitatively visualizes the cell and sub-cellular structures; FCM images the spatial distribution of fluorescently labeled molecules. Here, we review the characteristics of SXT-FCM, and briefly discuss how this method compares with existing correlative imaging techniques. We also describe how the incorporation of a cryo-rotation stage into a cryogenic fluorescence microscope allows acquisition of fluorescence cryogenic tomography (FCT) data. FCT is optimally suited for correlation with SXT, since both techniques image the specimen in 3-D, potentially with similar, isotropic spatial resolution. © 2013 Elsevier B.V. All rights reserved.

Demonstration of lipofuscin and Nissl bodies in crystal violet stained sections using a fluorescence technique or pyronin Y stain.

PubMed

Terr, L I

1986-09-01

This paper presents two simple, reliable methods for identification of lipofuscin and Nissl bodies in the same section. One method shows that lipofuscin stained with crystal violet retains its ability to fluoresce and can be observed under the fluorescence microscope after the stain has faded. Fading is accompanied by a gradual increase in the intensity of the fluorescence and is complete in about 5 min. Exciting illumination from this part of the spectrum also substantially fades staining of other autofluorescing tissue elements, such as lipids. Nonfluorescing structures, such as Nissl bodies, remain stained. By changing from transillumination with tungsten light to epifluorescent illumination and vice versa, both types of structures--Nissl bodies and lipofuscin--can be identified in the same section. The second technique uses pyronin Y for staining Nissl bodies in preparations previously stained with crystal violet. Nissl bodies are stained pink but lipofuscin remains violet. Lipofuscin in these sections also remains autofluorescent after the crystal violet stain has faded under violet or near-UV light.

Eu3+-doped Gd2O3 nanoparticles as reporters for optical detection and visualization of antibodies patterned by microcontact printing.

PubMed

Nichkova, Mikaela; Dosev, Dosi; Perron, Richard; Gee, Shirley J; Hammock, Bruce D; Kennedy, Ian M

2006-02-01

Lanthanide oxide nanoparticles are promising luminescent probes in bioanalysis, because of their unique spectral properties, photostability, and low-cost synthesis. We report for the first time the application of europium-doped gadolinium oxide (Eu:Gd2O3) nanoparticles to the optical imaging of antibody micropatterns. The nanoparticles were synthesized by spray pyrolysis and coated with antibody (IgG) molecules by physical adsorption. Our experiments showed that the Eu:Gd2O3 is a good biocompatible solid support for antibody immobilization. The antibodies (anti-rabbit IgG) immobilized on the nanoparticles had excellent biological activity in the specific recognition reaction with rabbit IgG patterned in line strips (10 micromx10 microm) on a glass substrate by use of a micro-contact printing technique. The specific immunoreaction was confirmed by two independent microscopic techniques-fluorescence and scanning electron microscopy (SEM). Both microscopic images revealed that the nanoparticles were organized into designated structures as defined by the microcontact printing process with negligible non-specific binding. The nanoparticles can be used as fluorescent markers in a variety of immunosensing applications in a microscale format.

A setup for combined multiphoton laser scanning microscopic and multi-electrode patch clamp experiments on brain slices

NASA Astrophysics Data System (ADS)

Helm, P. Johannes; Reppen, Trond; Heggelund, Paul

2009-02-01

Multi Photon Laser Scanning Microscopy (MPLSM) appears today as one of the most powerful experimental tools in cellular neurophysiology, notably in studies of the functional dynamics of signal processing in single neurons. Simultaneous recording of fluorescence signals at high spatial and temporal resolution and electric signals by means of multi electrode patch clamp techniques have provided new paths for the systematic investigation of neuronal mechanisms. In particular, this approach has opened for direct studies of dendritic signal processing in neurons. We report about a setup optimized for simultaneous electrophysiological multi electrode patch clamp and multi photon laser scanning fluorescence microscopic experiments on brain slices. The microscopic system is based on a modified commercially available confocal scanning laser microscope (CLSM). From a technical and operational point of view, two developments are important: Firstly, in order to reduce the workload for the experimentalist, who in general is forced to concentrate on controlling the electrophysiological parameters during the recordings, a system of shutters has been installed together with dedicated electronic modules protecting the photo detectors against destructive light levels caused by erroneous opening or closing of microscopic light paths by the experimentalist. Secondly, the standard detection unit has been improved by installing the photomultiplier tubes (PMT) in a Peltier cooled thermal box shielding the detector from both room temperature and distortions caused by external electromagnetic fields. The electrophysiological system is based on an industrial standard multi patch clamp unit ergonomically arranged around the microscope stage. The electrophysiological and scanning processes can be time coordinated by standard trigger electronics.

Parallel excitation-emission multiplexed fluorescence lifetime confocal microscopy for live cell imaging

PubMed Central

Zhao, Ming; Li, Yu; Peng, Leilei

2014-01-01

We present a novel excitation-emission multiplexed fluorescence lifetime microscopy (FLIM) method that surpasses current FLIM techniques in multiplexing capability. The method employs Fourier multiplexing to simultaneously acquire confocal fluorescence lifetime images of multiple excitation wavelength and emission color combinations at 44,000 pixels/sec. The system is built with low-cost CW laser sources and standard PMTs with versatile spectral configuration, which can be implemented as an add-on to commercial confocal microscopes. The Fourier lifetime confocal method allows fast multiplexed FLIM imaging, which makes it possible to monitor multiple biological processes in live cells. The low cost and compatibility with commercial systems could also make multiplexed FLIM more accessible to biological research community. PMID:24921725

Hard x-ray phase contrastmicroscopy - techniques and applications

NASA Astrophysics Data System (ADS)

Holzner, Christian

In 1918, Einstein provided the first description of the nature of the refractive index for X-rays, showing that phase contrast effects are significant. A century later, most x-ray microscopy and nearly all medical imaging remains based on absorption contrast, even though phase contrast offers orders of magnitude improvements in contrast and reduced radiation exposure at multi-keV x-ray energies. The work presented is concerned with developing practical and quantitative methods of phase contrast for x-ray microscopy. A theoretical framework for imaging in phase contrast is put forward; this is used to obtain quantitative images in a scanning microscope using a segmented detector, and to correct for artifacts in a commercial phase contrast x-ray nano-tomography system. The principle of reciprocity between scanning and full-field microscopes is then used to arrive at a novel solution: Zernike contrast in a scanning microscope. These approaches are compared on a theoretical and experimental basis in direct connection with applications using multi-keV x-ray microscopes at the Advanced Photon Source at Argonne National Laboratory. Phase contrast provides the best means to image mass and ultrastructure of light elements that mainly constitute biological matter, while stimulated x-ray fluorescence provides high sensitivity for studies of the distribution of heavier trace elements, such as metals. These approaches are combined in a complementary way to yield quantitative maps of elemental concentration from 2D images, with elements placed in their ultrastructural context. The combination of x-ray fluorescence and phase contrast poses an ideal match for routine, high resolution tomographic imaging of biological samples in the future. The presented techniques and demonstration experiments will help pave the way for this development.

Design and performance of an X-ray scanning microscope at the Hard X-ray Nanoprobe beamline of NSLS-II

DOE PAGES

Nazaretski, E.; Yan, H.; Lauer, K.; ...

2017-10-05

A hard X-ray scanning microscope installed at the Hard X-ray Nanoprobe beamline of the National Synchrotron Light Source II has been designed, constructed and commissioned. The microscope relies on a compact, high stiffness, low heat dissipation approach and utilizes two types of nanofocusing optics. It is capable of imaging with ~15 nm × 15 nm spatial resolution using multilayer Laue lenses and 25 nm × 26 nm resolution using zone plates. Fluorescence, diffraction, absorption, differential phase contrast, ptychography and tomography are available as experimental techniques. The microscope is also equipped with a temperature regulation system which allows the temperature ofmore » a sample to be varied in the range between 90 K and 1000 K. The constructed instrument is open for general users and offers its capabilities to the material science, battery research and bioscience communities.« less

Design and performance of an X-ray scanning microscope at the Hard X-ray Nanoprobe beamline of NSLS-II

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nazaretski, E.; Yan, H.; Lauer, K.

A hard X-ray scanning microscope installed at the Hard X-ray Nanoprobe beamline of the National Synchrotron Light Source II has been designed, constructed and commissioned. The microscope relies on a compact, high stiffness, low heat dissipation approach and utilizes two types of nanofocusing optics. It is capable of imaging with ~15 nm × 15 nm spatial resolution using multilayer Laue lenses and 25 nm × 26 nm resolution using zone plates. Fluorescence, diffraction, absorption, differential phase contrast, ptychography and tomography are available as experimental techniques. The microscope is also equipped with a temperature regulation system which allows the temperature ofmore » a sample to be varied in the range between 90 K and 1000 K. The constructed instrument is open for general users and offers its capabilities to the material science, battery research and bioscience communities.« less

Calibration and validation of projection lithography in chemically amplified resist systems using fluorescence imaging

NASA Astrophysics Data System (ADS)

Mason, Michael D.; Ray, Krishanu; Feke, Gilbert D.; Grober, Robert D.; Pohlers, Gerd; Cameron, James F.

2003-05-01

Coumarin 6 (C6), a pH sensitive fluorescent molecule were doped into commercial resist systems to demonstrate a cost-effective fluorescence microscopy technique for detecting latent photoacid images in exposed chemically amplified resist films. The fluorescenec image contrast is optimized by carefully selecting optical filters to match the spectroscopic properties of C6 in the resist matrices. We demonstrate the potential of this technique for two sepcific non-invasive applications. First, a fast, conventient, fluorescence technique is demonstrated for determination of quantum yeidsl of photo-acid generation. Since the Ka of C6 in the 193nm resist system lies wihtin the range of acid concentrations that can be photogenerated, we have used this technique to evaluate the acid generation efficiency of various photo-acid generators (PAGs). The technique is based on doping the resist formulations containing the candidate PAGs with C6, coating one wafer per PAG, patterning the wafer with a dose ramp and spectroscopically imaging the wafers. The fluorescence of each pattern in the dose ramp is measured as a single image and analyzed with the optical titration model. Second, a nondestructive in-line diagnostic technique is developed for the focus calibration and validation of a projection lithography system. Our experimental results show excellent correlation between the fluorescence images and scanning electron microscope analysis of developed features. This technique has successfully been applied in both deep UV resists e.g., Shipley UVIIHS resist and 193 nm resists e.g., Shipley Vema-type resist. This method of focus calibration has also been extended to samples with feature sizes below the diffraction limit where the pitch between adjacent features is on the order of 300 nm. Image capture, data analysis, and focus latitude verification are all computer controlled from a single hardware/software platform. Typical focus calibration curves can be obtained within several minutes.

Modular low-light microscope for imaging cellular bioluminescence and radioluminescence

PubMed Central

Kim, Tae Jin; Türkcan, Silvan; Pratx, Guillem

2017-01-01

Low-light microscopy methods are receiving increased attention as new applications have emerged. One such application is to allow longitudinal imaging of light-sensitive cells with no phototoxicity and no photobleaching of fluorescent biomarkers. Another application is for imaging signals that are inherently dim and undetectable using standard microscopy, such as bioluminescence, chemiluminescence, or radioluminescence. In this protocol, we provide instructions on how to build a modular low-light microscope (1-4 d) by coupling two microscope objective lenses, back-to-back from each other, using standard optomechanical components. We also provide directions on how to image dim signals such as radioluminescence (1-1.5 h), bioluminescence (∼30 min) and low-excitation fluorescence (∼15 min). In particular, radioluminescence microscopy is explained in detail as it is a newly developed technique, which enables the study of small molecule transport (eg. radiolabeled drugs, metabolic precursors, and nuclear medicine contrast agents) by single cells without perturbing endogenous biochemical processes. In this imaging technique, a scintillator crystal (eg. CdWO4) is placed in close proximity to the radiolabeled cells, where it converts the radioactive decays into optical flashes detectable using a sensitive camera. Using the image reconstruction toolkit provided in this protocol, the flashes can be reconstructed to yield high-resolution image of the radiotracer distribution. With appropriate timing, the three aforementioned imaging modalities may be performed altogether on a population of live cells, allowing the user to perform parallel functional studies of cell heterogeneity at the single-cell level. PMID:28426025

Faster and less phototoxic 3D fluorescence microscopy using a versatile compressed sensing scheme

PubMed Central

Woringer, Maxime; Darzacq, Xavier; Zimmer, Christophe

2017-01-01

Three-dimensional fluorescence microscopy based on Nyquist sampling of focal planes faces harsh trade-offs between acquisition time, light exposure, and signal-to-noise. We propose a 3D compressed sensing approach that uses temporal modulation of the excitation intensity during axial stage sweeping and can be adapted to fluorescence microscopes without hardware modification. We describe implementations on a lattice light sheet microscope and an epifluorescence microscope, and show that images of beads and biological samples can be reconstructed with a 5-10 fold reduction of light exposure and acquisition time. Our scheme opens a new door towards faster and less damaging 3D fluorescence microscopy. PMID:28788909

Biological applications of an LCoS-based programmable array microscope (PAM)

NASA Astrophysics Data System (ADS)

Hagen, Guy M.; Caarls, Wouter; Thomas, Martin; Hill, Andrew; Lidke, Keith A.; Rieger, Bernd; Fritsch, Cornelia; van Geest, Bert; Jovin, Thomas M.; Arndt-Jovin, Donna J.

2007-02-01

We report on a new generation, commercial prototype of a programmable array optical sectioning fluorescence microscope (PAM) for rapid, light efficient 3D imaging of living specimens. The stand-alone module, including light source(s) and detector(s), features an innovative optical design and a ferroelectric liquid-crystal-on-silicon (LCoS) spatial light modulator (SLM) instead of the DMD used in the original PAM design. The LCoS PAM (developed in collaboration with Cairn Research, Ltd.) can be attached to a port of a(ny) unmodified fluorescence microscope. The prototype system currently operated at the Max Planck Institute incorporates a 6-position high-intensity LED illuminator, modulated laser and lamp light sources, and an Andor iXon emCCD camera. The module is mounted on an Olympus IX71 inverted microscope with 60-150X objectives with a Prior Scientific x,y, and z high resolution scanning stages. Further enhancements recently include: (i) point- and line-wise spectral resolution and (ii) lifetime imaging (FLIM) in the frequency domain. Multiphoton operation and other nonlinear techniques should be feasible. The capabilities of the PAM are illustrated by several examples demonstrating single molecule as well as lifetime imaging in live cells, and the unique capability to perform photoconversion with arbitrary patterns and high spatial resolution. Using quantum dot coupled ligands we show real-time binding and subsequent trafficking of individual ligand-growth factor receptor complexes on and in live cells with a temporal resolution and sensitivity exceeding those of conventional CLSM systems. The combined use of a blue laser and parallel LED or visible laser sources permits photoactivation and rapid kinetic analysis of cellular processes probed by photoswitchable visible fluorescent proteins such as DRONPA.

Measurement of pH micro-heterogeneity in natural cheese matrices by fluorescence lifetime imaging

PubMed Central

Burdikova, Zuzana; Svindrych, Zdenek; Pala, Jan; Hickey, Cian D.; Wilkinson, Martin G.; Panek, Jiri; Auty, Mark A. E.; Periasamy, Ammasi; Sheehan, Jeremiah J.

2015-01-01

Cheese, a product of microbial fermentation may be defined as a protein matrix entrapping fat, moisture, minerals and solutes as well as dispersed bacterial colonies. The growth and physiology of bacterial cells in these colonies may be influenced by the microenvironment around the colony, or alternatively the cells within the colony may modify the microenvironment (e.g., pH, redox potential) due to their metabolic activity. While cheese pH may be measured at macro level there remains a significant knowledge gap relating to the degree of micro-heterogeneity of pH within the cheese matrix and its relationship with microbial, enzymatic and physiochemical parameters and ultimately with cheese quality, consistency and ripening patterns. The pH of cheese samples was monitored both at macroscopic scale and at microscopic scale, using a non-destructive microscopic technique employing C-SNARF-4 and Oregon Green 488 fluorescent probes. The objectives of this work were to evaluate the suitability of these dyes for microscale pH measurements in natural cheese matrices and to enhance the sensitivity and extend the useful pH range of these probes using fluorescence lifetime imaging (FLIM). In particular, fluorescence lifetime of Oregon Green 488 proved to be sensitive probe to map pH micro heterogeneity within cheese matrices. Good agreement was observed between macroscopic scale pH measurement by FLIM and by traditional pH methods, but in addition considerable localized microheterogeneity in pH was evident within the curd matrix with pH range between 4.0 and 5.5. This technique provides significant potential to further investigate the relationship between cheese matrix physico-chemistry and bacterial metabolism during cheese manufacture and ripening. PMID:25798136

Measurement of pH micro-heterogeneity in natural cheese matrices by fluorescence lifetime imaging.

PubMed

Burdikova, Zuzana; Svindrych, Zdenek; Pala, Jan; Hickey, Cian D; Wilkinson, Martin G; Panek, Jiri; Auty, Mark A E; Periasamy, Ammasi; Sheehan, Jeremiah J

2015-01-01

Cheese, a product of microbial fermentation may be defined as a protein matrix entrapping fat, moisture, minerals and solutes as well as dispersed bacterial colonies. The growth and physiology of bacterial cells in these colonies may be influenced by the microenvironment around the colony, or alternatively the cells within the colony may modify the microenvironment (e.g., pH, redox potential) due to their metabolic activity. While cheese pH may be measured at macro level there remains a significant knowledge gap relating to the degree of micro-heterogeneity of pH within the cheese matrix and its relationship with microbial, enzymatic and physiochemical parameters and ultimately with cheese quality, consistency and ripening patterns. The pH of cheese samples was monitored both at macroscopic scale and at microscopic scale, using a non-destructive microscopic technique employing C-SNARF-4 and Oregon Green 488 fluorescent probes. The objectives of this work were to evaluate the suitability of these dyes for microscale pH measurements in natural cheese matrices and to enhance the sensitivity and extend the useful pH range of these probes using fluorescence lifetime imaging (FLIM). In particular, fluorescence lifetime of Oregon Green 488 proved to be sensitive probe to map pH micro heterogeneity within cheese matrices. Good agreement was observed between macroscopic scale pH measurement by FLIM and by traditional pH methods, but in addition considerable localized microheterogeneity in pH was evident within the curd matrix with pH range between 4.0 and 5.5. This technique provides significant potential to further investigate the relationship between cheese matrix physico-chemistry and bacterial metabolism during cheese manufacture and ripening.

A New Low Cost Wide-Field Illumination Method for Photooxidation of Intracellular Fluorescent Markers

PubMed Central

da Silva Filho, Manoel; Santos, Daniel Valle Vasconcelos; Costa, Kauê Machado

2013-01-01

Analyzing cell morphology is crucial in the fields of cell biology and neuroscience. One of the main methods for evaluating cell morphology is by using intracellular fluorescent markers, including various commercially available dyes and genetically encoded fluorescent proteins. These markers can be used as free radical sources in photooxidation reactions, which in the presence of diaminobenzidine (DAB) forms an opaque and electron-dense precipitate that remains localized within the cellular and organelle membranes. This method confers many methodological advantages for the investigator, including absence of photo-bleaching, high visual contrast and the possibility of correlating optical imaging with electron microscopy. However, current photooxidation techniques require the continuous use of fluorescent or confocal microscopes, which wastes valuable mercury lamp lifetime and limits the conversion process to a few cells at a time. We developed a low cost optical apparatus for performing photooxidation reactions and propose a new procedure that solves these methodological restrictions. Our “photooxidizer” consists of a high power light emitting diode (LED) associated with a custom aluminum and acrylic case and a microchip-controlled current source. We demonstrate the efficacy of our method by converting intracellular DiI in samples of developing rat neocortex and post-mortem human retina. DiI crystals were inserted in the tissue and allowed to diffuse for 20 days. The samples were then processed with the new photooxidation technique and analyzed under optical microscopy. The results show that our protocols can unveil the fine morphology of neurons in detail. Cellular structures such as axons, dendrites and spine-like appendages were well defined. In addition to its low cost, simplicity and reliability, our method precludes the use of microscope lamps for photooxidation and allows the processing of many labeled cells simultaneously in relatively large tissue samples with high efficacy. PMID:23441199

Diagnostic testing for Giardia infections.

PubMed

Heyworth, Martin F

2014-03-01

The traditional method for diagnosing Giardia infections involves microscopic examination of faecal specimens for Giardia cysts. This method is subjective and relies on observer experience. From the 1980s onwards, objective techniques have been developed for diagnosing Giardia infections, and are superseding diagnostic techniques reliant on microscopy. Detection of Giardia antigen(s) by immunoassay is the basis of commercially available diagnostic kits. Various nucleic acid amplification techniques (NAATs) can demonstrate DNA of Giardia intestinalis, and have the potential to become standard approaches for diagnosing Giardia infections. Of such techniques, methods involving either fluorescent microspheres (Luminex) or isothermal amplification of DNA (loop-mediated isothermal amplification; LAMP) are especially promising.

Simultaneous dual-color fluorescence microscope: a characterization study.

PubMed

Li, Zheng; Chen, Xiaodong; Ren, Liqiang; Song, Jie; Li, Yuhua; Zheng, Bin; Liu, Hong

2013-01-01

High spatial resolution and geometric accuracy is crucial for chromosomal analysis of clinical cytogenetic applications. High resolution and rapid simultaneous acquisition of multiple fluorescent wavelengths can be achieved by utilizing concurrent imaging with multiple detectors. However, such class of microscopic systems functions differently from traditional fluorescence microscopes. To develop a practical characterization framework to assess and optimize the performance of a high resolution and dual-color fluorescence microscope designed for clinical chromosomal analysis. A dual-band microscopic imaging system utilizes a dichroic mirror, two sets of specially selected optical filters, and two detectors to simultaneously acquire two fluorescent wavelengths. The system's geometric distortion, linearity, the modulation transfer function, and the dual detectors' alignment were characterized. Experiment results show that the geometric distortion at lens periphery is less than 1%. Both fluorescent channels show linear signal responses, but there exists discrepancy between the two due to the detectors' non-uniform response ratio to different wavelengths. In terms of the spatial resolution, the two contrast transfer function curves trend agreeably with the spatial frequency. The alignment measurement allows quantitatively assessing the cameras' alignment. A result image of adjusted alignment is demonstrated to show the reduced discrepancy by using the alignment measurement method. In this paper, we present a system characterization study and its methods for a specially designed imaging system for clinical cytogenetic applications. The presented characterization methods are not only unique to this dual-color imaging system but also applicable to evaluation and optimization of other similar multi-color microscopic image systems for improving their clinical utilities for future cytogenetic applications.

Vibrational and UV spectroscopic studies of 2-coumaranone by experimental and density functional theory calculations

NASA Astrophysics Data System (ADS)

Priya, Y. Sushma; Rao, K. Ramachandra; Chalapathi, P. V.; Satyavani, M.; Veeraiah, A.

2017-09-01

The vibrational and electronic properties of 2-coumaranone have been reported in the ground state using experimental techniques (FT-IR, FT-Raman, UV spectra and fluorescence microscopic imaging) and density functional theory (DFT) employing B3LYP correlation with the 6-31G(d, p) basis set. The theoretically reported optimized parameters, vibrational frequencies etc., were compared with the experimental values, which yielded good concurrence between the experimental and calculated values. The assignments of the vibrational spectra were done with the help of normal co-ordinate analysis (NCA) following the Scaled Quantum Mechanical Force Field(SQMFF) methodology. The whole assignments of fundamental modes were based on the potential energy distribution (PED) matrix. The electric dipole moment and the first order hyperpolarizability of the 2-coumaranone have been computed using quantum mechanical calculations. NBO and HOMO, LUMO analyses have been carried out. UV spectrum of 2-coumaranone was recorded in the region 100-300 nm and compared with the theoretical UV spectrum using TD-DFT and SAC-CI methods by which a good agreement is observed. Fluorescence microscopic imaging study reflects that the compound fluoresces in the green-yellow region.

Smart textile framework: Photochromic and fluorescent cellulosic fabric printed by strontium aluminate pigment.

PubMed

Khattab, Tawfik A; Rehan, Mohamed; Hamouda, Tamer

2018-09-01

Smart clothing can be defined as textiles that respond to a certain stimulus accompanied by a change in their properties. A specific class herein is the photochromic and fluorescent textiles that change color with light. A photochromic and fluorescent cotton fabric based on pigment printing is obtained. Such fabric is prepared by aqueous-based pigment-binder printing formulation containing inorganic pigment phosphor characterized by good photo- and thermal stability. It exhibits optimal excitation wavelength (365 nm) results in color and fluorescence change of the fabric surface. To prepare the transparent pigment-binder composite film, the phosphor pigment must be well-dispersed via physical immobilization without their aggregation. The pigment-binder paste is applied successfully onto cotton fabric using screen printing technique followed by thermal fixation. After screen-printing, a homogenous photochromic film is assembled on a cotton substrate surface, which represents substantial greenish-yellow color development as indicated by CIE Lab color space measurements under ultraviolet light, even at a pigment concentration of 0.08 wt% of the printing paste. The photochromic cotton fabric exhibit three excitation peaks at 272, 325 and 365 nm and three emission peaks at 418, 495 and 520 nm. The fluorescent optical microscope, scanning electron microscope, elemental mapping, energy dispersive X-ray spectroscopy, fluorescence emission and UV/Vis absorption spectroscopic data of the printed cotton fabric are described. The printed fabric showed a reversible and rapid photochromic response during ultra-violet excitation without fatigue. The fastness properties including washing, crocking, perspiration, sublimation/heat, and light are described. Copyright © 2018 Elsevier Ltd. All rights reserved.

Time-Domain Microfluidic Fluorescence Lifetime Flow Cytometry for High-Throughput Förster Resonance Energy Transfer Screening

PubMed Central

Nedbal, Jakub; Visitkul, Viput; Ortiz-Zapater, Elena; Weitsman, Gregory; Chana, Prabhjoat; Matthews, Daniel R; Ng, Tony; Ameer-Beg, Simon M

2015-01-01

Sensing ion or ligand concentrations, physico-chemical conditions, and molecular dimerization or conformation change is possible by assays involving fluorescent lifetime imaging. The inherent low throughput of imaging impedes rigorous statistical data analysis on large cell numbers. We address this limitation by developing a fluorescence lifetime-measuring flow cytometer for fast fluorescence lifetime quantification in living or fixed cell populations. The instrument combines a time-correlated single photon counting epifluorescent microscope with microfluidics cell-handling system. The associated computer software performs burst integrated fluorescence lifetime analysis to assign fluorescence lifetime, intensity, and burst duration to each passing cell. The maximum safe throughput of the instrument reaches 3,000 particles per minute. Living cells expressing spectroscopic rulers of varying peptide lengths were distinguishable by Förster resonant energy transfer measured by donor fluorescence lifetime. An epidermal growth factor (EGF)-stimulation assay demonstrated the technique's capacity to selectively quantify EGF receptor phosphorylation in cells, which was impossible by measuring sensitized emission on a standard flow cytometer. Dual-color fluorescence lifetime detection and cell-specific chemical environment sensing were exemplified using di-4-ANEPPDHQ, a lipophilic environmentally sensitive dye that exhibits changes in its fluorescence lifetime as a function of membrane lipid order. To our knowledge, this instrument opens new applications in flow cytometry which were unavailable due to technological limitations of previously reported fluorescent lifetime flow cytometers. The presented technique is sensitive to lifetimes of most popular fluorophores in the 0.5–5 ns range including fluorescent proteins and is capable of detecting multi-exponential fluorescence lifetime decays. This instrument vastly enhances the throughput of experiments involving fluorescence lifetime measurements, thereby providing statistically significant quantitative data for analysis of large cell populations. © 2014 International Society for Advancement of Cytometry PMID:25523156

Differential polarization nonlinear optical microscopy with adaptive optics controlled multiplexed beams.

PubMed

Samim, Masood; Sandkuijl, Daaf; Tretyakov, Ian; Cisek, Richard; Barzda, Virginijus

2013-09-09

Differential polarization nonlinear optical microscopy has the potential to become an indispensable tool for structural investigations of ordered biological assemblies and microcrystalline aggregates. Their microscopic organization can be probed through fast and sensitive measurements of nonlinear optical signal anisotropy, which can be achieved with microscopic spatial resolution by using time-multiplexed pulsed laser beams with perpendicular polarization orientations and photon-counting detection electronics for signal demultiplexing. In addition, deformable membrane mirrors can be used to correct for optical aberrations in the microscope and simultaneously optimize beam overlap using a genetic algorithm. The beam overlap can be achieved with better accuracy than diffraction limited point-spread function, which allows to perform polarization-resolved measurements on the pixel-by-pixel basis. We describe a newly developed differential polarization microscope and present applications of the differential microscopy technique for structural studies of collagen and cellulose. Both, second harmonic generation, and fluorescence-detected nonlinear absorption anisotropy are used in these investigations. It is shown that the orientation and structural properties of the fibers in biological tissue can be deduced and that the orientation of fluorescent molecules (Congo Red), which label the fibers, can be determined. Differential polarization microscopy sidesteps common issues such as photobleaching and sample movement. Due to tens of megahertz alternating polarization of excitation pulses fast data acquisition can be conveniently applied to measure changes in the nonlinear signal anisotropy in dynamically changing in vivo structures.

Nanoscale characterization of local structures and defects in photonic crystals using synchrotron-based transmission soft X-ray microscopy

PubMed Central

Nho, Hyun Woo; Kalegowda, Yogesh; Shin, Hyun-Joon; Yoon, Tae Hyun

2016-01-01

For the structural characterization of the polystyrene (PS)-based photonic crystals (PCs), fast and direct imaging capabilities of full field transmission X-ray microscopy (TXM) were demonstrated at soft X-ray energy. PS-based PCs were prepared on an O2-plasma treated Si3N4 window and their local structures and defects were investigated using this label-free TXM technique with an image acquisition speed of ~10 sec/frame and marginal radiation damage. Micro-domains of face-centered cubic (FCC (111)) and hexagonal close-packed (HCP (0001)) structures were dominantly found in PS-based PCs, while point and line defects, FCC (100), and 12-fold symmetry structures were also identified as minor components. Additionally, in situ observation capability for hydrated samples and 3D tomographic reconstruction of TXM images were also demonstrated. This soft X-ray full field TXM technique with faster image acquisition speed, in situ observation, and 3D tomography capability can be complementally used with the other X-ray microscopic techniques (i.e., scanning transmission X-ray microscopy, STXM) as well as conventional characterization methods (e.g., electron microscopic and optical/fluorescence microscopic techniques) for clearer structure identification of self-assembled PCs and better understanding of the relationship between their structures and resultant optical properties. PMID:27087141

Coherent beam control through inhomogeneous media in multi-photon microscopy

NASA Astrophysics Data System (ADS)

Paudel, Hari Prasad

Multi-photon fluorescence microscopy has become a primary tool for high-resolution deep tissue imaging because of its sensitivity to ballistic excitation photons in comparison to scattered excitation photons. The imaging depth of multi-photon microscopes in tissue imaging is limited primarily by background fluorescence that is generated by scattered light due to the random fluctuations in refractive index inside the media, and by reduced intensity in the ballistic focal volume due to aberrations within the tissue and at its interface. We built two multi-photon adaptive optics (AO) correction systems, one for combating scattering and aberration problems, and another for compensating interface aberrations. For scattering correction a MEMS segmented deformable mirror (SDM) was inserted at a plane conjugate to the objective back-pupil plane. The SDM can pre-compensate for light scattering by coherent combination of the scattered light to make an apparent focus even at a depths where negligible ballistic light remains (i.e. ballistic limit). This problem was approached by investigating the spatial and temporal focusing characteristics of a broad-band light source through strongly scattering media. A new model was developed for coherent focus enhancement through or inside the strongly media based on the initial speckle contrast. A layer of fluorescent beads under a mouse skull was imaged using an iterative coherent beam control method in the prototype two-photon microscope to demonstrate the technique. We also adapted an AO correction system to an existing in three-photon microscope in a collaborator lab at Cornell University. In the second AO correction approach a continuous deformable mirror (CDM) is placed at a plane conjugate to the plane of an interface aberration. We demonstrated that this "Conjugate AO" technique yields a large field-of-view (FOV) advantage in comparison to Pupil AO. Further, we showed that the extended FOV in conjugate AO is maintained over a relatively large axial misalignment of the conjugate planes of the CDM and the aberrating interface. This dissertation advances the field of microscopy by providing new models and techniques for imaging deeply within strongly scattering tissue, and by describing new adaptive optics approaches to extending imaging FOV due to sample aberrations.

Imaging Live Drosophila Brain with Two-Photon Fluorescence Microscopy

NASA Astrophysics Data System (ADS)

Ahmed, Syeed Ehsan

Two-photon fluorescence microscopy is an imaging technique which delivers distinct benefits for in vivo cellular and molecular imaging. Cyclic adenosine monophosphate (cAMP), a second messenger molecule, is responsible for triggering many physiological changes in neural system. However, the mechanism by which this molecule regulates responses in neuron cells is not yet clearly understood. When cAMP binds to a target protein, it changes the structure of that protein. Therefore, studying this molecular structure change with fluorescence resonance energy transfer (FRET) imaging can shed light on the cAMP functioning mechanism. FRET is a non-radiative dipole-dipole coupling which is sensitive to small distance change in nanometer scale. In this study we have investigated the effect of dopamine in cAMP dynamics in vivo. In our study two-photon fluorescence microscope was used for imaging mushroom bodies inside live Drosophila melanogaster brain and we developed a method for studying the change in cyclic AMP level.

Life under the Microscope: Single-Molecule Fluorescence Highlights the RNA World.

PubMed

Ray, Sujay; Widom, Julia R; Walter, Nils G

2018-04-25

The emergence of single-molecule (SM) fluorescence techniques has opened up a vast new toolbox for exploring the molecular basis of life. The ability to monitor individual biomolecules in real time enables complex, dynamic folding pathways to be interrogated without the averaging effect of ensemble measurements. In parallel, modern biology has been revolutionized by our emerging understanding of the many functions of RNA. In this comprehensive review, we survey SM fluorescence approaches and discuss how the application of these tools to RNA and RNA-containing macromolecular complexes in vitro has yielded significant insights into the underlying biology. Topics covered include the three-dimensional folding landscapes of a plethora of isolated RNA molecules, their assembly and interactions in RNA-protein complexes, and the relation of these properties to their biological functions. In all of these examples, the use of SM fluorescence methods has revealed critical information beyond the reach of ensemble averages.

Simultaneous multiview capture and fusion improves spatial resolution in wide-field and light-sheet microscopy

PubMed Central

Wu, Yicong; Chandris, Panagiotis; Winter, Peter W.; Kim, Edward Y.; Jaumouillé, Valentin; Kumar, Abhishek; Guo, Min; Leung, Jacqueline M.; Smith, Corey; Rey-Suarez, Ivan; Liu, Huafeng; Waterman, Clare M.; Ramamurthi, Kumaran S.; La Riviere, Patrick J.; Shroff, Hari

2016-01-01

Most fluorescence microscopes are inefficient, collecting only a small fraction of the emitted light at any instant. Besides wasting valuable signal, this inefficiency also reduces spatial resolution and causes imaging volumes to exhibit significant resolution anisotropy. We describe microscopic and computational techniques that address these problems by simultaneously capturing and subsequently fusing and deconvolving multiple specimen views. Unlike previous methods that serially capture multiple views, our approach improves spatial resolution without introducing any additional illumination dose or compromising temporal resolution relative to conventional imaging. When applying our methods to single-view wide-field or dual-view light-sheet microscopy, we achieve a twofold improvement in volumetric resolution (~235 nm × 235 nm × 340 nm) as demonstrated on a variety of samples including microtubules in Toxoplasma gondii, SpoVM in sporulating Bacillus subtilis, and multiple protein distributions and organelles in eukaryotic cells. In every case, spatial resolution is improved with no drawback by harnessing previously unused fluorescence. PMID:27761486

A comparison of two micro-beam X-ray emission techniques for actinide elemental distribution in microscopic particles originating from the hydrogen bombs involved in the Palomares (Spain) and Thule (Greenland) accidents

NASA Astrophysics Data System (ADS)

Jimenez-Ramos, M. C.; Eriksson, M.; García-López, J.; Ranebo, Y.; García-Tenorio, R.; Betti, M.; Holm, E.

2010-09-01

In order to validate and to gain confidence in two micro-beam techniques: particle induced X-ray emission with nuclear microprobe technique (μ-PIXE) and synchrotron radiation induced X-ray fluorescence in a confocal alignment (confocal SR μ-XRF) for characterization of microscopic particles containing actinide elements (mixed plutonium and uranium) a comparative study has been performed. Inter-comparison of the two techniques is essential as the X-ray production cross-sections for U and Pu are different for protons and photons and not well defined in the open literature, especially for Pu. The particles studied consisted of nuclear weapons material, and originate either in the so called Palomares accident in Spain, 1966 or in the Thule accident in Greenland, 1968. In the determination of the average Pu/U mass ratios (not corrected by self-absorption) in the analysed microscopic particles the results from both techniques show a very good agreement. In addition, the suitability of both techniques for the analysis with good resolution (down to a few μm) of the Pu/U distribution within the particles has been proved. The set of results obtained through both techniques has allowed gaining important information concerning the characterization of the remaining fissile material in the areas affected by the aircraft accidents. This type of information is essential for long-term impact assessments of contaminated sites.

Azimuthal phase retardation microscope for visualizing actin filaments of biological cells

NASA Astrophysics Data System (ADS)

Shin, In Hee; Shin, Sang-Mo

2011-09-01

We developed a new theory-based azimuthal phase retardation microscope to visualize distributions of actin filaments in biological cells without having them with exogenous dyes, fluorescence labels, or stains. The azimuthal phase retardation microscope visualizes distributions of actin filaments by measuring the intensity variations of each pixel of a charge coupled device camera while rotating a single linear polarizer. Azimuthal phase retardation δ between two fixed principal axes was obtained by calculating the rotation angles of the polarizer at the intensity minima from the acquired intensity data. We have acquired azimuthal phase retardation distributions of human breast cancer cell, MDA MB 231 by our microscope and compared the azimuthal phase retardation distributions with the fluorescence image of actin filaments by the commercial fluorescence microscope. Also, we have observed movement of human umbilical cord blood derived mesenchymal stem cells by measuring azimuthal phase retardation distributions.

All-near-infrared multiphoton microscopy interrogates intact tissues at deeper imaging depths than conventional single- and two-photon near-infrared excitation microscopes

PubMed Central

Sarder, Pinaki; Yazdanfar, Siavash; Akers, Walter J.; Tang, Rui; Sudlow, Gail P.; Egbulefu, Christopher

2013-01-01

Abstract. The era of molecular medicine has ushered in the development of microscopic methods that can report molecular processes in thick tissues with high spatial resolution. A commonality in deep-tissue microscopy is the use of near-infrared (NIR) lasers with single- or multiphoton excitations. However, the relationship between different NIR excitation microscopic techniques and the imaging depths in tissue has not been established. We compared such depth limits for three NIR excitation techniques: NIR single-photon confocal microscopy (NIR SPCM), NIR multiphoton excitation with visible detection (NIR/VIS MPM), and all-NIR multiphoton excitation with NIR detection (NIR/NIR MPM). Homologous cyanine dyes provided the fluorescence. Intact kidneys were harvested after administration of kidney-clearing cyanine dyes in mice. NIR SPCM and NIR/VIS MPM achieved similar maximum imaging depth of ∼100  μm. The NIR/NIR MPM enabled greater than fivefold imaging depth (>500  μm) using the harvested kidneys. Although the NIR/NIR MPM used 1550-nm excitation where water absorption is relatively high, cell viability and histology studies demonstrate that the laser did not induce photothermal damage at the low laser powers used for the kidney imaging. This study provides guidance on the imaging depth capabilities of NIR excitation-based microscopic techniques and reveals the potential to multiplex information using these platforms. PMID:24150231

Is fluorescein-guided technique able to help in resection of high-grade gliomas?

PubMed

Acerbi, Francesco; Broggi, Morgan; Eoli, Marica; Anghileri, Elena; Cavallo, Claudio; Boffano, Carlo; Cordella, Roberto; Cuppini, Lucia; Pollo, Bianca; Schiariti, Marco; Visintini, Sergio; Orsi, Chiara; La Corte, Emanuele; Broggi, Giovanni; Ferroli, Paolo

2014-02-01

Fluorescein, a dye that is widely used as a fluorescent tracer, accumulates in cerebral areas where the blood-brain barrier is damaged. This quality makes it an ideal dye for the intraoperative visualization of high-grade gliomas (HGGs). The authors report their experience with a new fluorescein-guided technique for the resection of HGGs using a dedicated filter on the surgical microscope. The authors initiated a prospective Phase II trial (FLUOGLIO) in September 2011 with the objective of evaluating the safety of fluorescein-guided surgery for HGGs and obtaining preliminary evidence regarding its efficacy for this purpose. To be eligible for participation in the study, a patient had to have suspected HGG amenable to complete resection of the contrast-enhancing area. The present report is based on the analysis of the short- and long-term results in 20 consecutive patients with HGGs (age range 45-74 years), enrolled in the study since September 2011. In all cases fluorescein (5-10 mg/kg) was injected intravenously after intubation. Tumor resection was performed with microsurgical technique and fluorescence visualization by means of BLUE 400 or YELLOW 560 filters on a Pentero microscope. The median preoperative tumor volume was 30.3 cm(3) (range 2.4-87.8 cm(3)). There were no adverse reactions related to fluorescein administration. Complete removal of contrast-enhanced tumor was achieved in 80% of the patients. The median duration of follow-up was 10 months. The 6-months progression-free survival rate was 71.4% and the median survival was 11 months. Analysis of these 20 cases suggested that fluorescein-guided technique with a dedicated filter on the surgical microscope is safe and allows a high rate of complete resection of contrast-enhanced tumor as determined on early postoperative MRI. Clinical trial registration no.: 2011-002527-18 (EudraCT).

Intravital Fluorescence Videomicroscopy to Study Tumor Angiogenesis and Microcirculation1

PubMed Central

Vajkoczy, Peter; Ullrich, Axel; Meager, Michael D

2000-01-01

Abstract Angiogenesis and microcirculation play a central role in growth and metastasis of human neoplasms, and, thus, represent a major target for novel treatment strategies. Mechanistic analysis of processes involved in tumor vascularization, however, requires sophisticated in vivo experimental models and techniques. Intravital microscopy allows direct assessment of tumor angiogenesis, microcirculation and overall perfusion. Its application to the study of tumor-induced neovascularization further provides information on molecular transport and delivery, intra- and extravascular cell-to-cell and cell-to-matrix interaction, as well as tumor oxygenation and metabolism. With the recent advances in the field of bioluminescence and fluorescent reporter genes, appropriate for in vivo imaging, the intravital fluorescent microscopic approach has to be considered a powerful tool to study microvascular, cellular and molecular mechanisms of tumor growth. PMID:10933068

Fluorescein sodium-guided surgery of a brain abscess: A case report

PubMed Central

Höhne, Julius; Brawanski, Alexander; Schebesch, Karl-Michael

2016-01-01

Background: Up to now, the feasibility and benefit of using fluorescein sodium under a dedicated surgical microscope filter (YE560, YELLOW 560 nm filter, Carl Zeiss Meditec, Germany) has never been clinically evaluated in infectious disorders of the brain. Case Description: Here, we report the case of a male patient with a brain abscess in the right parietal lobe that was removed under fluorescence-guidance (intravenous administration of fluorescein sodium 10%, 5 mg/kg bodyweight). The abscess capsule showed intensive yellow fluorescent staining, while − under white light − the cortex appeared normal. Conclusion: This technique may improve the identification and surgical removal of brain abscesses. PMID:28031990

Single-frame 3D fluorescence microscopy with ultraminiature lensless FlatScope

PubMed Central

Adams, Jesse K.; Boominathan, Vivek; Avants, Benjamin W.; Vercosa, Daniel G.; Ye, Fan; Baraniuk, Richard G.; Robinson, Jacob T.; Veeraraghavan, Ashok

2017-01-01

Modern biology increasingly relies on fluorescence microscopy, which is driving demand for smaller, lighter, and cheaper microscopes. However, traditional microscope architectures suffer from a fundamental trade-off: As lenses become smaller, they must either collect less light or image a smaller field of view. To break this fundamental trade-off between device size and performance, we present a new concept for three-dimensional (3D) fluorescence imaging that replaces lenses with an optimized amplitude mask placed a few hundred micrometers above the sensor and an efficient algorithm that can convert a single frame of captured sensor data into high-resolution 3D images. The result is FlatScope: perhaps the world’s tiniest and lightest microscope. FlatScope is a lensless microscope that is scarcely larger than an image sensor (roughly 0.2 g in weight and less than 1 mm thick) and yet able to produce micrometer-resolution, high–frame rate, 3D fluorescence movies covering a total volume of several cubic millimeters. The ability of FlatScope to reconstruct full 3D images from a single frame of captured sensor data allows us to image 3D volumes roughly 40,000 times faster than a laser scanning confocal microscope while providing comparable resolution. We envision that this new flat fluorescence microscopy paradigm will lead to implantable endoscopes that minimize tissue damage, arrays of imagers that cover large areas, and bendable, flexible microscopes that conform to complex topographies. PMID:29226243

Visualization of Electrical Field of Electrode Using Voltage-Controlled Fluorescence Release

PubMed Central

Jia, Wenyan; Wu, Jiamin; Gao, Di; Wang, Hao; Sun, Mingui

2016-01-01

In this study we propose an approach to directly visualize electrical current distribution at the electrode-electrolyte interface of a biopotential electrode. High-speed fluorescent microscopic images are acquired when an electric potential is applied across the interface to trigger the release of fluorescent material from the surface of the electrode. These images are analyzed computationally to obtain the distribution of the electric field from the fluorescent intensity of each pixel. Our approach allows direct observation of microscopic electrical current distribution around the electrode. Experiments are conducted to validate the feasibility of the fluorescent imaging method. PMID:27253615

Scanning light-sheet microscopy in the whole mouse brain with HiLo background rejection.

PubMed

Mertz, Jerome; Kim, Jinhyun

2010-01-01

It is well known that light-sheet illumination can enable optically sectioned wide-field imaging of macroscopic samples. However, the optical sectioning capacity of a light-sheet macroscope is undermined by sample-induced scattering or aberrations that broaden the thickness of the sheet illumination. We present a technique to enhance the optical sectioning capacity of a scanning light-sheet microscope by out-of-focus background rejection. The technique, called HiLo microscopy, makes use of two images sequentially acquired with uniform and structured sheet illumination. An optically sectioned image is then synthesized by fusing high and low spatial frequency information from both images. The benefits of combining light-sheet macroscopy and HiLo background rejection are demonstrated in optically cleared whole mouse brain samples, using both green fluorescent protein (GFP)-fluorescence and dark-field scattered light contrast.

Scanning light-sheet microscopy in the whole mouse brain with HiLo background rejection

NASA Astrophysics Data System (ADS)

Mertz, Jerome; Kim, Jinhyun

2010-01-01

It is well known that light-sheet illumination can enable optically sectioned wide-field imaging of macroscopic samples. However, the optical sectioning capacity of a light-sheet macroscope is undermined by sample-induced scattering or aberrations that broaden the thickness of the sheet illumination. We present a technique to enhance the optical sectioning capacity of a scanning light-sheet microscope by out-of-focus background rejection. The technique, called HiLo microscopy, makes use of two images sequentially acquired with uniform and structured sheet illumination. An optically sectioned image is then synthesized by fusing high and low spatial frequency information from both images. The benefits of combining light-sheet macroscopy and HiLo background rejection are demonstrated in optically cleared whole mouse brain samples, using both green fluorescent protein (GFP)-fluorescence and dark-field scattered light contrast.

Facile synthesis of SiO{sub 2} nanoparticles for biomedical applications

DOE Office of Scientific and Technical Information (OSTI.GOV)

Scano, A., E-mail: alescano80@tiscali.it; Pilloni, M., E-mail: alescano80@tiscali.it; Cabras, V., E-mail: alescano80@tiscali.it

Silica nanoparticles (SiO{sub 2} NPs) for biomedical applications have been prepared by using a facile modified Stöber-synthesis. Potassium borohydride (KBH{sub 4}) has been introduced in the synthesis procedure in order to control NP size. Several samples have been prepared varying tetraethylorthosilicate (TEOS) concentration, and using different process conditions (temperature, reaction time and atmosphere). In order to study the influence of the process conditions on the NP size, morphology and properties, several characterization techniques were used. Size and morphology of the as-prepared SiO{sub 2} NPs have been studied by using Transmission Electron Microscope (TEM) and Dynamic Light Scattering (DLS) techniques. Structuralmore » characterization was carried out by X-ray powder diffraction. To investigate the SiO{sub 2} NP fluorescence emission properties the fluorescence spectroscopy was also used.« less

A portable fluorescence microscopic imaging system for cholecystectomy

NASA Astrophysics Data System (ADS)

Ye, Jian; Yang, Chaoyu; Gan, Qi; Ma, Rong; Zhang, Zeshu; Chang, Shufang; Shao, Pengfei; Zhang, Shiwu; Liu, Chenhai; Xu, Ronald

2016-03-01

In this paper we proposed a portable fluorescence microscopic imaging system to prevent iatrogenic biliary injuries from occurring during cholecystectomy due to misidentification of the cystic structures. The system consisted of a light source module, a CMOS camera, a Raspberry Pi computer and a 5 inch HDMI LCD. Specifically, the light source module was composed of 690 nm and 850 nm LEDs, allowing the CMOS camera to simultaneously acquire both fluorescence and background images. The system was controlled by Raspberry Pi using Python programming with the OpenCV library under Linux. We chose Indocyanine green(ICG) as a fluorescent contrast agent and then tested fluorescence intensities of the ICG aqueous solution at different concentration levels by our fluorescence microscopic system compared with the commercial Xenogen IVIS system. The spatial resolution of the proposed fluorescence microscopic imaging system was measured by a 1951 USAF resolution target and the dynamic response was evaluated quantitatively with an automatic displacement platform. Finally, we verified the technical feasibility of the proposed system in mouse models of bile duct, performing both correct and incorrect gallbladder resection. Our experiments showed that the proposed system can provide clear visualization of the confluence between the cystic duct and common bile duct or common hepatic duct, suggesting that this is a potential method for guiding cholecystectomy. The proposed portable system only cost a total of $300, potentially promoting its use in resource-limited settings.

Aro: a machine learning approach to identifying single molecules and estimating classification error in fluorescence microscopy images.

PubMed

Wu, Allison Chia-Yi; Rifkin, Scott A

2015-03-27

Recent techniques for tagging and visualizing single molecules in fixed or living organisms and cell lines have been revolutionizing our understanding of the spatial and temporal dynamics of fundamental biological processes. However, fluorescence microscopy images are often noisy, and it can be difficult to distinguish a fluorescently labeled single molecule from background speckle. We present a computational pipeline to distinguish the true signal of fluorescently labeled molecules from background fluorescence and noise. We test our technique using the challenging case of wide-field, epifluorescence microscope image stacks from single molecule fluorescence in situ experiments on nematode embryos where there can be substantial out-of-focus light and structured noise. The software recognizes and classifies individual mRNA spots by measuring several features of local intensity maxima and classifying them with a supervised random forest classifier. A key innovation of this software is that, by estimating the probability that each local maximum is a true spot in a statistically principled way, it makes it possible to estimate the error introduced by image classification. This can be used to assess the quality of the data and to estimate a confidence interval for the molecule count estimate, all of which are important for quantitative interpretations of the results of single-molecule experiments. The software classifies spots in these images well, with >95% AUROC on realistic artificial data and outperforms other commonly used techniques on challenging real data. Its interval estimates provide a unique measure of the quality of an image and confidence in the classification.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kuhlmann, Andreas V.; Houel, Julien; Warburton, Richard J.

Optically active quantum dots, for instance self-assembled InGaAs quantum dots, are potentially excellent single photon sources. The fidelity of the single photons is much improved using resonant rather than non-resonant excitation. With resonant excitation, the challenge is to distinguish between resonance fluorescence and scattered laser light. We have met this challenge by creating a polarization-based dark-field microscope to measure the resonance fluorescence from a single quantum dot at low temperature. We achieve a suppression of the scattered laser exceeding a factor of 10{sup 7} and background-free detection of resonance fluorescence. The same optical setup operates over the entire quantum dotmore » emission range (920–980 nm) and also in high magnetic fields. The major development is the outstanding long-term stability: once the dark-field point has been established, the microscope operates for days without alignment. The mechanical and optical designs of the microscope are presented, as well as exemplary resonance fluorescence spectroscopy results on individual quantum dots to underline the microscope's excellent performance.« less

Multiresolution multiscale active mask segmentation of fluorescence microscope images

NASA Astrophysics Data System (ADS)

Srinivasa, Gowri; Fickus, Matthew; Kovačević, Jelena

2009-08-01

We propose an active mask segmentation framework that combines the advantages of statistical modeling, smoothing, speed and flexibility offered by the traditional methods of region-growing, multiscale, multiresolution and active contours respectively. At the crux of this framework is a paradigm shift from evolving contours in the continuous domain to evolving multiple masks in the discrete domain. Thus, the active mask framework is particularly suited to segment digital images. We demonstrate the use of the framework in practice through the segmentation of punctate patterns in fluorescence microscope images. Experiments reveal that statistical modeling helps the multiple masks converge from a random initial configuration to a meaningful one. This obviates the need for an involved initialization procedure germane to most of the traditional methods used to segment fluorescence microscope images. While we provide the mathematical details of the functions used to segment fluorescence microscope images, this is only an instantiation of the active mask framework. We suggest some other instantiations of the framework to segment different types of images.

A wavelength-dispersive instrument for characterizing fluorescence and scattering spectra of individual aerosol particles on a substrate

NASA Astrophysics Data System (ADS)

Huffman, Donald R.; Swanson, Benjamin E.; Huffman, J. Alex

2016-08-01

We describe a novel, low-cost instrument to acquire both elastic and inelastic (fluorescent) scattering spectra from individual supermicron-size particles in a multi-particle collection on a microscope slide. The principle of the device is based on a slitless spectroscope that is often employed in astronomy to determine the spectra of individual stars in a star cluster but had not been applied to atmospheric particles. Under excitation, most commonly by either a 405 nm diode laser or a UV light-emitting diode (LED), fluorescence emission spectra of many individual particles can be determined simultaneously. The instrument can also acquire elastic scattering spectra from particles illuminated by a white-light source. The technique also provides the ability to detect and rapidly estimate the number fraction of fluorescent particles that could contaminate a collection of non-fluorescent material, even without analyzing full spectra. Advantages and disadvantages of using black-and-white cameras compared to color cameras are given. The primary motivation for this work has been to develop an inexpensive technique to characterize fluorescent biological aerosol particles, especially particles such as pollen and mold spores that can cause allergies. An example of an iPhone-enabled device is also shown as a means for collecting data on biological aerosols at lower cost or by utilizing citizen scientists for expanded data collection.

Superresolution Imaging with Standard Fluorescent Probes

PubMed Central

Burnette, Dylan T.; Lippincott-Schwartz, Jennifer; Kachar, Bechara

2013-01-01

For more than 100 years, the ultimate resolution of a light microscope (~200 nm) has been constrained by the fundamental physical phenomenon of diffraction, as described by Ernst Abbe in 1873. While this limitation is just as applicable to today’s light microscopes, it is the combination of high-end optics, clever methods of sample illumination, and computational techniques that has enabled researchers to access high-resolution information an order of magnitude greater than once thought possible. This combination, broadly termed superresolution microscopy, has been increasingly practical for many labs to implement from both a hardware and software standpoint, but as with many cutting-edge techniques, it also comes with limitations. One of the current drawbacks to superresolution microscopy is the limited number of probes and conditions that have been suitable for imaging. Here, a technique termed bleaching/blinking assisted localization microscopy (BaLM) makes use of almost all fluorophore’s inherent blinking and bleaching properties as a means to generate superresolution images. PMID:24510788

Radioluminescence Microscopy: Measuring the Heterogeneous Uptake of Radiotracers in Single Living Cells

PubMed Central

Pratx, Guillem; Chen, Kai; Sun, Conroy; Martin, Lynn; Carpenter, Colin M.; Olcott, Peter D.; Xing, Lei

2012-01-01

Radiotracers play an important role in interrogating molecular processes both in vitro and in vivo. However, current methods are limited to measuring average radiotracer uptake in large cell populations and, as a result, lack the ability to quantify cell-to-cell variations. Here we apply a new technique, termed radioluminescence microscopy, to visualize radiotracer uptake in single living cells, in a standard fluorescence microscopy environment. In this technique, live cells are cultured sparsely on a thin scintillator plate and incubated with a radiotracer. Light produced following beta decay is measured using a highly sensitive microscope. Radioluminescence microscopy revealed strong heterogeneity in the uptake of [18F]fluoro-deoxyglucose (FDG) in single cells, which was found consistent with fluorescence imaging of a glucose analog. We also verified that dynamic uptake of FDG in single cells followed the standard two-tissue compartmental model. Last, we transfected cells with a fusion PET/fluorescence reporter gene and found that uptake of FHBG (a PET radiotracer for transgene expression) coincided with expression of the fluorescent protein. Together, these results indicate that radioluminescence microscopy can visualize radiotracer uptake with single-cell resolution, which may find a use in the precise characterization of radiotracers. PMID:23056276

Applications of rigid and flexible GRIN-endoscopes

NASA Astrophysics Data System (ADS)

Schenkl, Selma; Ehlers, Alexander; Riemann, Iris; Messerschmidt, Bernhard; Bückle, Rainer; König, Karsten

2007-02-01

Multiphoton autofluorescence imaging became an important technique for minimal invasive examination of cells in biological tissue. Rigid and flexible endoscopes based on gradient index lenses (GRIN-lenses) extend this minimalinvasive technique to deep lying cell layers, inner body and specimens, difficult to access. In the rigid endoscope, a GRIN-lens overcomes the limited depth range, given by the working distance of the microscope objective. The focus of the conventional laser scanning tomography is reproduced tens of millimeters in the specimen under study by the GRIN-lens (diameter 1.8 and 3 μm). We will present images of fluorescent beads, proteins cells and skin tissue, as well as first in vivo measurements on human skin. The autofluorescence signal stems from the endogenous fluorophore elastin and SHG from collagen. The flexible endoscope dispenses completely the need of a microscope next to the specimen of interest. The excitation laser pulses is delivered via a well-characterized photonic crystal fiber and subsequently focused by a newly designed GRIN-lens system. The fluorescence, also transferred by a fiber is detected by a PMT detector. We will show the excellent imaging qualities of a newly developed GRIN-lens system with high-resolution images of proteins, cells and plant tissue and give an out-look on multiphoton endoscopy.

Automated imaging of cellular spheroids with selective plane illumination microscopy on a chip (Conference Presentation)

NASA Astrophysics Data System (ADS)

Paiè, Petra; Bassi, Andrea; Bragheri, Francesca; Osellame, Roberto

2017-02-01

Selective plane illumination microscopy (SPIM) is an optical sectioning technique that allows imaging of biological samples at high spatio-temporal resolution. Standard SPIM devices require dedicated set-ups, complex sample preparation and accurate system alignment, thus limiting the automation of the technique, its accessibility and throughput. We present a millimeter-scaled optofluidic device that incorporates selective plane illumination and fully automatic sample delivery and scanning. To this end an integrated cylindrical lens and a three-dimensional fluidic network were fabricated by femtosecond laser micromachining into a single glass chip. This device can upgrade any standard fluorescence microscope to a SPIM system. We used SPIM on a CHIP to automatically scan biological samples under a conventional microscope, without the need of any motorized stage: tissue spheroids expressing fluorescent proteins were flowed in the microchannel at constant speed and their sections were acquired while passing through the light sheet. We demonstrate high-throughput imaging of the entire sample volume (with a rate of 30 samples/min), segmentation and quantification in thick (100-300 μm diameter) cellular spheroids. This optofluidic device gives access to SPIM analyses to non-expert end-users, opening the way to automatic and fast screening of a high number of samples at subcellular resolution.

Fluorescence-guided tumor visualization using a custom designed NIR attachment to a surgical microscope for high sensitivity imaging (Conference Presentation)

NASA Astrophysics Data System (ADS)

Kittle, David S.; Patil, Chirag G.; Mamelak, Adam; Hansen, Stacey; Perry, Jeff; Ishak, Laura; Black, Keith L.; Butte, Pramod V.

2016-03-01

Current surgical microscopes are limited in sensitivity for NIR fluorescence. Recent developments in tumor markers attached with NIR dyes require newer, more sensitive imaging systems with high resolution to guide surgical resection. We report on a small, single camera solution enabling advanced image processing opportunities previously unavailable for ultra-high sensitivity imaging of these agents. The system captures both visible reflectance and NIR fluorescence at 300 fps while displaying full HD resolution video at 60 fps. The camera head has been designed to easily mount onto the Zeiss Pentero microscope head for seamless integration into surgical procedures.

EVALUATION OF AN ALTERNATIVE IMS DISSOCIATION PROCEDURE FOR USE WITH METHOD 1622: DETECTION OF CRYPTOSPORIDIUM IN WATER

EPA Science Inventory

U.S. EPA Method 1623 is used to detect and quantify Cruptosporidum spp. oocysts in ater. The protocol consists of filtration, immunomagnetic separation (IMS), staining with a fluorescent antibody, and microscopic analysis. Microscopic analysis includes detection by fluorescent ...

Chip-scale fluorescence microscope based on a silo-filter complementary metal-oxide semiconductor image sensor.

PubMed

Ah Lee, Seung; Ou, Xiaoze; Lee, J Eugene; Yang, Changhuei

2013-06-01

We demonstrate a silo-filter (SF) complementary metal-oxide semiconductor (CMOS) image sensor for a chip-scale fluorescence microscope. The extruded pixel design with metal walls between neighboring pixels guides fluorescence emission through the thick absorptive filter to the photodiode of a pixel. Our prototype device achieves 13 μm resolution over a wide field of view (4.8 mm × 4.4 mm). We demonstrate bright-field and fluorescence longitudinal imaging of living cells in a compact, low-cost configuration.

Multimodal biophotonic workstation for live cell analysis.

PubMed

Esseling, Michael; Kemper, Björn; Antkowiak, Maciej; Stevenson, David J; Chaudet, Lionel; Neil, Mark A A; French, Paul W; von Bally, Gert; Dholakia, Kishan; Denz, Cornelia

2012-01-01

A reliable description and quantification of the complex physiology and reactions of living cells requires a multimodal analysis with various measurement techniques. We have investigated the integration of different techniques into a biophotonic workstation that can provide biological researchers with these capabilities. The combination of a micromanipulation tool with three different imaging principles is accomplished in a single inverted microscope which makes the results from all the techniques directly comparable. Chinese Hamster Ovary (CHO) cells were manipulated by optical tweezers while the feedback was directly analyzed by fluorescence lifetime imaging, digital holographic microscopy and dynamic phase-contrast microscopy. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Near-Membrane Refractometry Using Supercritical Angle Fluorescence.

PubMed

Brunstein, Maia; Roy, Lopamudra; Oheim, Martin

2017-05-09

Total internal reflection fluorescence (TIRF) microscopy and its variants are key technologies for visualizing the dynamics of single molecules or organelles in live cells. Yet truly quantitative TIRF remains problematic. One unknown hampering the interpretation of evanescent-wave excited fluorescence intensities is the undetermined cell refractive index (RI). Here, we use a combination of TIRF excitation and supercritical angle fluorescence emission detection to directly measure the average RI in the "footprint" region of the cell during image acquisition. Our RI measurement is based on the determination on a back-focal plane image of the critical angle separating evanescent and far-field fluorescence emission components. We validate our method by imaging mouse embryonic fibroblasts and BON cells. By targeting various dyes and fluorescent-protein chimeras to vesicles, the plasma membrane, as well as mitochondria and the endoplasmic reticulum, we demonstrate local RI measurements with subcellular resolution on a standard TIRF microscope, with a removable Bertrand lens as the only modification. Our technique has important applications for imaging axial vesicle dynamics and the mitochondrial energy state or detecting metabolically more active cancer cells. Copyright © 2017. Published by Elsevier Inc.

Two-photon microscopy and spectroscopy based on a compact confocal scanning head

NASA Astrophysics Data System (ADS)

Diaspro, Alberto; Chirico, Giberto; Federici, Federico; Cannone, Fabio; Beretta, Sabrina; Robello, Mauro; Olivini, Francesca; Ramoino, Paola

2001-07-01

We have combined a confocal laser scanning head modified for TPE (two-photon excitation) microscopy with some spectroscopic modules to study single molecules and molecular aggregates. The behavior of the TPE microscope unit has been characterized by means of point spread function measurements and of the demonstration of its micropatterning abilities. One-photon and two-photon mode can be simply accomplished by switching from a mono-mode optical fiber (one-photon) coupled to conventional laser sources to an optical module that allows IR laser beam (two- photon/TPE) delivery to the confocal laser scanning head. We have then described the characterization of the two-photon microscope for spectroscopic applications: fluorescence correlation, lifetime and fluorescence polarization anisotropy measurements. We describe the measurement of the response of the two-photon microscope to the light polarization and discuss fluorescence polarization anisotropy measurements on Rhodamine 6G as a function of the viscosity and on a globular protein, the Beta-lactoglobulin B labeled with Alexa 532 at very high dilutions. The average rotational and translational diffusion coefficients measured with fluorescence polarization anisotropy and fluorescence correlation methods are in good agreement with the protein size, therefore validating the use of the microscope for two-photon spectroscopy on biomolecules.

A new technique for the rapid screening and selection of large pieces of tissue for ultrastructural evaluation.

PubMed

Dalley, B K; Seliger, W G

1980-05-01

A simple and rapid technique is described for the screening of Epon embedded organ slices for the location, isolation, and removal of small specific sites for ultrastructural study with the transmission electron microscope. This procedure consists of perfusion fixation followed by making 1 to 21/2 mm thick slices of relatively large pieces of the organs, control of the degree and evenness of the osmium staining by addition of 3% sodium iodate, and infiltration with a fluorescent dye prior to embedment in Epon. Tissue slices are embedded in wafer-shaped blocks, generally with several slices in one "wafer", and are examined in a controlled manner using a rapid form of serial surface polishing. Each level of the polished wafer is examined using an epi-illuminated fluorescence microscope, and selected sites are chosen at each level for ultrastructural study. Methods are also described for marking each selected site using a conventional slide marker, and for the removal of the selected site in the form of a small disc of Epon, after which the Epon wafer can be further serially polished and the examination continued. Areas to be thin-sectioned are removed using a core drill mounted on a model-maker's drill press. The technique is simple, does not require the destruction of remaining tissues to evaluate more critically a single small site, allows for the easy maintenance of tissue orientation, and the most time-consuming portions of the technique can be quickly taught to a person with no previous histological training.

Dual functionalized graphene oxide serves as a carrier for delivering oligohistidine- and biotin-tagged biomolecules into cells.

PubMed

Jana, Batakrishna; Mondal, Goutam; Biswas, Atanu; Chakraborty, Indrani; Saha, Abhijit; Kurkute, Prashant; Ghosh, Surajit

2013-11-01

A versatile method of dual chemical functionalization of graphene oxide (GO) with Tris-[nitrilotris(acetic acid)] (Tris-NTA) and biotin for cellular delivery of oligohistidine- and biotin-tagged biomolecules is reported. Orthogonally functionalized GO surfaces with Tris-NTA and biotin to obtain a dual-functionalized GO (DFGO) are prepared and characterized by various spectroscopic and microscopic techniques. Fluorescence microscopic images reveal that DFGO surfaces are capable of binding oligohistidine-tagged biomolecules/proteins and avidin/biotin-tagged biomolecules/proteins orthogonally. The DFGO nanoparticles are non-cytotoxic in nature and can deliver oligohistidine- and biotin-tagged biomolecules simultaneously into the cell. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Light and Life in Baltimore—and Beyond

PubMed Central

Edidin, Michael

2015-01-01

Baltimore has been the home of numerous biophysical studies using light to probe cells. One such study, quantitative measurement of lateral diffusion of rhodopsin, set the standard for experiments in which recovery after photobleaching is used to measure lateral diffusion. Development of this method from specialized microscopes to commercial scanning confocal microscopes has led to widespread use of the technique to measure lateral diffusion of membrane proteins and lipids, and as well diffusion and binding interactions in cell organelles and cytoplasm. Perturbation of equilibrium distributions by photobleaching has also been developed into a robust method to image molecular proximity in terms of fluorescence resonance energy transfer between donor and acceptor fluorophores. PMID:25650914

A Low-Cost Digital Microscope with Real-Time Fluorescent Imaging Capability.

PubMed

Hasan, Md Mehedi; Alam, Mohammad Wajih; Wahid, Khan A; Miah, Sayem; Lukong, Kiven Erique

2016-01-01

This paper describes the development of a prototype of a low-cost digital fluorescent microscope built from commercial off-the-shelf (COTS) components. The prototype was tested to detect malignant tumor cells taken from a living organism in a preclinical setting. This experiment was accomplished by using Alexa Fluor 488 conjugate dye attached to the cancer cells. Our prototype utilizes a torch along with an excitation filter as a light source for fluorophore excitation, a dichroic mirror to reflect the excitation and pass the emitted green light from the sample under test and a barrier filter to permit only appropriate wavelength. The system is designed out of a microscope using its optical zooming property and an assembly of exciter filter, dichroic mirror and transmitter filter. The microscope is connected to a computer or laptop through universal serial bus (USB) that allows real-time transmission of captured florescence images; this also offers real-time control of the microscope. The designed system has comparable features of high-end commercial fluorescent microscopes while reducing cost, power, weight and size.

A Low-Cost Digital Microscope with Real-Time Fluorescent Imaging Capability

PubMed Central

Hasan, Md. Mehedi; Wahid, Khan A.; Miah, Sayem; Lukong, Kiven Erique

2016-01-01

This paper describes the development of a prototype of a low-cost digital fluorescent microscope built from commercial off-the-shelf (COTS) components. The prototype was tested to detect malignant tumor cells taken from a living organism in a preclinical setting. This experiment was accomplished by using Alexa Fluor 488 conjugate dye attached to the cancer cells. Our prototype utilizes a torch along with an excitation filter as a light source for fluorophore excitation, a dichroic mirror to reflect the excitation and pass the emitted green light from the sample under test and a barrier filter to permit only appropriate wavelength. The system is designed out of a microscope using its optical zooming property and an assembly of exciter filter, dichroic mirror and transmitter filter. The microscope is connected to a computer or laptop through universal serial bus (USB) that allows real-time transmission of captured florescence images; this also offers real-time control of the microscope. The designed system has comparable features of high-end commercial fluorescent microscopes while reducing cost, power, weight and size. PMID:27977709

Assessing delivery and quantifying efficacy of small interfering ribonucleic acid therapeutics in the skin using a dual-axis confocal microscope

NASA Astrophysics Data System (ADS)

Ra, Hyejun; Gonzalez-Gonzalez, Emilio; Smith, Bryan R.; Gambhir, Sanjiv S.; Kino, Gordon S.; Solgaard, Olav; Kaspar, Roger L.; Contag, Christopher H.

2010-05-01

Transgenic reporter mice and advances in imaging instrumentation are enabling real-time visualization of cellular mechanisms in living subjects and accelerating the development of novel therapies. Innovative confocal microscope designs are improving their utility for microscopic imaging of fluorescent reporters in living animals. We develop dual-axis confocal (DAC) microscopes for such in vivo studies and create mouse models where fluorescent proteins are expressed in the skin for the purpose of advancing skin therapeutics and transdermal delivery tools. Three-dimensional image volumes, through the different skin compartments of the epidermis and dermis, can be acquired in several seconds with the DAC microscope in living mice, and are comparable to histologic analyses of reporter protein expression patterns in skin sections. Intravital imaging with the DAC microscope further enables visualization of green fluorescent protein (GFP) reporter gene expression in the skin over time, and quantification of transdermal delivery of small interfering RNA (siRNA) and therapeutic efficacy. Visualization of transdermal delivery of nucleic acids will play an important role in the development of innovative strategies for treating skin pathologies.

Parallel-multiplexed excitation light-sheet microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Xu, Dongli; Zhou, Weibin; Peng, Leilei

2017-02-01

Laser scanning light-sheet imaging allows fast 3D image of live samples with minimal bleach and photo-toxicity. Existing light-sheet techniques have very limited capability in multi-label imaging. Hyper-spectral imaging is needed to unmix commonly used fluorescent proteins with large spectral overlaps. However, the challenge is how to perform hyper-spectral imaging without sacrificing the image speed, so that dynamic and complex events can be captured live. We report wavelength-encoded structured illumination light sheet imaging (λ-SIM light-sheet), a novel light-sheet technique that is capable of parallel multiplexing in multiple excitation-emission spectral channels. λ-SIM light-sheet captures images of all possible excitation-emission channels in true parallel. It does not require compromising the imaging speed and is capable of distinguish labels by both excitation and emission spectral properties, which facilitates unmixing fluorescent labels with overlapping spectral peaks and will allow more labels being used together. We build a hyper-spectral light-sheet microscope that combined λ-SIM with an extended field of view through Bessel beam illumination. The system has a 250-micron-wide field of view and confocal level resolution. The microscope, equipped with multiple laser lines and an unlimited number of spectral channels, can potentially image up to 6 commonly used fluorescent proteins from blue to red. Results from in vivo imaging of live zebrafish embryos expressing various genetic markers and sensors will be shown. Hyper-spectral images from λ-SIM light-sheet will allow multiplexed and dynamic functional imaging in live tissue and animals.

Visualization of DNA molecules in time during electrophoresis

NASA Technical Reports Server (NTRS)

Lubega, Seth

1991-01-01

For several years individual DNA molecules have been observed and photographed during agarose gel electrophoresis. The DNA molecule is clearly the largest molecule known. Nevertheless, the largest molecule is still too small to be seen using a microscope. A technique developed by Morikawa and Yanagida has made it possible to visualize individual DNA molecules. When these long molecules are labeled with appropriate fluorescence dyes and observed under a fluorescence microscope, although it is not possible to directly visualize the local ultrastructure of the molecules, yet because they are long light emitting chains, their microscopic dynamical behavior can be observed. This visualization works in the same principle that enables one to observe a star through a telescope because it emits light against a dark background. The dynamics of individual DNA molecules migrating through agarose matrix during electrophoresis have been described by Smith et al. (1989), Schwartz and Koval (1989), and Bustamante et al. (1990). DNA molecules during agarose gel electrophoresis advance lengthwise thorough the gel in an extended configuration. They display an extension-contraction motion and tend to bunch up in their leading ends as the 'heads' find new pores through the gel. From time to time they get hooked on obstacles in the gel to form U-shaped configurations before they resume their linear configuration.

AOTF microscope for imaging with increased speed and spectral versatility.

PubMed Central

Wachman, E S; Niu, W; Farkas, D L

1997-01-01

We have developed a new fluorescence microscope that addresses the spectral and speed limitations of current light microscopy instrumentation. In the present device, interference and neutral density filters normally used for fluorescence excitation and detection are replaced by acousto-optic tunable filters (AOTFs). Improvements are described, including the use of a dispersing prism in conjunction with the imaging AOTF and an oblique-illumination excitation scheme, which together enable the AOTF microscope to produce images comparable to those obtained with conventional fluorescence instruments. The superior speed and spectral versatility of the AOTF microscope are demonstrated by a ratio image pair acquired in 3.5 ms and a micro-spectral absorbance measurement of hemoglobin through a cranial window in a living mouse. Images FIGURE 1 FIGURE 2 FIGURE 4 FIGURE 5 FIGURE 6 FIGURE 7 PMID:9284289

Characterization of the binding of shikonin to human immunoglobulin using scanning electron microscope, molecular modeling and multi-spectroscopic methods.

PubMed

He, Wenying; Ye, Xinyu; Yao, Xiaojun; Wu, Xiuli; Lin, Qiang; Huang, Guolei; Hua, Yingjie; Hui, Yang

2015-11-05

Shikonin, one of the active components isolated from the root of Arnebia euchroma (Royle) Johnst, have anti-tumor, anti-bacterial and anti-inflammatory activities and has been used clinically in phlebitis and vascular purpura. In the present work, the interaction of human immunoglobulin (HIg) with shikonin has been investigated by using scanning electron microscope (SEM), Fourier transform infrared (FT-IR) spectroscopy, fluorescence polarization, synchronous and 3D fluorescence spectroscopy in combination with molecular modeling techniques under physiological conditions with drug concentrations of 3.33-36.67 μM. The results of SEM exhibited visually the special effect on aggregation behavior of the complex formed between HIg and shikonin. The fluorescence polarization values indicated that shikonin molecules were found in a motionally unrestricted environment introduced by HIg. Molecular docking showed the shikonin moiety bound to the hydrophobic cavity of HIg, and there are four hydrogen-bonding interactions between shikonin and the residues of protein. The synchronous and 3D fluorescence spectra confirmed that shikonin could quench the intrinsic fluorescence of HIg and has an effect on the microenvironment around HIg in aqueous solution. The changes in the secondary structure of HIg were estimated by qualitative and quantitative FT-IR spectroscopic analysis. The binding constants and thermodynamic parameters for shikonin-HIg systems were obtained under different temperatures (300 K, 310 K and 320 K). The above results revealed the binding mechanism of shikonin and HIg at the ultrastructure and molecular level. Copyright © 2015 Elsevier B.V. All rights reserved.

A Comparative Study of Microscopic Images Captured by a Box Type Digital Camera Versus a Standard Microscopic Photography Camera Unit

PubMed Central

Desai, Nandini J.; Gupta, B. D.; Patel, Pratik Narendrabhai

2014-01-01

Introduction: Obtaining images of slides viewed by a microscope can be invaluable for both diagnosis and teaching.They can be transferred among technologically-advanced hospitals for further consultation and evaluation. But a standard microscopic photography camera unit (MPCU)(MIPS-Microscopic Image projection System) is costly and not available in resource poor settings. The aim of our endeavour was to find a comparable and cheaper alternative method for photomicrography. Materials and Methods: We used a NIKON Coolpix S6150 camera (box type digital camera) with Olympus CH20i microscope and a fluorescent microscope for the purpose of this study. Results: We got comparable results for capturing images of light microscopy, but the results were not as satisfactory for fluorescent microscopy. Conclusion: A box type digital camera is a comparable, less expensive and convenient alternative to microscopic photography camera unit. PMID:25478350

Radiation Dosimetry via Automated Fluorescence Microscopy

NASA Technical Reports Server (NTRS)

Castleman, Kenneth R.; Schulze, Mark

2005-01-01

A developmental instrument for assessment of radiation-induced damage in human lymphocytes includes an automated fluorescence microscope equipped with a one or more chargecoupled- device (CCD) video camera(s) and circuitry to digitize the video output. The microscope is also equipped with a three-axis translation stage that includes a rotation stage, and a rotary tray that holds as many as thirty specimen slides. The figure depicts one version of the instrument. Once the slides have been prepared and loaded into the tray, the instrument can operate unattended. A computer controls the operation of the stage, tray, and microscope, and processes the digital fluorescence-image data to recognize and count chromosomes that have been broken, presumably by radiation. The design and method of operation of the instrument exploit fluorescence in situ hybridization (FISH) of metaphase chromosome spreads, which is a technique that has been found to be valuable for monitoring the radiation dose to circulating lymphocytes. In the specific FISH protocol used to prepare specimens for this instrument, metaphase lymphocyte cultures are chosen for high mitotic index and highly condensed chromosomes, then several of the largest chromosomes are labeled with three of four differently colored whole-chromosome-staining dyes. The three dyes, which are used both individually and in various combinations, are fluorescein isothiocyanate (FITC), Texas Red (or equivalent), and Cy5 (or equivalent); The fourth dye 4',6-diamidino- 2-phenylindole (DAPI) is used as a counterstain. Under control by the computer, the microscope is automatically focused on the cells and each slide is scanned while the computer analyzes the DAPI-fluorescence images to find the metaphases. Each metaphase field is recentered in the field of view and refocused. Then a four-color image (more precisely, a set of images of the same view in the fluorescent colors of the four dyes) is acquired. By use of pattern-recognition software developed specifically for this instrument, the images in the various colors are processed to recognize the metaphases and count the chromosome fragments of each color within the metaphases. The intermediate results are then further processed to estimate the proportion of cells that have suffered genetic damage. The prototype instrument scans at an average areal rate of 4.7 mm2/h in unattended operation, finding about 14 metaphases per hour. The false-alarm rate is typically less than 3 percent, and the metaphase-miss rate has been estimated to be less than 5 percent. The counts of chromosomes and fragments thereof are 50 to 70 percent accurate.

BHHST: An improved lanthanide chelate for time-resolved fluorescence applications

NASA Astrophysics Data System (ADS)

Connally, Russell; Jin, Dayong; Piper, James

2005-04-01

The detection of the waterborne pathogens Giardia lamblia and Cryptosporidium parvum in environmental water bodies requires concentration of large volumes of water due to the low dose required for infection. The highly concentrated (10,000-fold) water sample is often rich in strongly autofluorescent algae, organic debris and mineral particles that can obscure immunofluorescently labeled (oo)cysts during analysis. Time-resolved fluorescence techniques exploit the long fluorescence lifetimes of lanthanide chelates (ms) to differentiate target fluorescence from background autofluorescence (ns). Relatively simple instrumentation can be used to enhance the signal-to-noise ratio (S/N) of labelled target. Time-resolved fluorescence techniques exploit the large difference in lifetime by briefly exciting fluorescence from the sample using a pulsed excitation source. Capture of the resulting fluorescence emission is delayed until the more rapidly decaying autofluorescence has faded beyond detection, whereon the much stronger and slower fading emission from labelled target is collected. BHHCT is a tetradentate beta-diketone chelate that is activated to bind with protein (antibody) as the chlorosulfonate. The high activity of this residue makes conjugations difficult to control and can lead to the formation of unstable immunoconjugates. To overcome these limitations a 5-atom hydrophylic molecular tether was attached to BHHCT via the chlorosulfonate and the BHHCT derivative was then activated to bind to proteins as the succinimide. The new compound (BHHST) could be prepared in high purity and was far more stable than the chlorosulfonate on storage. A high activity immunocojugate was prepared against Cryptosporidium that yielded an 8-fold increase in SNR using a lab-built time-resolved fluorescence microscope.

Development and Optical Testing of the Camera, Hand Lens, and Microscope Probe with Scannable Laser Spectroscopy (CHAMP-SLS)

NASA Technical Reports Server (NTRS)

Mungas, Greg S.; Gursel, Yekta; Sepulveda, Cesar A.; Anderson, Mark; La Baw, Clayton; Johnson, Kenneth R.; Deans, Matthew; Beegle, Luther; Boynton, John

2008-01-01

Conducting high resolution field microscopy with coupled laser spectroscopy that can be used to selectively analyze the surface chemistry of individual pixels in a scene is an enabling capability for next generation robotic and manned spaceflight missions, civil, and military applications. In the laboratory, we use a range of imaging and surface preparation tools that provide us with in-focus images, context imaging for identifying features that we want to investigate at high magnification, and surface-optical coupling that allows us to apply optical spectroscopic analysis techniques for analyzing surface chemistry particularly at high magnifications. The camera, hand lens, and microscope probe with scannable laser spectroscopy (CHAMP-SLS) is an imaging/spectroscopy instrument capable of imaging continuously from infinity down to high resolution microscopy (resolution of approx. 1 micron/pixel in a final camera format), the closer CHAMP-SLS is placed to a feature, the higher the resultant magnification. At hand lens to microscopic magnifications, the imaged scene can be selectively interrogated with point spectroscopic techniques such as Raman spectroscopy, microscopic Laser Induced Breakdown Spectroscopy (micro-LIBS), laser ablation mass-spectrometry, Fluorescence spectroscopy, and/or Reflectance spectroscopy. This paper summarizes the optical design, development, and testing of the CHAMP-SLS optics.

Fluorescent antibody detection of microorganisms in terrestrial environments

NASA Technical Reports Server (NTRS)

Schmidt, E. L.

1972-01-01

The fluorescent antibody technique and its use in direct microscopic examination of the soil is discussed. Feasibility analyses were made to determine if the method could be used to simultaneously observe and recognize microorganisms in the soil. Some data indicate this may be possible. Data are also given on two related problems involving the interaction of soil microorganisms with plant roots to form symbiotic structures. One was concerned with the developmental ecology and biology of the root nodule of alder and the second was concerned with the ectotrophic mycorrhizal structure on forest trees, especially pines. In both, the fluorescent antibody detection of the microbial symbiont both as a free living form in soil, and as a root inhabiting form in the higher plant was emphasized. A third aspect of the research involved the detection of autotrophic ammonia oxidizing microorganisms in soil.

Three-dimensional fluorescent microscopy via simultaneous illumination and detection at multiple planes.

PubMed

Ma, Qian; Khademhosseinieh, Bahar; Huang, Eric; Qian, Haoliang; Bakowski, Malina A; Troemel, Emily R; Liu, Zhaowei

2016-08-16

The conventional optical microscope is an inherently two-dimensional (2D) imaging tool. The objective lens, eyepiece and image sensor are all designed to capture light emitted from a 2D 'object plane'. Existing technologies, such as confocal or light sheet fluorescence microscopy have to utilize mechanical scanning, a time-multiplexing process, to capture a 3D image. In this paper, we present a 3D optical microscopy method based upon simultaneously illuminating and detecting multiple focal planes. This is implemented by adding two diffractive optical elements to modify the illumination and detection optics. We demonstrate that the image quality of this technique is comparable to conventional light sheet fluorescent microscopy with the advantage of the simultaneous imaging of multiple axial planes and reduced number of scans required to image the whole sample volume.

Fluorescence Microscopy Gets Faster and Clearer: Roles of Photochemistry and Selective Illumination

PubMed Central

Wolenski, Joseph S.; Julich, Doerthe

2014-01-01

Significant advances in fluorescence microscopy tend be a balance between two competing qualities wherein improvements in resolution and low light detection are typically accompanied by losses in acquisition rate and signal-to-noise, respectively. These trade-offs are becoming less of a barrier to biomedical research as recent advances in optoelectronic microscopy and developments in fluorophore chemistry have enabled scientists to see beyond the diffraction barrier, image deeper into live specimens, and acquire images at unprecedented speed. Selective plane illumination microscopy has provided significant gains in the spatial and temporal acquisition of fluorescence specimens several mm in thickness. With commercial systems now available, this method promises to expand on recent advances in 2-photon deep-tissue imaging with improved speed and reduced photobleaching compared to laser scanning confocal microscopy. Superresolution microscopes are also available in several modalities and can be coupled with selective plane illumination techniques. The combination of methods to increase resolution, acquisition speed, and depth of collection are now being married to common microscope systems, enabling scientists to make significant advances in live cell and in situ imaging in real time. We show that light sheet microscopy provides significant advantages for imaging live zebrafish embryos compared to laser scanning confocal microscopy. PMID:24600334

New light on ion channel imaging by total internal reflection fluorescence (TIRF) microscopy.

PubMed

Yamamura, Hisao; Suzuki, Yoshiaki; Imaizumi, Yuji

2015-05-01

Ion channels play pivotal roles in a wide variety of cellular functions; therefore, their physiological characteristics, pharmacological responses, and molecular structures have been extensively investigated. However, the mobility of an ion channel itself in the cell membrane has not been examined in as much detail. A total internal reflection fluorescence (TIRF) microscope allows fluorophores to be imaged in a restricted region within an evanescent field of less than 200 nm from the interface of the coverslip and plasma membrane in living cells. Thus the TIRF microscope is useful for selectively visualizing the plasmalemmal surface and subplasmalemmal zone. In this review, we focused on a single-molecule analysis of the dynamic movement of ion channels in the plasma membrane using TIRF microscopy. We also described two single-molecule imaging techniques under TIRF microscopy: fluorescence resonance energy transfer (FRET) for the identification of molecules that interact with ion channels, and subunit counting for the determination of subunit stoichiometry in a functional channel. TIRF imaging can also be used to analyze spatiotemporal Ca(2+) events in the subplasmalemma. Single-molecule analyses of ion channels and localized Ca(2+) signals based on TIRF imaging provide beneficial pharmacological and physiological information concerning the functions of ion channels. Copyright © 2015 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

Fluorescence fibre-optic confocal microscopy of skin in vivo: microscope and fluorophores.

PubMed

Suihko, Christian; Swindle, Lucinda D; Thomas, Steven G; Serup, Jørgen

2005-11-01

Fibre-optic confocal imaging in vivo is a new approach in the assessment of human skin. The objective is to describe a novel instrument and its operation and use in combination with fluorophores. The Stratum is a fibre-optic fluorescence confocal microscope especially developed for the study of skin and mucous membranes. The system is flexible and any body site can be studied with a hand-held scanner. The light source is a 488 nm argon ion laser. Horizontal (en face) images of the epidermis and outer dermis are produced with cellular resolution. Magnification is approximately 1000 x . Fluorescein sodium is routinely used as fluorophore (intradermal injection or application to the skin surface). This fluorophore is safe for human use in vivo, but other substances (rhodamine B, Acridine Orange, green fluorescent protein, curcumin) have also been studied. The instrument produces sharp images of epidermal cell layers from the epidermal surface to the sub-papillary dermis, with sub-cellular resolution. The scanner is flexible in use. The technique of intradermal fluorophore injection requires some skill. We consider this fibre-optic instrument a potentially important tool in skin research for non-invasive optical biopsy of primarily the epidermis. Present use is focussed on research applications, where the fluorophore distribution in the skin may illustrate morphological changes in the epidermis.

Biocompatible near-infrared fluorescent nanoparticles for macro and microscopic in vivo functional bioimaging

PubMed Central

Chu, Liliang; Wang, Shaowei; Li, Kanghui; Xi, Wang; Zhao, Xinyuan; Qian, Jun

2014-01-01

Near-infrared (NIR) imaging technology has been widely used for biomedical research and applications, since it can achieve deep penetration in biological tissues due to less absorption and scattering of NIR light. In our research, polymer nanoparticles with NIR fluorophores doped were synthesized. The morphology, absorption/emission features and chemical stability of the fluorescent nanoparticles were characterized, separately. NIR fluorescent nanoparticles were then utilized as bright optical probes for macro in vivo imaging of mice, including sentinel lymph node (SLN) mapping, as well as distribution and excretion monitoring of nanoparticles in animal body. Furthermore, we applied the NIR fluorescent nanoparticles in in vivo microscopic bioimaging via a confocal microscope. Under the 635 nm-CW excitation, the blood vessel architecture in the ear and the brain of mice, which were administered with nanoparticles, was visualized very clearly. The imaging depth of our one-photon microscopy, which was assisted with NIR fluorescent nanoprobes, can reach as deep as 500 μm. Our experiments show that NIR fluorescent nanoparticles have great potentials in various deep-tissue imaging applications. PMID:25426331

Towards automated early cancer detection: Non-invasive, fluorescence-based approaches for quantitative assessment of cells and tissue to identify pre-cancers

NASA Astrophysics Data System (ADS)

Levitt, Jonathan Michael

Cancer is the second leading cause of death globally, second only to heart disease. As in many diseases, patient survival is directly related to how early lesions are detected. Using conventional screening methods, the early changes associated with cancer, which occur on the microscopic scale, can easily go overlooked. Due to the inherent drawbacks of conventional techniques we present non-invasive, optically based methods to acquire high resolution images from live samples and assess cellular function associated with the onset of disease. Specifically, we acquired fluorescence images from NADH and FAD to quantify morphology and metabolic activity. We first conducted studies to monitor monolayers of keratinocytes in response to apoptosis which has been shown to be disrupted during cancer progression. We found that as keratinocytes undergo apoptosis there are populations of mitochondria that exhibit a higher metabolic activity that become progressively confined to a gradually smaller perinuclear region. To further assess the changes associated with early cancer growth we developed automated methods to rapidly quantify fluorescence images and extract morphological and metabolic information from life tissue. In this study, we simultaneously quantified mitochondrial organization, metabolic activity, nuclear size distribution, and the localization of the structural protein keratin, to differentiate between normal and pre-cancerous engineered tissues. We found the degree mitochondrial organization, as determined from the fractal derived Hurst parameter, was well correlated to level of cellular differentiation. We also found that the metabolic activity in the pre-cancerous cells was greater and more consistent throughout tissue depths in comparison to normal tissue. Keratin localization, also quantified from the fluorescence images, we found it to be confined to the uppermost layers of normal tissue while it was more evenly distributed in the precancerous tissues. To allow for evaluation of the early cancerous changes in vivo, we developed video-rate confocal reflectance/multi-photon fluorescence microscope as a clinical prototype. This device was specifically designed to rapidly acquire and assess non-invasively acquire fluorescence images using the automated methods we have developed. We have demonstrated the ability of this microscope to simultaneously acquire fluorescence, confocal reflectance, and second-harmonic generation images as well as assess blood flow in vivo.

Near-isotropic 3D optical nanoscopy with photon-limited chromophores

PubMed Central

Tang, Jianyong; Akerboom, Jasper; Vaziri, Alipasha; Looger, Loren L.; Shank, Charles V.

2010-01-01

Imaging approaches based on single molecule localization break the diffraction barrier of conventional fluorescence microscopy, allowing for bioimaging with nanometer resolution. It remains a challenge, however, to precisely localize photon-limited single molecules in 3D. We have developed a new localization-based imaging technique achieving almost isotropic subdiffraction resolution in 3D. A tilted mirror is used to generate a side view in addition to the front view of activated single emitters, allowing their 3D localization to be precisely determined for superresolution imaging. Because both front and side views are in focus, this method is able to efficiently collect emitted photons. The technique is simple to implement on a commercial fluorescence microscope, and especially suitable for biological samples with photon-limited chromophores such as endogenously expressed photoactivatable fluorescent proteins. Moreover, this method is relatively resistant to optical aberration, as it requires only centroid determination for localization analysis. Here we demonstrate the application of this method to 3D imaging of bacterial protein distribution and neuron dendritic morphology with subdiffraction resolution. PMID:20472826

Real-time single cell analysis of molecular mechanism of apoptosis and proliferation using FRET technique

NASA Astrophysics Data System (ADS)

Chen, Tongsheng; Xing, Da; Gao, Xuejuan; Wang, Fang

2006-09-01

Bcl-2 family proteins (such as Bid and Bak/Bax) and 14-3-3 proteins play a key role in the mitochondria-mediated cell apoptosis induced by cell death factors such as TNF-α and lower power laser irradiation (LPLI). In this report, fluorescence resonance energy transfer (FRET) has been used to study the molecular mechanism of apoptosis in living cells on a fluorescence scanning confocal microscope. Based on the genetic code technique and the green fluorescent proteins (GFPs), single-cell dynamic analysis of caspase3 activation, caspase8 activation, and PKCs activation are performed during apoptosis induced by laser irradiation in real-time. To investigate the cellular effect and mechanism of laser irradiation, human lung adenocarcinoma cells (ASTC-a-1) transfected with plasmid SCAT3 (pSCAT3)/ CKAR FRET reporter, were irradiated and monitored noninvasively with both FRET imaging. Our results show that high fluence lower power laser irradiation (HFLPLI) can induce an increase of caspase3 activation and a decrease of PKCs activation, and that LPLI induces the ASTC-a-1 cell proliferation by specifically activating PKCs.

Microdose fluorescence imaging of ABY-029 on an operating microscope adapted by custom illumination and imaging modules.

PubMed

Elliott, Jonathan T; Dsouza, Alisha V; Marra, Kayla; Pogue, Brian W; Roberts, David W; Paulsen, Keith D

2016-09-01

Fluorescence guided surgery has the potential to positively impact surgical oncology; current operating microscopes and stand-alone imaging systems are too insensitive or too cumbersome to maximally take advantage of new tumor-specific agents developed through the microdose pathway. To this end, a custom-built illumination and imaging module enabling picomolar-sensitive near-infrared fluorescence imaging on a commercial operating microscope is described. The limits of detection and system specifications are characterized, and in vivo efficacy of the system in detecting ABY-029 is evaluated in a rat orthotopic glioma model following microdose injections, showing the suitability of the device for microdose phase 0 clinical trials.

Microdose fluorescence imaging of ABY-029 on an operating microscope adapted by custom illumination and imaging modules

PubMed Central

Dsouza, Alisha V.; Marra, Kayla; Pogue, Brian W.; Roberts, David W.; Paulsen, Keith D.

2016-01-01

Fluorescence guided surgery has the potential to positively impact surgical oncology; current operating microscopes and stand-alone imaging systems are too insensitive or too cumbersome to maximally take advantage of new tumor-specific agents developed through the microdose pathway. To this end, a custom-built illumination and imaging module enabling picomolar-sensitive near-infrared fluorescence imaging on a commercial operating microscope is described. The limits of detection and system specifications are characterized, and in vivo efficacy of the system in detecting ABY-029 is evaluated in a rat orthotopic glioma model following microdose injections, showing the suitability of the device for microdose phase 0 clinical trials. PMID:27699098

Scanning light-sheet microscopy in the whole mouse brain with HiLo background rejection

PubMed Central

Mertz, Jerome; Kim, Jinhyun

2010-01-01

It is well known that light-sheet illumination can enable optically sectioned wide-field imaging of macroscopic samples. However, the optical sectioning capacity of a light-sheet macroscope is undermined by sample-induced scattering or aberrations that broaden the thickness of the sheet illumination. We present a technique to enhance the optical sectioning capacity of a scanning light-sheet microscope by out-of-focus background rejection. The technique, called HiLo microscopy, makes use of two images sequentially acquired with uniform and structured sheet illumination. An optically sectioned image is then synthesized by fusing high and low spatial frequency information from both images. The benefits of combining light-sheet macroscopy and HiLo background rejection are demonstrated in optically cleared whole mouse brain samples, using both green fluorescent protein (GFP)-fluorescence and dark-field scattered light contrast. PMID:20210471

Miniaturized fiber-coupled confocal fluorescence microscope with an electrowetting variable focus lens using no moving parts

PubMed Central

Ozbay, Baris N.; Losacco, Justin T.; Cormack, Robert; Weir, Richard; Bright, Victor M.; Gopinath, Juliet T.; Restrepo, Diego; Gibson, Emily A.

2015-01-01

We report a miniature, lightweight fiber-coupled confocal fluorescence microscope that incorporates an electrowetting variable focus lens to provide axial scanning for full three-dimensional (3D) imaging. Lateral scanning is accomplished by coupling our device to a laser-scanning confocal microscope through a coherent imaging fiber-bundle. The optical components of the device are combined in a custom 3D-printed adapter with an assembled weight of <2 g that can be mounted onto the head of a mouse. Confocal sectioning provides an axial resolution of ~12 µm and an axial scan range of ~80 µm. The lateral field-of-view is 300 µm, and the lateral resolution is 1.8 µm. We determined these parameters by imaging fixed sections of mouse neuronal tissue labeled with green fluorescent protein (GFP) and fluorescent bead samples in agarose gel. To demonstrate viability for imaging intact tissue, we resolved multiple optical sections of ex vivo mouse olfactory nerve fibers expressing yellow fluorescent protein (YFP). PMID:26030555

Multicolor fluorescence microscopic imaging of cancer cells on the plasmonic chip (Presentation Recording)

NASA Astrophysics Data System (ADS)

Tawa, Keiko; Sasakawa, Chisato; Yamamura, Shohei; Shibata, Izumi; Kataoka, Masatoshi

2015-09-01

A plasmonic chip which is a metal coated substrate with grating structure can provide the enhanced fluorescence by the grating-coupled surface plasmon field. In our previous studies, bright epi-fluorescence microscopic imaging of neuron cells and sensitive immunosesnsing have been reported. In this study, two kinds of breast cancer cells, MCF-7 and MDA-MB231, were observed with epi-fluorescence microscope on the plasmonic chip with 2D hole-arrays . They were multicolor stained with 4', 6-diamidino-2-phenylindole (DAPI) and allophycocyanin (APC)-labeled anti-epithelial cell adhesion molecule (EpCAM) antibody. Our plasmonic chip provided the brighter fluorescence images of these cells compared with the glass slide. Even in the cells including few EpCAM, the distribution of EpCAM was clearly observed in the cell membrane. It was found that the plasmonic chip can be one of the powerful tools to detect the marker protein existing around the chip surface even at low concentration.

Time-resolved wide-field optically sectioned fluorescence microscopy

NASA Astrophysics Data System (ADS)

Dupuis, Guillaume; Benabdallah, Nadia; Chopinaud, Aurélien; Mayet, Céline; Lévêque-Fort, Sandrine

2013-02-01

We present the implementation of a fast wide-field optical sectioning technique called HiLo microscopy on a fluorescence lifetime imaging microscope. HiLo microscopy is based on the fusion of two images, one with structured illumination and another with uniform illumination. Optically sectioned images are then digitally generated thanks to a fusion algorithm. HiLo images are comparable in quality with confocal images but they can be acquired faster over larger fields of view. We obtain 4D imaging by combining HiLo optical sectioning, time-gated detection, and z-displacement. We characterize the performances of this set-up in terms of 3D spatial resolution and time-resolved capabilities in both fixed- and live-cell imaging modes.

Free-radical sensing by using naphthalimide based mesoporous silica (MCM-41) nanoparticles: A combined fluorescence and cellular imaging study

NASA Astrophysics Data System (ADS)

Jha, Gaurav; Roy, Subhasis; Sahu, Prabhat Kumar; Banerjee, Somnath; Anoop, N.; Rahaman, Abdur; Sarkar, Moloy

2018-01-01

Keeping in mind the advantages of material-based systems over simple molecule-based systems, we have designed and developed three inorganic-organic hybrid systems by anchoring 1,8-naphthalimide derivatives to mesoporous silica nanoparticles for detection of free radicals. Prior to photophysical study, systems are characterized by spectroscopic, microscopic and thermo-gravimetric techniques. Steady state and time-resolved fluorescence studies demonstrate that the hydrazine based system is senstive towards detection of various free radicals. Cellular imaging study reveals cell permeability and toxicity study demonstrates the non-toxic nature of the material. These studies have suggested that present system has the potential to be used in various biological applications.

Water-soluble, neutral 3,5-diformyl-BODIPY with extended fluorescence lifetime in a self-healable chitosan hydrogel.

PubMed

Belali, Simin; Emandi, Ganapathi; Cafolla, Atillio A; O'Connell, Barry; Haffner, Benjamin; Möbius, Matthias E; Karimi, Alireza; Senge, Mathias O

2017-11-08

3,5-Diformyl-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (3,5-diformyl-BODIPY) can be used as an efficient biofunctional cross-linker to generate a new class of chitosan-based hydrogels with fluorescence resonance energy transfer (FRET) dynamics and good solubility in water. The hydrogel was fully characterized by FT-IR, UV-vis, fluorescence, FE-SEM, AFM, rheology and picosecond time-resolved spectroscopic techniques. The self-healing ability was demonstrated by rheological recovery and macroscopic and microscopic observations. The fluorescence lifetime was found to increase in aqueous solution of the BODIPY-chitosan hydrogel compared to the 3,5-diformyl-BODIPY monomer. Calculations based on experimental results such as red-shift and decreased intensity of the emission spectrum of highly dye-concentrated hydrogel in comparison to dilute hydrogels, together with changes in the fluorescence lifetime of the hydrogel at different concentration of dyes, suggest that the BDP-CS hydrogels fluorescence dynamics obey the Förster resonance energy transfer (FRET). Improvements in mechanical and photochemical properties and the acceptable values of BODIPY fluorescence lifetime in the hydrogel matrix indicate the utility of the newly synthesized hydrogels for biomedical applications.

A total internal reflection-fluorescence correlation spectroscopy setup with pulsed diode laser excitation

NASA Astrophysics Data System (ADS)

Weger, Lukas; Hoffmann-Jacobsen, Kerstin

2017-09-01

Fluorescence correlation spectroscopy (FCS) measures fluctuations in a (sub-)femtoliter volume to analyze the diffusive behavior of fluorescent particles. This highly sensitive method has proven to be useful for the analysis of dynamic biological systems as well as in chemistry, physics, and material sciences. It is routinely performed with commercial fluorescence microscopes, which provide a confined observation volume by the confocal technique. The evanescent wave of total internal reflectance (TIR) is used in home-built systems to permit a surface sensitive FCS analysis. We present a combined confocal and TIR-FCS setup which uses economic low-power pulsed diode lasers for excitation. Excitation and detection are coupled to time-correlated photon counting hardware. This allows simultaneous fluorescence lifetime and FCS measurements in a surface-sensitive mode. Moreover, the setup supports fluorescence lifetime correlation spectroscopy at surfaces. The excitation can be easily switched between TIR and epi-illumination to compare the surface properties with those in liquid bulk. The capabilities of the presented setup are demonstrated by measuring the diffusion coefficients of a free dye molecule, a labeled polyethylene glycol, and a fluorescent nanoparticle in confocal as well as in TIR-FCS.

Phase Sensitive Demodulation in Multiphoton Microscopy

NASA Astrophysics Data System (ADS)

Fisher, Walt G.; Piston, David W.; Wachter, Eric A.

2002-06-01

Multiphoton laser scanning microscopy offers advantages in depth of penetration into intact samples over other optical sectioning techniques. To achieve these advantages it is necessary to detect the emitted light without spatial filtering. In this nondescanned (nonconfocal) approach, ambient room light can easily contaminate the signal, forcing experiments to be performed in absolute darkness. For multiphoton microscope systems employing mode-locked lasers, signal processing can be used to reduce such problems by taking advantage of the pulsed characteristics of such lasers. Specifically, by recovering fluorescence generated at the mode-locked frequency, interference from stray light and other ambient noise sources can be significantly reduced. This technology can be adapted to existing microscopes by inserting demodulation circuitry between the detector and data collection system. The improvement in signal-to-noise ratio afforded by this approach yields a more robust microscope system and opens the possibility of moving multiphoton microscopy from the research lab to more demanding settings, such as the clinic.

Fluorescence-guided resection of intracranial VX2 tumor in a preclinical model using 5-aminolevulinic acid (ALA): preliminary results

NASA Astrophysics Data System (ADS)

Bogaards, Arjen; Varma, Abhay; Moriyama, Eduardo H.; Lin, Annie; Giles, Anoja; Bisland, Stuart K.; Lilge, Lothar D.; Bilbao, G. M.; Muller, Paul J.; Wilson, Brian C.

2003-06-01

Fluorescence-guided brain tumor resection may help the neurosurgeon to identify tumor margins that merge imperceptibly into the normal brain tissue and are difficult to identify under white light illumination even using an operating microscope. We compared the amount of residual tumor after white light resection using an operating microscope versus that after fluorescnece-guided resection of an intracranial VX2 tumor in a preclinical model using our previously developed co-axial fluorscence imaging and spectroscopy system, exciting and detecting PpIX fluorescence at 405nm and 635nm respectively. Preliminary results: No fluorescence was present in 3 non-tumor-bearing animals. Fluorescence was present in all 15 tumor-bearing animals after white light resection was completed. To date in 4 rabbits, a decrease in residual tumor was found when using additional fluorescence guided resection compared to white light resection only. Conclusions: ALA induced PpIX fluorescence detects tumor margins not seen under an operation microscope using while light. Using fluorescence imaging to guide tumor resection resulted in a 3-fold decrease in the amount of residual timor. However, these preliminary results indicate that also an additional amount of normal brain is resected, which will be further investigated.

Ultrahigh resolution multicolor colocalization of single fluorescent probes

DOEpatents

Weiss, Shimon; Michalet, Xavier; Lacoste, Thilo D.

2005-01-18

A novel optical ruler based on ultrahigh-resolution colocalization of single fluorescent probes is described. Two unique families of fluorophores are used, namely energy-transfer fluorescent beads and semiconductor nanocrystal (NC) quantum dots, that can be excited by a single laser wavelength but emit at different wavelengths. A novel multicolor sample-scanning confocal microscope was constructed which allows one to image each fluorescent light emitter, free of chromatic aberrations, by scanning the sample with nanometer scale steps using a piezo-scanner. The resulting spots are accurately localized by fitting them to the known shape of the excitation point-spread-function of the microscope.

Multi-scale fluorescence imaging of bacterial infections in animal models

NASA Astrophysics Data System (ADS)

Bixler, Joel N.; Kong, Ying; Cirillo, Jeffrey D.; Maitland, Kristen C.

2013-03-01

Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), currently affects roughly one-third of the world's population. Drug resistant strains of Mtb decrease the effectiveness of current therapeutics and demand the development of new antimicrobial therapies. In addition, the current vaccine, Bacille Calmette Guérin (BCG), has variable efficacy for disease prevention in different populations. Animal studies are often limited by the need to sacrifice at discrete time points for pathology and tissue homogenization, which greatly reduces spatial and temporal resolution. Optical imaging offers the potential for a minimally-invasive solution to imaging on a macroscopic and microscopic scale, allowing for high resolution study of infection. We have integrated a fluorescence microendoscope into a whole-animal optical imaging system, allowing for simultaneous microscopic and macroscopic imaging of tdTomato expressing BCG in vivo. A 535 nm LED was collimated and launched into a 10,000 element fiber bundle with an outer diameter of 0.66 mm. The fiber bundle can be inserted through an intra-tracheal catheter into the lung of a mouse. Fluorescence emission can either be (1) collected by the bundle and imaged onto the surface of a CCD camera for localized detection or (2) the fluorescence can be imaged by the whole animal imaging system providing macroscopic information. Results from internal localized excitation and external whole body detection indicate the potential for imaging bacterial infections down to 100 colony forming units. This novel imaging technique has the potential to allow for functional studies, enhancing the ability to assess new therapeutic agents.

Time-Lapse Monitoring of DNA Damage Colocalized With Particle Tracks in Single Living Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

McFadden, Conor H.; Hallacy, Timothy M.; Department of Physics and Astronomy, Rice University, Houston, Texas

2016-09-01

Purpose: Understanding the DNA damage and repair induced by hadron therapy (HT) beams is crucial for developing novel strategies to maximize the use of HT beams to treat cancer patients. However, spatiotemporal studies of DNA damage and repair for beam energies relevant to HT have been challenging. We report a technique that enables spatiotemporal measurement of radiation-induced damage in live cells and colocalization of this damage with charged particle tracks over a broad range of clinically relevant beam energies. The technique uses novel fluorescence nuclear track detectors with fluorescence confocal laser scanning microscopy in the beam line to visualize particlemore » track traversals within the subcellular compartments of live cells within seconds after injury. Methods and Materials: We designed and built a portable fluorescence confocal laser scanning microscope for use in the beam path, coated fluorescence nuclear track detectors with fluorescent-tagged live cells (HT1080 expressing enhanced green fluorescent protein tagged to XRCC1, a single-strand break repair protein), placed the entire assembly into a proton therapy beam line, and irradiated the cells with a fluence of ∼1 × 10{sup 6} protons/cm{sup 2}. Results: We successfully obtained confocal images of proton tracks and foci of DNA single-strand breaks immediately after irradiation. Conclusions: This technique represents an innovative method for analyzing biological responses in any HT beam line at energies and dose rates relevant to therapy. It allows precise determination of the number of tracks traversing a subcellular compartment and monitoring the cellular damage therein, and has the potential to measure the linear energy transfer of each track from therapeutic beams.« less

A portable confocal hyperspectral microscope without any scan or tube lens and its application in fluorescence and Raman spectral imaging

NASA Astrophysics Data System (ADS)

Li, Jingwei; Cai, Fuhong; Dong, Yongjiang; Zhu, Zhenfeng; Sun, Xianhe; Zhang, Hequn; He, Sailing

2017-06-01

In this study, a portable confocal hyperspectral microscope is developed. In traditional confocal laser scanning microscopes, scan lens and tube lens are utilized to achieve a conjugate relationship between the galvanometer and the back focal plane of the objective, in order to achieve a better resolution. However, these lenses make it difficult to scale down the volume of the system. In our portable confocal hyperspectral microscope (PCHM), the objective is placed directly next to the galvomirror. Thus, scan lens and tube lens are not included in our system and the size of this system is greatly reduced. Furthermore, the resolution is also acceptable in many biomedical and food-safety applications. Through reducing the optical length of the system, the signal detection efficiency is enhanced. This is conducive to realizing both the fluorescence and Raman hyperspectral imaging. With a multimode fiber as a pinhole, an improved image contrast is also achieved. Fluorescent spectral images for HeLa cells/fingers and Raman spectral images of kumquat pericarp are present. The spectral resolution and spatial resolutions are about 0.4 nm and 2.19 μm, respectively. These results demonstrate that this portable hyperspectral microscope can be used in in-vivo fluorescence imaging and in situ Raman spectral imaging.

Early quantitative method for measuring germination in non-green spores of Dryopteris paleacea using an epifluorescence-microscope technique

NASA Technical Reports Server (NTRS)

Scheuerlein, R.; Wayne, R.; Roux, S. J.

1988-01-01

A method is described to determine germination by blue-light excited red fluorescence in the positively photoblastic spores of Dryopteris paleacea Sw. This fluorescence is due to chlorophyll as evidenced from 1) a fluorescence-emission spectrum in vivo, where a bright fluorescence around 675 nm is obtained only in red light (R)-irradiated spores and 2) in vitro measurements with acetone extracts prepared from homogenized spores. Significant amounts of chlorophyll can be found only in R-treated spores; this chlorophyll exhibits an emission band around 668 nm, when irradiated with 430 nm light at 21 degrees C. Compared to other criteria for germination, such as swelling of the cell, coat splitting, greening, and rhizoid formation, which require longer periods after induction for their expression, chlorophyll fluorescence can be used to quantify germination after two days. This result is confirmed by fluence-response curves for R-induced spore germination; the same relationship between applied R and germination is obtained by the evaluation with the epifluorescence method 2 days after the light treatment as compared with the evaluation with bright-field microscopy 5 days after the inducing R. Using this technique we show for the first time that Ca2+ contributes to the signal-transduction chain in phytochrome-mediated chlorophyll synthesis in spores of Dryopteris paleacea.

Dual-modal three-dimensional imaging of single cells with isometric high resolution using an optical projection tomography microscope

NASA Astrophysics Data System (ADS)

Miao, Qin; Rahn, J. Richard; Tourovskaia, Anna; Meyer, Michael G.; Neumann, Thomas; Nelson, Alan C.; Seibel, Eric J.

2009-11-01

The practice of clinical cytology relies on bright-field microscopy using absorption dyes like hematoxylin and eosin in the transmission mode, while the practice of research microscopy relies on fluorescence microscopy in the epi-illumination mode. The optical projection tomography microscope is an optical microscope that can generate 3-D images of single cells with isometric high resolution both in absorption and fluorescence mode. Although the depth of field of the microscope objective is in the submicron range, it can be extended by scanning the objective's focal plane. The extended depth of field image is similar to a projection in a conventional x-ray computed tomography. Cells suspended in optical gel flow through a custom-designed microcapillary. Multiple pseudoprojection images are taken by rotating the microcapillary. After these pseudoprojection images are further aligned, computed tomography methods are applied to create 3-D reconstruction. 3-D reconstructed images of single cells are shown in both absorption and fluorescence mode. Fluorescence spatial resolution is measured at 0.35 μm in both axial and lateral dimensions. Since fluorescence and absorption images are taken in two different rotations, mechanical error may cause misalignment of 3-D images. This mechanical error is estimated to be within the resolution of the system.

Optical-sectioning microscopy of protoporphyrin IX fluorescence in human gliomas: standardization and quantitative comparison with histology

NASA Astrophysics Data System (ADS)

Wei, Linpeng; Chen, Ye; Yin, Chengbo; Borwege, Sabine; Sanai, Nader; Liu, Jonathan T. C.

2017-04-01

Systemic delivery of 5-aminolevulinic acid leads to enhanced fluorescence image contrast in many tumors due to the increased accumulation of protoporphyrin IX (PpIX), a fluorescent porphyrin that is associated with tumor burden and proliferation. The value of PpIX-guided resection of malignant gliomas has been demonstrated in prospective randomized clinical studies in which a twofold greater extent of resection and improved progression-free survival have been observed. In low-grade gliomas and at the diffuse infiltrative margins of all gliomas, PpIX fluorescence is often too weak to be detected with current low-resolution surgical microscopes that are used in operating rooms. However, it has been demonstrated that high-resolution optical-sectioning microscopes are capable of detecting the sparse and punctate accumulations of PpIX that are undetectable via conventional low-power surgical fluorescence microscopes. To standardize the performance of high-resolution optical-sectioning devices for future clinical use, we have developed an imaging phantom and methods to ensure that the imaging of PpIX-expressing brain tissues can be performed reproducibly. Ex vivo imaging studies with a dual-axis confocal microscope demonstrate that these methods enable the acquisition of images from unsectioned human brain tissues that quantitatively and consistently correlate with images of histologically processed tissue sections.

Fluorescence Live Cell Imaging

PubMed Central

Ettinger, Andreas

2014-01-01

Fluorescence microscopy of live cells has become an integral part of modern cell biology. Fluorescent protein tags, live cell dyes, and other methods to fluorescently label proteins of interest provide a range of tools to investigate virtually any cellular process under the microscope. The two main experimental challenges in collecting meaningful live cell microscopy data are to minimize photodamage while retaining a useful signal-to-noise ratio, and to provide a suitable environment for cells or tissues to replicate physiological cell dynamics. This chapter aims to give a general overview on microscope design choices critical for fluorescence live cell imaging that apply to most fluorescence microscopy modalities, and on environmental control with a focus on mammalian tissue culture cells. In addition, we provide guidance on how to design and evaluate fluorescent protein constructs by spinning disk confocal microscopy. PMID:24974023

Maximizing the Biochemical Resolving Power of Fluorescence Microscopy

PubMed Central

Esposito, Alessandro; Popleteeva, Marina; Venkitaraman, Ashok R.

2013-01-01

Most recent advances in fluorescence microscopy have focused on achieving spatial resolutions below the diffraction limit. However, the inherent capability of fluorescence microscopy to non-invasively resolve different biochemical or physical environments in biological samples has not yet been formally described, because an adequate and general theoretical framework is lacking. Here, we develop a mathematical characterization of the biochemical resolution in fluorescence detection with Fisher information analysis. To improve the precision and the resolution of quantitative imaging methods, we demonstrate strategies for the optimization of fluorescence lifetime, fluorescence anisotropy and hyperspectral detection, as well as different multi-dimensional techniques. We describe optimized imaging protocols, provide optimization algorithms and describe precision and resolving power in biochemical imaging thanks to the analysis of the general properties of Fisher information in fluorescence detection. These strategies enable the optimal use of the information content available within the limited photon-budget typically available in fluorescence microscopy. This theoretical foundation leads to a generalized strategy for the optimization of multi-dimensional optical detection, and demonstrates how the parallel detection of all properties of fluorescence can maximize the biochemical resolving power of fluorescence microscopy, an approach we term Hyper Dimensional Imaging Microscopy (HDIM). Our work provides a theoretical framework for the description of the biochemical resolution in fluorescence microscopy, irrespective of spatial resolution, and for the development of a new class of microscopes that exploit multi-parametric detection systems. PMID:24204821

New design of a cryostat-mounted scanning near-field optical microscope for single molecule spectroscopy

NASA Astrophysics Data System (ADS)

Durand, Yannig; Woehl, Jörg C.; Viellerobe, Bertrand; Göhde, Wolfgang; Orrit, Michel

1999-02-01

Due to the weakness of the fluorescence signal from a single fluorophore, a scanning near-field optical microscope for single molecule spectroscopy requires a very efficient setup for the collection and detection of emitted photons. We have developed a home-built microscope for operation in a l-He cryostat which uses a solid parabolic mirror in order to optimize the fluorescence collection efficiency. This microscope works with Al-coated, tapered optical fibers in illumination mode. The tip-sample separation is probed by an optical shear-force detection. First results demonstrate the capability of the microscope to image single molecules and achieve a topographical resolution of a few nanometers vertically and better than 50 nm laterally.

Multidimensional custom-made non-linear microscope: from ex-vivo to in-vivo imaging

NASA Astrophysics Data System (ADS)

Cicchi, R.; Sacconi, L.; Jasaitis, A.; O'Connor, R. P.; Massi, D.; Sestini, S.; de Giorgi, V.; Lotti, T.; Pavone, F. S.

2008-09-01

We have built a custom-made multidimensional non-linear microscope equipped with a combination of several non-linear laser imaging techniques involving fluorescence lifetime, multispectral two-photon and second-harmonic generation imaging. The optical system was mounted on a vertical honeycomb breadboard in an upright configuration, using two galvo-mirrors relayed by two spherical mirrors as scanners. A double detection system working in non-descanning mode has allowed both photon counting and a proportional regime. This experimental setup offering high spatial (micrometric) and temporal (sub-nanosecond) resolution has been used to image both ex-vivo and in-vivo biological samples, including cells, tissues, and living animals. Multidimensional imaging was used to spectroscopically characterize human skin lesions, as malignant melanoma and naevi. Moreover, two-color detection of two photon excited fluorescence was applied to in-vivo imaging of living mice intact neocortex, as well as to induce neuronal microlesions by femtosecond laser burning. The presented applications demonstrate the capability of the instrument to be used in a wide range of biological and biomedical studies.

A Cost-Effective Fluorescence Mini-Microscope with Adjustable Magnifications for Biomedical Applications

PubMed Central

Zhang, Yu Shrike; Ribas, João; Nadhman, Akhtar; Aleman, Julio; Selimović, Šeila; Lesher-Perez, Sasha Cai; Wang, Ting; Manoharan, Vijayan; Shin, Su-Ryon; Damilano, Alessia; Annabi, Nasim; Dokmeci, Mehmet Remzi; Takayama, Shuichi; Khademhosseini, Ali

2015-01-01

We have designed and fabricated a miniature microscope from off-the-shelf components and webcam, with built-in fluorescence capability for biomedical applications. The mini-microscope was able to detect both biochemical parameters such as cell/tissue viability (e.g. Live/Dead assay), and biophysical properties of the microenvironment such as oxygen levels in microfabricated tissues based on an oxygen-sensitive fluorescent dye. This mini-microscope has adjustable magnifications from 8-60X, achieves a resolution as high as <2 μm, and possesses a long working distance of 4.5 mm (at a magnification of 8X). The mini-microscope was able to chronologically monitor cell migration and analyze beating of microfluidic liver and cardiac bioreactors in real time, respectively. The mini-microscope system is cheap, and its modularity allows convenient integration with a wide variety of pre-existing platforms including but not limited to, cell culture plates, microfluidic devices, and organs-on-a-chip systems. Therefore, we envision its widespread applications in cell biology, tissue engineering, biosensing, microfluidics, and organs-on-chips, which can potentially replace conventional bench-top microscopy where long-term in situ and large-scale imaging/analysis is required. PMID:26282117

A cost-effective fluorescence mini-microscope for biomedical applications.

PubMed

Zhang, Yu Shrike; Ribas, João; Nadhman, Akhtar; Aleman, Julio; Selimović, Šeila; Lesher-Perez, Sasha Cai; Wang, Ting; Manoharan, Vijayan; Shin, Su-Ryon; Damilano, Alessia; Annabi, Nasim; Dokmeci, Mehmet Remzi; Takayama, Shuichi; Khademhosseini, Ali

2015-01-01

We have designed and fabricated a miniature microscope from off-the-shelf components and a webcam, with built-in fluorescence capability for biomedical applications. The mini-microscope was able to detect both biochemical parameters, such as cell/tissue viability (e.g. live/dead assay), and biophysical properties of the microenvironment such as oxygen levels in microfabricated tissues based on an oxygen-sensitive fluorescent dye. This mini-microscope has adjustable magnifications from 8-60×, achieves a resolution as high as <2 μm, and possesses a long working distance of 4.5 mm (at a magnification of 8×). The mini-microscope was able to chronologically monitor cell migration and analyze beating of microfluidic liver and cardiac bioreactors in real time, respectively. The mini-microscope system is cheap, and its modularity allows convenient integration with a wide variety of pre-existing platforms including, but not limited to, cell culture plates, microfluidic devices, and organs-on-a-chip systems. Therefore, we envision its widespread application in cell biology, tissue engineering, biosensing, microfluidics, and organs-on-chips, which can potentially replace conventional bench-top microscopy where long-term in situ and large-scale imaging/analysis is required.

Maximizing fluorescence collection efficiency in multiphoton microscopy

PubMed Central

Zinter, Joseph P.; Levene, Michael J.

2011-01-01

Understanding fluorescence propagation through a multiphoton microscope is of critical importance in designing high performance systems capable of deep tissue imaging. Optical models of a scattering tissue sample and the Olympus 20X 0.95NA microscope objective were used to simulate fluorescence propagation as a function of imaging depth for physiologically relevant scattering parameters. The spatio-angular distribution of fluorescence at the objective back aperture derived from these simulations was used to design a simple, maximally efficient post-objective fluorescence collection system. Monte Carlo simulations corroborated by data from experimental tissue phantoms demonstrate collection efficiency improvements of 50% – 90% over conventional, non-optimized fluorescence collection geometries at large imaging depths. Imaging performance was verified by imaging layer V neurons in mouse cortex to a depth of 850 μm. PMID:21934897

Selective plane illumination microscopy (SPIM) with time-domain fluorescence lifetime imaging microscopy (FLIM) for volumetric measurement of cleared mouse brain samples

NASA Astrophysics Data System (ADS)

Funane, Tsukasa; Hou, Steven S.; Zoltowska, Katarzyna Marta; van Veluw, Susanne J.; Berezovska, Oksana; Kumar, Anand T. N.; Bacskai, Brian J.

2018-05-01

We have developed an imaging technique which combines selective plane illumination microscopy with time-domain fluorescence lifetime imaging microscopy (SPIM-FLIM) for three-dimensional volumetric imaging of cleared mouse brains with micro- to mesoscopic resolution. The main features of the microscope include a wavelength-adjustable pulsed laser source (Ti:sapphire) (near-infrared) laser, a BiBO frequency-doubling photonic crystal, a liquid chamber, an electrically focus-tunable lens, a cuvette based sample holder, and an air (dry) objective lens. The performance of the system was evaluated with a lifetime reference dye and micro-bead phantom measurements. Intensity and lifetime maps of three-dimensional human embryonic kidney (HEK) cell culture samples and cleared mouse brain samples expressing green fluorescent protein (GFP) (donor only) and green and red fluorescent protein [positive Förster (fluorescence) resonance energy transfer] were acquired. The results show that the SPIM-FLIM system can be used for sample sizes ranging from single cells to whole mouse organs and can serve as a powerful tool for medical and biological research.

Using Carbon Nanotubes for Nanometer-Scale Energy Transfer Microscopy

NASA Astrophysics Data System (ADS)

Johnston, Jessica; Shafran, Eyal; Mangum, Ben; Mu, Chun; Gerton, Jordan

2009-10-01

We investigate optical energy transfer between fluorophores and carbon nanotubes (CNTs). CNTs are grown on Si-oxide wafers by chemical vapor deposition (CVD), lifted off substrates by atomic force microscope (AFM) tips via Van der Waals forces, then shortened by electrical pulses. The tip-attached CNTs are scanned over fluorescent CdSe-ZnS quantum dots (QDs) with sub-nm precision while recording the fluorescence rate. A novel photon counting technique enables us to produce 3D maps of the QD-CNT coupling, revealing nanoscale lateral and vertical features. All CNTs tested (>50) strongly quenched the QD fluorescence, apparently independent of chirality. In some data, a delay in the recovery of QD fluorescence following CNT-QD contact was observed, suggesting possible charge transfer in this system. In the future, we will perform time-resolved studies to quantify the rate of energy and charge transfer processes and study the possible differences in fluorescence quenching and nanotube-QD energy transfer when comparing single-walled (SW) versus multi-walled (MW) CNTs, attempting to grow substrates consisting primarily of SW or MWCNTs and characterizing the structure of tip-attached CNTs using optical spectroscopy.

Pulse-Shaping-Based Nonlinear Microscopy: Development and Applications

NASA Astrophysics Data System (ADS)

Flynn, Daniel Christopher

The combination of optical microscopy and ultrafast spectroscopy make the spatial characterization of chemical kinetics on the femtosecond time scale possible. Commercially available octave-spanning Ti:Sapphire oscillators with sub-8 fs pulse durations can drive a multitude of nonlinear transitions across a significant portion of the visible spectrum with minimal average power. Unfortunately, dispersion from microscope objectives broadens pulse durations, decreases temporal resolution and lowers the peak intensities required for driving nonlinear transitions. In this dissertation, pulse shaping is used to compress laser pulses after the microscope objective. By using a binary genetic algorithm, pulse-shapes are designed to enable selective two-photon excitation. The pulse-shapes are demonstrated in two-photon fluorescence of live COS-7 cells expressing GFP-variants mAmetrine and tdTomato. The pulse-shaping approach is applied to a new multiphoton fluorescence resonance energy transfer (FRET) stoichiometry method that quantifies donor and acceptor molecules in complex, as well as the ratio of total donor to acceptor molecules. Compared to conventional multi-photon imaging techniques that require laser tuning or multiple laser systems to selectively excite individual fluorophores, the pulse-shaping approach offers rapid selective multifluorphore imaging at biologically relevant time scales. By splitting the laser beam into two beams and building a second pulse shaper, a pulse-shaping-based pump-probe microscope is developed. The technique offers multiple imaging modalities, such as excited state absorption (ESA), ground state bleach (GSB), and stimulated emission (SE), enhancing contrast of structures via their unique quantum pathways without the addition of contrast agents. Pulse-shaping based pump-probe microscopy is demonstrated for endogenous chemical-contrast imaging of red blood cells. In the second section of this dissertation, ultrafast spectroscopic techniques are used to characterize structure-function relationships of two-photon absorbing GFP-type probes and optical limiting materials. Fluorescence lifetimes of GFP-type probes are shown to depend on functional group substitution position, therefore, enabling the synthesis of designer probes for the possible study of conformation changes and aggregation in biological systems. Similarly, it is determined that small differences in the structure and dimensionality of organometallic macrocycles result in a diverse set of optical properties, which serves as a basis for the molecular level design of nonlinear optical materials.

Behavior of platelets stained by 5,6-CF-encapsulated PEGylated liposomes after laser irradiation of vessel wall: an in-vivo model for studying site-selective delivery of diagnostic or therapeutic agents

NASA Astrophysics Data System (ADS)

Mordon, Serge R.; Begu, Sylvie; Buys, Bruno; Tourne-Peteilh, Corine; Devoisselle, Jean-Marie

2001-05-01

Vascular endothelium serves as an extensive interface between circulating blood and various tissues and organs of the body. As such, it offers an accessible target for blood-borne pharmacological and genetic manipulations that can mediate both local and systemic effects. Thus, targeting of liposomes to activated vascular endothelial cells may provide a strategy for site-selective delivery in the vascular system with broad therapeutic applicability. This study aimed to evaluate an intravital fluorescence imaging technique to visualize in-situ and in real-time the activation of platelets after staining by 5,6-CF- encapsulated PEGylated liposomes injected intravenously. The study was performed on skin by using a dorsal skin-fold chamber implanted in golden hamsters using intravital microscopy. The skin micro circulation was observed with an intravital microscope (using x25 and x40 magnification) fitted with a Xenon light source and an epi-fluorescence assembly. An ultra-high sensitivity video-camera mounted on the microscope projected the image onto a monitor, and the images were recorded for play-back analysis with a digital video cassette recorder. An inflammatory response was induced by an Argon laser emitting at 514.5nm. The 80micrometers laser beam was focused on a vessel and its position was controlled with the microscope imaging system, it was possible to see individual platelets flowing in blood vessels. As liposomes were labeled with a fluorescent probe which was hydrophilic (located in the aqueous phase), the fluorescence of platelets was due only to the uptake of liposomes. After laser irradiation, platelets activation at sites of vascular injury was obtained. Tethering, translocation of some platelets inside the irradiated zone were clearly seen. At last, detachment and extravasation of platelets were observed. A perivascular fluorescence confirmed that platelets migrated across the basal lamina into the dermal connective tissue. In conclusion, staining of platelets using 5,6-CF-encapsulated PEGylated liposomes injected intravenously presents the following advantages: i) in-situ labeling, ii) use of hydrophilic marker located in an aqueous compartment within the platelet, iii) as the release of the fluorescence marker is slow due to the formulation of liposomes, labeling of platelets could be observed during the whole experiment. Laser irradiation of blood vessels in vivo can induce the different phases of platelet activation: i) recruitment, ii) adhesion, iii) detachment, iv) transmigration. The combination of these techniques (platelet staining with PEGylated liposomes, intravital fluorescence microscopy, laser irradiation) provides a powerful tool to study local inflammation, platelet activation and behavior of liposomes in situ and in real time at an inflammation site. These observations could be considered as a preliminary approach to study the targeting of drugs to an endothelium under inflammation environment.

Use of fluorescence and scanning electron microscopy as tools in teaching biology

NASA Astrophysics Data System (ADS)

Ghosh, Nabarun; Silva, Jessica; Vazquez, Aracely; Das, A. B.; Smith, Don W.

2011-06-01

Recent nationwide surveys reveal significant decline in students' interest in Math and Sciences. The objective of this project was to inspire young minds in using various techniques involved in Sciences including Scanning Electron Microscopy. We used Scanning Electron Microscope in demonstrating various types of Biological samples. An SEM Tabletop model in the past decade has revolutionized the use of Scanning Electron Microscopes. Using SEM Tabletop model TM 1000 we studied biological specimens of fungal spores, pollen grains, diatoms, plant fibers, dust mites, insect parts and leaf surfaces. We also used fluorescence microscopy to view, to record and analyze various specimens with an Olympus BX40 microscope equipped with FITC and TRITC fluorescent filters, a mercury lamp source, DP-70 digital camera with Image Pro 6.0 software. Micrographs were captured using bright field microscopy, the fluoresceinisothiocyanate (FITC) filter, and the tetramethylrhodamine (TRITC) filter settings at 40X. A high pressure mercury lamp or UV source was used to excite the storage molecules or proteins which exhibited autofluorescence. We used fluorescent microscopy to confirm the localization of sugar beet viruses in plant organs by viewing the vascular bundles in the thin sections of the leaves and other tissues. We worked with the REU summer students on sample preparation and observation on various samples utilizing the SEM. Critical Point Drying (CPD) and metal coating with the sputter coater was followed before observing some cultured specimen and the samples that were soft in textures with high water content. SEM Top allowed investigating the detailed morphological features that can be used for classroom teaching. Undergraduate and graduate researchers studied biological samples of Arthropods, pollen grains and teeth collected from four species of snakes using SEM. This project inspired the research students to pursue their career in higher studies in science and 45% of the undergraduates participated in this project entered Graduate school.

7 CFR 353.9 - Standards for accreditation of non-government facilities to perform laboratory seed health...

Code of Federal Regulations, 2013 CFR

2013-01-01

... seed requires a stereo microscope. Visual examination of tissue requires a compound light microscope... equipment; fluorescent microscopes; plate readers; spectrophotometers; and the appropriate assay materials...

7 CFR 353.9 - Standards for accreditation of non-government facilities to perform laboratory seed health...

Code of Federal Regulations, 2011 CFR

2011-01-01

... seed requires a stereo microscope. Visual examination of tissue requires a compound light microscope... equipment; fluorescent microscopes; plate readers; spectrophotometers; and the appropriate assay materials...

7 CFR 353.9 - Standards for accreditation of non-government facilities to perform laboratory seed health...

Code of Federal Regulations, 2010 CFR

2010-01-01

... seed requires a stereo microscope. Visual examination of tissue requires a compound light microscope... equipment; fluorescent microscopes; plate readers; spectrophotometers; and the appropriate assay materials...

7 CFR 353.9 - Standards for accreditation of non-government facilities to perform laboratory seed health...

Code of Federal Regulations, 2014 CFR

2014-01-01

... seed requires a stereo microscope. Visual examination of tissue requires a compound light microscope... equipment; fluorescent microscopes; plate readers; spectrophotometers; and the appropriate assay materials...

eduSPIM: Light Sheet Microscopy in the Museum

PubMed Central

Schmid, Benjamin; Weber, Michael; Huisken, Jan

2016-01-01

Light Sheet Microscopy in the Museum Light sheet microscopy (or selective plane illumination microscopy) is an important imaging technique in the life sciences. At the same time, this technique is also ideally suited for community outreach projects, because it produces visually appealing, highly dynamic images of living organisms and its working principle can be understood with basic optics knowledge. Still, the underlying concepts are widely unknown to the non-scientific public. On the occasion of the UNESCO International Year of Light, a technical museum in Dresden, Germany, launched a special, interactive exhibition. We built a fully functional, educational selective plane illumination microscope (eduSPIM) to demonstrate how developments in microscopy promote discoveries in biology. Design Principles of an Educational Light Sheet Microscope To maximize educational impact, we radically reduced a standard light sheet microscope to its essential components without compromising functionality and incorporated stringent safety concepts beyond those needed in the lab. Our eduSPIM system features one illumination and one detection path and a sealed sample chamber. We image fixed zebrafish embryos with fluorescent vasculature, because the structure is meaningful to laymen and visualises the optical principles of light sheet microscopy. Via a simplified interface, visitors acquire fluorescence and transmission data simultaneously. The eduSPIM Design Is Tailored Easily to Fit Numerous Applications The universal concepts presented here may also apply to other scientific approaches that are communicated to laymen in interactive settings. The specific eduSPIM design is adapted easily for various outreach and teaching activities. eduSPIM may even prove useful for labs needing a simple SPIM. A detailed parts list and schematics to rebuild eduSPIM are provided. PMID:27560188

3D on-chip microscopy of optically cleared tissue

NASA Astrophysics Data System (ADS)

Zhang, Yibo; Shin, Yoonjung; Sung, Kevin; Yang, Sam; Chen, Harrison; Wang, Hongda; Teng, Da; Rivenson, Yair; Kulkarni, Rajan P.; Ozcan, Aydogan

2018-02-01

Traditional pathology relies on tissue biopsy, micro-sectioning, immunohistochemistry and microscopic imaging, which are relatively expensive and labor-intensive, and therefore are less accessible in resource-limited areas. Low-cost tissue clearing techniques, such as the simplified CLARITY method (SCM), are promising to potentially reduce the cost of disease diagnosis by providing 3D imaging and phenotyping of thicker tissue samples with simpler preparation steps. However, the mainstream imaging approach for cleared tissue, fluorescence microscopy, suffers from high-cost, photobleaching and signal fading. As an alternative approach to fluorescence, here we demonstrate 3D imaging of SCMcleared tissue using on-chip holography, which is based on pixel-super-resolution and multi-height phase recovery algorithms to digitally compute the sample's amplitude and phase images at various z-slices/depths through the sample. The tissue clearing procedures and the lens-free imaging system were jointly optimized to find the best illumination wavelength, tissue thickness, staining solution pH, and the number of hologram heights to maximize the imaged tissue volume, minimize the amount of acquired data, while maintaining a high contrast-to-noise ratio for the imaged cells. After this optimization, we achieved 3D imaging of a 200-μm thick cleared mouse brain tissue over a field-of-view of <20mm2 , and the resulting 3D z-stack agrees well with the images acquired with a scanning lens-based microscope (20× 0.75NA). Moreover, the lens-free microscope achieves an order-of-magnitude better data efficiency compared to its lens-based counterparts for volumetric imaging of samples. The presented low-cost and high-throughput lens-free tissue imaging technique enabled by CLARITY can be used in various biomedical applications in low-resource-settings.

Membrane filtration – Fluorescent antibody staining procedure for detecting and quantifying Renibacterium salmoninarum in coelomic fluid of Chinook Salmon (Oncorhynchus tshawytscha)

USGS Publications Warehouse

Elliott, D.G.; Barila, T.Y.

1987-01-01

We developed a rapid method for detecting and quantifying the pathogen Renibacterium salmoninarum in coelomic fluid of spring chinook salmon (Oncorhynchus tshawytscha) by concentrating the bacteria on 0.2-μm polycarbonate filters and staining them with specific fluorescein-labeled antibody. Centrifugation of samples and resuspension of the sedimented material in phosphate-buffered saline containing Triton X-100 increased the ease of filtration. Background fluorescence was reduced by counterstaining filters with Eriochrome black T. Postfiltration staining, rinsing, and counterstaining were done in the syringe-mounted filter holders, reducing handling of the filters and possible loss of bacteria. The number of bacteria detected by the filtration – fluorescent antibody technique in a broth culture of R. salmoninarum ranged from 6.7 × 107to7.6 × 107/mL and was slightly higher than that determined by plate count (9.6 × 106/mL). Increasing the sample dilution or decreasing the number of microscope fields examined generally increased the variability of filter counts of R. salmoninarum. Using the filtration – fluorescent antibody technique, we detected the bacterium in the coelomic fluid of 85% of spawning female spring chinook salmon sampled from a hatchery population.

Immunomagnetic cell separation, imaging, and analysis using Captivate ferrofluids

NASA Astrophysics Data System (ADS)

Jones, Laurie; Beechem, Joseph M.

2002-05-01

We have developed applications of CaptivateTM ferrofluids, paramagnetic particles (approximately 200 nm diameter), for isolating and analyzing cell populations in combination with fluorescence-based techniques. Using a microscope-mounted magnetic yoke and sample insertion chamber, fluorescent images of magnetically captured cells were obtained in culture media, buffer, or whole blood, while non-magnetically labeled cells sedimented to the bottom of the chamber. We combined this immunomagnetic cell separation and imaging technique with fluorescent staining, spectroscopy, and analysis to evaluate cell surface receptor-containing subpopulations, live/dead cell ratios, apoptotic/dead cell ratios, etc. The acquired images were analyzed using multi-color parameters, as produced by nucleic acid staining, esterase activity, or antibody labeling. In addition, the immunomagnetically separated cell fractions were assessed through microplate analysis using the CyQUANT Cell Proliferation Assay. These methods should provide an inexpensive alternative to some flow cytometric measurements. The binding capacities of the streptavidin- labled Captivate ferrofluid (SA-FF) particles were determined to be 8.8 nmol biotin/mg SA-FF, using biotin-4- fluorescein, and > 106 cells/mg SA-FF, using several cell types labeled with biotinylated probes. For goat anti- mouse IgG-labeled ferrofluids (GAM-FF), binding capacities were established to be approximately 0.2 - 7.5 nmol protein/mg GAM-FF using fluorescent conjugates of antibodies, protein G, and protein A.

Light and life in Baltimore--and beyond.

PubMed

Edidin, Michael

2015-02-03

Baltimore has been the home of numerous biophysical studies using light to probe cells. One such study, quantitative measurement of lateral diffusion of rhodopsin, set the standard for experiments in which recovery after photobleaching is used to measure lateral diffusion. Development of this method from specialized microscopes to commercial scanning confocal microscopes has led to widespread use of the technique to measure lateral diffusion of membrane proteins and lipids, and as well diffusion and binding interactions in cell organelles and cytoplasm. Perturbation of equilibrium distributions by photobleaching has also been developed into a robust method to image molecular proximity in terms of fluorescence resonance energy transfer between donor and acceptor fluorophores. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

On-Chip Biomedical Imaging

PubMed Central

Göröcs, Zoltán; Ozcan, Aydogan

2012-01-01

Lab-on-a-chip systems have been rapidly emerging to pave the way toward ultra-compact, efficient, mass producible and cost-effective biomedical research and diagnostic tools. Although such microfluidic and micro electromechanical systems achieved high levels of integration, and are capable of performing various important tasks on the same chip, such as cell culturing, sorting and staining, they still rely on conventional microscopes for their imaging needs. Recently several alternative on-chip optical imaging techniques have been introduced, which have the potential to substitute conventional microscopes for various lab-on-a-chip applications. Here we present a critical review of these recently emerging on-chip biomedical imaging modalities, including contact shadow imaging, lensfree holographic microscopy, fluorescent on-chip microscopy and lensfree optical tomography. PMID:23558399

Ultrawidefield microscope for high-speed fluorescence imaging and targeted optogenetic stimulation.

PubMed

Werley, Christopher A; Chien, Miao-Ping; Cohen, Adam E

2017-12-01

The rapid increase in the number and quality of fluorescent reporters and optogenetic actuators has yielded a powerful set of tools for recording and controlling cellular state and function. To achieve the full benefit of these tools requires improved optical systems with high light collection efficiency, high spatial and temporal resolution, and patterned optical stimulation, in a wide field of view (FOV). Here we describe our 'Firefly' microscope, which achieves these goals in a Ø6 mm FOV. The Firefly optical system is optimized for simultaneous photostimulation and fluorescence imaging in cultured cells. All but one of the optical elements are commercially available, yet the microscope achieves 10-fold higher light collection efficiency at its design magnification than the comparable commercially available microscope using the same objective. The Firefly microscope enables all-optical electrophysiology ('Optopatch') in cultured neurons with a throughput and information content unmatched by other neuronal phenotyping systems. This capability opens possibilities in disease modeling and phenotypic drug screening. We also demonstrate applications of the system to voltage and calcium recordings in human induced pluripotent stem cell derived cardiomyocytes.

Ultrawidefield microscope for high-speed fluorescence imaging and targeted optogenetic stimulation

PubMed Central

Werley, Christopher A.; Chien, Miao-Ping; Cohen, Adam E.

2017-01-01

The rapid increase in the number and quality of fluorescent reporters and optogenetic actuators has yielded a powerful set of tools for recording and controlling cellular state and function. To achieve the full benefit of these tools requires improved optical systems with high light collection efficiency, high spatial and temporal resolution, and patterned optical stimulation, in a wide field of view (FOV). Here we describe our ‘Firefly’ microscope, which achieves these goals in a Ø6 mm FOV. The Firefly optical system is optimized for simultaneous photostimulation and fluorescence imaging in cultured cells. All but one of the optical elements are commercially available, yet the microscope achieves 10-fold higher light collection efficiency at its design magnification than the comparable commercially available microscope using the same objective. The Firefly microscope enables all-optical electrophysiology (‘Optopatch’) in cultured neurons with a throughput and information content unmatched by other neuronal phenotyping systems. This capability opens possibilities in disease modeling and phenotypic drug screening. We also demonstrate applications of the system to voltage and calcium recordings in human induced pluripotent stem cell derived cardiomyocytes. PMID:29296505

The Mechanosensitive Ca2+ Channel as a Central Regulator of Prostate Tumor Cell Migration and Invasiveness

DTIC Science & Technology

2010-01-01

Use time-lapse videomicroscopy and patch-clamp techniques to characterize the motility of eGFP-transfected PC-3 cells in which MScCa/TRPC1 has been...except for GsmTx-4 (Peptides International, Louisville, KY) and fluorescent agents (Invitrogen/Molecular Probes, Carlsbad, CA). Videomicroscopy ...and Ca2+-imaging. Cell migration was monitored at 37oC by time-lapse videomicroscopy using Nomarski optics with an Epifluorescent microscope (Nikon

SPIM-fluid: open source light-sheet based platform for high-throughput imaging

PubMed Central

Gualda, Emilio J.; Pereira, Hugo; Vale, Tiago; Estrada, Marta Falcão; Brito, Catarina; Moreno, Nuno

2015-01-01

Light sheet fluorescence microscopy has recently emerged as the technique of choice for obtaining high quality 3D images of whole organisms/embryos with low photodamage and fast acquisition rates. Here we present an open source unified implementation based on Arduino and Micromanager, which is capable of operating Light Sheet Microscopes for automatized 3D high-throughput imaging on three-dimensional cell cultures and model organisms like zebrafish, oriented to massive drug screening. PMID:26601007

A simple and fast method for fixation of cultured cell lines that preserves cellular structures containing gamma-tubulin.

PubMed

Alvarado-Kristensson, Maria

2018-01-01

When using fluorescence microscope techniques to study cells, it is essential that the cell structure and contents are preserved after preparation of the samples, and that the preparation method employed does not create artefacts that can be perceived as cellular structure/components. γ-Tubulin forms filaments that in some cases are immunostained with an anti-γ-tubulin antibody, but this immunostaining is not reproducible [[1], [2

Supramolecular interactions of nonsteroidal anti-inflammatory drug in nanochannels of molecular containers: a spectroscopic, thermogravimetric and microscopic investigation.

PubMed

Maity, Banibrata; Chatterjee, Aninda; Ahmed, Sayeed Ashique; Seth, Debabrata

2014-11-10

Supramolecular host-guest complexation between the nonsteroidal anti-inflammatory drug indomethacin (IMC) and molecular containers were investigated. The weakly fluorescent drug molecule becomes highly fluorescent on complexation with different molecular containers, and time-resolved fluorescence emission spectroscopy reveals that the lifetime components of IMC significantly increase in the presence of molecular containers, compared with the lifetimes in neat water. The respective solid host-guest complexes were synthesised and characterised by Fourier transform infrared and (1) H nuclear magnetic resonance spectroscopic analysis. Microscopy techniques were used to analyse modifications of the surface morphology, owing to the formation of supramolecular complexes. The effect of the molecular container on the optical properties of IMC has also been investigated to determine the effect of nanochannels of different size and structure. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Wide-field depth-sectioning fluorescence microscopy using projector-generated patterned illumination

NASA Astrophysics Data System (ADS)

Delica, Serafin; Mar Blanca, Carlo

2007-10-01

We present a simple and cost-effective wide-field, depth-sectioning, fluorescence microscope utilizing a commercial multimedia projector to generate excitation patterns on the sample. Highly resolved optical sections of fluorescent pollen grains at 1.9 μm axial resolution are constructed using the structured illumination technique. This requires grid excitation patterns to be scanned across the sample, which is straightforwardly implemented by creating slideshows of gratings at different phases, projecting them onto the sample, and synchronizing camera acquisition with slide transition. In addition to rapid dynamic pattern generation, the projector provides high illumination power and spectral excitation selectivity. We exploit these properties by imaging mouse neural cells in cultures multistained with Alexa 488 and Cy3. The spectral and structural neural information is effectively resolved in three dimensions. The flexibility and commercial availability of this light source is envisioned to open multidimensional imaging to a broader user base.

Whole mount nuclear fluorescent imaging: convenient documentation of embryo morphology

PubMed Central

Sandell, Lisa L.; Kurosaka, Hiroshi; Trainor, Paul A.

2012-01-01

Here we describe a relatively inexpensive and easy method to produce high quality images that reveal fine topological details of vertebrate embryonic structures. The method relies on nuclear staining of whole mount embryos in combination with confocal microscopy or conventional widefield fluorescent microscopy. In cases where confocal microscopy is used in combination with whole mount nuclear staining, the resulting embryo images can rival the clarity and resolution of images of similar specimens produced by Scanning Electron Microscopy (SEM). The fluorescent nuclear staining may be performed with a variety of cell permeable nuclear dyes, enabling the technique to be performed with multiple standard microscope/illumination or confocal/laser systems. The method may be used to document morphology of embryos of a variety of organisms, as well as individual organs and tissues. Nuclear stain imaging imposes minimal impact on embryonic specimens, enabling imaged specimens to be utilized for additional assays. PMID:22930523

Whole mount nuclear fluorescent imaging: convenient documentation of embryo morphology.

PubMed

Sandell, Lisa L; Kurosaka, Hiroshi; Trainor, Paul A

2012-11-01

Here, we describe a relatively inexpensive and easy method to produce high quality images that reveal fine topological details of vertebrate embryonic structures. The method relies on nuclear staining of whole mount embryos in combination with confocal microscopy or conventional wide field fluorescent microscopy. In cases where confocal microscopy is used in combination with whole mount nuclear staining, the resulting embryo images can rival the clarity and resolution of images produced by scanning electron microscopy (SEM). The fluorescent nuclear staining may be performed with a variety of cell permeable nuclear dyes, enabling the technique to be performed with multiple standard microscope/illumination or confocal/laser systems. The method may be used to document morphology of embryos of a variety of organisms, as well as individual organs and tissues. Nuclear stain imaging imposes minimal impact on embryonic specimens, enabling imaged specimens to be utilized for additional assays. Copyright © 2012 Wiley Periodicals, Inc.

Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

PubMed Central

Gulati, Srishti; Cao, Vania Y.; Otte, Stephani

2017-01-01

In vivo circuit and cellular level functional imaging is a critical tool for understanding the brain in action. High resolution imaging of mouse cortical neurons with two-photon microscopy has provided unique insights into cortical structure, function and plasticity. However, these studies are limited to head fixed animals, greatly reducing the behavioral complexity available for study. In this paper, we describe a procedure for performing chronic fluorescence microscopy with cellular-resolution across multiple cortical layers in freely behaving mice. We used an integrated miniaturized fluorescence microscope paired with an implanted prism probe to simultaneously visualize and record the calcium dynamics of hundreds of neurons across multiple layers of the somatosensory cortex as the mouse engaged in a novel object exploration task, over several days. This technique can be adapted to other brain regions in different animal species for other behavioral paradigms. PMID:28654056

Sensorless adaptive optics for isoSTED nanoscopy

NASA Astrophysics Data System (ADS)

Antonello, Jacopo; Hao, Xiang; Allgeyer, Edward S.; Bewersdorf, Joerg; Rittscher, Jens; Booth, Martin J.

2018-02-01

The presence of aberrations is a major concern when using fluorescence microscopy to image deep inside tissue. Aberrations due to refractive index mismatch and heterogeneity of the specimen under investigation cause severe reduction in the amount of fluorescence emission that is collected by the microscope. Furthermore, aberrations adversely affect the resolution, leading to loss of fine detail in the acquired images. These phenomena are particularly troublesome for super-resolution microscopy techniques such as isotropic stimulated-emission-depletion microscopy (isoSTED), which relies on accurate control of the shape and co-alignment of multiple excitation and depletion foci to operate as expected and to achieve the super-resolution effect. Aberrations can be suppressed by implementing sensorless adaptive optics techniques, whereby aberration correction is achieved by maximising a certain image quality metric. In confocal microscopy for example, one can employ the total image brightness as an image quality metric. Aberration correction is subsequently achieved by iteratively changing the settings of a wavefront corrector device until the metric is maximised. This simplistic approach has limited applicability to isoSTED microscopy where, due to the complex interplay between the excitation and depletion foci, maximising the total image brightness can lead to introducing aberrations in the depletion foci. In this work we first consider the effects that different aberration modes have on isoSTED microscopes. We then propose an iterative, wavelet-based aberration correction algorithm and evaluate its benefits.

Characterizing Fibrosis in Mouse Kidney using Label Free Fluorescence Lifetime and Second Harmonic Generation Imaging Microscopy in Unilateral Ureteral Obstruction Model

PubMed Central

Ranjit, Suman; Dobrinskikh, Evgenia; Montford, John; Dvornikov, Alexander; Lehman, Allison; Orlicky, David J.; Nemenoff, Raphael; Gratton, Enrico; Levi, Moshe; Furgeson, Seth

2017-01-01

All forms of progressive renal diseases develop a final pathway of tubulointerstitial fibrosis and glomerulosclerosis. Renal fibrosis is usually quantified using histological staining, a process that is time-consuming and pathologist dependent. The work described here shows the development of a fast and operator-independent method to measure fibrosis. To study renal fibrosis, the unilateral ureteral obstruction (UUO) model was chosen. Mice develop a time-dependent increase in obstructed kidneys; contralateral kidneys are used as controls. After UUO, kidneys were analyzed at three time points: 7 days, 14 days, and 21 days. Fibrosis was investigated using FLIM (Fluorescence Lifetime Imaging) and SHG (Second Harmonic Generation) in the deep tissue imaging microscope called DIVER (Deep Imaging via Enhanced photon Recovery). This microscope was developed for deep tissue and SHG and THG (Third Harmonic Generation) imaging and has extraordinary sensitivity towards harmonic generation. SHG data suggests the presence of more fibrillar collagen in the diseased kidneys. The combinations of short wavelength FLIM and SHG analysis results in a robust analysis procedure independent of observer interpretation and let us create a criterion to quantify the extent of fibrosis directly from the image. The progression of fibrosis in UUO model has been studied using this new FLIM-SHG technique and it shows remarkable improvement in quantification of fibrosis compared to standard histological techniques. PMID:27555119

Two-photon imaging and analysis of neural network dynamics

NASA Astrophysics Data System (ADS)

Lütcke, Henry; Helmchen, Fritjof

2011-08-01

The glow of a starry night sky, the smell of a freshly brewed cup of coffee or the sound of ocean waves breaking on the beach are representations of the physical world that have been created by the dynamic interactions of thousands of neurons in our brains. How the brain mediates perceptions, creates thoughts, stores memories and initiates actions remains one of the most profound puzzles in biology, if not all of science. A key to a mechanistic understanding of how the nervous system works is the ability to measure and analyze the dynamics of neuronal networks in the living organism in the context of sensory stimulation and behavior. Dynamic brain properties have been fairly well characterized on the microscopic level of individual neurons and on the macroscopic level of whole brain areas largely with the help of various electrophysiological techniques. However, our understanding of the mesoscopic level comprising local populations of hundreds to thousands of neurons (so-called 'microcircuits') remains comparably poor. Predominantly, this has been due to the technical difficulties involved in recording from large networks of neurons with single-cell spatial resolution and near-millisecond temporal resolution in the brain of living animals. In recent years, two-photon microscopy has emerged as a technique which meets many of these requirements and thus has become the method of choice for the interrogation of local neural circuits. Here, we review the state-of-research in the field of two-photon imaging of neuronal populations, covering the topics of microscope technology, suitable fluorescent indicator dyes, staining techniques, and in particular analysis techniques for extracting relevant information from the fluorescence data. We expect that functional analysis of neural networks using two-photon imaging will help to decipher fundamental operational principles of neural microcircuits.

Evaluation of mobile digital light-emitting diode fluorescence microscopy in Hanoi, Viet Nam.

PubMed

Chaisson, L H; Reber, C; Phan, H; Switz, N; Nilsson, L M; Myers, F; Nhung, N V; Luu, L; Pham, T; Vu, C; Nguyen, H; Nguyen, A; Dinh, T; Nahid, P; Fletcher, D A; Cattamanchi, A

2015-09-01

Hanoi Lung Hospital, Hanoi, Viet Nam. To compare the accuracy of CellScopeTB, a manually operated mobile digital fluorescence microscope, with conventional microscopy techniques. Patients referred for sputum smear microscopy to the Hanoi Lung Hospital from May to September 2013 were included. Ziehl-Neelsen (ZN) smear microscopy, conventional light-emitting diode (LED) fluorescence microscopy (FM), CellScopeTB-based LED FM and Xpert(®) MTB/RIF were performed on sputum samples. The sensitivity and specificity of microscopy techniques were determined in reference to Xpert results, and differences were compared using McNemar's paired test of proportions. Of 326 patients enrolled, 93 (28.5%) were Xpert-positive for TB. The sensitivity of ZN microscopy, conventional LED FM, and CellScopeTB-based LED FM was respectively 37.6% (95%CI 27.8-48.3), 41.9% (95%CI 31.8-52.6), and 35.5% (95%CI 25.8-46.1). The sensitivity of CellScopeTB was similar to that of conventional LED FM (difference -6.5%, 95%CI -18.2 to 5.3, P = 0.33) and ZN microscopy (difference -2.2%, 95%CI -9.2 to 4.9, P = 0.73). The specificity was >99% for all three techniques. CellScopeTB performed similarly to conventional microscopy techniques in the hands of experienced TB microscopists. However, the sensitivity of all sputum microscopy techniques was low. Options enabled by digital microscopy, such as automated imaging with real-time computerized analysis, should be explored to increase sensitivity.

Laser-induced fluorescence microscopic system using an optical parametric oscillator for tunable detection in microchip analysis.

PubMed

Kumemura, Momoko; Odake, Tamao; Korenaga, Takashi

2005-06-01

A laser-induced fluorescence microscopic system based on optical parametric oscillation has been constructed as a tunable detector for microchip analysis. The detection limit of sulforhodamine B (Ex. 520 nm, Em. 570 nm) was 0.2 mumol, which was approximately eight orders of magnitude better than with a conventional fluorophotometer. The system was applied to the determination of fluorescence-labeled DNA (Ex. 494 nm, Em. 519 nm) in a microchannel and the detection limit reached a single molecule. These results showed the feasibility of this system as a highly sensitive and tunable fluorescence detector for microchip analysis.

Improving confocal microscopy with solid-state semiconductor excitation sources

NASA Astrophysics Data System (ADS)

Sivers, Nelson L.

To efficiently excite the fluorescent dyes used in imaging biological samples with a confocal microscope, the wavelengths of the exciting laser must be near the fluorochrome absorption peak. However, this causes imaging problems when the fluorochrome absorption and emission spectra overlap significantly, i.e. have small Stokes shifts, which is the case for most fluorochromes that emit in the red to infrared. As a result, the reflected laser excitation cannot be distinguished from the information-containing fluorescence signal. However, cryogenically cooling the exciting laser diode enabled the laser emission wavelengths to be tuned to shorter wavelengths, decreasing the interference between the laser and the fluorochrome's fluorescence. This reduced the amount of reflected laser light in the confocal image. However, the cooled laser diode's shorter wavelength signal resulted in slightly less efficient fluorochrome excitation. Spectrophotometric analysis showed that as the laser diodes were cooled, their output power increased, which more than compensated for the lower fluorochrome excitation and resulted in significantly more intense fluorescence. Thus, by tuning the laser diode emission wavelengths away from the fluorescence signal, less reflected laser light and more fluorescence information reached the detector, creating images with better signal to noise ratios. Additionally, new, high, luminous flux, light-emitting diodes (LEDs) are now powerful enough to create confocal fluorescence signals comparable to those produced by the traditional laser excitation sources in fluorescence confocal microscopes. The broader LED spectral response effectively excited the fluorochrome, yet was spectrally limited enough for standard filter sets to separate the LED excitation from the fluorochrome fluorescence signal. Spectrophotometric analysis of the excitation and fluorescence spectra of several fluorochromes showed that high-powered, LED-induced fluorescence contained the same spectral information and could be more intense than that produced by lasers. An alternative, LED-based, confocal microscope is proposed in this thesis that would be capable of exciting multiple fluorochromes in a single specimen, producing images of several distinct cellular components simultaneously. The inexpensive, LED-based, confocal microscope would require lower peak excitation intensities to produce fluorescence signals equal to those produced by laser excitation, reducing cellular damage and slowing fluorochrome photobleaching.

pH and chloride recordings in living cells using two-photon fluorescence lifetime imaging microscopy

NASA Astrophysics Data System (ADS)

Lahn, Mattes; Hille, Carsten; Koberling, Felix; Kapusta, Peter; Dosche, Carsten

2010-02-01

Today fluorescence lifetime imaging microscopy (FLIM) has become an extremely powerful technique in life sciences. The independency of the fluorescence decay time on fluorescence dye concentration and emission intensity circumvents many artefacts arising from intensity based measurements. To minimize cell damage and improve scan depth, a combination with two-photon (2P) excitation is quite promising. Here, we describe the implementation of a 2P-FLIM setup for biological applications. For that we used a commercial fluorescence lifetime microscope system. 2P-excitation at 780nm was achieved by a non-tuneable, but inexpensive and easily manageable mode-locked fs-fiber laser. Time-resolved fluorescence image acquisition was performed by objective-scanning with the reversed time-correlated single photon counting (TCSPC) technique. We analyzed the suitability of the pH-sensitive dye BCECF and the chloride-sensitive dye MQAE for recordings in an insect tissue. Both parameters are quite important, since they affect a plethora of physiological processes in living tissues. We performed a straight forward in situ calibration method to link the fluorescence decay time with the respective ion concentration and carried out spatially resolved measurements under resting conditions. BCECF still offered only a limited dynamic range regarding fluorescence decay time changes under physiologically pH values. However, MQAE proofed to be well suited to record chloride concentrations in the physiologically relevant range. Subsequently, several chloride transport pathways underlying the intracellular chloride homeostasis were investigated pharmacologically. In conclusion, 2P-FLIM is well suited for ion detection in living tissues due to precise and reproducible decay time measurements in combination with reduced cell and dye damages.

Delineating Normal from Diseased Brain by Aminolevulinic Acid-Induced Fluorescence

NASA Astrophysics Data System (ADS)

Stepp, Herbert; Stummer, Walter

5-Aminolevulinic acid (5-ALA) as a precursor of protoporphyrin IX (PpIX) has been established as an orally applied drug to guide surgical resection of malignant brain tumors by exciting the red fluorescence of PpIX. The accumulation of PpIX in glioblastoma multiforme (GBM) is highly selective and provides excellent contrast to normal brain when using surgical microscopes with appropriately filtered light sources and cameras. The positive predictive value of fluorescent tissue is very high, enabling safe gross total resection of GBM and other brain tumors and improving prognosis of patients. Compared to other intraoperative techniques that have been developed with the aim of increasing the rate of safe gross total resections of malignant gliomas, PpIX fluorescence is considerably simpler, more cost effective, and comparably reliable. We present the basics of 5-ALA-based fluorescence-guided resection, and discuss the clinical results obtained for GBM and the experience with the fluorescence staining of other primary brain tumors and metastases as well as the results for spinal cord tumors. The phototoxicity of PpIX, increasingly used for photodynamic therapy of brain tumors, is mentioned briefly in this chapter.

Development of UV-curable liquid for in-liquid fluorescence alignment in ultraviolet nanoimprint lithography

NASA Astrophysics Data System (ADS)

Ochiai, Kento; Kikuchi, Eri; Ishito, Yota; Kumagai, Mari; Nakamura, Takahiro; Nakagawa, Masaru

2018-06-01

We studied a fluorescent UV-curable resin suitable for fluorescence alignment in UV nanoimprinting. The addition of a cationic fluorescent dye caused radical photopolymerization of a UV-curable resin by exposure to visible excitation light for fluorescence microscope observation. The microscope observation of a resin film prepared by pressing resin droplets on a silica substrate with a fluorinated silica superstrate revealed that the cationic dye molecules were preferably adsorbed onto the silica surface. It was indicated that the dye molecules concentrated on the silica surface may cause the photocuring. A nonionic fluorescent dye was selected owing to its low polar symmetrical structure and its solubility parameter close to monomers. The fluorescent UV-curable resin with the nonionic dye showed uncured stability to exposure to visible excitation light for 30 min with a light intensity of 8.5 mW cm‑2 detected at 530 nm.

Ratiometric Imaging of Extracellular pH in Dental Biofilms.

PubMed

Schlafer, Sebastian; Dige, Irene

2016-03-09

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites are not distributed evenly in dental biofilms. A complex interplay of sorption to and reaction with organic matter in the biofilm reduces the diffusion paths of solutes and creates steep gradients of reactive molecules, including organic acids, across the biofilm. Quantitative fluorescent microscopic methods, such as fluorescence life time imaging or pH ratiometry, can be employed to visualize pH in different microenvironments of dental biofilms. pH ratiometry exploits a pH-dependent shift in the fluorescent emission of pH-sensitive dyes. Calculation of the emission ratio at two different wavelengths allows determining local pH in microscopic images, irrespective of the concentration of the dye. Contrary to microelectrodes the technique allows monitoring both vertical and horizontal pH gradients in real-time without mechanically disturbing the biofilm. However, care must be taken to differentiate accurately between extra- and intracellular compartments of the biofilm. Here, the ratiometric dye, seminaphthorhodafluor-4F 5-(and-6) carboxylic acid (C-SNARF-4) is employed to monitor extracellular pH in in vivo grown dental biofilms of unknown species composition. Upon exposure to glucose the dye is up-concentrated inside all bacterial cells in the biofilms; it is thus used both as a universal bacterial stain and as a marker of extracellular pH. After confocal microscopic image acquisition, the bacterial biomass is removed from all pictures using digital image analysis software, which permits to exclusively calculate extracellular pH. pH ratiometry with the ratiometric dye is well-suited to study extracellular pH in thin biofilms of up to 75 µm thickness, but is limited to the pH range between 4.5 and 7.0.

Design and construction of a modular low-cost epifluorescence upright microscope for neuron visualized recording and fluorescence detection.

PubMed

Beltran-Parrazal, Luis; Morgado-Valle, Consuelo; Serrano, Raul E; Manzo, Jorge; Vergara, Julio L

2014-03-30

One of the limitations when establishing an electrophysiology setup, particularly in low resource settings, is the high cost of microscopes. The average cost for a microscope equipped with the optics for infrared (IR) contrast or microfluorometry is $40,000. We hypothesized that optical elements and features included in commercial microscopes are not necessary to IR video-visualize neurons or for microfluorometry. We present instructions for building a low-cost epifluorescence upright microscope suitable for visualized patch-clamp recording and fluorescence detection using mostly catalog-available parts. This microscope supports applications such as visualized whole-cell recording using IR oblique illumination (IR-OI), or more complex applications such as microfluorometry using a photodiode. In both IR-OI and fluorescence, actual resolution measured with 2-μm latex beads is close to theoretical resolution. The lack of movable parts to switch configurations ensures stability when doing intracellular recording. The low cost is a significant advantage of this microscope compared to existent custom-built microscopes. The cost of the simplest configuration with IR-OI is ∼$2000, whereas the cost of the configuration with epifluorescence is ∼$5000. Since this design does not use pieces discarded from commercial microscopes, it is completely reproducible. We suggest that this microscope is a viable alternative for doing in vitro electrophysiology and microfluorometry in low-resource settings. Characteristics such as an open box design, easy assembly, and low-cost make this microscope a useful instrument for science education and teaching for topics such as optics, biology, neuroscience, and for scientific "hands-on" workshops. Copyright © 2014 Elsevier B.V. All rights reserved.

Multimodal Spectral Imaging of Cells Using a Transmission Diffraction Grating on a Light Microscope

PubMed Central

Isailovic, Dragan; Xu, Yang; Copus, Tyler; Saraswat, Suraj; Nauli, Surya M.

2011-01-01

A multimodal methodology for spectral imaging of cells is presented. The spectral imaging setup uses a transmission diffraction grating on a light microscope to concurrently record spectral images of cells and cellular organelles by fluorescence, darkfield, brightfield, and differential interference contrast (DIC) spectral microscopy. Initially, the setup was applied for fluorescence spectral imaging of yeast and mammalian cells labeled with multiple fluorophores. Fluorescence signals originating from fluorescently labeled biomolecules in cells were collected through triple or single filter cubes, separated by the grating, and imaged using a charge-coupled device (CCD) camera. Cellular components such as nuclei, cytoskeleton, and mitochondria were spatially separated by the fluorescence spectra of the fluorophores present in them, providing detailed multi-colored spectral images of cells. Additionally, the grating-based spectral microscope enabled measurement of scattering and absorption spectra of unlabeled cells and stained tissue sections using darkfield and brightfield or DIC spectral microscopy, respectively. The presented spectral imaging methodology provides a readily affordable approach for multimodal spectral characterization of biological cells and other specimens. PMID:21639978

Practical three color live cell imaging by widefield microscopy

PubMed Central

Xia, Jianrun; Kim, Song Hon H.; Macmillan, Susan

2006-01-01

Live cell fluorescence microscopy using fluorescent protein tags derived from jellyfish and coral species has been a successful tool to image proteins and dynamics in many species. Multi-colored aequorea fluorescent protein (AFP) derivatives allow investigators to observe multiple proteins simultaneously, but overlapping spectral properties sometimes require the use of sophisticated and expensive microscopes. Here, we show that the aequorea coerulescens fluorescent protein derivative, PS-CFP2 has excellent practical properties as a blue fluorophore that are distinct from green or red fluorescent proteins and can be imaged with standard filter sets on a widefield microscope. We also find that by widefield illumination in live cells, that PS-CFP2 is very photostable. When fused to proteins that form concentrated puncta in either the cytoplasm or nucleus, PSCFP2 fusions do not artifactually interact with other AFP fusion proteins, even at very high levels of over-expression. PSCFP2 is therefore a good blue fluorophore for distinct three color imaging along with eGFP and mRFP using a relatively simple and inexpensive microscope. PMID:16909160

Micro-fractures produced in the Cadalso de los Vidrios granite (Madrid) subjected to Freeze-Thaw Durability Testing

NASA Astrophysics Data System (ADS)

Freire-Lista, D. M.; Varas-Muriel, M. J.; Fort, R.

2012-04-01

A specific leucogranite (fine to medium grain sized) from Cadalso de los Vidrios, Madrid, Spain, from where it takes the name of the stone variety, which is a traditional heritage building material used in Central Spain, was subjected to freezing-thaw durability tests or accelerated artificial ageing tests (according to Spanish standard EN 12371:2001) to assess its durability by means of ultrasonic velocity measurements (a non-destructive technique), and optical and fluorescence petrography using a polarized optical microscope (destructive technique), both techniques used before, during and after laboratory ageing tests, or in other words, what is determined is the improvement or deterioration in some properties. The measurement of the ultrasonic velocity in the leucogranite cubic test specimens along the freezing-thaw cycles shown that the velocity diminishes with the number of cycles, in relation to the decay that the stones were experiencing. This deterioration can be observed by the loss of crystalline minerals in the surface of the analyzed samples and by the micro-fractures appearance up to one centimeter deep, which have been detected by the petrographic techniques previously mentioned. The images taken by means of the fluorescence microscope clearly show the micro-fractures generated during the durability test. These images have been processed and analyzed by the UTHSCSA Image Tool program with the purpose of being able to quantify the degree of decay that this type of crystalline materials undergone, when subjected to a number of freezing-thaw test cycles. It is therefore an effective, reliable and complementary technique to that of the petrography analysis, both optical and fluorescence ones. In the first cycles of the ageing test, the micro-fractures propagate along crystals edges and during the last cycles of the test, intracrystalline micro-fractures are generated, which are developed in different ways depending on the mineralogy of the crystals. Thus, the quartz crystals are those that undergo more intracrystalline micro-fractures, whereas the biotites, behave in a more ductile form and they are not micro-fractured. Both analytical techniques give information of this granite deterioration, showing a relation between the number of freezing-thaw cycles, the superficial micro-fractures proliferation and the decrease of ultrasonic waves propagation velocity produced by the ageing cycles.

Effects of a common worldwide drink (Beer) on L-Phenylalanine and L-Tyrosine fibrillar assemblies

NASA Astrophysics Data System (ADS)

Banik, Debasis; Banerjee, Pavel; Sabeehuddin, Ghazi; Sarkar, Nilmoni

2017-11-01

In this letter, small amount of beer [0.42-2.08% (v/v)] is employed to investigate the fibril inhibition kinetics of 1 mM L-Phenylalanine and L-Tyrosine (relevant to disease condition) using Fluorescence Lifetime imaging Microscopy (FLIM), Field Emission Scanning Electron Microscopy (FESEM) and High Resolution Transmission Electron Microscopic (HR-TEM) techniques. Our results indicate that 1.67 and 0.42% of beer is sufficient for effective breakdown of L-Phe and L-Tyr assemblies, respectively. Quantitative information about fibril inhibition is obtained from Fluorescence Correlation Spectroscopic (FCS) measurements. We have shown that the morphology of L-Phe changes to L-Tyr in presence of 2,2‧-Bipyridine-3,3‧-diol (BP(OH)2).

Moving Toward the Light: Using New Technology to Answer Old Questions

PubMed Central

LUCITTI, JENNIFER L.; DICKINSON, MARY E.

2006-01-01

Fluorescence microscopy has become a principle methodology in the field of developmental biology. Recent technological advances have led to the design of high-speed and high-resolution confocal and multiphoton microscopes that enable researchers to obtain three- and four-dimensional information in living cells and whole embryos. Paralleling this progress, the development of stable and bright vital fluorescent probes has revolutionized the ability to track individual cells in vitro and in vivo and to visualize intercellular and subcellular molecular interactions in real time. Combining imaging modalities and labeling techniques that are increasingly unobtrusive to cell and whole animal function, our understanding of how proteins interact, tissues take form, and organs synchronize to create a functioning animal is reaching a whole new level. PMID:16690954

Fluorescence microscopy.

PubMed

Sanderson, Michael J; Smith, Ian; Parker, Ian; Bootman, Martin D

2014-10-01

Fluorescence microscopy is a major tool with which to monitor cell physiology. Although the concepts of fluorescence and its optical separation using filters remain similar, microscope design varies with the aim of increasing image contrast and spatial resolution. The basics of wide-field microscopy are outlined to emphasize the selection, advantages, and correct use of laser scanning confocal microscopy, two-photon microscopy, scanning disk confocal microscopy, total internal reflection, and super-resolution microscopy. In addition, the principles of how these microscopes form images are reviewed to appreciate their capabilities, limitations, and constraints for operation. © 2014 Cold Spring Harbor Laboratory Press.

Fluorescence Microscopy

PubMed Central

Sanderson, Michael J.; Smith, Ian; Parker, Ian; Bootman, Martin D.

2016-01-01

Fluorescence microscopy is a major tool with which to monitor cell physiology. Although the concepts of fluorescence and its optical separation using filters remain similar, microscope design varies with the aim of increasing image contrast and spatial resolution. The basics of wide-field microscopy are outlined to emphasize the selection, advantages, and correct use of laser scanning confocal microscopy, two-photon microscopy, scanning disk confocal microscopy, total internal reflection, and super-resolution microscopy. In addition, the principles of how these microscopes form images are reviewed to appreciate their capabilities, limitations, and constraints for operation. PMID:25275114

Observation of microscopic dynamics of phase transition in ferroelectric crystals using fluorescence spectroscopy

NASA Astrophysics Data System (ADS)

Sedarous, Salah S.

1996-03-01

Despite the large quantity of data on the macroscopic changes in the physical properties of ferroelectric crystals during phase transition, there is a continued need for understanding their microscopic origin. Here we describe a novel method for examining the microscopic dynamics of the ferroelectric phase transition using time-resolved fluorescence spectroscopy. The fluorescence properties of organic chromophores embedded in the ferroelectric crystals triglycine sulfate and potassium dihydrogen phosphate are altered in response to the structural phase transitions. The lifetime and the fractional intensity decay show large changes around Tc and the order of the phase transition is readily recovered (first or second order). To explain the fluorescence lifetime data we present a novel theoretical model based on the concept of polaritons in these crystals. Deactivation of the excited state chromophore involves the participation of the vibrational modes of the chromophore. These modes are coupled to the polarization dispersion of the matrix and facilitate the coupling of the excited state to the collective modes in the crystal. The net result is the flow of energy from the excited state chromophore to the lattice phonon. The data indicate that changes in fluorescence lifetime can be used to examine directly the collective modes in these crystals. Our work provides important insight into the emergence of macroscopic phase transition behavior out of microscopic fluctuations.

Isometric multimodal photoacoustic microscopy based on optically transparent micro-ring ultrasonic detection.

PubMed

Dong, Biqin; Li, Hao; Zhang, Zhen; Zhang, Kevin; Chen, Siyu; Sun, Cheng; Zhang, Hao F

2015-01-01

Photoacoustic microscopy (PAM) is an attractive imaging tool complementary to established optical microscopic modalities by providing additional molecular specificities through imaging optical absorption contrast. While the development of optical resolution photoacoustic microscopy (ORPAM) offers high lateral resolution, the acoustically-determined axial resolution is limited due to the constraint in ultrasonic detection bandwidth. ORPAM with isometric spatial resolution along both axial and lateral direction is yet to be developed. Although recently developed sophisticated optical illumination and reconstruction methods offer improved axial resolution in ORPAM, the image acquisition procedures are rather complicated, limiting their capabilities for high-speed imaging and being easily integrated with established optical microscopic modalities. Here we report an isometric ORPAM based on an optically transparent micro-ring resonator ultrasonic detector and a commercial inverted microscope platform. Owing to the superior spatial resolution and the ease of integrating our ORPAM with established microscopic modalities, single cell imaging with extrinsic fluorescence staining, intrinsic autofluorescence, and optical absorption can be achieved simultaneously. This technique holds promise to greatly improve the accessibility of PAM to the broader biomedical researchers.

Diagnosis of viral agents associated with neonatal calf diarrhea.

PubMed Central

Marsolais, G; Assaf, R; Montpetit, C; Marois, P

1978-01-01

During this study, 134 samples have been examined for the detection of the viruses associated with neonatal calf diarrhea. The presence of Nebraska viruses (rotavirus and coronavirus) has been demonstrated by using the electron microscope and the fluorescent antibody techniques while the presence of other viruses has been detected by the observation of a cytopathic effect on monolayer cells of calf testis. The Nebraska viruses have been demonstrated in 107 (80%) out of 134 field case specimens. An association of rotaviruses and coronaviruses was found in 58 cases (54%) whilst the coronaviruses and the rotavirus were found singly in 34 cases (53%) and in 15 cases (14%) respectively. Four bovine virus diarrhea viruses, two infectious bovine rhinotracheitis viruses and two enteroviruses have also been isolated in the preceding 107 Nebraska positive specimens. For the detection of the Nebraska viruses, the fluorescent antibody techniques were more sensitive than the electron microscopy. However, those two techniques must be used simultaneously for a better detection of a greatest possible number of cases. Images Fig. 1. Fig. 2. PMID:208735

Hyperspectral imaging fluorescence excitation scanning for colon cancer detection

NASA Astrophysics Data System (ADS)

Leavesley, Silas J.; Walters, Mikayla; Lopez, Carmen; Baker, Thomas; Favreau, Peter F.; Rich, Thomas C.; Rider, Paul F.; Boudreaux, Carole W.

2016-10-01

Optical spectroscopy and hyperspectral imaging have shown the potential to discriminate between cancerous and noncancerous tissue with high sensitivity and specificity. However, to date, these techniques have not been effectively translated to real-time endoscope platforms. Hyperspectral imaging of the fluorescence excitation spectrum represents new technology that may be well suited for endoscopic implementation. However, the feasibility of detecting differences between normal and cancerous mucosa using fluorescence excitation-scanning hyperspectral imaging has not been evaluated. The goal of this study was to evaluate the initial feasibility of using fluorescence excitation-scanning hyperspectral imaging for measuring changes in fluorescence excitation spectrum concurrent with colonic adenocarcinoma using a small pre-pilot-scale sample size. Ex vivo analysis was performed using resected pairs of colorectal adenocarcinoma and normal mucosa. Adenocarcinoma was confirmed by histologic evaluation of hematoxylin and eosin (H&E) permanent sections. Specimens were imaged using a custom hyperspectral imaging fluorescence excitation-scanning microscope system. Results demonstrated consistent spectral differences between normal and cancerous tissues over the fluorescence excitation range of 390 to 450 nm that could be the basis for wavelength-dependent detection of colorectal cancers. Hence, excitation-scanning hyperspectral imaging may offer an alternative approach for discriminating adenocarcinoma from surrounding normal colonic mucosa, but further studies will be required to evaluate the accuracy of this approach using a larger patient cohort.

A surface science compatible epifluorescence microscope for inspection of samples under ultra high vacuum and cryogenic conditions.

PubMed

Marquardt, Christian; Paulheim, Alexander; Rohbohm, Nils; Merkel, Rudolf; Sokolowski, Moritz

2017-08-01

We modified an epi-illumination light microscope and mounted it on an ultra high vacuum chamber for investigating samples used in a surface science experiment. For easy access and bake out, all optical components are placed outside the vacuum and the sample is imaged through a glass window. The microscope can be operated in reflection brightfield or epifluorescence mode to image the sample surface or fluorescent dye molecules adsorbed on it. The homemade sample mounting was made compatible for the use under the microscope; sample temperatures as low as 6 K can be achieved. The performance of the microscope is demonstrated on two model samples: Brightfield-images of a well-prepared Ag(100) surface show a macroscopic corrugation of the surface, although low energy electron diffraction data indicate a highly ordered crystalline surface. The surface shows macroscopic protrusions with flat regions, about 20-200 μm in diameter, in between. Fluorescence images of diluted 3,4,9,10-perylene tetracarboxylicacid dianhydride (PTCDA) molecules adsorbed on an ultrathin epitaxial KCl film on the Ag(100) surface show a shading effect at surface protrusions due to an inclined angle of incidence of the PTCDA beam during deposition. For some preparations, the distribution of the fluorescence intensity is inhomogeneous and shows a dense network of bright patches about 5 μm in diameter related to the macroscopic corrugation of the surface. We propose that such a light microscope can aid many surface science experiments, especially those dealing with epitaxial growth or fluorescent materials.

Optical depth localization of nitrogen-vacancy centers in diamond with nanometer accuracy.

PubMed

Häußler, Andreas J; Heller, Pascal; McGuinness, Liam P; Naydenov, Boris; Jelezko, Fedor

2014-12-01

Precise positioning of nitrogen-vacancy (NV) centers is crucial for their application in sensing and quantum information. Here we present a new purely optical technique enabling determination of the NV position with nanometer resolution. We use a confocal microscope to determine the position of individual emitters along the optical axis. Using two separate detection channels, it is possible to simultaneously measure reflected light from the diamond surface and fluorescent light from the NV center and statistically evaluate both signals. An accuracy of 2.6 nm for shallow NV centers was achieved and is consistent with other techniques for depth determination.

Chemical analyses of fossil bone.

PubMed

Zheng, Wenxia; Schweitzer, Mary Higby

2012-01-01

The preservation of microstructures consistent with soft tissues, cells, and other biological components in demineralized fragments of dinosaur bone tens of millions of years old was unexpected, and counter to current hypotheses of tissue, cellular, and molecular degradation. Although the morphological similarity of these tissues to extant counterparts was unmistakable, after at least 80 million years exposed to geochemical influences, morphological similarity is insufficient to support an endogenous source. To test this hypothesis, and to characterize these materials at a molecular level, we applied multiple independent chemical, molecular, and microscopic analyses to identify the presence of original components produced by the extinct organisms. Microscopic techniques included field emission scanning electron microscopy, analytical transmission electron microscopy, transmitted light microscopy (LM), and fluorescence microscopy (FM). The chemical and molecular techniques include enzyme-linked immunosorbant assay, sodium dodecyl sulfate polyacrylamide gel electrophoresis, western blot (immunoblot), and attenuated total reflectance infrared spectroscopy. In situ analyses performed directly on tissues included immunohistochemistry and time-of-flight secondary ion mass spectrometry. The details of sample preparation and methodology are described in detail herein.

Microscopic quantification of bacterial invasion by a novel antibody-independent staining method.

PubMed

Agerer, Franziska; Waeckerle, Stephanie; Hauck, Christof R

2004-10-01

Microscopic discrimination between extracellular and invasive, intracellular bacteria is a valuable technique in microbiology and immunology. We describe a novel fluorescence staining protocol, called FITC-biotin-avidin (FBA) staining, which allows the differentiation between extracellular and intracellular bacteria and is independent of specific antibodies directed against the microorganisms. FBA staining of eukaryotic cells infected with Gram-negative bacteria of the genus Neisseria or the Gram-positive pathogen Staphylococcus aureus are employed to validate the novel technique. The quantitative evaluation of intracellular pathogens by the FBA staining protocol yields identical results compared to parallel samples stained with conventional, antibody-dependent methods. FBA staining eliminates the need for cell permeabilization resulting in robust and rapid detection of invasive microbes. Taken together, FBA staining provides a reliable and convenient alternative for the differential detection of intracellular and extracellular bacteria and should be a valuable technical tool for the quantitative analysis of the invasive properties of pathogenic bacteria and other microorganisms.

Liquid-Phase Circulation and Mixing in Multicomponent Droplets Vaporizing in a Laminar Convective Environment

DTIC Science & Technology

1993-10-15

included an f/2.8 dual port long-distance microscope coupled to a black d•rl white CCD video camera. A long-pass filter (with a cut-off at 530 nm) was...evaporation rates of multicomponent droplets is needed for the calibration of exciplex -based vapor/liquid visualization techniques that are employed today in...Publishing Co., Houston. Texas. Hanlon. T. R.. and Melton. L. A. (1992). Exciplex fluorescence thermometry of falling hexadecane droplets. Journal of Heat

Remote microscopy and volumetric imaging on the surface of icy satellites

NASA Astrophysics Data System (ADS)

Soto, Alejandro; Nowicki, Keith; Howett, Carly; Feldkhun, Daniel; Retherford, Kurt D.

2017-10-01

With NASA PIDDP support we have applied recent advancements in Fourier-domain microscopy to develop an instrument capable of microscopic imaging from meter-scale distances for use on a planetary lander on the surface of an icy satellite or other planetary bodies. Without moving parts, our instrument projects dynamic patterns of laser light onto a distant target using a lightweight large-aperture reflector, which then collects the light scattered or fluoresced by the target on a fast photon-bucket detector. Using Fourier Transform based techniques, we reconstruct an image from the detected light. The remote microscope has been demonstrated to produce 2D images with better than 15 micron lateral resolution for targets at a distance of 5 meters and is capable of linearly proportionally higher resolution at shorter distances. The remote microscope is also capable of providing three-dimensional (3D) microscopic imaging capabilities, allowing future surface scientists to explore the morphology of microscopic features in surface ices, for example. The instrument enables microscopic in-situ imaging during day or night without the use of a robotic arm, greatly facilitating the surface operations for a lander or rover while expanding the area of investigation near a landing site for improved science targeting. We are developing this remote microscope for in-situ planetary exploration as a collaboration between the Southwest Research Institute, LambdaMetrics, and the University of Colorado.

LabVIEW interface with Tango control system for a multi-technique X-ray spectrometry IAEA beamline end-station at Elettra Sincrotrone Trieste

NASA Astrophysics Data System (ADS)

Wrobel, P. M.; Bogovac, M.; Sghaier, H.; Leani, J. J.; Migliori, A.; Padilla-Alvarez, R.; Czyzycki, M.; Osan, J.; Kaiser, R. B.; Karydas, A. G.

2016-10-01

A new synchrotron beamline end-station for multipurpose X-ray spectrometry applications has been recently commissioned and it is currently accessible by end-users at the XRF beamline of Elettra Sincrotrone Trieste. The end-station consists of an ultra-high vacuum chamber that includes as main instrument a seven-axis motorized manipulator for sample and detectors positioning, different kinds of X-ray detectors and optical cameras. The beamline end-station allows performing measurements in different X-ray spectrometry techniques such as Microscopic X-Ray Fluorescence analysis (μXRF), Total Reflection X-Ray Fluorescence analysis (TXRF), Grazing Incidence/Exit X-Ray Fluorescence analysis (GI-XRF/GE-XRF), X-Ray Reflectometry (XRR), and X-Ray Absorption Spectroscopy (XAS). A LabVIEW Graphical User Interface (GUI) bound with Tango control system consisted of many custom made software modules is utilized as a user-friendly tool for control of the entire end-station hardware components. The present work describes this advanced Tango and LabVIEW software platform that utilizes in an optimal synergistic manner the merits and functionality of these well-established programming and equipment control tools.

Semiautomated confocal imaging of fungal pathogenesis on plants: Microscopic analysis of macroscopic specimens.

PubMed

Minker, Katharine R; Biedrzycki, Meredith L; Kolagunda, Abhishek; Rhein, Stephen; Perina, Fabiano J; Jacobs, Samuel S; Moore, Michael; Jamann, Tiffany M; Yang, Qin; Nelson, Rebecca; Balint-Kurti, Peter; Kambhamettu, Chandra; Wisser, Randall J; Caplan, Jeffrey L

2018-02-01

The study of phenotypic variation in plant pathogenesis provides fundamental information about the nature of disease resistance. Cellular mechanisms that alter pathogenesis can be elucidated with confocal microscopy; however, systematic phenotyping platforms-from sample processing to image analysis-to investigate this do not exist. We have developed a platform for 3D phenotyping of cellular features underlying variation in disease development by fluorescence-specific resolution of host and pathogen interactions across time (4D). A confocal microscopy phenotyping platform compatible with different maize-fungal pathosystems (fungi: Setosphaeria turcica, Cochliobolus heterostrophus, and Cercospora zeae-maydis) was developed. Protocols and techniques were standardized for sample fixation, optical clearing, species-specific combinatorial fluorescence staining, multisample imaging, and image processing for investigation at the macroscale. The sample preparation methods presented here overcome challenges to fluorescence imaging such as specimen thickness and topography as well as physiological characteristics of the samples such as tissue autofluorescence and presence of cuticle. The resulting imaging techniques provide interesting qualitative and quantitative information not possible with conventional light or electron 2D imaging. Microsc. Res. Tech., 81:141-152, 2018. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Multimodal Light Microscopy Approaches to Reveal Structural and Functional Properties of Promyelocytic Leukemia Nuclear Bodies.

PubMed

Hoischen, Christian; Monajembashi, Shamci; Weisshart, Klaus; Hemmerich, Peter

2018-01-01

The promyelocytic leukemia ( pml ) gene product PML is a tumor suppressor localized mainly in the nucleus of mammalian cells. In the cell nucleus, PML seeds the formation of macromolecular multiprotein complexes, known as PML nuclear bodies (PML NBs). While PML NBs have been implicated in many cellular functions including cell cycle regulation, survival and apoptosis their role as signaling hubs along major genome maintenance pathways emerged more clearly. However, despite extensive research over the past decades, the precise biochemical function of PML in these pathways is still elusive. It remains a big challenge to unify all the different previously suggested cellular functions of PML NBs into one mechanistic model. With the advent of genetically encoded fluorescent proteins it became possible to trace protein function in living specimens. In parallel, a variety of fluorescence fluctuation microscopy (FFM) approaches have been developed which allow precise determination of the biophysical and interaction properties of cellular factors at the single molecule level in living cells. In this report, we summarize the current knowledge on PML nuclear bodies and describe several fluorescence imaging, manipulation, FFM, and super-resolution techniques suitable to analyze PML body assembly and function. These include fluorescence redistribution after photobleaching, fluorescence resonance energy transfer, fluorescence correlation spectroscopy, raster image correlation spectroscopy, ultraviolet laser microbeam-induced DNA damage, erythrocyte-mediated force application, and super-resolution microscopy approaches. Since most if not all of the microscopic equipment to perform these techniques may be available in an institutional or nearby facility, we hope to encourage more researches to exploit sophisticated imaging tools for their research in cancer biology.

Discriminative detection and enumeration of microbial life in marine subsurface sediments.

PubMed

Morono, Yuki; Terada, Takeshi; Masui, Noriaki; Inagaki, Fumio

2009-05-01

Detection and enumeration of microbial life in natural environments provide fundamental information about the extent of the biosphere on Earth. However, it has long been difficult to evaluate the abundance of microbial cells in sedimentary habitats because non-specific binding of fluorescent dye and/or auto-fluorescence from sediment particles strongly hampers the recognition of cell-derived signals. Here, we show a highly efficient and discriminative detection and enumeration technique for microbial cells in sediments using hydrofluoric acid (HF) treatment and automated fluorescent image analysis. Washing of sediment slurries with HF significantly reduced non-biological fluorescent signals such as amorphous silica and enhanced the efficiency of cell detachment from the particles. We found that cell-derived SYBR Green I signals can be distinguished from non-biological backgrounds by dividing green fluorescence (band-pass filter: 528/38 nm (center-wavelength/bandwidth)) by red (617/73 nm) per image. A newly developed automated microscope system could take a wide range of high-resolution image in a short time, and subsequently enumerate the accurate number of cell-derived signals by the calculation of green to red fluorescence signals per image. Using our technique, we evaluated the microbial population in deep marine sediments offshore Peru and Japan down to 365 m below the seafloor, which provided objective digital images as evidence for the quantification of the prevailing microbial life. Our method is hence useful to explore the extent of sub-seafloor life in the future scientific drilling, and moreover widely applicable in the study of microbial ecology.

Photonic engineering for biological study

NASA Astrophysics Data System (ADS)

Wu, Fei

My dissertation focuses on designing and developing prototypes of optical tools in the laboratory that can facilitate practical medical therapies. More specifically, this dissertation examines two novel biophotonic techniques: (1) a frequency multiplexed confocal microscope with the potential to provide rational therapy of congestive heart failure (CHF), and (2) the "optical comb" with the potential to improve results of retina reattachment surgery and accelerate post surgical recovery. Next, I will discuss the background, design and initial experimental results of each study individually. Part I: The Frequency Multiplexed Confocal Microscope. To overcome the limitations of existing confocal microscope technology, this dissertation proposes a non-scanning, real-time, high resolution technique (a multi-point frequency multiplexed confocal microscope) to measure 3-D intracellular calcium ion concentration in a living cardiac myocyte. This method can be also applied to measure the intracellular sodium ion concentration, or other ions in which high quantum-yield fluorescent probes are available. The novelty of the proposed research lies in the introduction of carrier frequency multiplexing techniques which can differentiate fluorescence emitted at different spatial locations in cardiac myocyte by their modulated frequency. It therefore opens the possibility to visualize the transient dynamics of intracellular dynamics at multiple locations in cells simultaneously, which will shine a new light on our understanding of CHF. The procedure for frequency multiplexing proposed is described below. Multiple incident laser beams are focused onto different locations in an isolated rat cardiac myocyte with each beam modulated at a different carrier frequency. The fluorescence emission at each location therefore bears the same modulated frequency as the stimulation laser beam. Each fluorescence signal is sent to the photo multiplier tube (PMT) after being spatially filtered by a single mode fiber (functioning as a pinhole). Since each signal has a different carrier frequency, only one signal detector is required to collect multiple signal streams which eliminates the errors introduced by difference of multiple detectors. After taking the Fourier Transform of the collected data, multiple peaks can be found in the frequency domain. Each peak refers to a corresponding location in the sample. The temporal information of the fluorescence signal variation at each location can be obtained by demodulating the low frequency information from the carrier frequency, followed by an inverse Fourier transform. Part II: The "Optical Comb". Retinal detachment refers to separation of the inner layers of the retina from the underlying retinal pigment epithelium. It can cause degeneration of the retina and may lead to permanent vision loss if not promptly treated and hence is considered an ocular emergency. Currently, the only treatment available for retinal detachment is surgical reattachment. The idea of an "optical comb" is developed from the general working principle of the well known "optical tweezers" in the optical literature, which can pull micro-objects through the trapping force produced by a focused laser beam. If we can manage to incident the focused laser beam onto the misaligned photoreceptors and further scan it back and forth, trapping forces that produced may be able to "comb" the photoreceptors to be aligned, and thereby help with post surgery recovery. A series of experiments have been carried out to demonstrate the plausibility of this idea. First, several micro glass rods with size similar to human's photoreceptors (6 microns in diameter and 30 microns in length) were used. We observed that when the laser beam is focused close to one end of the micro rod originally laid on a glass coverslip, the rod is pulled to stand upright successfully, and we can manipulate the direction it faces by controlling its relative position to the laser beam. We are now experimenting with this combing technique with detached bovine retina samples to further verify its feasibility over live animal cells. (Abstract shortened by UMI.)

A Clinical Wide-Field Fluorescence Endoscopic Device for Molecular Imaging Demonstrating Cathepsin Protease Activity in Colon Cancer.

PubMed

Sensarn, Steven; Zavaleta, Cristina L; Segal, Ehud; Rogalla, Stephan; Lee, Wansik; Gambhir, Sanjiv S; Bogyo, Matthew; Contag, Christopher H

2016-12-01

Early and effective detection of cancers of the gastrointestinal tract will require novel molecular probes and advances in instrumentation that can reveal functional changes in dysplastic and malignant tissues. Here, we describe adaptation of a wide-field clinical fiberscope to perform wide-field fluorescence imaging while preserving its white-light capability for the purpose of providing wide-field fluorescence imaging capability to point-of-care microscopes. We developed and used a fluorescent fiberscope to detect signals from a quenched probe, BMV109, that becomes fluorescent when cleaved by, and covalently bound to, active cathepsin proteases. Cathepsins are expressed in inflammation- and tumor-associated macrophages as well as directly from tumor cells and are a promising target for cancer imaging. The fiberscope has a 1-mm outer diameter enabling validation via endoscopic exams in mice, and therefore we evaluated topically applied BMV109 for the ability to detect colon polyps in an azoxymethane-induced colon tumor model in mice. This wide-field endoscopic imaging device revealed consistent and clear fluorescence signals from BMV109 that specifically localized to the polypoid regions as opposed to the normal adjacent colon tissue (p < 0.004) in the murine colon carcinoma model. The sensitivity of detection of BMV109 with the fluorescence fiberscope suggested utility of these tools for early detection at hard-to-reach sites. The fiberscope was designed to be used in conjunction with miniature, endoscope-compatible fluorescence microscopes for dual wide-field and microscopic cancer detection.

Planetary surface exploration using Raman spectroscopy for minerals and organics

NASA Astrophysics Data System (ADS)

Blacksberg, J.; Alerstam, E.; Maruyama, Y.; Charbon, E.; Rossman, G. R.; Shkolyar, S.; Farmer, J. D.

2013-12-01

Raman spectroscopy has been identified as one of the primary techniques for planetary surface mineralogy. It is widely used as a laboratory technique since it can identify nearly all crystalline mineral phases. Using a small spot size on the surface (on the order of a micron), mineral phases can be mapped onto microscopic images preserving information about surface morphology. As a result, this technique has been steadily gaining support for in situ exploration of a variety of target bodies, for example Mars, the Moon, Venus, asteroids, and comets. In addition to in situ exploration, Raman spectroscopy has been identified as a feasible means for pre-selection of samples on Mars for subsequent return to Earth. This is in part due to the fact that Raman can detect many organics in addition to minerals. As a result, the most relevant rock samples containing organics (potentially fossil biosignatures) may potentially be selected for return to Earth. We present a next-generation instrument that builds on the widely used 532 nm Raman technique to provide a means for performing Raman spectroscopy without the background noise that is often generated by fluorescence of minerals and organics. We use time-resolved laser spectroscopy to eliminate this fluorescence interference that can often make it difficult or impossible to obtain Raman spectra. We will discuss significant advances leading to the feasibility of a compact time-resolved spectrometer, including the development of a new solid-state detector capable of sub-ns temporal resolution. We will address the challenges of analyzing surface materials, often organics, that exhibit short-lifetime fluorescence. We will present result on planetary analog samples to demonstrate the instrument performance including fluorescence rejection.

Analysis of the chicken retina with an adaptive optics multiphoton microscope.

PubMed

Bueno, Juan M; Giakoumaki, Anastasia; Gualda, Emilio J; Schaeffel, Frank; Artal, Pablo

2011-06-01

The structure and organization of the chicken retina has been investigated with an adaptive optics multiphoton imaging microscope in a backward configuration. Non-stained flat-mounted retinal tissues were imaged at different depths, from the retinal nerve fiber layer to the outer segment, by detecting the intrinsic nonlinear fluorescent signal. From the stacks of images corresponding to the different retinal layers, volume renderings of the entire retina were reconstructed. The density of photoreceptors and ganglion cells layer were directly estimated from the images as a function of the retinal eccentricity. The maximum anatomical resolving power at different retinal eccentricities was also calculated. This technique could be used for a better characterization of retinal alterations during myopia development, and may be useful for visualization of retinal pathologies and intoxication during pharmacological studies.

Microsphere-aided optical microscopy and its applications for super-resolution imaging

NASA Astrophysics Data System (ADS)

Upputuri, Paul Kumar; Pramanik, Manojit

2017-12-01

The spatial resolution of a standard optical microscope (SOM) is limited by diffraction. In visible spectrum, SOM can provide ∼ 200 nm resolution. To break the diffraction limit several approaches were developed including scanning near field microscopy, metamaterial super-lenses, nanoscale solid immersion lenses, super-oscillatory lenses, confocal fluorescence microscopy, techniques that exploit non-linear response of fluorophores like stimulated emission depletion microscopy, stochastic optical reconstruction microscopy, etc. Recently, photonic nanojet generated by a dielectric microsphere was used to break the diffraction limit. The microsphere-approach is simple, cost-effective and can be implemented under a standard microscope, hence it has gained enormous attention for super-resolution imaging. In this article, we briefly review the microsphere approach and its applications for super-resolution imaging in various optical imaging modalities.

Biophotonics and Bone Biology

NASA Technical Reports Server (NTRS)

Zimmerli, Gregory; Fischer, David; Asipauskas, Marius; Chauhan, Chirag; Compitello, Nicole; Burke, Jamie; Tate, Melissa Knothe

2004-01-01

One of the more serious side effects of extended space flight is an accelerated bone loss. Rates of bone loss are highest in the weight-bearing bones of the hip and spine regions, and the average rate of bone loss as measured by bone mineral density measurements is around 1.2% per month for persons in a microgravity environment. It is well known that bone remodeling responds to mechanical forces. We are developing two-photon microscopy techniques to study bone tissue and bone cell cultures to better understand the fundamental response mechanism in bone remodeling. Osteoblast and osteoclast cell cultures are being studied, and the goal is to use molecular biology techniques in conjunction with Fluorescence Lifetime Imaging Microscopy (FLIM) to study the physiology of in-vitro cell cultures in response to various stimuli, such as fluid flow induced shear stress and mechanical stress. We have constructed a two-photon fluorescence microscope for these studies, and are currently incorporating FLIM detection. Current progress will be reviewed. This work is supported by the NASA John Glenn Biomedical Engineering Consortium.

Photothermal imaging of skeletal muscle mitochondria.

PubMed

Tomimatsu, Toru; Miyazaki, Jun; Kano, Yutaka; Kobayashi, Takayoshi

2017-06-01

The morphology and topology of mitochondria provide useful information about the physiological function of skeletal muscle. Previous studies of skeletal muscle mitochondria are based on observation with transmission, scanning electron microscopy or fluorescence microscopy. In contrast, photothermal (PT) microscopy has advantages over the above commonly used microscopic techniques because of no requirement for complex sample preparation by fixation or fluorescent-dye staining. Here, we employed the PT technique using a simple diode laser to visualize skeletal muscle mitochondria in unstained and stained tissues. The fine mitochondrial network structures in muscle fibers could be imaged with the PT imaging system, even in unstained tissues. PT imaging of tissues stained with toluidine blue revealed the structures of subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria and the swelling behavior of mitochondria in damaged muscle fibers with sufficient image quality. PT image analyses based on fast Fourier transform (FFT) and Grey-level co-occurrence matrix (GLCM) were performed to derive the characteristic size of mitochondria and to discriminate the image patterns of normal and damaged fibers.

3D quantitative phase imaging of neural networks using WDT

NASA Astrophysics Data System (ADS)

Kim, Taewoo; Liu, S. C.; Iyer, Raj; Gillette, Martha U.; Popescu, Gabriel

2015-03-01

White-light diffraction tomography (WDT) is a recently developed 3D imaging technique based on a quantitative phase imaging system called spatial light interference microscopy (SLIM). The technique has achieved a sub-micron resolution in all three directions with high sensitivity granted by the low-coherence of a white-light source. Demonstrations of the technique on single cell imaging have been presented previously; however, imaging on any larger sample, including a cluster of cells, has not been demonstrated using the technique. Neurons in an animal body form a highly complex and spatially organized 3D structure, which can be characterized by neuronal networks or circuits. Currently, the most common method of studying the 3D structure of neuron networks is by using a confocal fluorescence microscope, which requires fluorescence tagging with either transient membrane dyes or after fixation of the cells. Therefore, studies on neurons are often limited to samples that are chemically treated and/or dead. WDT presents a solution for imaging live neuron networks with a high spatial and temporal resolution, because it is a 3D imaging method that is label-free and non-invasive. Using this method, a mouse or rat hippocampal neuron culture and a mouse dorsal root ganglion (DRG) neuron culture have been imaged in order to see the extension of processes between the cells in 3D. Furthermore, the tomogram is compared with a confocal fluorescence image in order to investigate the 3D structure at synapses.

Enhanced optical coupling and Raman scattering via microscopic interface engineering

NASA Astrophysics Data System (ADS)

Thompson, Jonathan V.; Hokr, Brett H.; Kim, Wihan; Ballmann, Charles W.; Applegate, Brian E.; Jo, Javier A.; Yamilov, Alexey; Cao, Hui; Scully, Marlan O.; Yakovlev, Vladislav V.

2017-11-01

Spontaneous Raman scattering is an extremely powerful tool for the remote detection and identification of various chemical materials. However, when those materials are contained within strongly scattering or turbid media, as is the case in many biological and security related systems, the sensitivity and range of Raman signal generation and detection is severely limited. Here, we demonstrate that through microscopic engineering of the optical interface, the optical coupling of light into a turbid material can be substantially enhanced. This improved coupling facilitates the enhancement of the Raman scattering signal generated by molecules within the medium. In particular, we detect at least two-orders of magnitude more spontaneous Raman scattering from a sample when the pump laser light is focused into a microscopic hole in the surface of the sample. Because this approach enhances both the interaction time and interaction region of the laser light within the material, its use will greatly improve the range and sensitivity of many spectroscopic techniques, including Raman scattering and fluorescence emission detection, inside highly scattering environments.

Nanometric depth resolution from multi-focal images in microscopy.

PubMed

Dalgarno, Heather I C; Dalgarno, Paul A; Dada, Adetunmise C; Towers, Catherine E; Gibson, Gavin J; Parton, Richard M; Davis, Ilan; Warburton, Richard J; Greenaway, Alan H

2011-07-06

We describe a method for tracking the position of small features in three dimensions from images recorded on a standard microscope with an inexpensive attachment between the microscope and the camera. The depth-measurement accuracy of this method is tested experimentally on a wide-field, inverted microscope and is shown to give approximately 8 nm depth resolution, over a specimen depth of approximately 6 µm, when using a 12-bit charge-coupled device (CCD) camera and very bright but unresolved particles. To assess low-flux limitations a theoretical model is used to derive an analytical expression for the minimum variance bound. The approximations used in the analytical treatment are tested using numerical simulations. It is concluded that approximately 14 nm depth resolution is achievable with flux levels available when tracking fluorescent sources in three dimensions in live-cell biology and that the method is suitable for three-dimensional photo-activated localization microscopy resolution. Sub-nanometre resolution could be achieved with photon-counting techniques at high flux levels.

Nanometric depth resolution from multi-focal images in microscopy

PubMed Central

Dalgarno, Heather I. C.; Dalgarno, Paul A.; Dada, Adetunmise C.; Towers, Catherine E.; Gibson, Gavin J.; Parton, Richard M.; Davis, Ilan; Warburton, Richard J.; Greenaway, Alan H.

2011-01-01

We describe a method for tracking the position of small features in three dimensions from images recorded on a standard microscope with an inexpensive attachment between the microscope and the camera. The depth-measurement accuracy of this method is tested experimentally on a wide-field, inverted microscope and is shown to give approximately 8 nm depth resolution, over a specimen depth of approximately 6 µm, when using a 12-bit charge-coupled device (CCD) camera and very bright but unresolved particles. To assess low-flux limitations a theoretical model is used to derive an analytical expression for the minimum variance bound. The approximations used in the analytical treatment are tested using numerical simulations. It is concluded that approximately 14 nm depth resolution is achievable with flux levels available when tracking fluorescent sources in three dimensions in live-cell biology and that the method is suitable for three-dimensional photo-activated localization microscopy resolution. Sub-nanometre resolution could be achieved with photon-counting techniques at high flux levels. PMID:21247948

Development and Applications of Laminar Optical Tomography for In Vivo Imaging

NASA Astrophysics Data System (ADS)

Burgess, Sean A.

Laminar optical tomography (LOT) is an optical imaging technique capable of making depth-resolved measurements of absorption and fluorescence contrast in scattering tissue. LOT was first demonstrated in 2004 by Hillman et al [1]. The technique combines a non-contact laser scanning geometry, similar to a low magnification confocal microscope, with the imaging principles of diffuse optical tomography (DOT). This thesis describes the development and application of a second generation LOT system, which acquires both fluorescence and multi-wavelength measurements simultaneously and is better suited for in vivo measurements. Chapter 1 begins by reviewing the interactions of light with tissue that form the foundation of optical imaging. A range of related optical imaging techniques and the basic principles of LOT imaging are then described. In Chapter 2, the development of the new LOT imaging system is described including the implementation of a series of interfaces to allow clinical imaging. System performance is then evaluated on a range of imaging phantoms. Chapter 3 describes two in vivo imaging applications explored using the second generation LOT system, first in a clinical setting where skin lesions were imaged, and then in a laboratory setting where LOT imaging was performed on exposed rat cortex. The final chapter provides a brief summary and describes future directions for LOT. LOT has the potential to find applications in medical diagnostics, surgical guidance, and in-situ monitoring owing to its sensitivity to absorption and fluorescence contrast as well as its ability to provide depth sensitive measures. Optical techniques can characterize blood volume and oxygenation, two important biological parameters, through measurements at different wavelengths. Fluorescence measurements, either from autofluorescence or fluorescent dyes, have shown promise for identifying and analyzing lesions in various epithelial tissues including skin [2, 3], colon [4], esophagus [5, 6], oral mucosa [7, 8], and cervix [9]. The desire to capture these types of measurements with LOT motivated much of the work presented here.

Optics clustered to output unique solutions: A multi-laser facility for combined single molecule and ensemble microscopy

NASA Astrophysics Data System (ADS)

Clarke, David T.; Botchway, Stanley W.; Coles, Benjamin C.; Needham, Sarah R.; Roberts, Selene K.; Rolfe, Daniel J.; Tynan, Christopher J.; Ward, Andrew D.; Webb, Stephen E. D.; Yadav, Rahul; Zanetti-Domingues, Laura; Martin-Fernandez, Marisa L.

2011-09-01

Optics clustered to output unique solutions (OCTOPUS) is a microscopy platform that combines single molecule and ensemble imaging methodologies. A novel aspect of OCTOPUS is its laser excitation system, which consists of a central core of interlocked continuous wave and pulsed laser sources, launched into optical fibres and linked via laser combiners. Fibres are plugged into wall-mounted patch panels that reach microscopy end-stations in adjacent rooms. This allows multiple tailor-made combinations of laser colours and time characteristics to be shared by different end-stations minimising the need for laser duplications. This setup brings significant benefits in terms of cost effectiveness, ease of operation, and user safety. The modular nature of OCTOPUS also facilitates the addition of new techniques as required, allowing the use of existing lasers in new microscopes while retaining the ability to run the established parts of the facility. To date, techniques interlinked are multi-photon/multicolour confocal fluorescence lifetime imaging for several modalities of fluorescence resonance energy transfer (FRET) and time-resolved anisotropy, total internal reflection fluorescence, single molecule imaging of single pair FRET, single molecule fluorescence polarisation, particle tracking, and optical tweezers. Here, we use a well-studied system, the epidermal growth factor receptor network, to illustrate how OCTOPUS can aid in the investigation of complex biological phenomena.

Using DNA origami nanorulers as traceable distance measurement standards and nanoscopic benchmark structures.

PubMed

Raab, Mario; Jusuk, Ija; Molle, Julia; Buhr, Egbert; Bodermann, Bernd; Bergmann, Detlef; Bosse, Harald; Tinnefeld, Philip

2018-01-29

In recent years, DNA origami nanorulers for superresolution (SR) fluorescence microscopy have been developed from fundamental proof-of-principle experiments to commercially available test structures. The self-assembled nanostructures allow placing a defined number of fluorescent dye molecules in defined geometries in the nanometer range. Besides the unprecedented control over matter on the nanoscale, robust DNA origami nanorulers are reproducibly obtained in high yields. The distances between their fluorescent marks can be easily analysed yielding intermark distance histograms from many identical structures. Thus, DNA origami nanorulers have become excellent reference and training structures for superresolution microscopy. In this work, we go one step further and develop a calibration process for the measured distances between the fluorescent marks on DNA origami nanorulers. The superresolution technique DNA-PAINT is used to achieve nanometrological traceability of nanoruler distances following the guide to the expression of uncertainty in measurement (GUM). We further show two examples how these nanorulers are used to evaluate the performance of TIRF microscopes that are capable of single-molecule localization microscopy (SMLM).

Intraoperative spatial frequency domain diffuse optical tomography with indo-cyanine green (ICG) fluorescence contrast (Conference Presentation)

NASA Astrophysics Data System (ADS)

Chong, Sang Hoon; Parthasarathy, Ashwin B.; Kavuri, Venkaiah C.; Moscatelli, Frank A.; Singhal, Sunil; Yodh, Arjun G.

2017-02-01

Surgical resection is the most effective treatment strategy for solid tumors, but complete removal of the tumor is critical for post-surgical recovery/long-term survival and is dependent on correct identification of the tumor margin and accurate excision of microscopic residual tumor in the surgical field. Fluorescence image guided surgery is an emerging technique that has shown promise for intraoperative location of tumors and tumor margins. Current versions of such intraoperative fluorescence imaging, however, are generally limited to 2D near-surface images, i.e., without information about tumor depth. Here we present an intraoperative fluorescence imaging system for 3D volumetric imaging of tumors; the system uses spatial frequency domain diffuse optical tomography with an analytic inversion reconstruction method. The new instrument can derive depth-sensitive 3D tumor images at depths up to 1 cm, and it employs compact epi-imaging and illumination suitable for the operating room, with quasi-real-time image reconstruction for surgical visualization. We present experimental results with FDA-approved Indocynanine Green using an extensive array of tissue phantoms and in a pilot in-vivo study.

Benchtop and animal validation of a portable fluorescence microscopic imaging system for potential use in cholecystectomy

NASA Astrophysics Data System (ADS)

Ye, Jian; Liu, Guanghui; Liu, Peng; Zhang, Shiwu; Shao, Pengfei; Smith, Zachary J.; Liu, Chenhai; Xu, Ronald X.

2018-02-01

We propose a portable fluorescence microscopic imaging system (PFMS) for intraoperative display of biliary structure and prevention of iatrogenic injuries during cholecystectomy. The system consists of a light source module, a camera module, and a Raspberry Pi computer with an LCD. Indocyanine green (ICG) is used as a fluorescent contrast agent for experimental validation of the system. Fluorescence intensities of the ICG aqueous solution at different concentration levels are acquired by our PFMS and compared with those of a commercial Xenogen IVIS system. We study the fluorescence detection depth by superposing different thicknesses of chicken breast on an ICG-loaded agar phantom. We verify the technical feasibility for identifying potential iatrogenic injury in cholecystectomy using a rat model in vivo. The proposed PFMS system is portable, inexpensive, and suitable for deployment in resource-limited settings.

Automatic tracking of cells for video microscopy in patch clamp experiments

PubMed Central

2014-01-01

Background Visualisation of neurons labeled with fluorescent proteins or compounds generally require exposure to intense light for a relatively long period of time, often leading to bleaching of the fluorescent probe and photodamage of the tissue. Here we created a technique to drastically shorten light exposure and improve the targeting of fluorescent labeled cells that is specially useful for patch-clamp recordings. We applied image tracking and mask overlay to reduce the time of fluorescence exposure and minimise mistakes when identifying neurons. Methods Neurons are first identified according to visual criteria (e.g. fluorescence protein expression, shape, viability etc.) and a transmission microscopy image Differential Interference Contrast (DIC) or Dodt contrast containing the cell used as a reference for the tracking algorithm. A fluorescence image can also be acquired later to be used as a mask (that can be overlaid on the target during live transmission video). As patch-clamp experiments require translating the microscope stage, we used pattern matching to track reference neurons in order to move the fluorescence mask to match the new position of the objective in relation to the sample. For the image processing we used the Open Source Computer Vision (OpenCV) library, including the Speeded-Up Robust Features (SURF) for tracking cells. The dataset of images (n = 720) was analyzed under normal conditions of acquisition and with influence of noise (defocusing and brightness). Results We validated the method in dissociated neuronal cultures and fresh brain slices expressing Enhanced Yellow Fluorescent Protein (eYFP) or Tandem Dimer Tomato (tdTomato) proteins, which considerably decreased the exposure to fluorescence excitation, thereby minimising photodamage. We also show that the neuron tracking can be used in differential interference contrast or Dodt contrast microscopy. Conclusion The techniques of digital image processing used in this work are an important addition to the set of microscopy tools used in modern electrophysiology, specially in experiments with neuron cultures and brain slices. PMID:24946774

Automatic tracking of cells for video microscopy in patch clamp experiments.

PubMed

Peixoto, Helton M; Munguba, Hermany; Cruz, Rossana M S; Guerreiro, Ana M G; Leao, Richardson N

2014-06-20

Visualisation of neurons labeled with fluorescent proteins or compounds generally require exposure to intense light for a relatively long period of time, often leading to bleaching of the fluorescent probe and photodamage of the tissue. Here we created a technique to drastically shorten light exposure and improve the targeting of fluorescent labeled cells that is specially useful for patch-clamp recordings. We applied image tracking and mask overlay to reduce the time of fluorescence exposure and minimise mistakes when identifying neurons. Neurons are first identified according to visual criteria (e.g. fluorescence protein expression, shape, viability etc.) and a transmission microscopy image Differential Interference Contrast (DIC) or Dodt contrast containing the cell used as a reference for the tracking algorithm. A fluorescence image can also be acquired later to be used as a mask (that can be overlaid on the target during live transmission video). As patch-clamp experiments require translating the microscope stage, we used pattern matching to track reference neurons in order to move the fluorescence mask to match the new position of the objective in relation to the sample. For the image processing we used the Open Source Computer Vision (OpenCV) library, including the Speeded-Up Robust Features (SURF) for tracking cells. The dataset of images (n = 720) was analyzed under normal conditions of acquisition and with influence of noise (defocusing and brightness). We validated the method in dissociated neuronal cultures and fresh brain slices expressing Enhanced Yellow Fluorescent Protein (eYFP) or Tandem Dimer Tomato (tdTomato) proteins, which considerably decreased the exposure to fluorescence excitation, thereby minimising photodamage. We also show that the neuron tracking can be used in differential interference contrast or Dodt contrast microscopy. The techniques of digital image processing used in this work are an important addition to the set of microscopy tools used in modern electrophysiology, specially in experiments with neuron cultures and brain slices.

Fluorescence Molecular Tomography: Principles and Potential for Pharmaceutical Research

PubMed Central

Stuker, Florian; Ripoll, Jorge; Rudin, Markus

2011-01-01

Fluorescence microscopic imaging is widely used in biomedical research to study molecular and cellular processes in cell culture or tissue samples. This is motivated by the high inherent sensitivity of fluorescence techniques, the spatial resolution that compares favorably with cellular dimensions, the stability of the fluorescent labels used and the sophisticated labeling strategies that have been developed for selectively labeling target molecules. More recently, two and three-dimensional optical imaging methods have also been applied to monitor biological processes in intact biological organisms such as animals or even humans. These whole body optical imaging approaches have to cope with the fact that biological tissue is a highly scattering and absorbing medium. As a consequence, light propagation in tissue is well described by a diffusion approximation and accurate reconstruction of spatial information is demanding. While in vivo optical imaging is a highly sensitive method, the signal is strongly surface weighted, i.e., the signal detected from the same light source will become weaker the deeper it is embedded in tissue, and strongly depends on the optical properties of the surrounding tissue. Derivation of quantitative information, therefore, requires tomographic techniques such as fluorescence molecular tomography (FMT), which maps the three-dimensional distribution of a fluorescent probe or protein concentration. The combination of FMT with a structural imaging method such as X-ray computed tomography (CT) or Magnetic Resonance Imaging (MRI) will allow mapping molecular information on a high definition anatomical reference and enable the use of prior information on tissue's optical properties to enhance both resolution and sensitivity. Today many of the fluorescent assays originally developed for studies in cellular systems have been successfully translated for experimental studies in animals. The opportunity of monitoring molecular processes non-invasively in the intact organism is highly attractive from a diagnostic point of view but even more so for the drug developer, who can use the techniques for proof-of-mechanism and proof-of-efficacy studies. This review shall elucidate the current status and potential of fluorescence tomography including recent advances in multimodality imaging approaches for preclinical and clinical drug development. PMID:24310495

eduSPIM: Light Sheet Microscopy in the Museum.

PubMed

Jahr, Wiebke; Schmid, Benjamin; Weber, Michael; Huisken, Jan

2016-01-01

Light sheet microscopy (or selective plane illumination microscopy) is an important imaging technique in the life sciences. At the same time, this technique is also ideally suited for community outreach projects, because it produces visually appealing, highly dynamic images of living organisms and its working principle can be understood with basic optics knowledge. Still, the underlying concepts are widely unknown to the non-scientific public. On the occasion of the UNESCO International Year of Light, a technical museum in Dresden, Germany, launched a special, interactive exhibition. We built a fully functional, educational selective plane illumination microscope (eduSPIM) to demonstrate how developments in microscopy promote discoveries in biology. To maximize educational impact, we radically reduced a standard light sheet microscope to its essential components without compromising functionality and incorporated stringent safety concepts beyond those needed in the lab. Our eduSPIM system features one illumination and one detection path and a sealed sample chamber. We image fixed zebrafish embryos with fluorescent vasculature, because the structure is meaningful to laymen and visualises the optical principles of light sheet microscopy. Via a simplified interface, visitors acquire fluorescence and transmission data simultaneously. The universal concepts presented here may also apply to other scientific approaches that are communicated to laymen in interactive settings. The specific eduSPIM design is adapted easily for various outreach and teaching activities. eduSPIM may even prove useful for labs needing a simple SPIM. A detailed parts list and schematics to rebuild eduSPIM are provided.

Fluorescent probes as a tool for labelling and tracking the amphibian chytrid fungus Batrachochytrium dendrobatidis.

PubMed

Herbert, Sarah M; Leung, Tommy L F; Bishop, Phillip J

2011-09-09

The dissemination of the virulent pathogen Batrachochytrium dendrobatidis (Bd) has contributed to the decline and extinction of many amphibian species worldwide. Several different strains have been identified, some of which are sympatric. Interactions between co-infecting strains of a pathogen can have significant influences on disease epidemiology and evolution; therefore the dynamics of multi-strain infections is an important area of research. We stained Bd cells with 2 fluorescent BODIPY fatty acid probes to determine whether these can potentially be used to distinguish and track Bd cell lines in multi-strain experiments. Bd cells in broth culture were stained with 5 concentrations of green-fluorescent BODIPY FL and red-fluorescent BODIPY 558/568 and visualised under an epifluorescent microscope for up to 16 d post-dye. Dyed strains were also assessed for growth inhibition. The most effective concentration for both dyes was 10 pM. This concentration of dye produced strong fluorescence for 12 to 16 d in Bd cultures held at 23 degrees C (3 to 4 generations), and did not inhibit Bd growth. Cells dyed with BODIPY FL and BODIPY 558/568 can be distinguished from each other on the basis of their fluorescence characteristics. Therefore, it is likely that this technique will be useful for research into multi-strain dynamics of Bd infections.

Lateral resolution testing of a novel developed confocal microscopic imaging system

NASA Astrophysics Data System (ADS)

Zhang, Xin; Zhang, Yunhai; Chang, Jian; Huang, Wei; Xue, Xiaojun; Xiao, Yun

2015-10-01

Laser scanning confocal microscope has been widely used in biology, medicine and material science owing to its advantages of high resolution and tomographic imaging. Based on a set of confirmatory experiments and system design, a novel confocal microscopic imaging system is developed. The system is composed of a conventional fluorescence microscope and a confocal scanning unit. In the scanning unit a laser beam coupling module provides four different wavelengths 405nm 488nm 561nm and 638nm which can excite a variety of dyes. The system works in spot-to-spot scanning mode with a two-dimensional galvanometer. A 50 microns pinhole is used to guarantee that stray light is blocked and only the fluorescence signal from the focal point can be received . The three-channel spectral splitter is used to perform fluorescence imaging at three different working wavelengths simultaneously. The rat kidney tissue slice is imaged using the developed confocal microscopic imaging system. Nucleues labeled by DAPI and kidney spherule curved pipe labeled by Alexa Fluor 488 can be imaged clearly and respectively, realizing the distinction between the different components of mouse kidney tissue. The three-dimensional tomographic imaging of mouse kidney tissue is reconstructed by several two-dimensional images obtained in different depths. At last the resolution of the confocal microscopic imaging system is tested quantitatively. The experimental result shows that the system can achieve lateral resolution priority to 230nm.

Halide (Cl(super -)) Quenching of Quinine Sulfate Fluorescence: A Time-Resolved Fluorescence Experiment for Physical Chemistry

ERIC Educational Resources Information Center

Gutow, Jonathan H.

2005-01-01

The time-resolved fluorescence experiment investigating the halide quenching of fluorescence from quinine sulfate in water is described. The objectives of the experiment include reinforcing student understanding of the kinetics of competing pathways, making connections with microscopic theories of kinetics through comparison of experimental and…

Chemical characterization of surface precipitates in La0.7Sr0.3Co0.2Fe0.8O3-δ as cathode material for solid oxide fuel cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Yang; Nikiforov, Alexey Y.; Kaspar, Tiffany C.

2016-11-01

In this study, a strontium doped lanthanum cobalt ferrite thin film with 30% Sr on A-site, denoted as La0.7Sr0.3Co0.2Fe0.8O3-δ or LSCF-7328, was investigated before and after annealing at 800 °C under CO2 containing atmosphere for 9 hours. The formation of secondary phases on surface of post-annealed LSCF-7328 has been observed using atomic force microscope (AFM) and scanning electron microscope (SEM). The extent of Sr segregation at the film surface was observed using the synchrotron-based total reflection X-ray fluorescence (TXRF) technique. The bonding environment of the secondary phases formed on the surface was investigated by synchrotron-based hard X-ray photoelectron spectroscopy (HAXPES).more » Transmission electron microscope (TEM) and related spectroscopy techniques were used for microstructural and quantitative elemental analyses of the secondary phases on surface. These studies revealed that the secondary phases on surface consisted of SrO covered with a capping layer of SrCO3. The formation of Co-rich phases has also been observed on the surface of post-annealed LSCF-7328.« less

Improved recognition of figures containing fluorescence microscope images in online journal articles using graphical models.

PubMed

Qian, Yuntao; Murphy, Robert F

2008-02-15

There is extensive interest in automating the collection, organization and analysis of biological data. Data in the form of images in online literature present special challenges for such efforts. The first steps in understanding the contents of a figure are decomposing it into panels and determining the type of each panel. In biological literature, panel types include many kinds of images collected by different techniques, such as photographs of gels or images from microscopes. We have previously described the SLIF system (http://slif.cbi.cmu.edu) that identifies panels containing fluorescence microscope images among figures in online journal articles as a prelude to further analysis of the subcellular patterns in such images. This system contains a pretrained classifier that uses image features to assign a type (class) to each separate panel. However, the types of panels in a figure are often correlated, so that we can consider the class of a panel to be dependent not only on its own features but also on the types of the other panels in a figure. In this article, we introduce the use of a type of probabilistic graphical model, a factor graph, to represent the structured information about the images in a figure, and permit more robust and accurate inference about their types. We obtain significant improvement over results for considering panels separately. The code and data used for the experiments described here are available from http://murphylab.web.cmu.edu/software.

An automated system for whole microscopic image acquisition and analysis.

PubMed

Bueno, Gloria; Déniz, Oscar; Fernández-Carrobles, María Del Milagro; Vállez, Noelia; Salido, Jesús

2014-09-01

The field of anatomic pathology has experienced major changes over the last decade. Virtual microscopy (VM) systems have allowed experts in pathology and other biomedical areas to work in a safer and more collaborative way. VMs are automated systems capable of digitizing microscopic samples that were traditionally examined one by one. The possibility of having digital copies reduces the risk of damaging original samples, and also makes it easier to distribute copies among other pathologists. This article describes the development of an automated high-resolution whole slide imaging (WSI) system tailored to the needs and problems encountered in digital imaging for pathology, from hardware control to the full digitization of samples. The system has been built with an additional digital monochromatic camera together with the color camera by default and LED transmitted illumination (RGB). Monochrome cameras are the preferred method of acquisition for fluorescence microscopy. The system is able to digitize correctly and form large high resolution microscope images for both brightfield and fluorescence. The quality of the digital images has been quantified using three metrics based on sharpness, contrast and focus. It has been proved on 150 tissue samples of brain autopsies, prostate biopsies and lung cytologies, at five magnifications: 2.5×, 10×, 20×, 40×, and 63×. The article is focused on the hardware set-up and the acquisition software, although results of the implemented image processing techniques included in the software and applied to the different tissue samples are also presented. © 2014 Wiley Periodicals, Inc.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

PubMed Central

Pino, Elizabeth C.; Webster, Christopher M.; Carr, Christopher E.; Soukas, Alexander A.

2013-01-01

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research. PMID:23568026

Ultra-compact fiber-optic two-photon microscope for functional fluorescence imaging in vivo.

PubMed

Engelbrecht, Christoph J; Johnston, Richard S; Seibel, Eric J; Helmchen, Fritjof

2008-04-14

We present a small, lightweight two-photon fiberscope and demonstrate its suitability for functional imaging in the intact brain. Our device consists of a hollow-core photonic crystal fiber for efficient delivery of near-IR femtosecond laser pulses, a spiral fiber-scanner for resonant beam steering, and a gradient-index lens system for fluorescence excitation, dichroic beam splitting, and signal collection. Fluorescence light is remotely detected using a standard photomultiplier tube. All optical components have 1 mm dimensions and the microscope's headpiece weighs only 0.6 grams. The instrument achieves micrometer resolution at frame rates of typically 25 Hz with a field-of-view of up to 200 microns. We demonstrate functional imaging of calcium signals in Purkinje cell dendrites in the cerebellum of anesthetized rats. The microscope will be easily portable by a rat or mouse and thus should enable functional imaging in freely behaving animals.

Confocal fluorescence microscope with dual-axis architecture and biaxial postobjective scanning

PubMed Central

Wang, Thomas D.; Contag, Christopher H.; Mandella, Michael J.; Chan, Ning Y.; Kino, Gordon S.

2007-01-01

We present a novel confocal microscope that has dual-axis architecture and biaxial postobjective scanning for the collection of fluorescence images from biological specimens. This design uses two low-numerical-aperture lenses to achieve high axial resolution and long working distance, and the scanning mirror located distal to the lenses rotates along the orthogonal axes to produce arc-surface images over a large field of view (FOV). With fiber optic coupling, this microscope can potentially be scaled down to millimeter dimensions via microelectromechanical systems (MEMS) technology. We demonstrate a benchtop prototype with a spatial resolution ≤4.4 μm that collects fluorescence images with a high SNR and a good contrast ratio from specimens expressing GFP. Furthermore, the scanning mechanism produces only small differences in aberrations over the image FOV. These results demonstrate proof of concept of the dual-axis confocal architecture for in vivo molecular and cellular imaging. PMID:15250760

Fluorescence intensity and bright spot analyses using a confocal microscope for photodynamic diagnosis of brain tumors.

PubMed

Yoneyama, Takeshi; Watanabe, Tetsuyo; Kagawa, Hiroyuki; Hayashi, Yutaka; Nakada, Mitsutoshi

2017-03-01

In photodynamic diagnosis using 5-aminolevulinic acid (5-ALA), discrimination between the tumor and normal tissue is very important for a precise resection. However, it is difficult to distinguish between infiltrating tumor and normal regions in the boundary area. In this study, fluorescent intensity and bright spot analyses using a confocal microscope is proposed for the precise discrimination between infiltrating tumor and normal regions. From the 5-ALA-resected brain tumor tissue, the red fluorescent and marginal regions were sliced for observation under a confocal microscope. Hematoxylin and eosin (H&E) staining were performed on serial slices of the same tissue. According to the pathological inspection of the H&E slides, the tumor and infiltrating and normal regions on confocal microscopy images were investigated. From the fluorescent intensity of the image pixels, a histogram of pixel number with the same fluorescent intensity was obtained. The fluorescent bright spot sizes and total number were compared between the marginal and normal regions. The fluorescence intensity distribution and average intensity in the tumor were different from those in the normal region. The probability of a difference from the dark enhanced the difference between the tumor and the normal region. The bright spot size and number in the infiltrating tumor were different from those in the normal region. Fluorescence intensity analysis is useful to distinguish a tumor region, and a bright spot analysis is useful to distinguish between infiltrating tumor and normal regions. These methods will be important for the precise resection or photodynamic therapy of brain tumors. Copyright © 2016 Elsevier B.V. All rights reserved.

A Clinical Wide-Field Fluorescence Endoscopic Device for Molecular Imaging Demonstrating Cathepsin Protease Activity in Colon Cancer

PubMed Central

Sensarn, Steven; Zavaleta, Cristina L.; Segal, Ehud; Rogalla, Stephan; Lee, Wansik; Gambhir, Sanjiv S.; Bogyo, Matthew; Contag, Christopher H.

2017-01-01

Purpose Early and effective detection of cancers of the gastrointestinal tract will require novel molecular probes and advances in instrumentation that can reveal functional changes in dysplastic and malignant tissues. Here, we describe adaptation of a wide-field clinical fiberscope to perform wide-field fluorescence imaging while preserving its white-light capability for the purpose of providing wide-field fluorescence imaging capability to point-of-care microscopes. Procedures We developed and used a fluorescent fiberscope to detect signals from a quenched probe, BMV109, that becomes fluorescent when cleaved by, and covalently bound to, active cathepsin proteases. Cathepsins are expressed in inflammation- and tumor-associated macrophages as well as directly from tumor cells and are a promising target for cancer imaging. The fiberscope has a 1-mm outer diameter enabling validation via endoscopic exams in mice, and therefore we evaluated topically applied BMV109 for the ability to detect colon polyps in an azoxymethane-induced colon tumor model in mice. Results This wide-field endoscopic imaging device revealed consistent and clear fluorescence signals from BMV109 that specifically localized to the polypoid regions as opposed to the normal adjacent colon tissue (p < 0.004) in the murine colon carcinoma model. Conclusions The sensitivity of detection of BMV109 with the fluorescence fiberscope suggested utility of these tools for early detection at hard-to-reach sites. The fiberscope was designed to be used in conjunction with miniature, endoscope-compatible fluorescence microscopes for dual wide-field and microscopic cancer detection. PMID:27154508

Dynamic Assessment of the Endothelialization of Tissue-Engineered Blood Vessels Using an Optical Coherence Tomography Catheter-Based Fluorescence Imaging System.

PubMed

Gurjarpadhye, Abhijit Achyut; DeWitt, Matthew R; Xu, Yong; Wang, Ge; Rylander, Marissa Nichole; Rylander, Christopher G

2015-07-01

Lumen endothelialization of bioengineered vascular scaffolds is essential to maintain small-diameter graft patency and prevent thrombosis postimplantation. Unfortunately, nondestructive imaging methods to visualize this dynamic process are lacking, thus slowing development and clinical translation of these potential tissue-engineering approaches. To meet this need, a fluorescence imaging system utilizing a commercial optical coherence tomography (OCT) catheter was designed to visualize graft endothelialization. C7 DragonFly™ intravascular OCT catheter was used as a channel for delivery and collection of excitation and emission spectra. Poly-dl-lactide (PDLLA) electrospun scaffolds were seeded with endothelial cells (ECs). Seeded cells were exposed to Calcein AM before imaging, causing the living cells to emit green fluorescence in response to blue laser. By positioning the catheter tip precisely over a specimen using high-fidelity electromechanical components, small regions of the specimen were excited selectively. The resulting fluorescence intensities were mapped on a two-dimensional digital grid to generate spatial distribution of fluorophores at single-cell-level resolution. Fluorescence imaging of endothelialization on glass and PDLLA scaffolds was performed using the OCT catheter-based imaging system as well as with a commercial fluorescence microscope. Cell coverage area was calculated for both image sets for quantitative comparison of imaging techniques. Tubular PDLLA scaffolds were maintained in a bioreactor on seeding with ECs, and endothelialization was monitored over 5 days using the OCT catheter-based imaging system. No significant difference was observed in images obtained using our imaging system to those acquired with the fluorescence microscope. Cell area coverage calculated using the images yielded similar values. Nondestructive imaging of endothelialization on tubular scaffolds showed cell proliferation with cell coverage area increasing from 15 ± 4% to 89 ± 6% over 5 days. In this study, we showed the capability of an OCT catheter-based imaging system to obtain single-cell resolution and to quantify endothelialization in tubular electrospun scaffolds. We also compared the resulting images with traditional microscopy, showing high fidelity in image capability. This imaging system, used in conjunction with OCT, could potentially be a powerful tool for in vitro optimization of scaffold cellularization, ensuring long-term graft patency postimplantation.

Observation of development of breast cancer cell lines in real time by fluorescence microscopy under simulated microgravity

NASA Astrophysics Data System (ADS)

Lavan, David; Valdivia-Silva, Julio E.; Sanabria, Gabriela; Orihuela, Diego; Suarez, Juan; Quispe, Marco; Chuchon, Mariano; Martin, David; Maroto, Marcos; Egea, Javier

2016-07-01

This project consist in the implementation of a fluorescence microscope for the in real time monitoring of biological labeled samples by several fluorophores in microgravity conditions keeping the temperature, humidity, and (CO)2 controlled by an electronic platform. The system (fluorescence microscope and incubator) is integrated to a microgravity simulator machine which was presented on the "30th Annual American Society for Gravitation and Space Research Meeting" October 2014 in Pasadena, CA, USA. Currently, we have the microgravity machine biologically validated by genetic expression studies in pupal stage of Drosophila melanogaster. The fluorescence microscope has a platform designed to hold a culture flask, and a fluorescence camera (Leica DFC3000 G) connected to an optical system (Fluorescence Light source Leica EL6000, optic fiber, fiber adapter, and fluorescence filter) in order to take images in real time. The mechanical system of the fluorescence microsc ope is designed to allow the displacement of the fluorescence camera through a parallel plane to the culture flask's plane and also the movement of the platform through a perpendicular axis to the culture flask in order to focus the samples to the optical system. The mechanical system is propelled by four DC moto-reductors with encoder (A-max 26 Maxon motor, GP 32S screw and MR encoder) that generate displacements in the order of micrometers. The angular position control of the DC motoreductor's shaft of all the DC moto-reductors is done by PWM signals based on the interpretation of the signals provided by the encoders during the movement. The system is remotely operated by a graphic interface installed on a personal computer or any mobile device (smartphone, laptop or tablet) by using the internet. Acknowledgments: Grant of INNOVATE PERU (Formerly FINCYT)

Labeling single cell for in-vivo study of cell fate mapping and lineage tracing

NASA Astrophysics Data System (ADS)

He, Sicong; Xu, Jin; Wu, Yi; Tian, Ye; Sun, Qiqi; Wen, Zilong; Qu, Jianan Y.

2018-02-01

Cell fate mapping and lineage tracing are significant ways to understanding the developmental origins of biological tissues. It requires labeling individual cells and tracing the development of their progeny. We develop an infrared laser-evoked gene operator heat-shock microscope system to achieve single-cell labeling in zebrafish. With a fluorescent thermometry technique, we measure the temperature increase in zebrafish tissues induced by infrared laser and identify the optimal heat shock conditions for single-cell gene induction in different types of zebrafish cells. We use this technique to study the fate mapping of T lymphocytes and discover the distinct waves of lymphopoiesis during the zebrafish development.

Visualizing and quantifying the in vivo structure and dynamics of the Arabidopsis cortical cytoskeleton using CLSM and VAEM.

PubMed

Rosero, Amparo; Zárský, Viktor; Cvrčková, Fatima

2014-01-01

The cortical microtubules, and to some extent also the actin meshwork, play a central role in the shaping of plant cells. Transgenic plants expressing fluorescent protein markers specifically tagging the two main cytoskeletal systems are available, allowing noninvasive in vivo studies. Advanced microscopy techniques, in particular confocal laser scanning microscopy (CLSM) and variable angle epifluorescence microscopy (VAEM), can be nowadays used for imaging the cortical cytoskeleton of living cells with unprecedented spatial and temporal resolution. With the aid of suitable computing techniques, quantitative information can be extracted from microscopic images and video sequences, providing insight into both architecture and dynamics of the cortical cytoskeleton.

Transmission X-ray microscopy for full-field nano-imaging of biomaterials

PubMed Central

ANDREWS, JOY C; MEIRER, FLORIAN; LIU, YIJIN; MESTER, ZOLTAN; PIANETTA, PIERO

2010-01-01

Imaging of cellular structure and extended tissue in biological materials requires nanometer resolution and good sample penetration, which can be provided by current full-field transmission X-ray microscopic techniques in the soft and hard X-ray regions. The various capabilities of full-field transmission X-ray microscopy (TXM) include 3D tomography, Zernike phase contrast, quantification of absorption, and chemical identification via X-ray fluorescence and X-ray absorption near edge structure (XANES) imaging. These techniques are discussed and compared in light of results from imaging of biological materials including microorganisms, bone and mineralized tissue and plants, with a focus on hard X-ray TXM at ≤ 40 nm resolution. PMID:20734414

Antibacterial potential of rutin conjugated with thioglycolic acid capped cadmium telluride quantum dots (TGA-CdTe QDs)

NASA Astrophysics Data System (ADS)

Ananth, Devanesan Arul; Rameshkumar, Angappan; Jeyadevi, Ramachandran; Jagadeeswari, Sivanadanam; Nagarajan, Natarajan; Renganathan, Rajalingam; Sivasudha, Thilagar

2015-03-01

Quantum dots not only act as nanocarrier but also act as stable and resistant natural fluorescent bio markers used in various in vitro and in vivo photolabelling and biological applications. In this study, the antimicrobial potential of TGA-CdTe QDs and commercial phenolics (rutin and caffeine) were investigated against Escherichiacoli. UV absorbance and fluorescence quenching study of TGA-CdTe QDs with rutin and caffeine complex was measured by spectroscopic technique. QDs-rutin conjugate exhibited excellent quenching property due to the -OH groups present in the rutin structure. But the same time caffeine has not conjugated with QDs because of lacking of -OH group in its structure. Photolabelling of E. coli with QDs-rutin and QDs-caffeine complex was analyzed by fluorescent microscopic method. Microbe E. coli cell membrane damage was assessed by atomic force (AFM) and confocal microscopy. Based on the results obtained, it is suggested that QDs-rutin conjugate enhance the antimicrobial activity more than the treatment with QDs, rutin and caffeine alone.

Immunomicrospheres - Reagents for cell labeling and separation

NASA Technical Reports Server (NTRS)

Rembaum, A.; Dreyer, W. J.

1980-01-01

Immunomicrospheres are specially designed microscopic particles that have antibodies or similar molecules chemically bound to their surfaces. The antibody-coated microspheres react in a highly specific way with target cells, viruses, or other antigenic agents. Immunomicrospheres may be synthesized so that they incorporate compounds that are highly radioactive, intensely fluorescent, magnetic, electron opaque, highly colored, or pharmacologically active. These various types of microspheres may be coated with pure, highly specific monoclonal antibodies obtained by the new hybridoma cell cloning techniques or with conventional antibody preparations. Some of the many present and potential applications for these new reagents are (1) new types of radioimmune or immunofluorescent assays, (2) improved fluorescence microscopy, (3) separation of cells on the basis of the fluorescent, electrophoretic, or magnetic properties of bound immunomicrospheres, (4) markers for use in several types of electron or standard light microscopy, and (5) delivery of lethal compouds to specific undesirable living cells. The combination of the various new types of synthetic microspheres and the newly available homogeneous antibodies offers new opportunities in research, diagnosis, and therapy.

Real-time hyperspectral fluorescence imaging of pancreatic β-cell dynamics with the image mapping spectrometer

PubMed Central

Elliott, Amicia D.; Gao, Liang; Ustione, Alessandro; Bedard, Noah; Kester, Robert; Piston, David W.; Tkaczyk, Tomasz S.

2012-01-01

Summary The development of multi-colored fluorescent proteins, nanocrystals and organic fluorophores, along with the resulting engineered biosensors, has revolutionized the study of protein localization and dynamics in living cells. Hyperspectral imaging has proven to be a useful approach for such studies, but this technique is often limited by low signal and insufficient temporal resolution. Here, we present an implementation of a snapshot hyperspectral imaging device, the image mapping spectrometer (IMS), which acquires full spectral information simultaneously from each pixel in the field without scanning. The IMS is capable of real-time signal capture from multiple fluorophores with high collection efficiency (∼65%) and image acquisition rate (up to 7.2 fps). To demonstrate the capabilities of the IMS in cellular applications, we have combined fluorescent protein (FP)-FRET and [Ca2+]i biosensors to measure simultaneously intracellular cAMP and [Ca2+]i signaling in pancreatic β-cells. Additionally, we have compared quantitatively the IMS detection efficiency with a laser-scanning confocal microscope. PMID:22854044

Characterization of Cytokinetic Mutants Using Small Fluorescent Probes.

PubMed

Smertenko, Andrei; Moschou, Panagiotis; Zhang, Laining; Fahy, Deirdre; Bozhkov, Peter

2016-01-01

Cytokinesis is a powerful paradigm for addressing fundamental questions of plant biology including molecular mechanisms of development, cell division, cell signaling, membrane trafficking, cell wall synthesis, and cytoskeletal dynamics. Genetics was instrumental in identification of proteins regulating cytokinesis. Characterization of mutant lines generated using forward or reverse genetics includes microscopic analysis for defects in cell division. Typically, failure of cytokinesis results in appearance of multinucleate cells, formation of cell wall stubs, and isotropic cell expansion in the root elongation zone. Small fluorescent probes served as a very effective tool for the detection of cytokinetic defects. Such probes stain living or formaldehyde-fixed specimens avoiding complex preparatory steps. Although resolution of the fluorescence probes is inferior to electron microscopy, the procedure is fast, easy, and does not require expensive materials or equipment. This chapter describes techniques for staining DNA with the probes DAPI and SYTO82, for staining membranes with FM4-64, and for staining cell wall with propidium iodide.

Biological applications of near-field scanning optical microscopy

NASA Astrophysics Data System (ADS)

Moers, Marco H. P.; Ruiter, A. G. T.; Jalocha, Alain; van Hulst, Niko F.; Kalle, W. H. J.; Wiegant, J. C. A. G.; Raap, A. K.

1995-09-01

Near-field Scanning Optical Microscopy (NSOM) is a true optical microscopic technique allowing fluorescence, absorption, reflection and polarization contrast with the additional advantage of nanometer lateral resolution, unlimited by diffraction and operation at ambient conditions. NSOM based on metal coated adiabatically tapered fibers, combined with shear force feedback and operated in illumination mode, has proven to be the most powerful NSOM arrangement, because of its true localization of the optical interaction, its various optical contrast possibilities and its sensitivity down to the single molecular level. In this paper applications of `aperture' NSOM to Fluorescence In Situ Hybridization of human metaphase chromosomes are presented, where the localized fluorescence allows to identify specific DNA sequences. All images are accompanied by the simultaneously acquired force image, enabling direct comparison of the optical contrast with the sample topography on nanometer scale, far beyond the diffraction limit. Thus the unique combination of high resolution, specific optical contrast and ambient operation offers many new direction possibilities in biological studies.

Supercritical angle fluorescence as a tool to study the interaction between lipid bilayer and peptides

NASA Astrophysics Data System (ADS)

Dubois, Valentin; Serrano, Diana; Seeger, Stefan

2017-06-01

The understanding of processes occurring at the interface between two media are of prior importance in various fields of research, from material sciences to biology. A custom-made microscope objective based on the supercritical angle technique was developed in our group, allowing to probe these interfacial events by carrying out surface-sensitive and low invasive spectroscopy of aqueous samples. A biological example of particular interest is the comprehension of neurodegenerative diseases which seem caused by the interaction of specific peptides with the membrane of the neurons. Taking advantage of our optical setup, we used supercritical angle fluorescence spectroscopy to specifically monitor the interaction between a supported lipid bilayer (SLB) and the Amyloid β peptide, notably responsible of the Alzheimer disease. Different forms of the peptide (40 and 42 amino acids composition) were tested and the interfacial fluorescence measured to get information about the lipid integrity and mobility. The adsorption of the peptide was also characterized in terms of kinetic and affinity.

Hyperspectral imaging fluorescence excitation scanning for colon cancer detection

PubMed Central

Leavesley, Silas J.; Walters, Mikayla; Lopez, Carmen; Baker, Thomas; Favreau, Peter F.; Rich, Thomas C.; Rider, Paul F.; Boudreaux, Carole W.

2016-01-01

Abstract. Optical spectroscopy and hyperspectral imaging have shown the potential to discriminate between cancerous and noncancerous tissue with high sensitivity and specificity. However, to date, these techniques have not been effectively translated to real-time endoscope platforms. Hyperspectral imaging of the fluorescence excitation spectrum represents new technology that may be well suited for endoscopic implementation. However, the feasibility of detecting differences between normal and cancerous mucosa using fluorescence excitation-scanning hyperspectral imaging has not been evaluated. The goal of this study was to evaluate the initial feasibility of using fluorescence excitation-scanning hyperspectral imaging for measuring changes in fluorescence excitation spectrum concurrent with colonic adenocarcinoma using a small pre-pilot-scale sample size. Ex vivo analysis was performed using resected pairs of colorectal adenocarcinoma and normal mucosa. Adenocarcinoma was confirmed by histologic evaluation of hematoxylin and eosin (H&E) permanent sections. Specimens were imaged using a custom hyperspectral imaging fluorescence excitation-scanning microscope system. Results demonstrated consistent spectral differences between normal and cancerous tissues over the fluorescence excitation range of 390 to 450 nm that could be the basis for wavelength-dependent detection of colorectal cancers. Hence, excitation-scanning hyperspectral imaging may offer an alternative approach for discriminating adenocarcinoma from surrounding normal colonic mucosa, but further studies will be required to evaluate the accuracy of this approach using a larger patient cohort. PMID:27792808

FLIMX: A Software Package to Determine and Analyze the Fluorescence Lifetime in Time-Resolved Fluorescence Data from the Human Eye.

PubMed

Klemm, Matthias; Schweitzer, Dietrich; Peters, Sven; Sauer, Lydia; Hammer, Martin; Haueisen, Jens

2015-01-01

Fluorescence lifetime imaging ophthalmoscopy (FLIO) is a new technique for measuring the in vivo autofluorescence intensity decays generated by endogenous fluorophores in the ocular fundus. Here, we present a software package called FLIM eXplorer (FLIMX) for analyzing FLIO data. Specifically, we introduce a new adaptive binning approach as an optimal tradeoff between the spatial resolution and the number of photons required per pixel. We also expand existing decay models (multi-exponential, stretched exponential, spectral global analysis, incomplete decay) to account for the layered structure of the eye and present a method to correct for the influence of the crystalline lens fluorescence on the retina fluorescence. Subsequently, the Holm-Bonferroni method is applied to FLIO measurements to allow for group comparisons between patients and controls on the basis of fluorescence lifetime parameters. The performance of the new approaches was evaluated in five experiments. Specifically, we evaluated static and adaptive binning in a diabetes mellitus patient, we compared the different decay models in a healthy volunteer and performed a group comparison between diabetes patients and controls. An overview of the visualization capabilities and a comparison of static and adaptive binning is shown for a patient with macular hole. FLIMX's applicability to fluorescence lifetime imaging microscopy is shown in the ganglion cell layer of a porcine retina sample, obtained by a laser scanning microscope using two-photon excitation.

Automatic vision system for analysis of microscopic behavior of flow and transport in porous media

NASA Astrophysics Data System (ADS)

Rashidi, Mehdi; Dehmeshki, Jamshid; Dickenson, Eric; Daemi, M. Farhang

1997-10-01

This paper describes the development of a novel automated and efficient vision system to obtain velocity and concentration measurement within a porous medium. An aqueous fluid lace with a fluorescent dye to microspheres flows through a transparent, refractive-index-matched column packed with transparent crystals. For illumination purposes, a planar sheet of laser passes through the column as a CCD camera records all the laser illuminated planes. Detailed microscopic velocity and concentration fields have been computed within a 3D volume of the column. For measuring velocities, while the aqueous fluid, laced with fluorescent microspheres, flows through the transparent medium, a CCD camera records the motions of the fluorescing particles by a video cassette recorder. The recorded images are acquired automatically frame by frame and transferred to the computer for processing, by using a frame grabber an written relevant algorithms through an RS-232 interface. Since the grabbed image is poor in this stage, some preprocessings are used to enhance particles within images. Finally, these enhanced particles are monitored to calculate velocity vectors in the plane of the beam. For concentration measurements, while the aqueous fluid, laced with a fluorescent organic dye, flows through the transparent medium, a CCD camera sweeps back and forth across the column and records concentration slices on the planes illuminated by the laser beam traveling simultaneously with the camera. Subsequently, these recorded images are transferred to the computer for processing in similar fashion to the velocity measurement. In order to have a fully automatic vision system, several detailed image processing techniques are developed to match exact images that have different intensities values but the same topological characteristics. This results in normalized interstitial chemical concentrations as a function of time within the porous column.

Benchtop and animal validation of a portable fluorescence microscopic imaging system for potential use in cholecystectomy.

PubMed

Ye, Jian; Liu, Guanghui; Liu, Peng; Zhang, Shiwu; Shao, Pengfei; Smith, Zachary J; Liu, Chenhai; Xu, Ronald X

2018-02-01

We propose a portable fluorescence microscopic imaging system (PFMS) for intraoperative display of biliary structure and prevention of iatrogenic injuries during cholecystectomy. The system consists of a light source module, a camera module, and a Raspberry Pi computer with an LCD. Indocyanine green (ICG) is used as a fluorescent contrast agent for experimental validation of the system. Fluorescence intensities of the ICG aqueous solution at different concentration levels are acquired by our PFMS and compared with those of a commercial Xenogen IVIS system. We study the fluorescence detection depth by superposing different thicknesses of chicken breast on an ICG-loaded agar phantom. We verify the technical feasibility for identifying potential iatrogenic injury in cholecystectomy using a rat model in vivo. The proposed PFMS system is portable, inexpensive, and suitable for deployment in resource-limited settings. (2018) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

Rapid in situ hybridization technique using 16S rRNA segments for detecting and differentiating the closely related gram-positive organisms Bacillus polymyxa and Bacillus macerans

NASA Technical Reports Server (NTRS)

Jurtshuk, R. J.; Blick, M.; Bresser, J.; Fox, G. E.; Jurtshuk, P. Jr

1992-01-01

A rapid, sensitive, inexpensive in situ hybridization technique, using 30-mer 16S rRNA probes, can specifically differentiate two closely related Bacillus spp., B. polymyxa and B. macerans. The 16S rRNA probes were labeled with a rhodamine derivative (Texas Red), and quantitative fluorescence measurements were made on individual bacterial cells. The microscopic fields analyzed were selected by phase-contrast microscopy, and the fluorescence imaging analyses were performed on 16 to 67 individual cells. The labeled 16S rRNA probe, POL, whose sequence was a 100% match with B. polymyxa 16S rRNA but only a 60% match with B. macerans 16S rRNA, gave quantitative fluorescence ratio measurements that were 34.8-fold higher for B. polymyxa cells than for B. macerans cells. Conversely, the labeled probe, MAC, which matched B. polymyxa 16S rRNA in 86.6% of its positions and B. macerans 16S rRNA in 100% of its positions, gave quantitative fluorescence measurements that were 59.3-fold higher in B. macerans cells than in B. polymyxa cells. Control probes, whose 16S rRNA sequence segment (P-M) was present in both B. polymyxa and B. macerans as well as a panprokaryotic probe (16S), having a 100% match with all known bacteria, hybridized equally well with both organisms. These latter hybridizations generated very high fluorescence signals, but their comparative fluorescence ratios (the differences between two organisms) were low. The control paneukaryotic probe (28S), which had less than 30% identity for both B. macerans and B. polymyxa, did not hybridize with either organism.

Structural locus of transmucosal albumin efflux in canine ileum. A fluorescent study.

PubMed

Granger, D N; Cook, B H; Taylor, A E

1976-12-01

This study demonstrates the effects of elevated intestinal venous pressure on the intestinal tissue spaces and the histological locus of the transmucosal albumin flux under such conditions. The authors were able to localize albumin in the tissues using an Evans blue-albumin fluorescence technique. This technique makes use of the fluorescence properties and albumin affinity of Evans blue dye (T-1824). Evans blue dye has a high affinity for albumin and emits a red-orange fluorescence at a wavelength of 720 nm. Evans blue was mixed with a solution of bovine serum albumin at concentrations that yield negligible amounts of free dye. Control ileal samples were obtained in order to visualize the natural tissue morphology and fluorescence. The Evans blue-albumin solution was injected and tissue samples were obtained 15 and 60 min postinjection, then venous outflow was occluded and after 15 and 60 min the tissues were sampled. Each sample was immediately frozen, freeze dried, embedded in paraffin, and 7-mu sections were made. The Evans blue-albumin was demonstrated histologically with a fluorescence microscope. No leakage sites were apparent at normal venous pressures. However, after elevation of venous pressure, Evans blue-albumin was observed in the interepithelial and/or intraepithelial spaces of villus tips, but no Evans blue-albumin was observed either between or within the epithelial cells of the crypts, or within the tubular crypt lumina. These results indicate that at elevated venous pressures, the transmucosal albumin flux occurs exclusively at the villus tip region, suggesting a great vulnerability of the cells found in this region to elevations in tissue pressure as compared to the crypt epithelial cells.

Multimodal Light Microscopy Approaches to Reveal Structural and Functional Properties of Promyelocytic Leukemia Nuclear Bodies

PubMed Central

Hoischen, Christian; Monajembashi, Shamci; Weisshart, Klaus; Hemmerich, Peter

2018-01-01

The promyelocytic leukemia (pml) gene product PML is a tumor suppressor localized mainly in the nucleus of mammalian cells. In the cell nucleus, PML seeds the formation of macromolecular multiprotein complexes, known as PML nuclear bodies (PML NBs). While PML NBs have been implicated in many cellular functions including cell cycle regulation, survival and apoptosis their role as signaling hubs along major genome maintenance pathways emerged more clearly. However, despite extensive research over the past decades, the precise biochemical function of PML in these pathways is still elusive. It remains a big challenge to unify all the different previously suggested cellular functions of PML NBs into one mechanistic model. With the advent of genetically encoded fluorescent proteins it became possible to trace protein function in living specimens. In parallel, a variety of fluorescence fluctuation microscopy (FFM) approaches have been developed which allow precise determination of the biophysical and interaction properties of cellular factors at the single molecule level in living cells. In this report, we summarize the current knowledge on PML nuclear bodies and describe several fluorescence imaging, manipulation, FFM, and super-resolution techniques suitable to analyze PML body assembly and function. These include fluorescence redistribution after photobleaching, fluorescence resonance energy transfer, fluorescence correlation spectroscopy, raster image correlation spectroscopy, ultraviolet laser microbeam-induced DNA damage, erythrocyte-mediated force application, and super-resolution microscopy approaches. Since most if not all of the microscopic equipment to perform these techniques may be available in an institutional or nearby facility, we hope to encourage more researches to exploit sophisticated imaging tools for their research in cancer biology. PMID:29888200

Total variation based image deconvolution for extended depth-of-field microscopy images

NASA Astrophysics Data System (ADS)

Hausser, F.; Beckers, I.; Gierlak, M.; Kahraman, O.

2015-03-01

One approach for a detailed understanding of dynamical cellular processes during drug delivery is the use of functionalized biocompatible nanoparticles and fluorescent markers. An appropriate imaging system has to detect these moving particles so as whole cell volumes in real time with high lateral resolution in a range of a few 100 nm. In a previous study Extended depth-of-field microscopy (EDF-microscopy) has been applied to fluorescent beads and tradiscantia stamen hair cells and the concept of real-time imaging has been proved in different microscopic modes. In principle a phase retardation system like a programmable space light modulator or a static waveplate is incorporated in the light path and modulates the wavefront of light. Hence the focal ellipsoid is smeared out and images seem to be blurred in a first step. An image restoration by deconvolution using the known point-spread-function (PSF) of the optical system is necessary to achieve sharp microscopic images of an extended depth-of-field. This work is focused on the investigation and optimization of deconvolution algorithms to solve this restoration problem satisfactorily. This inverse problem is challenging due to presence of Poisson distributed noise and Gaussian noise, and since the PSF used for deconvolution exactly fits in just one plane within the object. We use non-linear Total Variation based image restoration techniques, where different types of noise can be treated properly. Various algorithms are evaluated for artificially generated 3D images as well as for fluorescence measurements of BPAE cells.

eSIP: A Novel Solution-Based Sectioned Image Property Approach for Microscope Calibration

PubMed Central

Butzlaff, Malte; Weigel, Arwed; Ponimaskin, Evgeni; Zeug, Andre

2015-01-01

Fluorescence confocal microscopy represents one of the central tools in modern sciences. Correspondingly, a growing amount of research relies on the development of novel microscopic methods. During the last decade numerous microscopic approaches were developed for the investigation of various scientific questions. Thereby, the former qualitative imaging methods became replaced by advanced quantitative methods to gain more and more information from a given sample. However, modern microscope systems being as complex as they are, require very precise and appropriate calibration routines, in particular when quantitative measurements should be compared over longer time scales or between different setups. Multispectral beads with sub-resolution size are often used to describe the point spread function and thus the optical properties of the microscope. More recently, a fluorescent layer was utilized to describe the axial profile for each pixel, which allows a spatially resolved characterization. However, fabrication of a thin fluorescent layer with matching refractive index is technically not solved yet. Therefore, we propose a novel type of calibration concept for sectioned image property (SIP) measurements which is based on fluorescent solution and makes the calibration concept available for a broader number of users. Compared to the previous approach, additional information can be obtained by application of this extended SIP chart approach, including penetration depth, detected number of photons, and illumination profile shape. Furthermore, due to the fit of the complete profile, our method is less susceptible to noise. Generally, the extended SIP approach represents a simple and highly reproducible method, allowing setup independent calibration and alignment procedures, which is mandatory for advanced quantitative microscopy. PMID:26244982

Quantitative fluorescence microscopy and image deconvolution.

PubMed

Swedlow, Jason R

2013-01-01

Quantitative imaging and image deconvolution have become standard techniques for the modern cell biologist because they can form the basis of an increasing number of assays for molecular function in a cellular context. There are two major types of deconvolution approaches--deblurring and restoration algorithms. Deblurring algorithms remove blur but treat a series of optical sections as individual two-dimensional entities and therefore sometimes mishandle blurred light. Restoration algorithms determine an object that, when convolved with the point-spread function of the microscope, could produce the image data. The advantages and disadvantages of these methods are discussed in this chapter. Image deconvolution in fluorescence microscopy has usually been applied to high-resolution imaging to improve contrast and thus detect small, dim objects that might otherwise be obscured. Their proper use demands some consideration of the imaging hardware, the acquisition process, fundamental aspects of photon detection, and image processing. This can prove daunting for some cell biologists, but the power of these techniques has been proven many times in the works cited in the chapter and elsewhere. Their usage is now well defined, so they can be incorporated into the capabilities of most laboratories. A major application of fluorescence microscopy is the quantitative measurement of the localization, dynamics, and interactions of cellular factors. The introduction of green fluorescent protein and its spectral variants has led to a significant increase in the use of fluorescence microscopy as a quantitative assay system. For quantitative imaging assays, it is critical to consider the nature of the image-acquisition system and to validate its response to known standards. Any image-processing algorithms used before quantitative analysis should preserve the relative signal levels in different parts of the image. A very common image-processing algorithm, image deconvolution, is used to remove blurred signal from an image. There are two major types of deconvolution approaches, deblurring and restoration algorithms. Deblurring algorithms remove blur, but treat a series of optical sections as individual two-dimensional entities, and therefore sometimes mishandle blurred light. Restoration algorithms determine an object that, when convolved with the point-spread function of the microscope, could produce the image data. The advantages and disadvantages of these methods are discussed. Copyright © 1998 Elsevier Inc. All rights reserved.

Volumetric HiLo microscopy employing an electrically tunable lens.

PubMed

Philipp, Katrin; Smolarski, André; Koukourakis, Nektarios; Fischer, Andreas; Stürmer, Moritz; Wallrabe, Ulrike; Czarske, Jürgen W

2016-06-27

Electrically tunable lenses exhibit strong potential for fast motion-free axial scanning in a variety of microscopes. However, they also lead to a degradation of the achievable resolution because of aberrations and misalignment between illumination and detection optics that are induced by the scan itself. Additionally, the typically nonlinear relation between actuation voltage and axial displacement leads to over- or under-sampled frame acquisition in most microscopic techniques because of their static depth-of-field. To overcome these limitations, we present an Adaptive-Lens-High-and-Low-frequency (AL-HiLo) microscope that enables volumetric measurements employing an electrically tunable lens. By using speckle-patterned illumination, we ensure stability against aberrations of the electrically tunable lens. Its depth-of-field can be adjusted a-posteriori and hence enables to create flexible scans, which compensates for irregular axial measurement positions. The adaptive HiLo microscope provides an axial scanning range of 1 mm with an axial resolution of about 4 μm and sub-micron lateral resolution over the full scanning range. Proof of concept measurements at home-built specimens as well as zebrafish embryos with reporter gene-driven fluorescence in the thyroid gland are shown.

Hybrid Microscopy: Enabling Inexpensive High-Performance Imaging through Combined Physical and Optical Magnifications.

PubMed

Zhang, Yu Shrike; Chang, Jae-Byum; Alvarez, Mario Moisés; Trujillo-de Santiago, Grissel; Aleman, Julio; Batzaya, Byambaa; Krishnadoss, Vaishali; Ramanujam, Aishwarya Aravamudhan; Kazemzadeh-Narbat, Mehdi; Chen, Fei; Tillberg, Paul W; Dokmeci, Mehmet Remzi; Boyden, Edward S; Khademhosseini, Ali

2016-03-15

To date, much effort has been expended on making high-performance microscopes through better instrumentation. Recently, it was discovered that physical magnification of specimens was possible, through a technique called expansion microscopy (ExM), raising the question of whether physical magnification, coupled to inexpensive optics, could together match the performance of high-end optical equipment, at a tiny fraction of the price. Here we show that such "hybrid microscopy" methods--combining physical and optical magnifications--can indeed achieve high performance at low cost. By physically magnifying objects, then imaging them on cheap miniature fluorescence microscopes ("mini-microscopes"), it is possible to image at a resolution comparable to that previously attainable only with benchtop microscopes that present costs orders of magnitude higher. We believe that this unprecedented hybrid technology that combines expansion microscopy, based on physical magnification, and mini-microscopy, relying on conventional optics--a process we refer to as Expansion Mini-Microscopy (ExMM)--is a highly promising alternative method for performing cost-effective, high-resolution imaging of biological samples. With further advancement of the technology, we believe that ExMM will find widespread applications for high-resolution imaging particularly in research and healthcare scenarios in undeveloped countries or remote places.

Analysis of the chicken retina with an adaptive optics multiphoton microscope

PubMed Central

Bueno, Juan M.; Giakoumaki, Anastasia; Gualda, Emilio J.; Schaeffel, Frank; Artal, Pablo

2011-01-01

The structure and organization of the chicken retina has been investigated with an adaptive optics multiphoton imaging microscope in a backward configuration. Non-stained flat-mounted retinal tissues were imaged at different depths, from the retinal nerve fiber layer to the outer segment, by detecting the intrinsic nonlinear fluorescent signal. From the stacks of images corresponding to the different retinal layers, volume renderings of the entire retina were reconstructed. The density of photoreceptors and ganglion cells layer were directly estimated from the images as a function of the retinal eccentricity. The maximum anatomical resolving power at different retinal eccentricities was also calculated. This technique could be used for a better characterization of retinal alterations during myopia development, and may be useful for visualization of retinal pathologies and intoxication during pharmacological studies. PMID:21698025

Photonic crystal enhanced fluorescence immunoassay on diatom biosilica.

PubMed

Squire, Kenneth; Kong, Xianming; LeDuff, Paul; Rorrer, Gregory L; Wang, Alan X

2018-05-16

Fluorescence biosensing is one of the most established biosensing methods, particularly fluorescence spectroscopy and microscopy. These are two highly sensitive techniques but require high grade electronics and optics to achieve the desired sensitivity. Efforts have been made to implement these methods using consumer grade electronics and simple optical setups for applications such as point-of-care diagnostics, but the sensitivity inherently suffers. Sensing substrates, capable of enhancing fluorescence are thus needed to achieve high sensitivity for such applications. In this paper, we demonstrate a photonic crystal-enhanced fluorescence immunoassay biosensor using diatom biosilica, which consists of silica frustules with sub-100 nm periodic pores. Utilizing the enhanced local optical field, the Purcell effect and increased surface area from the diatom photonic crystals, we create ultrasensitive immunoassay biosensors that can significantly enhance fluorescence spectroscopy as well as fluorescence imaging. Using standard antibody-antigen-labeled antibody immunoassay protocol, we experimentally achieved 100× and 10× better detection limit with fluorescence spectroscopy and fluorescence imaging respectively. The limit of detection of the mouse IgG goes down to 10 -16 M (14 fg/mL) and 10 -15 M (140 fg/mL) for the two respective detection modalities, virtually sensing a single mouse IgG molecule on each diatom frustule. The effectively enhanced fluorescence imaging in conjunction with the simple hot-spot counting analysis method used in this paper proves the great potential of diatom fluorescence immunoassay for point-of-care biosensing. Scanning electron microscope image of biosilica diatom frustule that enables significant enhancement of fluorescence spectroscopy and fluorescence image. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Development of and Clinical Experience with a Simple Device for Performing Intraoperative Fluorescein Fluorescence Cerebral Angiography: Technical Notes.

PubMed

Ichikawa, Tsuyoshi; Suzuki, Kyouichi; Watanabe, Yoichi; Sato, Taku; Sakuma, Jun; Saito, Kiyoshi

2016-01-01

To perform intraoperative fluorescence angiography (FAG) under a microscope without an integrated FAG function with reasonable cost and sufficient quality for evaluation, we made a small and easy to use device for fluorescein FAG (FAG filter). We investigated the practical use of this FAG filter during aneurysm surgery, revascularization surgery, and brain tumor surgery. The FAG filter consists of two types of filters: an excitatory filter and a barrier filter. The excitatory filter excludes all wavelengths except for blue light and the barrier filter passes long waves except for blue light. By adding this FAG filter to a microscope without an integrated FAG function, light from the microscope illuminating the surgical field becomes blue, which is blocked by the barrier filter. We put the FAG filter on the objective lens of the operating microscope correctly and fluorescein sodium was injected intravenously or intra-arterially. Fluorescence (green light) from vessels in the surgical field and the dyed tumor were clearly observed through the microscope and recorded by a memory device. This method was easy and could be performed in a short time (about 10 seconds). Blood flow of small vessels deep in the surgical field could be observed. Blood flow stagnation could be evaluated. However, images from this method were inferior to those obtained by currently commercially available microscopes with an integrated FAG function. In brain tumor surgery, a stained tumor on the brain surface could be observed using this method. FAG could be performed with a microscope without an integrated FAG function easily with only this FAG filter.

Development of and Clinical Experience with a Simple Device for Performing Intraoperative Fluorescein Fluorescence Cerebral Angiography: Technical Notes

PubMed Central

ICHIKAWA, Tsuyoshi; SUZUKI, Kyouichi; WATANABE, Yoichi; SATO, Taku; SAKUMA, Jun; SAITO, Kiyoshi

2016-01-01

To perform intraoperative fluorescence angiography (FAG) under a microscope without an integrated FAG function with reasonable cost and sufficient quality for evaluation, we made a small and easy to use device for fluorescein FAG (FAG filter). We investigated the practical use of this FAG filter during aneurysm surgery, revascularization surgery, and brain tumor surgery. The FAG filter consists of two types of filters: an excitatory filter and a barrier filter. The excitatory filter excludes all wavelengths except for blue light and the barrier filter passes long waves except for blue light. By adding this FAG filter to a microscope without an integrated FAG function, light from the microscope illuminating the surgical field becomes blue, which is blocked by the barrier filter. We put the FAG filter on the objective lens of the operating microscope correctly and fluorescein sodium was injected intravenously or intra-arterially. Fluorescence (green light) from vessels in the surgical field and the dyed tumor were clearly observed through the microscope and recorded by a memory device. This method was easy and could be performed in a short time (about 10 seconds). Blood flow of small vessels deep in the surgical field could be observed. Blood flow stagnation could be evaluated. However, images from this method were inferior to those obtained by currently commercially available microscopes with an integrated FAG function. In brain tumor surgery, a stained tumor on the brain surface could be observed using this method. FAG could be performed with a microscope without an integrated FAG function easily with only this FAG filter. PMID:26597335

Localization of mitochondria in living cells with rhodamine 123.

PubMed Central

Johnson, L V; Walsh, M L; Chen, L B

1980-01-01

The laser dye rhodamine 123 is shown to be a specific probe for the localization of mitochondria in living cells. By virtue of its selectivity for mitochondria and its fluorescent properties, the detectability of mitochondria stained with rhodamine 123 is significantly improved over that provided by conventional light microscopic techniques. With the use of rhodamine 123, it is possible to detect alterations in mitochondrial distribution following transformation by Rous sarcoma virus and changes in the shape and organization of mitochondria induced by colchicine treatment. Images PMID:6965798

Expression of cystic fibrosis transmembrane conductance regulator corrects defective chloride channel regulation in cystic fibrosis airway epithelial cells

NASA Astrophysics Data System (ADS)

Rich, Devra P.; Anderson, Matthew P.; Gregory, Richard J.; Cheng, Seng H.; Paul, Sucharita; Jefferson, Douglas M.; McCann, John D.; Klinger, Katherine W.; Smith, Alan E.; Welsh, Michael J.

1990-09-01

The cystic fibrosis transmembrane conductance regulator (CFTR) was expressed in cultured cystic fibrosis airway epithelial cells and Cl- channel activation assessed in single cells using a fluorescence microscopic assay and the patch-clamp technique. Expression of CFTR, but not of a mutant form of CFTR (ΔF508), corrected the Cl- channel defect. Correction of the phenotypic defect demonstrates a causal relationship between mutations in the CFTR gene and defective Cl- transport which is the hallmark of the disease.

GFP as potential cellular viscosimeter.

PubMed

Visser, Antonie J W G; Westphal, Adrie H; Skakun, Victor V; Borst, Jan Willem

2016-08-18

The molecular dimensions of proteins such as green fluorescent protein (GFP) are large as compared to the ones of solvents like water or glycerol. The microscopic viscosity, which determines the resistance to diffusion of, e.g. GFP, is then the same as that determined from the resistance of the solvent to flow, which is known as macroscopic viscosity. GFP in water/glycerol mixtures senses this macroscopic viscosity, because the translational and rotational diffusion coefficients are proportional to the reciprocal value of the viscosity as predicted by the Stokes-Einstein equations. To test this hypothesis, we have performed time-resolved fluorescence anisotropy (reporting on rotational diffusion) and fluorescence correlation spectroscopy (reporting on translational diffusion) experiments of GFP in water/glycerol mixtures. When the solvent also contains macromolecules of similar or larger dimensions as GFP, the microscopic and macroscopic viscosities can be markedly different and the Stokes-Einstein relations must be adapted. It was established from previous dynamic fluorescence spectroscopy observations of diffusing proteins with dextran polysaccharides as co-solvents (Lavalette et al 2006 Eur. Biophys. J. 35 517-22), that rotation and translation sense a different microscopic viscosity, in which the one arising from rotation is always less than that from translation. A microscopic viscosity parameter is defined that depends on scaling factors between GFP and its immediate environment. The direct consequence is discussed for two reported diffusion coefficients of GFP in living cells.

Single-photon counting multicolor multiphoton fluorescence microscope.

PubMed

Buehler, Christof; Kim, Ki H; Greuter, Urs; Schlumpf, Nick; So, Peter T C

2005-01-01

We present a multicolor multiphoton fluorescence microscope with single-photon counting sensitivity. The system integrates a standard multiphoton fluorescence microscope, an optical grating spectrograph operating in the UV-Vis wavelength region, and a 16-anode photomultiplier tube (PMT). The major technical innovation is in the development of a multichannel photon counting card (mC-PhCC) for direct signal collection from multi-anode PMTs. The electronic design of the mC-PhCC employs a high-throughput, fully-parallel, single-photon counting scheme along with a high-speed electrical or fiber-optical link interface to the data acquisition computer. There is no electronic crosstalk among the detection channels of the mC-PhCC. The collected signal remains linear up to an incident photon rate of 10(8) counts per second. The high-speed data interface offers ample bandwidth for real-time readout: 2 MByte lambda-stacks composed of 16 spectral channels, 256 x 256 pixel image with 12-bit dynamic range can be transferred at 30 frames per second. The modular design of the mC-PhCC can be readily extended to accommodate PMTs of more anodes. Data acquisition from a 64-anode PMT has been verified. As a demonstration of system performance, spectrally resolved images of fluorescent latex spheres and ex-vivo human skin are reported. The multicolor multiphoton microscope is suitable for highly sensitive, real-time, spectrally-resolved three-dimensional imaging in biomedical applications.

GFP as potential cellular viscosimeter

NASA Astrophysics Data System (ADS)

Visser, Antonie J. W. G.; Westphal, Adrie H.; Skakun, Victor V.; Borst, Jan Willem

2016-09-01

The molecular dimensions of proteins such as green fluorescent protein (GFP) are large as compared to the ones of solvents like water or glycerol. The microscopic viscosity, which determines the resistance to diffusion of, e.g. GFP, is then the same as that determined from the resistance of the solvent to flow, which is known as macroscopic viscosity. GFP in water/glycerol mixtures senses this macroscopic viscosity, because the translational and rotational diffusion coefficients are proportional to the reciprocal value of the viscosity as predicted by the Stokes-Einstein equations. To test this hypothesis, we have performed time-resolved fluorescence anisotropy (reporting on rotational diffusion) and fluorescence correlation spectroscopy (reporting on translational diffusion) experiments of GFP in water/glycerol mixtures. When the solvent also contains macromolecules of similar or larger dimensions as GFP, the microscopic and macroscopic viscosities can be markedly different and the Stokes-Einstein relations must be adapted. It was established from previous dynamic fluorescence spectroscopy observations of diffusing proteins with dextran polysaccharides as co-solvents (Lavalette et al 2006 Eur. Biophys. J. 35 517-22), that rotation and translation sense a different microscopic viscosity, in which the one arising from rotation is always less than that from translation. A microscopic viscosity parameter is defined that depends on scaling factors between GFP and its immediate environment. The direct consequence is discussed for two reported diffusion coefficients of GFP in living cells.

Single-cell optoporation and transfection using femtosecond laser and optical tweezers.

PubMed

Waleed, Muhammad; Hwang, Sun-Uk; Kim, Jung-Dae; Shabbir, Irfan; Shin, Sang-Mo; Lee, Yong-Gu

2013-01-01

In this paper, we demonstrate a new single-cell optoporation and transfection technique using a femtosecond Gaussian laser beam and optical tweezers. Tightly focused near-infrared (NIR) femtosecond laser pulse was employed to transiently perforate the cellular membrane at a single point in MCF-7 cancer cells. A distinct technique was developed by trapping the microparticle using optical tweezers to focus the femtosecond laser precisely on the cell membrane to puncture it. Subsequently, an external gene was introduced in the cell by trapping and inserting the same plasmid-coated microparticle into the optoporated cell using optical tweezers. Various experimental parameters such as femtosecond laser exposure power, exposure time, puncture hole size, exact focusing of the femtosecond laser on the cell membrane, and cell healing time were closely analyzed to create the optimal conditions for cell viability. Following the insertion of plasmid-coated microparticles in the cell, the targeted cells exhibited green fluorescent protein (GFP) under the fluorescent microscope, hence confirming successful transfection into the cell. This new optoporation and transfection technique maximizes the level of selectivity and control over the targeted cell, and this may be a breakthrough method through which to induce controllable genetic changes in the cell.

Directional bilateral filters for smoothing fluorescence microscopy images

NASA Astrophysics Data System (ADS)

Venkatesh, Manasij; Mohan, Kavya; Seelamantula, Chandra Sekhar

2015-08-01

Images obtained through fluorescence microscopy at low numerical aperture (NA) are noisy and have poor resolution. Images of specimens such as F-actin filaments obtained using confocal or widefield fluorescence microscopes contain directional information and it is important that an image smoothing or filtering technique preserve the directionality. F-actin filaments are widely studied in pathology because the abnormalities in actin dynamics play a key role in diagnosis of cancer, cardiac diseases, vascular diseases, myofibrillar myopathies, neurological disorders, etc. We develop the directional bilateral filter as a means of filtering out the noise in the image without significantly altering the directionality of the F-actin filaments. The bilateral filter is anisotropic to start with, but we add an additional degree of anisotropy by employing an oriented domain kernel for smoothing. The orientation is locally adapted using a structure tensor and the parameters of the bilateral filter are optimized for within the framework of statistical risk minimization. We show that the directional bilateral filter has better denoising performance than the traditional Gaussian bilateral filter and other denoising techniques such as SURE-LET, non-local means, and guided image filtering at various noise levels in terms of peak signal-to-noise ratio (PSNR). We also show quantitative improvements in low NA images of F-actin filaments.

Silver nanoparticle-induced degranulation observed with quantitative phase microscopy

NASA Astrophysics Data System (ADS)

Yang, Wenzhong; Lee, Seungrag; Lee, Jiyong; Bae, Yoonsung; Kim, Dugyoung

2010-07-01

Monitoring a degranulation process in a live mast cell is a quite important issue in immunology and pharmacology. Because the size of a granule is normally much smaller than the resolution limit of an optical microscope system, there is no direct real-time live cell imaging technique for observing degranulation processes except for fluorescence imaging techniques. In this research, we propose optical quantitative phase microscopy (QPM) as a new observation tool to study degranulation processes in a live mast cell without any fluorescence labeling. We measure the cell volumes and the cross sectional profiles (x-z plane) of an RBL-2H3 cell and a HeLa cell, before and after they are exposed to calcium ionophore A23187 and silver nanoparticles (AgNPs). We verify that the volume and the cross sectional line profile of the RBL-2H3 cell were changed significantly when it was exposed to A23187. When 50 μg/mL of AgNP is used instead of A23187, the measurements of cell volume and cross sectional profiles indicate that RBL-2H3 cells also follow degranulation processes. Degranulation processes for these cells are verified by monitoring the increase of intracellular calcium ([Ca2+]i) and histamine with fluorescent methods.

An experimental approach to identify dynamical models of transcriptional regulation in living cells

NASA Astrophysics Data System (ADS)

Fiore, G.; Menolascina, F.; di Bernardo, M.; di Bernardo, D.

2013-06-01

We describe an innovative experimental approach, and a proof of principle investigation, for the application of System Identification techniques to derive quantitative dynamical models of transcriptional regulation in living cells. Specifically, we constructed an experimental platform for System Identification based on a microfluidic device, a time-lapse microscope, and a set of automated syringes all controlled by a computer. The platform allows delivering a time-varying concentration of any molecule of interest to the cells trapped in the microfluidics device (input) and real-time monitoring of a fluorescent reporter protein (output) at a high sampling rate. We tested this platform on the GAL1 promoter in the yeast Saccharomyces cerevisiae driving expression of a green fluorescent protein (Gfp) fused to the GAL1 gene. We demonstrated that the System Identification platform enables accurate measurements of the input (sugars concentrations in the medium) and output (Gfp fluorescence intensity) signals, thus making it possible to apply System Identification techniques to obtain a quantitative dynamical model of the promoter. We explored and compared linear and nonlinear model structures in order to select the most appropriate to derive a quantitative model of the promoter dynamics. Our platform can be used to quickly obtain quantitative models of eukaryotic promoters, currently a complex and time-consuming process.

An image-processing method to detect sub-optical features based on understanding noise in intensity measurements.

PubMed

Bhatia, Tripta

2018-07-01

Accurate quantitative analysis of image data requires that we distinguish between fluorescence intensity (true signal) and the noise inherent to its measurements to the extent possible. We image multilamellar membrane tubes and beads that grow from defects in the fluid lamellar phase of the lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine dissolved in water and water-glycerol mixtures by using fluorescence confocal polarizing microscope. We quantify image noise and determine the noise statistics. Understanding the nature of image noise also helps in optimizing image processing to detect sub-optical features, which would otherwise remain hidden. We use an image-processing technique "optimum smoothening" to improve the signal-to-noise ratio of features of interest without smearing their structural details. A high SNR renders desired positional accuracy with which it is possible to resolve features of interest with width below optical resolution. Using optimum smoothening, the smallest and the largest core diameter detected is of width [Formula: see text] and [Formula: see text] nm, respectively, discussed in this paper. The image-processing and analysis techniques and the noise modeling discussed in this paper can be used for detailed morphological analysis of features down to sub-optical length scales that are obtained by any kind of fluorescence intensity imaging in the raster mode.

Nucleoplasmic viscosity of living cells investigated by fluorescence correlation spectroscopy

NASA Astrophysics Data System (ADS)

Liang, Lifang; Xing, Da; Chen, Tongshen; Pei, Yihui

2007-11-01

Fluorescence correlation spectroscopy (FCS) is a new kind of real-time, high-speed and single-molecule technique. It is used to detect the kinetic characteristics of fluorescent dye such as diffusion coefficient in the aqueous solution. Combined with confocal microscope optics, it has been now widely applied in cell biological research. Through a time correlation analysis of spontaneous intensity fluctuations, this technique with EGFP as a probe is capable of determining viscosity of fluids according to Stokes-Einstein equation. Nucleoplasmic viscosity is an important physical parameter to quantify the rheological characteristics of the nucleoplasm. Investigation on nucleoplasmic viscosity plays an important role in further understanding intranuclear environment. In this paper, FCS is introduced to noninvasively investigate nucleoplasmic viscosity of living cells. The results show that nucleoplasmic viscosity of lung adenocarcinoma (ASTC-a-1) cells is 2.55+/-0.61 cP and nucleoplasmic viscosity is larger than cytoplasmic viscosity at 37 °C (pH 7.4). In addition, significant changes in nucleoplasmic viscosity are detected by FCS when cells are exposed to hyper or hypotonic medium. Our study suggests that FCS can be used to detect the kinetic characteristics of biomolecules in living cells and thus helps to investigate the dynamic changes of the microenvironment in the cell.

Confocal nanoscanning, bead picking (CONA): PickoScreen microscopes for automated and quantitative screening of one-bead one-compound libraries.

PubMed

Hintersteiner, Martin; Buehler, Christof; Uhl, Volker; Schmied, Mario; Müller, Jürgen; Kottig, Karsten; Auer, Manfred

2009-01-01

Solid phase combinatorial chemistry provides fast and cost-effective access to large bead based libraries with compound numbers easily exceeding tens of thousands of compounds. Incubating one-bead one-compound library beads with fluorescently labeled target proteins and identifying and isolating the beads which contain a bound target protein, potentially represents one of the most powerful generic primary high throughput screening formats. On-bead screening (OBS) based on this detection principle can be carried out with limited automation. Often hit bead detection, i.e. recognizing beads with a fluorescently labeled protein bound to the compound on the bead, relies on eye-inspection under a wide-field microscope. Using low resolution detection techniques, the identification of hit beads and their ranking is limited by a low fluorescence signal intensity and varying levels of the library beads' autofluorescence. To exploit the full potential of an OBS process, reliable methods for both automated quantitative detection of hit beads and their subsequent isolation are needed. In a joint collaborative effort with Evotec Technologies (now Perkin-Elmer Cellular Technologies Germany GmbH), we have built two confocal bead scanner and picker platforms PS02 and a high-speed variant PS04 dedicated to automated high resolution OBS. The PS0X instruments combine fully automated confocal large area scanning of a bead monolayer at the bottom of standard MTP plates with semiautomated isolation of individual hit beads via hydraulic-driven picker capillaries. The quantification of fluorescence intensities with high spatial resolution in the equatorial plane of each bead allows for a reliable discrimination between entirely bright autofluorescent beads and real hit beads which exhibit an increased fluorescence signal at the outer few micrometers of the bead. The achieved screening speed of up to 200,000 bead assayed in less than 7 h and the picking time of approximately 1 bead/min allow exploitation of one-bead one-compound libraries with high sensitivity, accuracy, and speed.

Enhanced simulator software for image validation and interpretation for multimodal localization super-resolution fluorescence microscopy

NASA Astrophysics Data System (ADS)

Erdélyi, Miklós; Sinkó, József; Gajdos, Tamás.; Novák, Tibor

2017-02-01

Optical super-resolution techniques such as single molecule localization have become one of the most dynamically developed areas in optical microscopy. These techniques routinely provide images of fixed cells or tissues with sub-diffraction spatial resolution, and can even be applied for live cell imaging under appropriate circumstances. Localization techniques are based on the precise fitting of the point spread functions (PSF) to the measured images of stochastically excited, identical fluorescent molecules. These techniques require controlling the rate between the on, off and the bleached states, keeping the number of active fluorescent molecules at an optimum value, so their diffraction limited images can be detected separately both spatially and temporally. Because of the numerous (and sometimes unknown) parameters, the imaging system can only be handled stochastically. For example, the rotation of the dye molecules obscures the polarization dependent PSF shape, and only an averaged distribution - typically estimated by a Gaussian function - is observed. TestSTORM software was developed to generate image stacks for traditional localization microscopes, where localization meant the precise determination of the spatial position of the molecules. However, additional optical properties (polarization, spectra, etc.) of the emitted photons can be used for further monitoring the chemical and physical properties (viscosity, pH, etc.) of the local environment. The image stack generating program was upgraded by several new features, such as: multicolour, polarization dependent PSF, built-in 3D visualization, structured background. These features make the program an ideal tool for optimizing the imaging and sample preparation conditions.

Nanoscale imaging of whole cells using a liquid enclosure and a scanning transmission electron microscope.

PubMed

Peckys, Diana B; Veith, Gabriel M; Joy, David C; de Jonge, Niels

2009-12-14

Nanoscale imaging techniques are needed to investigate cellular function at the level of individual proteins and to study the interaction of nanomaterials with biological systems. We imaged whole fixed cells in liquid state with a scanning transmission electron microscope (STEM) using a micrometer-sized liquid enclosure with electron transparent windows providing a wet specimen environment. Wet-STEM images were obtained of fixed E. coli bacteria labeled with gold nanoparticles attached to surface membrane proteins. Mammalian cells (COS7) were incubated with gold-tagged epidermal growth factor and fixed. STEM imaging of these cells resulted in a resolution of 3 nm for the gold nanoparticles. The wet-STEM method has several advantages over conventional imaging techniques. Most important is the capability to image whole fixed cells in a wet environment with nanometer resolution, which can be used, e.g., to map individual protein distributions in/on whole cells. The sample preparation is compatible with that used for fluorescent microscopy on fixed cells for experiments involving nanoparticles. Thirdly, the system is rather simple and involves only minimal new equipment in an electron microscopy (EM) laboratory.

Flexible non-diffractive vortex microscope for three-dimensional depth-enhanced super-localization of dielectric, metal and fluorescent nanoparticles

NASA Astrophysics Data System (ADS)

Bouchal, Petr; Bouchal, Zdeněk

2017-10-01

In the past decade, probe-based super-resolution using temporally resolved localization of emitters became a groundbreaking imaging strategy in fluorescence microscopy. Here we demonstrate a non-diffractive vortex microscope (NVM), enabling three-dimensional super-resolution fluorescence imaging and localization and tracking of metal and dielectric nanoparticles. The NVM benefits from vortex non-diffractive beams (NBs) creating a double-helix point spread function that rotates under defocusing while maintaining its size and shape unchanged. Using intrinsic properties of the NBs, the dark-field localization of weakly scattering objects is achieved in a large axial range exceeding the depth of field of the microscope objective up to 23 times. The NVM was developed using an upright microscope Nikon Eclipse E600 operating with a spiral lithographic mask optimized using Fisher information and built into an add-on imaging module or microscope objective. In evaluation of the axial localization accuracy the root mean square error below 18 nm and 280 nm was verified over depth ranges of 3.5 μm and 13.6 μm, respectively. Subwavelength gold and polystyrene beads were localized with isotropic precision below 10 nm in the axial range of 3.5 μm and the axial precision reduced to 30 nm in the extended range of 13.6 μm. In the fluorescence imaging, the localization with isotropic precision below 15 nm was demonstrated in the range of 2.5 μm, whereas in the range of 8.3 μm, the precision of 15 nm laterally and 30-50 nm axially was achieved. The tracking of nanoparticles undergoing Brownian motion was demonstrated in the volume of 14 × 10 × 16 μm3. Applicability of the NVM was tested by fluorescence imaging of LW13K2 cells and localization of cellular proteins.

Microscopic Mapping of Minerals and Organics: A Modular Pulsed Raman Spectrometer Adaptable to Both Small and Large Landed Planetary Missions

NASA Astrophysics Data System (ADS)

Blacksberg, J.; Alerstam, E.; Maruyama, Y.; Cochrane, C.; Rossman, G. R.

2016-12-01

Raman spectroscopy combined with microscopic imaging is a powerful technique used to interrogate geological materials. In the laboratory, Raman spectroscopy is commonly used in the geosciences for mapping both major and minor mineral and organic constituents on a fine scale. This technique has proven valuable in analyzing planetary materials, including meteorites and lunar samples. By simultaneously analyzing microtexture and mineralogy, micro-Raman spectroscopy can provide essential information for inferring geologic processes by which planetary surfaces have evolved. Because Raman can perform these capabilities in a way that is non-destructive, requiring no sample preparation, it is extremely well suited for deployment on a planetary lander or rover arm. The pulsed Raman spectrometer presented here has been designed for maximum flexibility using miniaturized modular components in order to remain easily adaptable and relevant to numerous planetary surface missions (e.g. asteroids, comets, Mars, Mars' moons, Europa, Titan). Building on the widely used 532 nm laser Raman technique, the pulsed Raman spectrometer takes advantage of recent developments in miniaturized pulsed lasers and detectors; the instrument uses sub-ns time gating to remove pervasive background interference caused by fluorescence inherent in many minerals and organics. This technique ensures acquisition of diagnostic Raman spectra, even in environments that have been known to severely challenge conventional methods (e.g. aqueously-formed minerals from similar environments on Earth). We present the architecture and performance of the pulsed Raman spectrometer, which relies on our single photon avalanche diode (SPAD) detector synchronized with our high-speed microchip laser, both custom-built for this application. It is these key technological developments that now make time-gated Raman spectroscopy possible for applications where miniaturization is crucial. We then discuss recent progress in laser performance that enhances Raman return, provides improved fluorescence rejection, and minimizes damage to sensitive samples.

Field portable mobile phone based fluorescence microscopy for detection of Giardia lamblia cysts in water samples

NASA Astrophysics Data System (ADS)

Ceylan Koydemir, Hatice; Gorocs, Zoltan; McLeod, Euan; Tseng, Derek; Ozcan, Aydogan

2015-03-01

Giardia lamblia is a waterborne parasite that causes an intestinal infection, known as giardiasis, and it is found not only in countries with inadequate sanitation and unsafe water but also streams and lakes of developed countries. Simple, sensitive, and rapid detection of this pathogen is important for monitoring of drinking water. Here we present a cost-effective and field portable mobile-phone based fluorescence microscopy platform designed for automated detection of Giardia lamblia cysts in large volume water samples (i.e., 10 ml) to be used in low-resource field settings. This fluorescence microscope is integrated with a disposable water-sampling cassette, which is based on a flow-through porous polycarbonate membrane and provides a wide surface area for fluorescence imaging and enumeration of the captured Giardia cysts on the membrane. Water sample of interest, containing fluorescently labeled Giardia cysts, is introduced into the absorbent pads that are in contact with the membrane in the cassette by capillary action, which eliminates the need for electrically driven flow for sample processing. Our fluorescence microscope weighs ~170 grams in total and has all the components of a regular microscope, capable of detecting individual fluorescently labeled cysts under light-emitting-diode (LED) based excitation. Including all the sample preparation, labeling and imaging steps, the entire measurement takes less than one hour for a sample volume of 10 ml. This mobile phone based compact and cost-effective fluorescent imaging platform together with its machine learning based cyst counting interface is easy to use and can even work in resource limited and field settings for spatio-temporal monitoring of water quality.

Protocol for chromosome-specific probe construction using PRINS, micromanipulation and DOP-PCR techniques.

PubMed

Passamani, Paulo Z; Carvalho, Carlos R; Soares, Fernanda A F

2018-01-01

Chromosome-specific probes have been widely used in molecular cytogenetics, being obtained with different methods. In this study, a reproducible protocol for construction of chromosome-specific probes is proposed which associates in situ amplification (PRINS), micromanipulation and degenerate oligonucleotide-primed PCR (DOP-PCR). Human lymphocyte cultures were used to obtain metaphases from male and female individuals. The chromosomes were amplified via PRINS, and subcentromeric fragments of the X chromosome were microdissected using microneedles coupled to a phase contrast microscope. The fragments were amplified by DOP-PCR and labeled with tetramethyl-rhodamine-5-dUTP. The probes were used in fluorescent in situ hybridization (FISH) procedure to highlight these specific regions in the metaphases. The results show one fluorescent red spot in male and two in female X chromosomes and interphase nuclei.

Visualization of DNA Replication in the Vertebrate Model System DT40 using the DNA Fiber Technique

PubMed Central

Schwab, Rebekka A.V.; Niedzwiedz, Wojciech

2011-01-01

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not only ensure faithful genome duplication in the face of endogenous and exogenous DNA damage but also prevent genomic instability, a recognized causative factor in tumor development. Here, we describe a simple and cost-effective fluorescence microscopy-based method to visualize DNA replication in the avian B-cell line DT40. This cell line provides a powerful tool to investigate protein function in vivo by reverse genetics in vertebrate cells1. DNA fiber fluorography in DT40 cells lacking a specific gene allows one to elucidate the function of this gene product in DNA replication and genome stability. Traditional methods to analyze replication fork dynamics in vertebrate cells rely on measuring the overall rate of DNA synthesis in a population of pulse-labeled cells. This is a quantitative approach and does not allow for qualitative analysis of parameters that influence DNA synthesis. In contrast, the rate of movement of active forks can be followed directly when using the DNA fiber technique2-4. In this approach, nascent DNA is labeled in vivo by incorporation of halogenated nucleotides (Fig 1A). Subsequently, individual fibers are stretched onto a microscope slide, and the labeled DNA replication tracts are stained with specific antibodies and visualized by fluorescence microscopy (Fig 1B). Initiation of replication as well as fork directionality is determined by the consecutive use of two differently modified analogues. Furthermore, the dual-labeling approach allows for quantitative analysis of parameters that influence DNA synthesis during the S-phase, i.e. replication structures such as ongoing and stalled forks, replication origin density as well as fork terminations. Finally, the experimental procedure can be accomplished within a day, and requires only general laboratory equipment and a fluorescence microscope. PMID:22064662

Quenching of cascade reaction between triplet and photochrome probes with nitroxide radicals. A novel labeling method in study of membranes and surface systems.

PubMed

Papper, V; Medvedeva, N; Fishov, I; Likhtenshtein, G I

2000-01-01

We proposed a new method for the study of molecular dynamics and fluidity of the living and model biomembranes and surface systems. The method is based on the measurements of the sensitized photoisomerization kinetics of a photochrome probe. The cascade triplet cis-trans photoisomerization of the excited stilbene derivative sensitized with the excited triplet Erythrosin B has been studied in a model liposome membrane. The photoisomerization reaction is depressed with nitroxide radicals quenching the excited triplet state of the sensitizer. The enhanced fluorescence polarization of the stilbene probe incorporated into liposome membranes indicates that the stilbene molecules are squeezed in a relatively viscous media of the phospholipids. Calibration of the "triple" cascade system is based on a previously proposed method that allows the measurement of the product of the quenching rate constant and the sensitizer's triplet lifetime, as well as the quantitative detection of the nitroxide radicals in the vicinity of the membrane surface. The experiment was conducted using the constant-illumination fluorescence technique. Sensitivity of the method using a standard commercial spectrofluorimeter is about 10(-12) mol of fluorescence molecules per sample and can be improved using an advanced fluorescence technique. The minimal local concentration of nitroxide radicals or any other quenchers being detected is about 10(-5) M. This method enables the investigation of any chemical and biological surface processes of microscopic scale when the minimal volume is about 10(-3) microL or less.

Two-Dimensional Standing Wave Total Internal Reflection Fluorescence Microscopy: Superresolution Imaging of Single Molecular and Biological Specimens

PubMed Central

Chung, Euiheon; Kim, Daekeun; Cui, Yan; Kim, Yang-Hyo; So, Peter T. C.

2007-01-01

The development of high resolution, high speed imaging techniques allows the study of dynamical processes in biological systems. Lateral resolution improvement of up to a factor of 2 has been achieved using structured illumination. In a total internal reflection fluorescence microscope, an evanescence excitation field is formed as light is total internally reflected at an interface between a high and a low index medium. The <100 nm penetration depth of evanescence field ensures a thin excitation region resulting in low background fluorescence. We present even higher resolution wide-field biological imaging by use of standing wave total internal reflection fluorescence (SW-TIRF). Evanescent standing wave (SW) illumination is used to generate a sinusoidal high spatial frequency fringe pattern on specimen for lateral resolution enhancement. To prevent thermal drift of the SW, novel detection and estimation of the SW phase with real-time feedback control is devised for the stabilization and control of the fringe phase. SW-TIRF is a wide-field superresolution technique with resolution better than a fifth of emission wavelength or ∼100 nm lateral resolution. We demonstrate the performance of the SW-TIRF microscopy using one- and two-directional SW illumination with a biological sample of cellular actin cytoskeleton of mouse fibroblast cells as well as single semiconductor nanocrystal molecules. The results confirm the superior resolution of SW-TIRF in addition to the merit of a high signal/background ratio from TIRF microscopy. PMID:17483188

An Assemblable, Multi-Angle Fluorescence and Ellipsometric Microscope

PubMed Central

Nguyen, Victoria; Rizzo, John

2016-01-01

We introduce a multi-functional microscope for research laboratories that have significant cost and space limitations. The microscope pivots around the sample, operating in upright, inverted, side-on and oblique geometries. At these geometries it is able to perform bright-field, fluorescence and qualitative ellipsometric imaging. It is the first single instrument in the literature to be able to perform all of these functionalities. The system can be assembled by two undergraduate students from a provided manual in less than a day, from off-the-shelf and 3D printed components, which together cost approximately $16k at 2016 market prices. We include a highly specified assembly manual, a summary of design methodologies, and all associated 3D-printing files in hopes that the utility of the design outlives the current component market. This open design approach prepares readers to customize the instrument to specific needs and applications. We also discuss how to select household LEDs as low-cost light sources for fluorescence microscopy. We demonstrate the utility of the microscope in varied geometries and functionalities, with particular emphasis on studying hydrated, solid-supported lipid films and wet biological samples. PMID:27907008

Compact plane illumination plugin device to enable light sheet fluorescence imaging of multi-cellular organisms on an inverted wide-field microscope

PubMed Central

Guan, Zeyi; Lee, Juhyun; Jiang, Hao; Dong, Siyan; Jen, Nelson; Hsiai, Tzung; Ho, Chih-Ming; Fei, Peng

2015-01-01

We developed a compact plane illumination plugin (PIP) device which enabled plane illumination and light sheet fluorescence imaging on a conventional inverted microscope. The PIP device allowed the integration of microscope with tunable laser sheet profile, fast image acquisition, and 3-D scanning. The device is both compact, measuring approximately 15 by 5 by 5 cm, and cost-effective, since we employed consumer electronics and an inexpensive device molding method. We demonstrated that PIP provided significant contrast and resolution enhancement to conventional microscopy through imaging different multi-cellular fluorescent structures, including 3-D branched cells in vitro and live zebrafish embryos. Imaging with the integration of PIP greatly reduced out-of-focus contamination and generated sharper contrast in acquired 2-D plane images when compared with the stand-alone inverted microscope. As a result, the dynamic fluid domain of the beating zebrafish heart was clearly segmented and the functional monitoring of the heart was achieved. Furthermore, the enhanced axial resolution established by thin plane illumination of PIP enabled the 3-D reconstruction of the branched cellular structures, which leads to the improvement on the functionality of the wide field microscopy. PMID:26819828

Compact plane illumination plugin device to enable light sheet fluorescence imaging of multi-cellular organisms on an inverted wide-field microscope.

PubMed

Guan, Zeyi; Lee, Juhyun; Jiang, Hao; Dong, Siyan; Jen, Nelson; Hsiai, Tzung; Ho, Chih-Ming; Fei, Peng

2016-01-01

We developed a compact plane illumination plugin (PIP) device which enabled plane illumination and light sheet fluorescence imaging on a conventional inverted microscope. The PIP device allowed the integration of microscope with tunable laser sheet profile, fast image acquisition, and 3-D scanning. The device is both compact, measuring approximately 15 by 5 by 5 cm, and cost-effective, since we employed consumer electronics and an inexpensive device molding method. We demonstrated that PIP provided significant contrast and resolution enhancement to conventional microscopy through imaging different multi-cellular fluorescent structures, including 3-D branched cells in vitro and live zebrafish embryos. Imaging with the integration of PIP greatly reduced out-of-focus contamination and generated sharper contrast in acquired 2-D plane images when compared with the stand-alone inverted microscope. As a result, the dynamic fluid domain of the beating zebrafish heart was clearly segmented and the functional monitoring of the heart was achieved. Furthermore, the enhanced axial resolution established by thin plane illumination of PIP enabled the 3-D reconstruction of the branched cellular structures, which leads to the improvement on the functionality of the wide field microscopy.

A multimodal imaging platform with integrated simultaneous photoacoustic microscopy, optical coherence tomography, optical Doppler tomography and fluorescence microscopy

NASA Astrophysics Data System (ADS)

Dadkhah, Arash; Zhou, Jun; Yeasmin, Nusrat; Jiao, Shuliang

2018-02-01

Various optical imaging modalities with different optical contrast mechanisms have been developed over the past years. Although most of these imaging techniques are being used in many biomedical applications and researches, integration of these techniques will allow researchers to reach the full potential of these technologies. Nevertheless, combining different imaging techniques is always challenging due to the difference in optical and hardware requirements for different imaging systems. Here, we developed a multimodal optical imaging system with the capability of providing comprehensive structural, functional and molecular information of living tissue in micrometer scale. This imaging system integrates photoacoustic microscopy (PAM), optical coherence tomography (OCT), optical Doppler tomography (ODT) and fluorescence microscopy in one platform. Optical-resolution PAM (OR-PAM) provides absorption-based imaging of biological tissues. Spectral domain OCT is able to provide structural information based on the scattering property of biological sample with no need for exogenous contrast agents. In addition, ODT is a functional extension of OCT with the capability of measurement and visualization of blood flow based on the Doppler effect. Fluorescence microscopy allows to reveal molecular information of biological tissue using autofluoresce or exogenous fluorophores. In-vivo as well as ex-vivo imaging studies demonstrated the capability of our multimodal imaging system to provide comprehensive microscopic information on biological tissues. Integrating all the aforementioned imaging modalities for simultaneous multimodal imaging has promising potential for preclinical research and clinical practice in the near future.

Genetically expressed voltage sensor ArcLight for imaging large scale cortical activity in the anesthetized and awake mouse

PubMed Central

Borden, Peter Y.; Ortiz, Alex D.; Waiblinger, Christian; Sederberg, Audrey J.; Morrissette, Arthur E.; Forest, Craig R.; Jaeger, Dieter; Stanley, Garrett B.

2017-01-01

Abstract. With the recent breakthrough in genetically expressed voltage indicators (GEVIs), there has been a tremendous demand to determine the capabilities of these sensors in vivo. Novel voltage sensitive fluorescent proteins allow for direct measurement of neuron membrane potential changes through changes in fluorescence. Here, we utilized ArcLight, a recently developed GEVI, and examined the functional characteristics in the widely used mouse somatosensory whisker pathway. We measured the resulting evoked fluorescence using a wide-field microscope and a CCD camera at 200 Hz, which enabled voltage recordings over the entire cortical region with high temporal resolution. We found that ArcLight produced a fluorescent response in the S1 barrel cortex during sensory stimulation at single whisker resolution. During wide-field cortical imaging, we encountered substantial hemodynamic noise that required additional post hoc processing through noise subtraction techniques. Over a period of 28 days, we found clear and consistent ArcLight fluorescence responses to a simple sensory input. Finally, we demonstrated the use of ArcLight to resolve cortical S1 sensory responses in the awake mouse. Taken together, our results demonstrate the feasibility of ArcLight as a measurement tool for mesoscopic, chronic imaging. PMID:28491905

Synthesis of Water Dispersible Fluorescent Carbon Nanocrystals from Syzygium cumini Fruits for the Detection of Fe3+ Ion in Water and Biological Samples and Imaging of Fusarium avenaceum Cells.

PubMed

Bhamore, Jigna R; Jha, Sanjay; Singhal, Rakesh Kumar; Kailasa, Suresh Kumar

2017-01-01

In this work, water dispersible fluorescent carbon nanocrystals (NCs) were synthesized by a simple, green and low cost hydrothermal method using Syzygium cumini (jamun) as a carbon source at 180 °C for 6 h. The average size of carbon NCs was found to be 2.1 ± 0.5 nm and shown bright blue fluorescence when excited at 365 nm under UV lamp. The carbon NCs were characterized by spectroscopic (UV-visible and fluorescence, Fourier transform infrared and dynamic light scattering) and high resolution transmission electron microscopic techniques. The quantum yield of carbon NCs was found to be ~5.9 % at 438 nm emission wavelength when excited at 360 nm. It was noticed that none of the metal ions quenched the fluorescence intensity of carbon NCs at 438 nm except for Fe 3+ , indicating the formation of Fe 3+ ion-carbon NCs complexes. The linear range was observed in the concentration range of 0.01-100 μM with the corresponding detection limits of 0.001 μM, respectively. Furthermore, the carbon NCs were used as probes for imaging of fungal (Fusarium avenaceum) cells.

Digital optical imaging of green fluorescent proteins for tracking vascular gene expression: feasibility study in rabbit and human cell models.

PubMed

Yang, X; Liu, H; Li, D; Zhou, X; Jung, W C; Deans, A E; Cui, Y; Cheng, L

2001-04-01

To investigate the feasibility of using a sensitive digital optical imaging technique to detect green fluorescent protein (GFP) expressed in rabbit vasculature and human arterial smooth muscle cells. A GFP plasmid was transfected into human arterial smooth muscle cells to obtain a GFP-smooth muscle cell solution. This solution was imaged in cell phantoms by using a prototype digital optical imaging system. For in vivo validation, a GFP-lentivirus vector was transfected during surgery into the carotid arteries of two rabbits, and GFP-targeted vessels were harvested for digital optical imaging ex vivo. Optical imaging of cell phantoms resulted in a spatial resolution of 25 microm/pixel. Fluorescent signals were detected as diffusely distributed bright spots. At ex vivo optical imaging of arterial tissues, the average fluorescent signal was significantly higher (P <.05) in GFP-targeted tissues (mean +/- SD, 9,357.3 absolute units of density +/- 1,001.3) than in control tissues (5,633.7 absolute units of density +/- 985.2). Both fluorescence microscopic and immunohistochemical findings confirmed these differences between GFP-targeted and control vessels. The digital optical imaging system was sensitive to GFPs and may potentially provide an in vivo imaging tool to monitor and track vascular gene transfer and expression in experimental investigations.

Conjugates of a Photoactivated Rhodamine with Biopolymers for Cell Staining

PubMed Central

Zaitsev, Sergei Yu.; Shaposhnikov, Mikhail N.; Solovyeva, Daria O.; Solovyeva, Valeria V.; Rizvanov, Albert A.

2014-01-01

Conjugates of the photoactivated rhodamine dyes with biopolymers (proteins, polysaccharides, and nucleic acids) are important tools for microscopic investigation of biological tissue. In this study, a precursor of the photoactivated fluorescent dye (PFD) has been successfully used for staining of numerous mammalian cells lines and for conjugate formation with chitosan (“Chitosan-PFD”) and histone H1 (“Histone H1.3-PFD”). The intensive fluorescence has been observed after photoactivation of these conjugates inside cells (A431, HaCaT, HEK239, HBL-100, and MDCK). Developed procedures and obtained data are important for further application of novel precursors of fluorescent dyes (“caged” dyes) for microscopic probing of biological objects. Thus, the synthesized “Chitosan-PFD” and “Histone H1-PFD” have been successfully applied in this study for intracellular transport visualization by fluorescent microscopy. PMID:25383365

Intensity calibration of a laser scanning confocal microscope based on concentrated dyes.

PubMed

Model, Michael A; Blank, James L

2006-10-01

To find water-soluble fluorescent dyes with absorption in various regions of the spectrum and investigate their utility as standards for laser scanning confocal microscopy. Several dyes were found to have characteristics required for fluorescence microscopy standards. The intensity of biological fluorescent specimens was measured against the emission of concentrated dyes. Results using different optics and different microscopes were compared. Slides based on concentrated dyes can be prepared in a highly reproducible manner and are stable under laser scanning. Normalized fluorescence of biological specimens remains consistent with different objective lenses and is tolerant to some mismatch in optical filters or imperfect pinhole alignment. Careful choice of scanning parameters is necessary to ensure linearity of intensity measurements. Concentrated dyes provide a robust and inexpensive intensity standard that can be used in basic research or clinical studies.

Lagrangian 3D tracking of fluorescent microscopic objects in motion

NASA Astrophysics Data System (ADS)

Darnige, T.; Figueroa-Morales, N.; Bohec, P.; Lindner, A.; Clément, E.

2017-05-01

We describe the development of a tracking device, mounted on an epi-fluorescent inverted microscope, suited to obtain time resolved 3D Lagrangian tracks of fluorescent passive or active micro-objects in microfluidic devices. The system is based on real-time image processing, determining the displacement of a x, y mechanical stage to keep the chosen object at a fixed position in the observation frame. The z displacement is based on the refocusing of the fluorescent object determining the displacement of a piezo mover keeping the moving object in focus. Track coordinates of the object with respect to the microfluidic device as well as images of the object are obtained at a frequency of several tenths of Hertz. This device is particularly well adapted to obtain trajectories of motile micro-organisms in microfluidic devices with or without flow.

Lagrangian 3D tracking of fluorescent microscopic objects in motion.

PubMed

Darnige, T; Figueroa-Morales, N; Bohec, P; Lindner, A; Clément, E

2017-05-01

We describe the development of a tracking device, mounted on an epi-fluorescent inverted microscope, suited to obtain time resolved 3D Lagrangian tracks of fluorescent passive or active micro-objects in microfluidic devices. The system is based on real-time image processing, determining the displacement of a x, y mechanical stage to keep the chosen object at a fixed position in the observation frame. The z displacement is based on the refocusing of the fluorescent object determining the displacement of a piezo mover keeping the moving object in focus. Track coordinates of the object with respect to the microfluidic device as well as images of the object are obtained at a frequency of several tenths of Hertz. This device is particularly well adapted to obtain trajectories of motile micro-organisms in microfluidic devices with or without flow.

In vitro monitoring of oxidative processes with self-aggregating gold nanoparticles using all-optical photoacoustic spectroscopy.

PubMed

Yasmin, Zannatul; Khachatryan, Edward; Lee, Yuan-Hao; Maswadi, Saher; Glickman, Randolph; Nash, Kelly L

2015-02-15

In this work, the assembly of gold nanoparticles of (AuNPs) is used to detect the presence of the biomolecule glutathione (GSH) using a novel technique called "all-optical photoacoustic spectroscopy" (AOPAS). The AOPAS technique coupled with AuNPs forms the basis of a biosensing technique capable of probing the dynamic evolution of nano-bio interfaces within a microscopic volume. Dynamic Light Scattering (DLS) and ultraviolet-visible (UV-vis) spectra were measured to describe the kinetics governing the interparticle interactions by monitoring the AuNPs assembly and evolution of the surface plasmon resonance (SPR) band. A comparison of the same dynamic evolution of AuNPs assembly was performed using the AOPAS technique to confirm the validity of this method. The fundamental study is complemented by a demonstration of the performance of this biosensing technique in the presence of cell culture medium containing fetal bovine serum (FBS), which forms a protein corona on the surface of the AuNPs. This work demonstrates that the in vitro monitoring capabilities of the AOPAS provides sensitive measurement at the microscopic level and low nanoparticle concentrations without the artifacts limiting the use of conventional biosensing methods, such as fluorescent indicators. The AOPAS technique not only provides a facile approach for in vitro biosensing, but also shed a light on the real-time detection of thiol containing oxidative stress biomarkers in live systems using AuNPs. Copyright © 2014 Elsevier B.V. All rights reserved.

Synthesis and characterization of ZnS@Fe3O4 fluorescent-magnetic bifunctional nanospheres

NASA Astrophysics Data System (ADS)

Koc, Kenan; Karakus, Baris; Rajar, Kausar; Alveroglu, Esra

2017-10-01

Herein, we synthesized and characterized fluorescent and super paramagnetic ZnS@Fe3O4 nanospheres. First, (3-mercaptopropyl) trimethoxysilane (MPS) capped ZnS quantum dots (QDs) and SiO2 coated Fe3O4 nanoparticles were synthesized separately by using solution growth and co-precipitation techniques. After synthesis and characterization of these two nanoparticles, they were conglutinated together in a nano sized sphere. The QDs were attached to the surface of the Fe3O4 nanoparticles by Sisbnd Osbnd Si bonds and so Sisbnd Osbnd Si bonds created a SiO2 network around the nanoparticles during the formation of the ZnS@Fe3O4 nanospheres. The synthesized MPS capped ZnS fluorescent QDs, SiO2 coated magnetite super paramagnetic nanoparticles and ZnS@Fe3O4 fluorescent-magnetic bifunctional nanospheres were characterized by using UV-Vis Absorption Spectroscopy, Fluorescence Spectroscopy, X-ray analysis, Vibrating Sample Magnetometer analysis, Attenuated Total Reflection-Fourier Transform Infrared Spectroscopy, Scanning Electron Microscope and Energy-dispersive X-ray spectroscopy. ZnS@Fe3O4 bifunctional nanospheres were shown to retain the magnetic properties of magnetite, while exhibiting the luminescent optical properties of ZnS nanoparticles. The combination of fluorescent and magnetic behaviors of nano composites make them useful for potential applications in the field of bio-medical and environmental.

A modular, open-source, slide-scanning microscope for diagnostic applications in resource-constrained settings

PubMed Central

Lu, Qiang; Liu, Guanghui; Xiao, Chuanli; Hu, Chuanzhen; Zhang, Shiwu; Xu, Ronald X.; Chu, Kaiqin; Xu, Qianming

2018-01-01

In this paper we report the development of a cost-effective, modular, open source, and fully automated slide-scanning microscope, composed entirely of easily available off-the-shelf parts, and capable of bright field and fluorescence modes. The automated X-Y stage is composed of two low-cost micrometer stages coupled to stepper motors operated in open-loop mode. The microscope is composed of a low-cost CMOS sensor and low-cost board lenses placed in a 4f configuration. The system has approximately 1 micron resolution, limited by the f/# of available board lenses. The microscope is compact, measuring just 25×25×30 cm, and has an absolute positioning accuracy of ±1 μm in the X and Y directions. A Z-stage enables autofocusing and imaging over large fields of view even on non-planar samples, and custom software enables automatic determination of sample boundaries and image mosaicking. We demonstrate the utility of our device through imaging of fluorescent- and transmission-dye stained blood and fecal smears containing human and animal parasites, as well as several prepared tissue samples. These results demonstrate image quality comparable to high-end commercial microscopes at a cost of less than US$400 for a bright-field system, with an extra US$100 needed for the fluorescence module. PMID:29543835

A versatile new technique to clear mouse and human brain

NASA Astrophysics Data System (ADS)

Costantini, Irene; Di Giovanna, Antonino Paolo; Allegra Mascaro, Anna Letizia; Silvestri, Ludovico; Müllenbroich, Marie Caroline; Sacconi, Leonardo; Pavone, Francesco S.

2015-07-01

Large volumes imaging with microscopic resolution is limited by light scattering. In the last few years based on refractive index matching, different clearing approaches have been developed. Organic solvents and water-based optical clearing agents have been used for optical clearing of entire mouse brain. Although these methods guarantee high transparency and preservation of the fluorescence, though present other non-negligible limitations. Tissue transformation by CLARITY allows high transparency, whole brain immunolabelling and structural and molecular preservation. This method however requires a highly expensive refractive index matching solution limiting practical applicability. In this work we investigate the effectiveness of a water-soluble clearing agent, the 2,2'-thiodiethanol (TDE) to clear mouse and human brain. TDE does not quench the fluorescence signal, is compatible with immunostaining and does not introduce any deformation at sub-cellular level. The not viscous nature of the TDE make it a suitable agent to perform brain slicing during serial two-photon (STP) tomography. In fact, by improving penetration depth it reduces tissue slicing, decreasing the acquisition time and cutting artefacts. TDE can also be used as a refractive index medium for CLARITY. The potential of this method has been explored by imaging a whole transgenic mouse brain with the light sheet microscope. Moreover we apply this technique also on blocks of dysplastic human brain tissue transformed with CLARITY and labeled with different antibody. This clearing approach significantly expands the application of single and two-photon imaging, providing a new useful method for quantitative morphological analysis of structure in mouse and human brain.

Novel flashlamp-based time-resolved fluorescence microscope reduces autofluorescence for 30-fold contrast enhancement in environmental samples

NASA Astrophysics Data System (ADS)

Connally, Russell; Veal, Duncan; Piper, James A.

2003-07-01

The abundance of naturally fluorescing components (autofluorophors) encountered in environmentally sourced samples can greatly hinder the detection and identification of fluorescently labeled target using fluorescence microscopy. Time-resolved fluorescence microscopy (TRFM) is a technique that reduces the effects of autofluorescence through precisely controlled time delays. Lanthanide chelates have fluorescence lifetimes many orders of magnitude greater than typical autofluorophors, and persist in their luminescence long after autofluorescence has ceased. An intense short pulse of (UV) light is used to excite fluorescence in the sample and after a short delay period the longer persisting fluorescence from the chelate is captured with an image-intensified CCD camera. The choice of pulsed excitation source for TRFM has a large impact on the price and performance of the instrument. A flashlamp with a short pulse duration was selected for our instrument because of the high spectral energy in the UV region and short pulse length. However, flash output decays with an approximate lifetime of 18μs and the TRFM requires a long-lived chelate to ensure probe fluorescence is still visible after decay of the flash plasma. We synthesized a recently reported fluorescent chelate (BHHCT) and conjugated it to a monoclonal antibody directed against the water-borne parasite Giardia lamblia. Fluorescence lifetime of the construct was determined to be 339μs +/- 14μs and provided a 45-fold enhancement of labeled Giardia over background using a gate delay of 100μs. Despite the sub-optimal decay characteristics of the light pulse, flashlamps have many advantages compared to optical chopper wheels and modulated lasers. Their low cost, lack of vibration, ease of interface and small footprint are important factors to consider in TRFM design.

Plasmonic Imaging of Electrochemical Reactions of Single Nanoparticles.

PubMed

Fang, Yimin; Wang, Hui; Yu, Hui; Liu, Xianwei; Wang, Wei; Chen, Hong-Yuan; Tao, N J

2016-11-15

Electrochemical reactions are involved in many natural phenomena, and are responsible for various applications, including energy conversion and storage, material processing and protection, and chemical detection and analysis. An electrochemical reaction is accompanied by electron transfer between a chemical species and an electrode. For this reason, it has been studied by measuring current, charge, or related electrical quantities. This approach has led to the development of various electrochemical methods, which have played an essential role in the understanding and applications of electrochemistry. While powerful, most of the traditional methods lack spatial and temporal resolutions desired for studying heterogeneous electrochemical reactions on electrode surfaces and in nanoscale materials. To overcome the limitations, scanning probe microscopes have been invented to map local electrochemical reactions with nanometer resolution. Examples include the scanning electrochemical microscope and scanning electrochemical cell microscope, which directly image local electrochemical reaction current using a scanning electrode or pipet. The use of a scanning probe in these microscopes provides high spatial resolution, but at the expense of temporal resolution and throughput. This Account discusses an alternative approach to study electrochemical reactions. Instead of measuring electron transfer electrically, it detects the accompanying changes in the reactant and product concentrations on the electrode surface optically via surface plasmon resonance (SPR). SPR is highly surface sensitive, and it provides quantitative information on the surface concentrations of reactants and products vs time and electrode potential, from which local reaction kinetics can be analyzed and quantified. The plasmonic approach allows imaging of local electrochemical reactions with high temporal resolution and sensitivity, making it attractive for studying electrochemical reactions in biological systems and nanoscale materials with high throughput. The plasmonic approach has two imaging modes: electrochemical current imaging and interfacial impedance imaging. The former images local electrochemical current associated with electrochemical reactions (faradic current), and the latter maps local interfacial impedance, including nonfaradic contributions (e.g., double layer charging). The plasmonic imaging technique can perform voltammetry (cyclic or square wave) in an analogous manner to the traditional electrochemical methods. It can also be integrated with bright field, dark field, and fluorescence imaging capabilities in one optical setup to provide additional capabilities. To date the plasmonic imaging technique has found various applications, including mapping of heterogeneous surface reactions, analysis of trace substances, detection of catalytic reactions, and measurement of graphene quantum capacitance. The plasmonic and other emerging optical imaging techniques (e.g., dark field and fluorescence microscopy), together with the scanning probe-based electrochemical imaging and single nanoparticle analysis techniques, provide new capabilities for one to study single nanoparticle electrochemistry with unprecedented spatial and temporal resolutions. In this Account, we focus on imaging of electrochemical reactions at single nanoparticles.

ReagentTF: a rapid and versatile optical clearing method for biological imaging(Conference Presentation)

NASA Astrophysics Data System (ADS)

Yu, Tingting; Zhu, Jingtan; Li, Yusha; Qi, Yisong; Xu, Jianyi; Gong, Hui; Luo, Qingming; Zhu, Dan

2017-02-01

The emergence of various optical clearing methods provides a great potential for imaging deep inside tissues by combining with multiple-labelling and microscopic imaging techniques. They were generally developed for specific imaging demand thus presented some non-negligible limitations such as long incubation time, tissue deformation, fluorescence quenching, incompatibility with immunostaining or lipophilic tracers. In this study, we developed a rapid and versatile clearing method, termed ReagentTF, for deep imaging of various fluorescent samples. This method can not only efficiently clear embryos, neonatal whole-brains and adult thick brain sections by simple immersion in aqueous mixtures with minimal volume change, but also can preserve fluorescence of various fluorescent proteins and simultaneously be compatible with immunostaining and lipophilic neuronal dyes. We demonstrate the effectiveness of this method in reconstructing the cell distributions of mouse hippocampus, visualizing the neural projection from CA1 (Cornu Ammonis 1) to HDB (nucleus of the horizontal limb of the diagonal band), and observing the growth of forelimb plexus in whole-mount embryos. These results suggest that ReagentTF is useful for large-volume imaging and will be an option for the deep imaging of biological tissues.

Correlative 3D superresolution fluorescence and electron microscopy reveal the relationship of mitochondrial nucleoids to membranes

PubMed Central

Kopek, Benjamin G.; Shtengel, Gleb; Xu, C. Shan; Clayton, David A.; Hess, Harald F.

2012-01-01

Microscopic images of specific proteins in their cellular context yield important insights into biological processes and cellular architecture. The advent of superresolution optical microscopy techniques provides the possibility to augment EM with nanometer-resolution fluorescence microscopy to access the precise location of proteins in the context of cellular ultrastructure. Unfortunately, efforts to combine superresolution fluorescence and EM have been stymied by the divergent and incompatible sample preparation protocols of the two methods. Here, we describe a protocol that preserves both the delicate photoactivatable fluorescent protein labels essential for superresolution microscopy and the fine ultrastructural context of EM. This preparation enables direct 3D imaging in 500- to 750-nm sections with interferometric photoactivatable localization microscopy followed by scanning EM images generated by focused ion beam ablation. We use this process to “colorize” detailed EM images of the mitochondrion with the position of labeled proteins. The approach presented here has provided a new level of definition of the in vivo nature of organization of mitochondrial nucleoids, and we expect this straightforward method to be applicable to many other biological questions that can be answered by direct imaging. PMID:22474357

Optimization of a cAMP response element signal pathway reporter system.

PubMed

Shan, Qiang; Storm, Daniel R

2010-08-15

A sensitive cAMP response element (CRE) reporter system is essential for studying the cAMP/protein kinase A/cAMP response element binding protein signal pathway. Here we have tested a few CRE promoters and found one with high sensitivity to external stimuli. Using this optimal CRE promoter and the enhanced green fluorescent protein as the reporter, we have established a CRE reporter cell line. This cell line can be used to study the signal pathway by fluorescent microscope, fluorescence-activated cell analysis and luciferase assay. This cell line's sensitivity to forskolin, using the technique of fluorescence-activated cell sorting, was increased to approximately seven times that of its parental HEK 293 cell line, which is currently the most commonly used cell line in the field for the signal pathway study. Therefore, this newly created cell line is potentially useful for studying the signal pathway's modulators, which generally have weaker effect than its mediators. Our research has also established a general procedure for optimizing transcription-based reporter cell lines, which might be useful in performing the same task when studying many other transcription-based signal pathways. (c) 2010 Elsevier B.V. All rights reserved.

Molecular cytogenetics using fluorescence in situ hybridization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gray, J.W.; Kuo, Wen-Lin; Lucas, J.

1990-12-07

Fluorescence in situ hybridization (FISH) with chromosome-specific probes enables several new areas of cytogenetic investigation by allowing visual determination of the presence and normality of specific genetic sequences in single metaphase or interphase cells. in this approach, termed molecular cytogenetics, the genetic loci to be analyzed are made microscopically visible in single cells using in situ hybridization with nucleic acid probes specific to these loci. To accomplish this, the DNA in the target cells is made single stranded by thermal denaturation and incubated with single-stranded, chemically modified probe under conditions where the probe will anneal only with DNA sequences tomore » which it has high DNA sequence homology. The bound probe is then made visible by treatment with a fluorescent reagent such as fluorescein that binds to the chemical modification carried by the probe. The DNA to which the probe does not bind is made visible by staining with a dye such as propidium iodide that fluoresces at a wavelength different from that of the reagent used for probe visualization. We show in this report that probes are now available that make this technique useful for biological dosimetry, prenatal diagnosis and cancer biology. 31 refs., 3 figs.« less

Green synthesis of multifunctional carbon dots from coriander leaves and their potential application as antioxidants, sensors and bioimaging agents.

PubMed

Sachdev, Abhay; Gopinath, P

2015-06-21

In the present study, a facile one-step hydrothermal treatment of coriander leaves for preparing carbon dots (CDs) has been reported. Optical and structural properties of the CDs have been extensively studied by UV-visible and fluorescence spectroscopic, microscopic (transmission electron microscopy, scanning electron microscopy) and X-ray diffraction techniques. Surface functionality and composition of the CDs have been illustrated by elemental analysis and Fourier transform infrared spectroscopy (FTIR). Quenching of the fluorescence of the CDs in the presence of metal ions is of prime significance, hence CDs have been used as a fluorescence probe for sensitive and selective detection of Fe(3+) ions. Eventually, biocompatibility and bioimaging aspects of CDs have been evaluated on lung normal (L-132) and cancer (A549) cell lines. Qualitative analysis of cellular uptake of CDs has been pursued through fluorescence microscopy, while quantitative analysis using a flow cytometer provided an insight into the concentration and cell-type dependent uptake of CDs. The article further investigates the antioxidant activity of CDs. Therefore, we have validated the practicality of CDs obtained from a herbal carbon source for versatile applications.

Nanoscale cellular imaging with scanning angle interference microscopy.

PubMed

DuFort, Christopher; Paszek, Matthew

2014-01-01

Fluorescence microscopy is among the most widely utilized tools in cell and molecular biology due to its ability to noninvasively obtain time-resolved images of live cells with molecule-specific contrast. In this chapter, we describe a simple high-resolution technique, scanning angle interference microscopy (SAIM), for the imaging and localization of fluorescent molecules with nanometer precision along the optical axis. In SAIM, samples above a reflective surface are sequentially scanned with an excitation laser at varying angles of incidence. Interference patterns generated between the incident and reflected lights result in an emission intensity that depends on the height of a fluorophore above the silicon surface and the angle of the incident radiation. The measured fluorescence intensities are then fit to an optical model to localize the labeled molecules along the z-axis with 5-10 nm precision and diffraction-limited lateral resolution. SAIM is easily implemented on widely available commercial total internal reflection fluorescence microscopes, offering potential for widespread use in cell biology. Here, we describe the setup of SAIM and its application for imaging cellular structures near (<1 μm) the sample substrate. © 2014 Elsevier Inc. All rights reserved.

Molecular Organization Induced Anisotropic Properties of Perylene - Silica Hybrid Nanoparticles.

PubMed

Sriramulu, Deepa; Turaga, Shuvan Prashant; Bettiol, Andrew Anthony; Valiyaveettil, Suresh

2017-08-10

Optically active silica nanoparticles are interesting owing to high stability and easy accessibility. Unlike previous reports on dye loaded silica particles, here we address an important question on how optical properties are dependent on the aggregation-induced segregation of perylene molecules inside and outside the silica nanoparticles. Three differentially functionalized fluorescent perylene - silica hybrid nanoparticles are prepared from appropriate ratios of perylene derivatives and tetraethyl orthosilicate (TEOS) and investigated the structure property correlation (P-ST, P-NP and P-SF). The particles differ from each other on the distribution, organization and intermolecular interaction of perylene inside or outside the silica matrix. Structure and morphology of all hybrid nanoparticles were characterized using a range of techniques such as electron microscope, optical spectroscopic measurements and thermal analysis. The organizations of perylene in three different silica nanoparticles were explored using steady-state fluorescence, fluorescence anisotropy, lifetime measurements and solid state polarized spectroscopic studies. The interactions and changes in optical properties of the silica nanoparticles in presence of different amines were tested and quantified both in solution and in vapor phase using fluorescence quenching studies. The synthesized materials can be regenerated after washing with water and reused for sensing of amines.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zeng, Yining; Yarbrough, John M.; Mittal, Ashutosh

Xylan constitutes a significant portion of biomass (e.g. 22% in corn stover used in this study). Xylan is also an important source of carbohydrates, besides cellulose, for renewable and sustainable energy applications. Currently used method for the localization of xylan in biomass is to use fluorescence confocal microscope to image the fluorescent dye labeled monoclonal antibody that specifically binds to xylan. With the rapid adoption of the Raman-based label-free chemical imaging techniques in biology, identifying Raman bands that are unique to xylan would be critical for the implementation of the above label-free techniques for in situ xylan imaging. Unlike ligninmore » and cellulose that have long be assigned fingerprint Raman bands, specific Raman bands for xylan remain unclear. The major challenge is the cellulose in plant cell wall, which has chemical units highly similar to that of xylan. Here we report using xylanase to specifically remove xylan from feedstock. Under various degree of xylan removal, with minimum impact to other major cell wall components, i.e. lignin and cellulose, we have identified Raman bands that could be further tested for chemical imaging of xylan in biomass in situ.« less

Fluorescence ratiometric classifier for the detection of skin pathologies

NASA Astrophysics Data System (ADS)

Anand, Suresh; Cicchi, Riccardo; Cosci, Alessandro; Rossari, Susanna; Kapsokalyvas, Dimitrios; Baria, Enrico; Maio, Vincenza; Massi, Daniela; De Giorgi, Vincenzo; Pimpinelli, Nicola; Pavone, Francesco S.

2015-07-01

Detection of pre-malignant lesions in skin could help in reducing the 5 year patient mortality rates and greatly advancing the quality of life. Current gold standard for the detection of skin pathologies is a tissue biopsy and followed by a series of steps before it is examined under a light microscope by a pathologist. The disadvantage with this method is its invasiveness. Light based biomedical point spectroscopic techniques offers an adjunct technique to invasive tissue pathology. In this context, we have implemented a simple multiplexed ratiometric approach (F470/F560 and F510/F580) based on fluorescence at two excitation wavelengths 378 nm and 445 nm respectively. The emission profile at these excitation wavelengths showed a shift towards the longer wavelengths for melanoma when compared with normal and nevus. At both excitation wavelengths, we observed an increased intensity ratios for normal, followed by nevus and melanoma. This intensity ratios provide a good diagnostic capability in differentiating normal, nevus and melanocytic skin lesions. This method could be applied in vivo because of the simplicity involved in discriminating normal and pathological skin tissues.

Sensitive and inexpensive digital DNA analysis by microfluidic enrichment of rolling circle amplified single-molecules

PubMed Central

Kühnemund, Malte; Hernández-Neuta, Iván; Sharif, Mohd Istiaq; Cornaglia, Matteo; Gijs, Martin A.M.

2017-01-01

Abstract Single molecule quantification assays provide the ultimate sensitivity and precision for molecular analysis. However, most digital analysis techniques, i.e. droplet PCR, require sophisticated and expensive instrumentation for molecule compartmentalization, amplification and analysis. Rolling circle amplification (RCA) provides a simpler means for digital analysis. Nevertheless, the sensitivity of RCA assays has until now been limited by inefficient detection methods. We have developed a simple microfluidic strategy for enrichment of RCA products into a single field of view of a low magnification fluorescent sensor, enabling ultra-sensitive digital quantification of nucleic acids over a dynamic range from 1.2 aM to 190 fM. We prove the broad applicability of our analysis platform by demonstrating 5-plex detection of as little as ∼1 pg (∼300 genome copies) of pathogenic DNA with simultaneous antibiotic resistance marker detection, and the analysis of rare oncogene mutations. Our method is simpler, more cost-effective and faster than other digital analysis techniques and provides the means to implement digital analysis in any laboratory equipped with a standard fluorescent microscope. PMID:28077562

Analysis of caspase-3 in ASTC-a-1 cells treated with mitomycin C using acceptor photobleaching techniques

NASA Astrophysics Data System (ADS)

Wang, Huiying; Chen, Tongsheng; Sun, Lei

2008-02-01

Caspase-3 is a key activated death protease, which catalyzes the specific cleavage of many cellular proteins and induces DNA cleavage eventually. In this report, cells were treated with mitomycin C (MMC) at different concentration and its activity was detected by cell counting kit (CCK-8). Based on results of CCK-8, cells were treated with 10μg/mL MMC and Hoechst 33258 has been used to observe cell apoptosis. Fluorescence resonance energy transfer (FRET) and confocal microscopy have been used to the effect of MMC on the caspase3 activation in living cells. Human lung adenocarcinoma cells (ASTC-a-1) was transfected with plasmid SCAT3 (pSCAT3)/CKAR FRET receptor. Acceptor photobleaching techniques of FRET plasmid has been used to destruct fluorophore of cells stably expressing SCAT3 reporter on a fluorescence confocal microscope. The activity of caspase3 can be analyzed by FRET dynamics of SCAT3 in living cells. Our results show that MM C can induce ASTC-a-1 cell apoptosis through activation of caspase3.

Eyecup scope—optical recordings of light stimulus-evoked fluorescence signals in the retina

PubMed Central

Hausselt, Susanne E.; Breuninger, Tobias; Castell, Xavier; Denk, Winfried; Margolis, David J.; Detwiler, Peter B.

2009-01-01

Dendritic signals play an essential role in processing visual information in the retina. To study them in neurites too small for electrical recording, we developed an instrument that combines a multi-photon (MP) microscope with a through-the-objective high-resolution visual stimulator. An upright microscope was designed that uses the objective lens for both MP imaging and delivery of visual stimuli to functionally intact retinal explants or eyecup preparations. The stimulator consists of a miniature liquid-crystal-on-silicon display coupled into the optical path of an infrared-excitation laser-scanning microscope. A pair of custom-made dichroic filters allows light from the excitation laser and three spectral bands (‘colors’) from the stimulator to reach the retina, leaving two intermediate bands for fluorescence imaging. Special optics allow displacement of the stimulator focus relative to the imaging focus. Spatially resolved changes in calcium-indicator fluorescence in response to visual stimuli were recorded in dendrites of different types of mammalian retinal neurons. PMID:19023590

Complementary studies of lipid membrane dynamics using iSCAT and super-resolved fluorescence correlation spectroscopy

NASA Astrophysics Data System (ADS)

Reina, Francesco; Galiani, Silvia; Shrestha, Dilip; Sezgin, Erdinc; de Wit, Gabrielle; Cole, Daniel; Lagerholm, B. Christoffer; Kukura, Philipp; Eggeling, Christian

2018-06-01

Observation techniques with high spatial and temporal resolution, such as single-particle tracking based on interferometric scattering (iSCAT) microscopy, and fluorescence correlation spectroscopy applied on a super-resolution STED microscope (STED-FCS), have revealed new insights of the molecular organization of membranes. While delivering complementary information, there are still distinct differences between these techniques, most prominently the use of fluorescent dye tagged probes for STED-FCS and a need for larger scattering gold nanoparticle tags for iSCAT. In this work, we have used lipid analogues tagged with a hybrid fluorescent tag–gold nanoparticle construct, to directly compare the results from STED-FCS and iSCAT measurements of phospholipid diffusion on a homogeneous supported lipid bilayer (SLB). These comparative measurements showed that while the mode of diffusion remained free, at least at the spatial (>40 nm) and temporal (50  ⩽  t  ⩽  100 ms) scales probed, the diffussion coefficient was reduced by 20- to 60-fold when tagging with 20 and 40 nm large gold particles as compared to when using dye tagged lipid analogues. These FCS measurements of hybrid fluorescent tag–gold nanoparticle labeled lipids also revealed that commercially supplied streptavidin-coated gold nanoparticles contain large quantities of free streptavidin. Finally, the values of apparent diffusion coefficients obtained by STED-FCS and iSCAT differed by a factor of 2–3 across the techniques, while relative differences in mobility between different species of lipid analogues considered were identical in both approaches. In conclusion, our experiments reveal that large and potentially cross-linking scattering tags introduce a significant slow-down in diffusion on SLBs but no additional bias, and our labeling approach creates a new way of exploiting complementary information from STED-FCS and iSCAT measurements.

Swept Field Laser Confocal Microscopy for Enhanced Spatial and Temporal Resolution in Live-Cell Imaging

PubMed Central

Castellano-Muñoz, Manuel; Peng, Anthony Wei; Salles, Felipe T.; Ricci, Anthony J.

2013-01-01

Confocal fluorescence microscopy is a broadly used imaging technique that enhances the signal-to-noise ratio by removing out of focal plane fluorescence. Confocal microscopes come with a variety of modifications depending on the particular experimental goals. Microscopes, illumination pathways, and light collection were originally focused upon obtaining the highest resolution image possible, typically on fixed tissue. More recently, live-cell confocal imaging has gained importance. Since measured signals are often rapid or transient, thus requiring higher sampling rates, specializations are included to enhance spatial and temporal resolution while maintaining tissue viability. Thus, a balance between image quality, temporal resolution, and tissue viability is needed. A subtype of confocal imaging, termed swept field confocal (SFC) microscopy, can image live cells at high rates while maintaining confocality. SFC systems can use a pinhole array to obtain high spatial resolution, similar to spinning disc systems. In addition, SFC imaging can achieve faster rates by using a slit to sweep the light across the entire image plane, thus requiring a single scan to generate an image. Coupled to a high-speed charge-coupled device camera and a laser illumination source, images can be obtained at greater than 1,000 frames per second while maintaining confocality. PMID:22831554

Immunodetection and intracellular localization of caldesmon-like proteins in Amoeba proteus.

PubMed

Gagola, M; Kłopocka, W; Greebecki, A; Makuch, R

2003-09-01

Caldesmon immunoanalogues were detected in Amoeba proteus cell homogenates by the Western blot technique. Three immunoreactive bands were recognized by polyclonal antibodies against the whole molecule of chicken gizzard caldesmon as well as by a monoclonal antibody against its C-terminal domain: one major and two minor bands corresponding to proteins with apparent molecular masses of 150, 69, and 60 kDa. The presence of caldesmon-like protein(s) in amoebae was revealed as well in single cells after their fixation, staining with the same antibodies, and recording their total fluorescence in a confocal laser scanning microscope. Proteins recognized by the antibodies bind to filamentous actin. This was established by a cosedimentation assay in cell homogenates and by colocalization of the caldesmon-related immunofluorescence with the fluorescence of filamentous actin stained with rhodamine-labelled phalloidin, demonstrated in optical sections of single cells in a confocal microscope. Caldesmon is colocalized with filamentous actin in the withdrawn cell regions where the cortical actomyosin network contracts and actin is depolymerized, in the frontal zone where actin is polymerized again and the cortical cytoskeleton is reconstructed, inside the nucleus and in the perinuclear cytoskeleton, and probably at the cell-to-substratum adhesion sites. The regulatory role of caldesmon in these functionally different regions of locomoting amoebae is discussed.

Correlative light and electron microscopic detection of GFP-labeled proteins using modular APEX.

PubMed

Ariotti, Nicholas; Hall, Thomas E; Parton, Robert G

2017-01-01

The use of green fluorescent protein (GFP) and related proteins has revolutionized light microscopy. Here we describe a rapid and simple method to localize GFP-tagged proteins in cells and in tissues by electron microscopy (EM) using a modular approach involving a small GFP-binding peptide (GBP) fused to the ascorbate peroxidase-derived APEX2 tag. We provide a method for visualizing GFP-tagged proteins by light and EM in cultured cells and in the zebrafish using modular APEX-GBP. Furthermore, we describe in detail the benefits of this technique over many of the currently available correlative light and electron microscopy approaches and demonstrate APEX-GBP is readily applicable to modern three-dimensional techniques. Copyright © 2017 Elsevier Inc. All rights reserved.

Transmission X-ray microscopy for full-field nano imaging of biomaterials.

PubMed

Andrews, Joy C; Meirer, Florian; Liu, Yijin; Mester, Zoltan; Pianetta, Piero

2011-07-01

Imaging of cellular structure and extended tissue in biological materials requires nanometer resolution and good sample penetration, which can be provided by current full-field transmission X-ray microscopic techniques in the soft and hard X-ray regions. The various capabilities of full-field transmission X-ray microscopy (TXM) include 3D tomography, Zernike phase contrast, quantification of absorption, and chemical identification via X-ray fluorescence and X-ray absorption near edge structure imaging. These techniques are discussed and compared in light of results from the imaging of biological materials including microorganisms, bone and mineralized tissue, and plants, with a focus on hard X-ray TXM at ≤ 40-nm resolution. Copyright © 2010 Wiley-Liss, Inc.

Photoluminescence study of carbon dots from ginger and galangal herbs using microwave technique

NASA Astrophysics Data System (ADS)

Isnaeni; Rahmawati, I.; Intan, R.; Zakaria, M.

2018-03-01

Carbon dots are new type of fluorescent nanoparticle that can be synthesis easily from natural sources. We have synthesized carbon dots from ginger and galangal herbs using microwave technique and studied their optical properties. We synthesized colloidal carbon dots in water solvent by varying microwave processing time. UV-Vis absorbance, photoluminescence, time-resolved photoluminescence, and transmission electron microscope were utilized to study properties of carbon dots. We found that microwave processing time significantly affect optical properties of synthesized carbon dots. UV-Vis absorbance spectra and time-resolved photoluminescence results show that luminescent of carbon dots is dominated by recombination process from n-π* surface energy level. With further development, these carbon dots are potential for several applications.

Quantitative orientation-independent differential interference contrast (DIC) microscopy

NASA Astrophysics Data System (ADS)

Shribak, Michael; LaFountain, James; Biggs, David; Inoué, Shinya

2007-02-01

We describe a new DIC technique, which records phase gradients within microscopic specimens independently of their orientation. The proposed system allows the generation of images representing the distribution of dry mass (optical path difference) in the specimen. Unlike in other forms of interference microscopes, this approach does not require a narrow illuminating cone. The orientation-independent differential interference contrast (OI-DIC) system can also be combined with orientation-independent polarization (OI-Pol) measurements to yield two complementary images: one showing dry mass distribution (which is proportional to refractive index) and the other showing distribution of birefringence (due to structural or internal anisotropy). With a model specimen used for this work -- living spermatocytes from the crane fly, Nephrotoma suturalis --- the OI-DIC image clearly reveals the detailed shape of the chromosomes while the polarization image quantitatively depicts the distribution of the birefringent microtubules in the spindle, both without any need for staining or other modifications of the cell. We present examples of a pseudo-color combined image incorporating both orientation-independent DIC and polarization images of a spermatocyte at diakinesis and metaphase of meiosis I. Those images provide clear evidence that the proposed technique can reveal fine architecture and molecular organization in live cells without perturbation associated with staining or fluorescent labeling. The phase image was obtained using optics having a numerical aperture 1.4, thus achieving a level of resolution never before achieved with any interference microscope.

Synthesis of di-functional ligand and fluorescently labeling SiO2 microspheres

NASA Astrophysics Data System (ADS)

Chen, Kexu; Kang, Ming; Liu, Min; Shen, Simin; Sun, Rong

2018-05-01

In order to complete the fluorescent labeling of SiO2 microspheres, a kind of di-functional ligand was synthesized and purified, which could not only coordinate rare earth ions but also react with the active groups to bond host materials with an alkoxysilane groups. Fourier transform infrared spectroscopy (FT-IR), 1H NMR spectra, MS spectra, field emission scanning electron microscope (FESEM), transmission electron microscope (TEM), X-ray photoelectron spectroscopy (XPS) and luminescence spectrophotometer were used to study the structure of di-functional ligand and properties of fluorescent coupling agent and fluorescent labeled SiO2 microspheres. The optimal experiment conditions were acquired as follows: molar ratio as 1: 4 (MDBM: MICPTES), reaction time at 6 h and reaction temperature as 65 °C (yield up to 40%) through the orthogonal experiment and purification process. The results indicated that fluorescent coupling agent presented red photoluminesence of Eu3+ ions at 610 nm, and the absolute quantum yield was 11%. On the other hand, the hydrolysis of the coupling agent reacted on the surface of SiO2 microspheres and presented fluorescent labeling homogeneously.

Hybrid fluorescence and electron cryo-microscopy for simultaneous electron and photon imaging.

PubMed

Iijima, Hirofumi; Fukuda, Yoshiyuki; Arai, Yoshihiro; Terakawa, Susumu; Yamamoto, Naoki; Nagayama, Kuniaki

2014-01-01

Integration of fluorescence light and transmission electron microscopy into the same device would represent an important advance in correlative microscopy, which traditionally involves two separate microscopes for imaging. To achieve such integration, the primary technical challenge that must be solved regards how to arrange two objective lenses used for light and electron microscopy in such a manner that they can properly focus on a single specimen. To address this issue, both lateral displacement of the specimen between two lenses and specimen rotation have been proposed. Such movement of the specimen allows sequential collection of two kinds of microscopic images of a single target, but prevents simultaneous imaging. This shortcoming has been made up by using a simple optical device, a reflection mirror. Here, we present an approach toward the versatile integration of fluorescence and electron microscopy for simultaneous imaging. The potential of simultaneous hybrid microscopy was demonstrated by fluorescence and electron sequential imaging of a fluorescent protein expressed in cells and cathodoluminescence imaging of fluorescent beads. Copyright © 2013 Elsevier Inc. All rights reserved.

Multimodal optoacoustic and multiphoton fluorescence microscopy

NASA Astrophysics Data System (ADS)

Sela, Gali; Razansky, Daniel; Shoham, Shy

2013-03-01

Multiphoton microscopy is a powerful imaging modality that enables structural and functional imaging with cellular and sub-cellular resolution, deep within biological tissues. Yet, its main contrast mechanism relies on extrinsically administered fluorescent indicators. Here we developed a system for simultaneous multimodal optoacoustic and multiphoton fluorescence 3D imaging, which attains both absorption and fluorescence-based contrast by integrating an ultrasonic transducer into a two-photon laser scanning microscope. The system is readily shown to enable acquisition of multimodal microscopic images of fluorescently labeled targets and cell cultures as well as intrinsic absorption-based images of pigmented biological tissue. During initial experiments, it was further observed that that detected optoacoustically-induced response contains low frequency signal variations, presumably due to cavitation-mediated signal generation by the high repetition rate (80MHz) near IR femtosecond laser. The multimodal system may provide complementary structural and functional information to the fluorescently labeled tissue, by superimposing optoacoustic images of intrinsic tissue chromophores, such as melanin deposits, pigmentation, and hemoglobin or other extrinsic particle or dye-based markers highly absorptive in the NIR spectrum.

3D Image Analysis of Geomaterials using Confocal Microscopy

NASA Astrophysics Data System (ADS)

Mulukutla, G.; Proussevitch, A.; Sahagian, D.

2009-05-01

Confocal microscopy is one of the most significant advances in optical microscopy of the last century. It is widely used in biological sciences but its application to geomaterials lingers due to a number of technical problems. Potentially the technique can perform non-invasive testing on a laser illuminated sample that fluoresces using a unique optical sectioning capability that rejects out-of-focus light reaching the confocal aperture. Fluorescence in geomaterials is commonly induced using epoxy doped with a fluorochrome that is impregnated into the sample to enable discrimination of various features such as void space or material boundaries. However, for many geomaterials, this method cannot be used because they do not naturally fluoresce and because epoxy cannot be impregnated into inaccessible parts of the sample due to lack of permeability. As a result, the confocal images of most geomaterials that have not been pre-processed with extensive sample preparation techniques are of poor quality and lack the necessary image and edge contrast necessary to apply any commonly used segmentation techniques to conduct any quantitative study of its features such as vesicularity, internal structure, etc. In our present work, we are developing a methodology to conduct a quantitative 3D analysis of images of geomaterials collected using a confocal microscope with minimal amount of prior sample preparation and no addition of fluorescence. Two sample geomaterials, a volcanic melt sample and a crystal chip containing fluid inclusions are used to assess the feasibility of the method. A step-by-step process of image analysis includes application of image filtration to enhance the edges or material interfaces and is based on two segmentation techniques: geodesic active contours and region competition. Both techniques have been applied extensively to the analysis of medical MRI images to segment anatomical structures. Preliminary analysis suggests that there is distortion in the shapes of the segmented vesicles, vapor bubbles, and void spaces due to the optical measurements, so corrective actions are being explored. This will establish a practical and reliable framework for an adaptive 3D image processing technique for the analysis of geomaterials using confocal microscopy.

Improved localization of cellular membrane receptors using combined fluorescence microscopy and simultaneous topography and recognition imaging

NASA Astrophysics Data System (ADS)

Duman, M.; Pfleger, M.; Zhu, R.; Rankl, C.; Chtcheglova, L. A.; Neundlinger, I.; Bozna, B. L.; Mayer, B.; Salio, M.; Shepherd, D.; Polzella, P.; Moertelmaier, M.; Kada, G.; Ebner, A.; Dieudonne, M.; Schütz, G. J.; Cerundolo, V.; Kienberger, F.; Hinterdorfer, P.

2010-03-01

The combination of fluorescence microscopy and atomic force microscopy has a great potential in single-molecule-detection applications, overcoming many of the limitations coming from each individual technique. Here we present a new platform of combined fluorescence and simultaneous topography and recognition imaging (TREC) for improved localization of cellular receptors. Green fluorescent protein (GFP) labeled human sodium-glucose cotransporter (hSGLT1) expressed Chinese Hamster Ovary (CHO) cells and endothelial cells (MyEnd) from mouse myocardium stained with phalloidin-rhodamine were used as cell systems to study AFM topography and fluorescence microscopy on the same surface area. Topographical AFM images revealed membrane features such as lamellipodia, cytoskeleton fibers, F-actin filaments and small globular structures with heights ranging from 20 to 30 nm. Combined fluorescence and TREC imaging was applied to detect density, distribution and localization of YFP-labeled CD1d molecules on α-galactosylceramide (αGalCer)-loaded THP1 cells. While the expression level, distribution and localization of CD1d molecules on THP1 cells were detected with fluorescence microscopy, the nanoscale distribution of binding sites was investigated with molecular recognition imaging by using a chemically modified AFM tip. Using TREC on the inverted light microscope, the recognition sites of cell receptors were detected in recognition images with domain sizes ranging from ~ 25 to ~ 160 nm, with the smaller domains corresponding to a single CD1d molecule.

Hyperspectral imaging fluorescence excitation scanning for detecting colorectal cancer: pilot study

NASA Astrophysics Data System (ADS)

Leavesley, Silas J.; Wheeler, Mikayla; Lopez, Carmen; Baker, Thomas; Favreau, Peter F.; Rich, Thomas C.; Rider, Paul F.; Boudreaux, Carole W.

2016-03-01

Optical spectroscopy and hyperspectral imaging have shown the theoretical potential to discriminate between cancerous and non-cancerous tissue with high sensitivity and specificity. To date, these techniques have not been able to be effectively translated to endoscope platforms. Hyperspectral imaging of the fluorescence excitation spectrum represents a new technology that may be well-suited for endoscopic implementation. However, the feasibility of detecting differences between normal and cancerous mucosa using fluorescence excitation-scanning hyperspectral imaging has not been evaluated. The objective of this pilot study was to evaluate the changes in the fluorescence excitation spectrum of resected specimen pairs of colorectal adenocarcinoma and normal colorectal mucosa. Patients being treated for colorectal adenocarcinoma were enrolled. Representative adenocarcinoma and normal colonic mucosa specimens were collected from each case. Specimens were flash frozen in liquid nitrogen. Adenocarcinoma was confirmed by histologic evaluation of H&E permanent sections. Hyperspectral image data of the fluorescence excitation of adenocarcinoma and surrounding normal tissue were acquired using a custom microscope configuration previously developed in our lab. Results demonstrated consistent spectral differences between normal and cancerous tissues over the fluorescence excitation spectral range of 390-450 nm. We conclude that fluorescence excitation-scanning hyperspectral imaging may offer an alternative approach for differentiating adenocarcinoma and surrounding normal mucosa of the colon. Future work will focus on expanding the number of specimen pairs analyzed and will utilize fresh tissues where possible, as flash freezing and reconstituting tissues may have altered the autofluorescence properties.

FLIMX: A Software Package to Determine and Analyze the Fluorescence Lifetime in Time-Resolved Fluorescence Data from the Human Eye

PubMed Central

Klemm, Matthias; Schweitzer, Dietrich; Peters, Sven; Sauer, Lydia; Hammer, Martin; Haueisen, Jens

2015-01-01

Fluorescence lifetime imaging ophthalmoscopy (FLIO) is a new technique for measuring the in vivo autofluorescence intensity decays generated by endogenous fluorophores in the ocular fundus. Here, we present a software package called FLIM eXplorer (FLIMX) for analyzing FLIO data. Specifically, we introduce a new adaptive binning approach as an optimal tradeoff between the spatial resolution and the number of photons required per pixel. We also expand existing decay models (multi-exponential, stretched exponential, spectral global analysis, incomplete decay) to account for the layered structure of the eye and present a method to correct for the influence of the crystalline lens fluorescence on the retina fluorescence. Subsequently, the Holm-Bonferroni method is applied to FLIO measurements to allow for group comparisons between patients and controls on the basis of fluorescence lifetime parameters. The performance of the new approaches was evaluated in five experiments. Specifically, we evaluated static and adaptive binning in a diabetes mellitus patient, we compared the different decay models in a healthy volunteer and performed a group comparison between diabetes patients and controls. An overview of the visualization capabilities and a comparison of static and adaptive binning is shown for a patient with macular hole. FLIMX’s applicability to fluorescence lifetime imaging microscopy is shown in the ganglion cell layer of a porcine retina sample, obtained by a laser scanning microscope using two-photon excitation. PMID:26192624

Improved localization of cellular membrane receptors using combined fluorescence microscopy and simultaneous topography and recognition imaging.

PubMed

Duman, M; Pfleger, M; Zhu, R; Rankl, C; Chtcheglova, L A; Neundlinger, I; Bozna, B L; Mayer, B; Salio, M; Shepherd, D; Polzella, P; Moertelmaier, M; Kada, G; Ebner, A; Dieudonne, M; Schütz, G J; Cerundolo, V; Kienberger, F; Hinterdorfer, P

2010-03-19

The combination of fluorescence microscopy and atomic force microscopy has a great potential in single-molecule-detection applications, overcoming many of the limitations coming from each individual technique. Here we present a new platform of combined fluorescence and simultaneous topography and recognition imaging (TREC) for improved localization of cellular receptors. Green fluorescent protein (GFP) labeled human sodium-glucose cotransporter (hSGLT1) expressed Chinese Hamster Ovary (CHO) cells and endothelial cells (MyEnd) from mouse myocardium stained with phalloidin-rhodamine were used as cell systems to study AFM topography and fluorescence microscopy on the same surface area. Topographical AFM images revealed membrane features such as lamellipodia, cytoskeleton fibers, F-actin filaments and small globular structures with heights ranging from 20 to 30 nm. Combined fluorescence and TREC imaging was applied to detect density, distribution and localization of YFP-labeled CD1d molecules on alpha-galactosylceramide (alphaGalCer)-loaded THP1 cells. While the expression level, distribution and localization of CD1d molecules on THP1 cells were detected with fluorescence microscopy, the nanoscale distribution of binding sites was investigated with molecular recognition imaging by using a chemically modified AFM tip. Using TREC on the inverted light microscope, the recognition sites of cell receptors were detected in recognition images with domain sizes ranging from approximately 25 to approximately 160 nm, with the smaller domains corresponding to a single CD1d molecule.

Wear effects on microscopic morphology and hyaluronan uptake in siloxane-hydrogel contact lenses.

PubMed

Tavazzi, Silvia; Tonveronachi, Martina; Fagnola, Matteo; Cozza, Federica; Ferraro, Lorenzo; Borghesi, Alessandro; Ascagni, Miriam; Farris, Stefano

2015-07-01

The purpose of this study was a comparison between new and worn siloxane-hydrogel contact lenses in terms of microscopic structure, surface morphology, and loading of hyaluronan. The analyses were performed by scanning electron microscopy, with the support of the freeze-drying technique, and by fluorescence confocal microscopy. Along the depth profile of new lenses, a thin porous top layer was observed, which corresponds to the region of hyaluronan penetration inside well-defined channels. The time evolution was followed from one day to two weeks of daily wear, when a completely different scenario was found. Clear experimental evidence of a buggy surface was observed with several crests and regions of swelling, which could be filled by the hyaluronan solution. The modifications are attributed to the progressive relaxation of the structure of the polymeric network. © 2014 Wiley Periodicals, Inc.

Imaging Chromosome Separation in Mouse Oocytes by Responsive 3D Confocal Timelapse Microscopy.

PubMed

Lane, Simon I R; Crouch, Stephen; Jones, Keith T

2017-01-01

Accurate chromosome segregation is necessary so that genetic material is equally shared among daughter cells. However, maturing mammalian oocytes are particularly prone to chromosome segregation errors, making them a valuable tool for identifying the causes of mis-segregation. Factors such as aging, cohesion loss, DNA damage, and the roles of a plethora of kinetochore and cell cycle-related proteins are involved. To study chromosome segregation in oocytes in a live setting is an imaging challenge that requires advanced techniques. Here we describe a method for examining chromosomes in live oocytes in detail as they undergo maturation. Our method is based on tracking the "center of brightness" of fluorescently labeled chromosomes. Here we describe how to set up our software and run experiments on a Leica TCS SP8 confocal microscope, but the method would be transferable to other microscopes with computer-aided microscopy.

A novel system for water soluble protein encapsulation with high efficiency: "micelles enhanced" polyelectrolyte capsules.

PubMed

Li, Xiaodong; Li, Xiaohui; Zhang, Jianxiang; Zhao, Shifang; Shen, Jiacong

2008-06-01

Novel "micelles enhanced" polyelectrolyte (PE) capsules based on functional templates of hybrid calcium carbonate were fabricated. Evidences suggested that the structure of capsule wall was different from that of conventional PE capsules, and the wall permeability of these PE capsules changed significantly. Lysozyme, a positively charged protein in neutral solution, was studied as a model protein to be encapsulated into the "micelles enhanced" PE capsules. Confocal laser scanning microscope was used to observe the entrapping process in real time, while UV-Vis spectroscope and scanning force microscope measurements suggested the high efficiency of encapsulation. In addition, the fluorescence recovery after photobleaching technique was employed to determine the existence form of deposited molecules. Further studies showed even negatively charged water-soluble peptides or proteins can be encapsulated into these hybrid capsules by modulating the pH value in bulk solution under its isoelectronic point as well. Copyright 2007 Wiley Periodicals, Inc.

Tapered fiber coupling of single photons emitted by a deterministically positioned single nitrogen vacancy center

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liebermeister, Lars, E-mail: lars.liebermeister@physik.uni-muenchen.de; Petersen, Fabian; Münchow, Asmus v.

2014-01-20

A diamond nano-crystal hosting a single nitrogen vacancy (NV) center is optically selected with a confocal scanning microscope and positioned deterministically onto the subwavelength-diameter waist of a tapered optical fiber (TOF) with the help of an atomic force microscope. Based on this nano-manipulation technique, we experimentally demonstrate the evanescent coupling of single fluorescence photons emitted by a single NV-center to the guided mode of the TOF. By comparing photon count rates of the fiber-guided and the free-space modes and with the help of numerical finite-difference time domain simulations, we determine a lower and upper bound for the coupling efficiency ofmore » (9.5 ± 0.6)% and (10.4 ± 0.7)%, respectively. Our results are a promising starting point for future integration of single photon sources into photonic quantum networks and applications in quantum information science.« less

Laser beam shaping for biomedical microscopy techniques

NASA Astrophysics Data System (ADS)

Laskin, Alexander; Kaiser, Peter; Laskin, Vadim; Ostrun, Aleksei

2016-04-01

Uniform illumination of a working field is very important in optical systems of confocal microscopy and various implementations of fluorescence microscopy like TIR, SSIM, STORM, PALM to enhance performance of these laser-based research techniques. Widely used TEM00 laser sources are characterized by essentially non-uniform Gaussian intensity profile which leads usually to non-uniform intensity distribution in a microscope working field or in a field of microlenses array of a confocal microscope optical system, this non-uniform illumination results in instability of measuring procedure and reducing precision of quantitative measurements. Therefore transformation of typical Gaussian distribution of a TEM00 laser to flat-top (top hat) profile is an actual technical task, it is solved by applying beam shaping optics. Due to high demands to optical image quality the mentioned techniques have specific requirements to a uniform laser beam: flatness of phase front and extended depth of field, - from this point of view the microscopy techniques are similar to holography and interferometry. There are different refractive and diffractive beam shaping approaches used in laser industrial and scientific applications, but only few of them are capable to fulfil the optimum conditions for beam quality required in discussed microscopy techniques. We suggest applying refractive field mapping beam shapers πShaper, which operational principle presumes almost lossless transformation of Gaussian to flat-top beam with flatness of output wavefront, conserving of beam consistency, providing collimated low divergent output beam, high transmittance, extended depth of field, negligible wave aberration, and achromatic design provides capability to work with several lasers with different wavelengths simultaneously. The main function of a beam shaper is transformation of laser intensity profile, further beam transformation to provide optimum for a particular technique spot size and shape has to be realized by an imaging optical system which can include microscope objectives and tube lenses. This paper will describe design basics of refractive beam shapers and optical layouts of their applying in microscopy systems. Examples of real implementations and experimental results will be presented as well.

Microscopy with multimode fibers

NASA Astrophysics Data System (ADS)

Moser, Christophe; Papadopoulos, Ioannis; Farahi, Salma; Psaltis, Demetri

2013-04-01

Microscopes are usually thought of comprising imaging elements such as objectives and eye-piece lenses. A different type of microscope, used for endoscopy, consists of waveguiding elements such as fiber bundles, where each fiber in the bundle transports the light corresponding to one pixel in the image. Recently a new type of microscope has emerged that exploits the large number of propagating modes in a single multimode fiber. We have successfully produced fluorescence images of neural cells with sub-micrometer resolution via a 200 micrometer core multimode fiber. The method for achieving imaging consists of using digital phase conjugation to reproduce a focal spot at the tip of the multimode fiber. The image is formed by scanning the focal spot digitally and collecting the fluorescence point by point.

Fluorescence histochemical and elec-ron microscopical observations on sympathetic ganglia of the chick embryo cultured with and without hydrocortisone.

PubMed

Hervonen, H; Eränkö, O

1975-01-01

Lumbar sympathetic ganglia of 12-day-old chick embryos were cultured in organ cultures for 14 days with 1, 10 or 100 mg/l of hydrocortisone or without it. Catecholamines were demonstrated by the formaldehyde-induced fluorescence method. For electron microscopy, the cultures were fixed with glutarialdehyde and osmium tetroxide. Two types of cells with catecholamine fluoresecence were observed in the control cultures: (1) weakly fluorescent sympathetic neurons and sympathicoblasts with long nerve fibres, which were the most common cell type in the explant, and (2) brightly fluorescent cells with or without fluorescent processes, which were less common and were scattered in the explant. Hydrocortisone caused a great increase in the number of the brightly fluorescent cells. With 10 mg/l of hydrocortisone the increase was about ten-fold as compared with the control cultures. There was no change in the morphology of the cells, nor could any change be observed in the fluorescence intensity by eye. Electron microscopically the mature neurons were the most common cell type on the surface of the culture, while more immature sympathicoblasts were seen in the deeper layers. Cells were also found which contained large numbers of catecholamine-strong granular vesicles 105-275 nm in diameter. These cells were infrequent. They had round vesicular nuclei and resembled also in other respects sympathicoblasts or young nerve cells. One such cell was found in mitotic division by electron microscopy. Hydrocortisone caused a marked increase in the number of these granule-containing cells and their processes. Cells which could have been classified as the small intensely fluorescent cells of the mammalian ganglion type or their electron microscopic equivalent, the granule-containing cells were found neither in the control cultures nor in the hydrocortisone-containing cultures. It is concluded that most brightly fluorescent cells in cultured sympathetic ganglia of the chick are nerve cells or sympathicoblasts rich in amine-storing granular vesicles.

Confocal Microscopy for the Histological Fluorescence Pattern of a Recurrent Atypical Meningioma: Case Report

PubMed Central

Whitson, Wesley J.; Valdes, Pablo A.; Harris, Brent T.; Paulsen, Keith D.; Roberts, David W.

2013-01-01

Background and Importance Fluorescence-guided resection with 5-aminolevulinic acid (5-ALA), which has shown promising results in the resection of malignant gliomas, has been used for meningioma resection in an attempt to more clearly delineate the tumor margin. However, no article has investigated the fluorescence pattern of meningiomas on a histological level. Understanding the microscopic pattern of fluorescence could help assess the precision and utility of using 5-ALA for these tumors. We present the case of a recurrent atypical meningioma operated on with 5-ALA fluorescence-guided resection for delineation of tumor tissue from surrounding uninvolved dura. Clinical Presentation A 53-year-old woman presented with recurrent atypical meningioma of the falx. Prior treatment included surgical resection 6 years earlier with subsequent fractionated radiation therapy and radiosurgery for tumor progression. The patient was given 5-ALA 20 mg/kg body weight dissolved in 100 mL water 3 hours before induction of anesthesia. Intraoperative fluorescence was coregistered with preoperative imaging. Neuropathological analysis of the resected falx with confocal microscopy enabled correlation of fluorescence with the extent of tumor on a histological level. Conclusion Fluorescence guidance allowed clear intraoperative delineation of tumor tissue from adjacent, uninvolved dura. On a microscopic level, there was a very close correlation of fluorescence with tumor, but some tumor cells did not fluoresce. PMID:21389893

Subtle trisomy 12q24.3 and subtle monosomy 22q13.3: three new cases and review.

PubMed

Rodríguez, Laura; Martínez Guardia, Nieves; Herens, Christian; Jamar, Mauricette; Verloes, Alain; López, Fermina; Santos Muñoz, José; Martínez-Frías, María Luisa

2003-10-01

The high resolution G-bands (850 bands) karyotype have made it possible to identify small chromosome anomalies (5 megabases) which are now microscopically visible. New techniques have been improved, such as the Fluorescent in situ hybridization (FISH) with subtelomeric probes, which can be employed to detect cryptic chromosome alterations not visible microscopically. We present three cases which had been remitted for a high resolution karyotype. The high resolution G-band karyotype and the FISH techniques led us to conclude that the three cases were carriers of a similar subtle chromosomal alteration. Case I is a new born female with developmental and psychomotor delay, hypotonia, and long limbs with arachnodactily. A high resolution G-band karyotype showed an abnormal chromosome 22. FISH techniques confirmed a der(22)t(12;22)(q24.31;q13.3). Case II is a 12-year-old girl, with growth retardation, long shaped face with thick eyebrows, smooth philtrum, and thin upper lip with severe mental retardation (still no language), with a phenotype very similar to that of his sister: long shaped face, thick eyebrows, smooth philtrum, and thin upper lip. A high resolution G-band karyotype also showed in Case II and III an abnormal chromosome 22, studied by FISH techniques which confirmed a der(22)t(12;22)(q24.3;q13.3) in both cases. Copyright 2003 Wiley-Liss, Inc.

Application of cytoplasmic Ca2+ fluorescence imaging techniques to study the molecular mechanisms of exercise-induced fatigue eliminated by Chinese medicine ginseng extract

NASA Astrophysics Data System (ADS)

Liu, Yi; Zhao, Yanping; Zhang, Heming; Liu, Songhao

2009-11-01

The exercise-induced fatigue eliminated by Chinese medicine offers advantages including good efficiency and smaller side-effects, however, the exact mechanisms have not been classified. A lot of literatures indicated the cytosolic free Ca2+ concentrations of skeletal muscle cells increased significantly during exercise-induced fatigue. This study is aimed to establish a rat skeletal muscle cell model of exercise-induced fatigue. We applied cytoplasmic Ca2+ fluorescence imaging techniques to study the molecular mechanisms of exercise-induced fatigue eliminated by Chinese medicine ginseng extract. In our research, the muscle tissues from the newborn 3 days rats were taken out and digested into cells. The cells were randomly divided into the ginseng extract group and the control group. The cells from the two groups were cultured in the medium respectively added 2mg/ml ginseng extract and 2mg/ml D-hanks solution. After differentiating into myotubes, the two groups of cells treated with a fluorescent probe Fluo-3 AM were put on the confocal microscope and the fluorescence intensity of cells pre- and post- stimulation with dexamethasone were detected. It was found that cytoplasmic Ca2+ concentrations of the two groups of cells both increased post-stimulation, however, the increasing amplitude of fluorescence intensity of the ginseng extract group was significantly lower than that of the control group. In conclusion, stimulating the cells with dexamethasone is a kind of workable cell models of exercise-induced fatigue, and the molecular mechanisms of exercise-induced fatigue eliminated by ginseng extract may be connected to regulatating cytosolic free Ca2+ concentrations.

Microscope-integrated quantitative analysis of intraoperative indocyanine green fluorescence angiography for blood flow assessment: first experience in 30 patients.

PubMed

Kamp, Marcel A; Slotty, Philipp; Turowski, Bernd; Etminan, Nima; Steiger, Hans-Jakob; Hänggi, Daniel; Stummer, Walter

2012-03-01

Intraoperative measurements of cerebral blood flow are of interest during vascular neurosurgery. Near-infrared indocyanine green (ICG) fluorescence angiography was introduced for visualizing vessel patency intraoperatively. However, quantitative information has not been available. To report our experience with a microscope with an integrated dynamic ICG fluorescence analysis system supplying semiquantitative information on blood flow. We recorded ICG fluorescence curves of cortex and cerebral vessels using software integrated into the surgical microscope (Flow 800 software; Zeiss Pentero) in 30 patients undergoing surgery for different pathologies. The following hemodynamic parameters were assessed: maximum intensity, rise time, time to peak, time to half-maximal fluorescence, cerebral blood flow index, and transit times from arteries to cortex. For patients without obvious perfusion deficit, maximum fluorescence intensity was 177.7 arbitrary intensity units (AIs; 5-mg ICG bolus), mean rise time was 5.2 seconds (range, 2.9-8.2 seconds; SD, 1.3 seconds), mean time to peak was 9.4 seconds (range, 4.9-15.2 seconds; SD, 2.5 seconds), mean cerebral blood flow index was 38.6 AI/s (range, 13.5-180.6 AI/s; SD, 36.9 seconds), and mean transit time was 1.5 seconds (range, 360 milliseconds-3 seconds; SD, 0.73 seconds). For 3 patients with impaired cerebral perfusion, time to peak, rise time, and transit time between arteries and cortex were markedly prolonged (>20, >9 , and >5 seconds). In single patients, the degree of perfusion impairment could be quantified by the cerebral blood flow index ratios between normal and ischemic tissue. Transit times also reflected blood flow perturbations in arteriovenous fistulas. Quantification of ICG-based fluorescence angiography appears to be useful for intraoperative monitoring of arterial patency and regional cerebral blood flow.

Hybrid nanocomposite coatings from metal (Mg alloy)-drug deposited onto medical implant by laser adaptive ablation deposition technique

NASA Astrophysics Data System (ADS)

Serbezov, Valery; Sotirov, Sotir; Serbezov, Svetlin

2013-03-01

Drug-eluting medical implants are active implants whose function is to create healing effects. The current requirements for active medical coatings for Drug-eluting medical implants are to be biocompatible, biodegradable, polymer free, mechanically stable and enable a controlled release of one or more drugs and defined degradation. This brings hybrid nanocomposite coatings into focus especially in the field of cardiovascular implants. We studied the properties of Metal (Mg alloy)-Paclitaxel coatings obtained by novel Laser Adaptive Ablation Deposition Technique (LAAD) onto cardiovascular stents from 316 LVM stainless steel material. The morphology and topology of coatings were studied by Bright field / Fluorescence optical microscope and Scanning Electron Microscope (SEM). Comparative measurements were made of the morphology and topology of hybrid, polymer free nanocomposite coatings deposited by LAAD and polymerdrug coatings deposited by classical spray technique. The coatings obtained by LAAD are homogeneous without damages and cracks. Metal nanoparticles with sizes from 40 nm to 230 nm were obtained in drug matrixes. Energy Dispersive X-ray Spectroscopy (EDX) was used for identification of metal nanoparticles presence in hybrid nanocomposites coatings. The new technology opens up possibilities to obtain new hybrid nanocomposite coatings with applications in medicine, pharmacy and biochemistry.

Spectroscopic identification of individual fluorophores using photoluminescence excitation spectra.

PubMed

Czerski, J; Colomb, W; Cannataro, F; Sarkar, S K

2018-01-25

The identity of a fluorophore can be ambiguous if other fluorophores or nonspecific fluorescent impurities have overlapping emission spectra. The presence of overlapping spectra makes it difficult to differentiate fluorescent species using discrete detection channels and unmixing of spectra. The unique absorption and emission signatures of fluorophores provide an opportunity for spectroscopic identification. However, absorption spectroscopy may be affected by scattering, whereas fluorescence emission spectroscopy suffers from signal loss by gratings or other dispersive optics. Photoluminescence excitation spectra, where excitation is varied and emission is detected at a fixed wavelength, allows hyperspectral imaging with a single emission filter for high signal-to-background ratio without any moving optics on the emission side. We report a high throughput method for measuring the photoluminescence excitation spectra of individual fluorophores using a tunable supercontinuum laser and prism-type total internal reflection fluorescence microscope. We used the system to measure and sort the photoluminescence excitation spectra of individual Alexa dyes, fluorescent nanodiamonds (FNDs), and fluorescent polystyrene beads. We used a Gaussian mixture model with maximum likelihood estimation to objectively separate the spectra. Finally, we spectroscopically identified different species of fluorescent nanodiamonds with overlapping spectra and characterized the heterogeneity of fluorescent nanodiamonds of varying size. © 2018 The Authors Journal of Microscopy © 2018 Royal Microscopical Society.

Studying aerosol light scattering based on aspect ratio distribution observed by fluorescence microscope.

PubMed

Li, Li; Zheng, Xu; Li, Zhengqiang; Li, Zhanhua; Dubovik, Oleg; Chen, Xingfeng; Wendisch, Manfred

2017-08-07

Particle shape is crucial to the properties of light scattered by atmospheric aerosol particles. A method of fluorescence microscopy direct observation was introduced to determine the aspect ratio distribution of aerosol particles. The result is comparable with that of the electron microscopic analysis. The measured aspect ratio distribution has been successfully applied in modeling light scattering and further in simulation of polarization measurements of the sun/sky radiometer. These efforts are expected to improve shape retrieval from skylight polarization by using directly measured aspect ratio distribution.

Nonlinear vibrational microscopy

DOEpatents

Holtom, Gary R.; Xie, Xiaoliang Sunney; Zumbusch, Andreas

2000-01-01

The present invention is a method and apparatus for microscopic vibrational imaging using coherent Anti-Stokes Raman Scattering or Sum Frequency Generation. Microscopic imaging with a vibrational spectroscopic contrast is achieved by generating signals in a nonlinear optical process and spatially resolved detection of the signals. The spatial resolution is attained by minimizing the spot size of the optical interrogation beams on the sample. Minimizing the spot size relies upon a. directing at least two substantially co-axial laser beams (interrogation beams) through a microscope objective providing a focal spot on the sample; b. collecting a signal beam together with a residual beam from the at least two co-axial laser beams after passing through the sample; c. removing the residual beam; and d. detecting the signal beam thereby creating said pixel. The method has significantly higher spatial resolution then IR microscopy and higher sensitivity than spontaneous Raman microscopy with much lower average excitation powers. CARS and SFG microscopy does not rely on the presence of fluorophores, but retains the resolution and three-dimensional sectioning capability of confocal and two-photon fluorescence microscopy. Complementary to these techniques, CARS and SFG microscopy provides a contrast mechanism based on vibrational spectroscopy. This vibrational contrast mechanism, combined with an unprecedented high sensitivity at a tolerable laser power level, provides a new approach for microscopic investigations of chemical and biological samples.

Structured illumination 3D microscopy using adaptive lenses and multimode fibers

NASA Astrophysics Data System (ADS)

Czarske, Jürgen; Philipp, Katrin; Koukourakis, Nektarios

2017-06-01

Microscopic techniques with high spatial and temporal resolution are required for in vivo studying biological cells and tissues. Adaptive lenses exhibit strong potential for fast motion-free axial scanning. However, they also lead to a degradation of the achievable resolution because of aberrations. This hurdle can be overcome by digital optical technologies. We present a novel High-and-Low-frequency (HiLo) 3D-microscope using structured illumination and an adaptive lens. Uniform illumination is used to obtain optical sectioning for the high-frequency (Hi) components of the image, and nonuniform illumination is needed to obtain optical sectioning for the low-frequency (Lo) components of the image. Nonuniform illumination is provided by a multimode fiber. It ensures robustness against optical aberrations of the adaptive lens. The depth-of-field of our microscope can be adjusted a-posteriori by computational optics. It enables to create flexible scans, which compensate for irregular axial measurement positions. The adaptive HiLo 3D-microscope provides an axial scanning range of 1 mm with an axial resolution of about 4 microns and sub-micron lateral resolution over the full scanning range. In result, volumetric measurements with high temporal and spatial resolution are provided. Demonstration measurements of zebrafish embryos with reporter gene-driven fluorescence in the thyroid gland are presented.

Practical guidelines for implementing adaptive optics in fluorescence microscopy

NASA Astrophysics Data System (ADS)

Wilding, Dean; Pozzi, Paolo; Soloviev, Oleg; Vdovin, Gleb; Verhaegen, Michel

2018-02-01

In life sciences, interest in the microscopic imaging of increasingly complex three dimensional samples, such as cell spheroids, zebrafish embryos, and in vivo applications in small animals, is growing quickly. Due to the increasing complexity of samples, more and more life scientists are considering the implementation of adaptive optics in their experimental setups. While several approaches to adaptive optics in microscopy have been reported, it is often difficult and confusing for the microscopist to choose from the array of techniques and equipment. In this poster presentation we offer a small guide to adaptive optics providing general guidelines for successful adaptive optics implementation.

Three-dimensional imaging of micro-specimen by optical scanning holography

NASA Astrophysics Data System (ADS)

Liu, Jung-Ping; Tsou, Cheng-Hao

2017-04-01

Optical scanning holography (OSH) is a scanning-type digital holographic technique. In OSH, a heterodyne interference pattern is generated to raster scan the object. OSH can be operated in the incoherent mode and thus is able to record a fluorescence hologram. In addition, resolution of the OSH is proportional to the density of the interference pattern. Here we use a high-NA microscope objective to generate a dynamic Fresnel zone plate to record a hologram of micro-specimen. The achieved transverse resolution and longitudinal resolution are 0.78μm and 3.1μm, respectively.

Picosecond absorption relaxation measured with nanosecond laser photoacoustics

PubMed Central

Danielli, Amos; Favazza, Christopher P.; Maslov, Konstantin; Wang, Lihong V.

2010-01-01

Picosecond absorption relaxation—central to many disciplines—is typically measured by ultrafast (femtosecond or picosecond) pump-probe techniques, which however are restricted to optically thin and weakly scattering materials or require artificial sample preparation. Here, we developed a reflection-mode relaxation photoacoustic microscope based on a nanosecond laser and measured picosecond absorption relaxation times. The relaxation times of oxygenated and deoxygenated hemoglobin molecules, both possessing extremely low fluorescence quantum yields, were measured at 576 nm. The added advantages in dispersion susceptibility, laser-wavelength availability, reflection sensing, and expense foster the study of natural—including strongly scattering and nonfluorescent—materials. PMID:21079726

Picosecond absorption relaxation measured with nanosecond laser photoacoustics.

PubMed

Danielli, Amos; Favazza, Christopher P; Maslov, Konstantin; Wang, Lihong V

2010-10-18

Picosecond absorption relaxation-central to many disciplines-is typically measured by ultrafast (femtosecond or picosecond) pump-probe techniques, which however are restricted to optically thin and weakly scattering materials or require artificial sample preparation. Here, we developed a reflection-mode relaxation photoacoustic microscope based on a nanosecond laser and measured picosecond absorption relaxation times. The relaxation times of oxygenated and deoxygenated hemoglobin molecules, both possessing extremely low fluorescence quantum yields, were measured at 576 nm. The added advantages in dispersion susceptibility, laser-wavelength availability, reflection sensing, and expense foster the study of natural-including strongly scattering and nonfluorescent-materials.