Application Of Fluorescence Microscopy Research Articles

Fourier transform measurement of two-photon excitation spectra: applications to microscopy and optimal control

We report a novel Fourier transform method for measuring two-photon excitation spectra. We demonstrate this method using simple dye molecules and discuss its applications in two-photon fluorescence microscopy and optimal control. This method facilitates an intuitive interpretation of recent control experiments in terms of tuning the nonlinear spectrum of the exciting laser source.

Total internal reflection fluorescence microscopy: technical innovations and novel applications

Recent years have seen the introduction of novel techniques and applications of total internal reflection fluorescence microscopy (TIRFM). Key technical achievements include miniaturization, enhanced depth resolution, reduction of detection volumes and the combination of TIRFM with other microscopic techniques. Novel applications have concentrated on single-molecule detection (e.g. of cellular receptors), imaging of exocytosis or endocytosis, measurements of adhesion foci of microtubules, and studies of the localization, activity and structural arrangement of specific ion channels. In addition to conventional fluorescent dyes, genetically engineered fluorescent proteins are increasingly being used to measure molecular conformations or intermolecular distances by fluorescence resonance energy transfer.

Direct fluorochrome labeling of phage display library clones for studying binding specificities: applications in flow cytometry and fluorescence microscopy

Phage display technology is increasingly employed to identify high-affinity peptides and single-chain antibodies with binding specificities for a diversity of target types. The analysis of phage-binding sensitivity and specificity typically employs directly labeled secondary antiphage antibodies and potentially tertiary labels, such as fluorochromes and enzymes, when biotinylated antibodies are used. However, secondary or tertiary reagents may not be feasible or desirable for some target types and applications. Here, we present a simple approach for directly labeling phage clones with two common amine-reactive fluorochromes. We show that these fluorochromes label the pVIII major coat protein and that the binding selectivity of peptides displayed on the pIII protein of several well-characterized phage clones is maintained in flow cytometric analysis and immunofluorescence microscopy. Uniquely, such labeled phage, in part, represent self-propagating reagents because conjugation does not impair the ability to efficiently reproduce in bacteria, although relabeling with fluorochrome would be necessary. Our data suggest that primary labeled phage clones may be used similarly to primary antibody conjugates.

Visualization of the antioxidative effects of melatonin at the mitochondrial level during oxidative stress-induced apoptosis of rat brain astrocytes.

Oxidative stress-induced mitochondrial dysfunction has been shown to play a crucial role in the pathogenesis of a wide range of diseases. Protecting mitochondrial function, therefore, is vital for cells to survive during these disease processes. In this study, we demonstrate that melatonin, a chief secretory product of the pineal gland, readily rescued mitochondria from oxidative stress-induced dysfunction and effectively prevented subsequent apoptotic events and death in rat brain astrocytes (RBA-1). The early protection provided by melatonin in mitochondria of intact living cells was investigated by the application of time-lapse conventional, confocal, and multiphoton fluorescent imaging microscopy coupled with noninvasive mitochondria-targeted fluorescent probes. In particular, we observed that melatonin effectively prevented exogenously applied H2O2-induced mitochondrial swelling in rat brain astrocytes at an early time point (within 10 min) and subsequently reduced apoptotic cell death (150 min later). Other early apoptotic events such as plasma membrane exposure of phosphatidyl serine and the positive YOPRO-1 staining of the early apoptotic nucleus were also prevented by melatonin. A mechanistic study at the mitochondrial level related to the early protection provided by melatonin revealed that the indole molecule significantly reduced mitochondrial reactive oxygen species (ROS) formation induced by H2O2 stress. Melatonin also prevented mitochondrial ROS generation caused by other organic hydroperoxides including tert-butyl hydroperoxide and cumene hydroperoxide. This antioxidative effect of melatonin is more potent than that of vitamin E. Via its ability to reduce mitochondrial ROS generation, melatonin prevented H2O2-induced mitochondrial calcium overload, mitochondrial membrane potential depolarization, and the opening of the mitochondrial permeability transition (MPT) pore. As a result, melatonin blocked MPT-dependent cytochrome c release, the downstream activation of caspase 3, the condensation and karyorrhexis of the nucleus and apoptotic fragmentation of nuclear DNA. Thus, the powerful mitochondrial protection provided by melatonin reinforces its therapeutic potential to combat a variety of oxidative stress-induced mitochondrial dysfunctions as well as mitochondria-mediated apoptosis in various diseases.

Photochemistry of dyes and fluorochromes used in biology and medicine: some physicochemical background and current applications

An overview of the basic principles of photochemistry is presented to facilitate discussion of fluorescence, quenching and quantum yields. These topics in turn provide the foundation for an account of fluorescence spectroscopy and its application to microscopy. A brief overview of light microscopy and the application of fluorescence microscopy is given. The influences of molecular features, such as aromatic character and substitution patterns, on color and fluorescence are described. The concept of color fading is considered with particular reference to its effect on microscopic preparations. A survey of representative fluorescent probes is provided, and their sensitivity, application, and limitations are described. The phototoxicity of fluorescent molecules is discussed using biomembranes and DNA as examples of targets of toxicity. Photodynamic therapy, a relatively new clinical application of phototoxicity, is described. Both anticancer and antimicrobial applications are noted, and an assessment is given of the current ideas on the ideal physicochemical properties of the sensitizing agents for such applications.

The application of total internal reflection fluorescence microscopy in single fluorophore molecules axial imaging

Here we present a method of modeling fluorophore molecules as spheres in evan escent fields mathematically instead of the assumption of tophat function dist r ibution. Based on this model, we discuss that it is possible to calculate relati ve separation distances of the molecules, and their axial positions and diameters with the total internal reflection fluorescence microscopy(TIRFM) due to its uni que intensity pattern in the z direction. In the whole process, we need mere ly to change the incident angle once, that is to get only two frame imaging of TIRFM.

Evaluation of Fluorescent Probe Surface Intensities as an Indicator of Transdermal Permeant Distributions Using Wide-Area Two-Photon Fluorescence Microscopy

The application of high-speed two-photon fluorescence microscopy (HTPM) to examine transdermal transport processes has enabled the noninvasive visualization of permeant spatial distributions over a larger, more clinically relevant wide area of the skin. Earlier studies demonstrated that the transdermal fluorescent probe distribution over a 2 × 2mm skin area was well represented by a significantly reduced sampling of the 400 microscale skin sites (100 × 100 μm) constituting the wide area. In the present study, the 400 microscale skin sites are considered individually, and the site-to-site variability in permeant distributions is used as a model to reflect the range in experimentally measured skin permeabilities resulting from the inherent stratum corneum structural heterogeneity. The correlation established between the permeant surface intensity and the corresponding permeant intensity gradient at each skin site provides an indication of the potential for screening transdermal permeant distributions solely based on the evaluation of microscale permeant surface intensities. The strong linear correlation between the intensity gradient and the surface intensity for the hydrophilic model permeant, sulforhodamine B, demonstrated that surface intensities provide a robust indicator of the corresponding transdermal probe distributions at the microscale. For the hydrophobic model permeant, rhodamine B hexyl ester, however, weak correlations were observed between these two parameters. This result suggests that the stratum corneum microscale surface intensity does not validly capture the corresponding intensity gradients for the entire range of skin permeabilities typically encountered as a result of the inherent stratum corneum heterogeneity. © 2003 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 92:2354–2365, 2003

Membrane Leftovers after Fusion of Vacuoles

Abstract In a clever application of time-lapse fluorescence microscopy, Li Wang, Scott Seeley, William Wickner, and Alexey Merz looked to see if there was membrane left over after vacuoles fused within a cell. Specifically, It is not known what happens when two vacuoles come together (this is referred to as “docking” ) and join together as one larger vacuole (“fusion“). For reference, the surfaces of the vacuoJes where they have joined is the “boundary” membrane, the membranes on the outside are the “outside” membranes, and the circular junction of the boundary and outside membranes is the “vertex.“

Visualization of Oleic Acid-induced Transdermal Diffusion Pathways Using Two-photon Fluorescence Microscopy

In a novel application of dual-channel high-speed two-photon fluorescence microscopy, the skin autofluores-cence and the transdermal fluorescent model drug spatial distributions were imaged simultaneously over precisely the same spatial coordinates. The dual channels enable the detection of the fluorescence emission wavelengths characteristic of the endogenous (intrinsic) skin fluorophores, as well as of the rhodamine-based model drug intensity emission at a different wavelength range of the fluorescence emission spectrum. These fluorescent model drugs delineate the oleic acid induced changes in permeant diffusion with respect to the skin structural features over the 0.3 mm by 0.3 mm skin area imaged per skin sample. The dual-channel high-speed two-photon fluorescence microscopy studies presented here provide evidence for the existence of intracorneocyte diffusion in addition to the commonly cited lipid multilamellar transdermal pathway. The image quantification analysis methodology introduced in this paper reveals that intracorneocyte diffusion exists for the hydrophobic (rhodamine B hexyl ester) and for the hydrophilic (sulforhodamine B) model drugs, in the absence of oleic acid chemical enhancer action. The mechanism of oleic acid chemical enhancer action, however, depends on the model drug physicochemical properties, where the oleic acid induces hydrophobic model drug localization to the lipid multilamellar region, while increasing the hydrophilic model drug lipid to corneocyte partitioning.

The power and prospects of fluorescence microscopies and spectroscopies.

Recent years have witnessed a renaissance of fluorescence microscopy techniques and applications, from live-animal multiphoton confocal microscopy to single-molecule fluorescence spectroscopy and imaging in living cells. These achievements have been made possible not so much because of improvements in microscope design, but rather because of development of new detectors, accessible continuous wave and pulsed laser sources, sophisticated multiparameter analysis on one hand, and the development of new probes and labeling chemistries on the other. This review tracks the lineage of ideas and the evolution of thinking that have led to the actual developments, and presents a comprehensive overview of the field, with emphasis put on our laboratory's interest in single-molecule microscopy and spectroscopy.

17] Filters and mirrors for applications in fluorescence microscopy

This chapter describes the filters and mirrors for applications in fluorescence microscopy. The chapter serves as a short summary of fluorescence; applications in fluorescence; and, specifically, the filters and mirrors required for some of the current methods of fluorescence microscopy. One of the most prominent changes in microscopy is the recent explosion of applications associated with fluorescence microscopy. Fluorescence is a phenomenon in which a substance (atom or molecule) absorbs light of a specific wavelength, and rapidly radiates light of another (usually longer) wavelength. The substances that exhibit the properties of fluorescence are called fluorescent molecules, fluorochromes, or fluorophores. The absorption of the initial photon is termed “excitation,” and functions to move the fluorophore from the ground state to an excited state. From this intermediate, excited singlet state, there are three principal options available: (1) intersystem crossing, which can lead to a prolonged emission of light called phosphorescence; (2) nonradiative conversion; or (3) emission of a photon as the electron falls back to the ground state.

Quantitative Fluorescence Microscopy and Image Deconvolution

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.

Application of fluorescence microscopy and image analysis for quantifying dynamics of maize pollen shed

ABSTRACTThe objective of this study was to develop a simple, rapid and accurate technique to quantify maize (Zea mays L.) pollen shed under field conditions, capitalizing on the capacity of pollen to fluoresce and recent improvements in microscopic methods for acquiring, processing, and analyzing fluorescence signals from biological systems. Pollen shed naturally by the tassels was captured daily on passive pollen traps placed at apical ear level. Fluorescence microscopy was used to generate digital images of the trapped pollen, and pollen density per unit area was counted using commercial imaging software. Visual confirmation of fluorescing pollen grains indicated high measurement accuracy (r2 = 0.99) for the entire range of pollen shed densities typically encountered in the field. Pollen was randomly distributed across the surface of the pollen trap, and six to eight images per trap provided greater than 95% confidence for the mean trap value. The entire process of sample preparation, image capture, and image counting required less than 6 min per trap. The accuracy and ease of use of this technique make it ideal for characterizing the pattern of maize pollen production and dispersal under field conditions.

Application of fluorescence microscopy for noninvasive detection of surface contamination and precursors to laser-induced damage.

We present an experimental investigation to evaluate fluorescence microscopy as a tool to detect surface contamination as well as reveal surface damage precursors on optical components for large-aperture laser systems. We performed fluorescence imaging experiments using 351-nm laser excitation, whereas in situ damage testing was performed at laser fluences well below the dielectric breakdown threshold of the pure material. The experimental results demonstrated the potential of this technique to address both aforementioned technical issues.

The Susceptibility of Non-UV Fluorescent Membrane Dyes to Dynamical Properties of Lipid Membranes

Fluorescence spectroscopy and microscopy are powerful techniques to detect dynamic properties in artificial and natural lipid membrane systems. Unfortunately, most fluorescent dyes that sense dynamically relevant membrane parameters are UV sensitive. Their major disadvantage is a high susceptibility to fluorescence bleaching. Additionally, the risk for hazardous damages in biological components generally increases with decreasing excitation wavelength. Therefore the use of non-UV–sensitive membrane dyes would provide significant advantage, particularly for applications in fluorescence microscopy, which usually implies high local excitation intensities. We applied steady-state fluorescence spectroscopy techniques to several UV and non-UV membrane dyes to detect and compare dynamically relevant excitation and emission characteristics. Small unilamellar liposomes (composed of egg yolk phosphatidylcholine) served as a model system for biological membranes. The dynamic properties of the membranes were varied by two independent parameters: the intrinsic cholesterol content (0–50 mol%) and temperature (10–50°C). We tested four non-UV–sensitive membrane dyes: 9-diethylamino-5H-benzophenoxazine-5-one (Nile Red), 4-(dicyanovinyl)julolidine (DCVJ), N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide (FM 4-64), and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiIC18). We also tested three derivatives of DiIC18: DiIC16 and DiIC12 differ in acyl chain length and Fast-DiIC18 provides double bonds between hydrocarbon atoms. The spectral results were compared to established fluorescence characteristics of four UV membrane dyes: the anisotropy of 1-6-phenyl-1,3,5,-hexatrien (DPH), two derivatives of DPH (TMA-DPH and COO−-DHP), and the generalized polarization of 6-dodecanoyl-2-dimethyl-aminonaphthalene (Laurdan). Our results indicate that the tested non-UV dyes do not reveal dynamically relevant membrane parameters in a direct manner. However, spectral characteristics make DiIC18, Nile Red, and DCVJ promising probes for the microscopic detection of lateral lipid organization, an indirect indicator of membrane dynamics. In particular, DiIC18 showed very selective shifts in the emission spectra at defined temperatures and cholesterol contents that have not been reported elsewhere.

Development and clinical application of near-infrared surgical microscope: preliminary report.

Visualization of near-infrared fluorescence through a surgical microscope can provide intraoperative information about deep-seated tissues. We studied the possibility of taking a near-infrared image through a surgical microscope after intravenous injection of indocyanine green (ICG). The surgical microscopes we used were Zeiss models OPMI MD and OPMI CS-NC. We used a halogen lamp as a light source, a band pass filter of 760 - 810 nm as an excitation filter, a laser light of 800 nm as an external light source, and a band pass filter of 820 - 920 nm as a barrier filter. Near-infrared fluorescence of vessels on the brain surface and of the dural sinus was visualized through the dura mater after intravenous injection of 25 mg ICG using a Hitachi KP-160 camera. Near-infrared fluorescence of vessels on the brain surface was observed through the dura mater. Venous images were clearer than were arterial ones. The dural sinus and the bridging veins were also clearly visualized. These results suggest that a clinical application of near-infrared fluorescence microscopy is possible. However, a stronger fluorescence emitted from ICG will be necessary for such a microscope to have practical use.

Spectral Imaging for Separation of Fluorescent Signals from Autofluorescence

Autofluorescence, also known as adventitious fluorescence or background fluorescence, often poses a significant problem in many applications of fluorescence microscopy. It contributes to unwanted noise and can overwhelm the desired signal. Particularly difficult samples to image include many pathology specimens that have been processed using crosslinking fixatives (typically formaldehyde). This procedure dramatically increase the autofluorescence level, leading to bright, broad spectrum emissions, particularly from connective tissue components. Unprocessed plant tissue and neuronal tissue also have extremely high levels of endogenous autofluorescence that can make many convenient labeling strategies, including most green fluorescent protein (GFP) labels, extremely problematic. Various solutions have been proposed for the reduction or elimination of autofluorescence. These include using narrow bandpass emission filters to try to isolate the desired fluorescence signal, the use of labels that can be excited at wavelengths that are much less likely to induce autofluorescence (moving the excitation towards the NIR is effective), and post-processing aldehyde-fixed samples with such reagents as sodium borohydride or toluidine blue to chemically suppress the autofluorescence signal. However, in many cases, these approaches are either infeasible or ineffective. Spectral imaging, that is, the acquisition of a high-resolution optical spectrum at every pixel of an image, offers another approach to elimination of the contribution of autofluorescence. This has recently become possible due to the development of a number of technologies that allow the collection of spectral datasets from fluorescently labeled samples using fluorescence microscopy. Such technologies include liquid crystal tunable filters (LCTFs) and Fourier transform imaging spectrometers, both of which are now commercially available.

In Vivo Pharmacology Down to the Single Molecule Level

Recent applications of ultra-sensitive fluorescence microscopy to living cells allowed, for the first time, the direct observation of cellular processes on the single molecule level [1,2]. The general approach of observing individual copies of a protein expressed on the plasma membrane of a cell by using fluorescence labeled ligands allows for an in vivo determination of its pharmacological profile on a single cell level. We report here the characterization of the binding properties of fluorescence labeled Hongotoxin to the potassium channel KV1.3 in human T-lymphocytes in terms of the dissociation constant, the off-rate, and by competitive inhibition with unlabeled toxin. The figure shows two images of the same cell at different labeling conditions: after incubation with 300pM (top) and 1nM Cy5 labeled Hongotoxin (bottom). As expected, the fluorescence increases with increasing concentration of the applied ligand. Quantitative analysis allows to determine the dissociation constant, yielding 280pM. In addition, the low expression of the KV1.3 on Jurkat cells allows to resolve single fluorescence labeled ion channels as diffraction limited peaks along the plasma membrane. The high positional accuracy of single molecule microscopy of 50nm can be utilized to determine a full 3D map of individual channel proteins on a single cell [3].

In Vitro Visualization and Quantification of Oleic Acid Induced Changes in Transdermal Transport Using Two-Photon Fluorescence Microscopy

In a novel application of two-photon scanning fluorescence microscopy, three-dimensional spatial distributions of the hydrophilic and hydrophobic fluorescent probes, sulforhodamine B and rhodamine B hexyl ester, in excised full-thickness human cadaver skin were visualized and quantified. Both sulforhodamine B and rhodamine B hexyl ester were observed to lie primarily in the lipid multilamellae region surrounding the corneocytes within the stratum corneum. From the two-photon scanning fluorescence microscopy scans, the changes in the concentration gradient and the vehicle to skin partition coefficient of each probe induced by the oleic acid enhancer action were calculated relative to the control sample (not exposed to oleic acid), and subsequently applied to theoretically derived mathematical expressions of transdermal transport to quantitatively characterize the oleic acid-induced relative changes in the skin diffusion coefficient and the skin barrier diffusion length of the permeant. For the hydrophobic probe rhodamine B hexyl ester, the permeability enhancement was primarily driven by an increase in the vehicle to skin partition coefficient, leading to an increase in the steepness of the concentration gradient across the skin. The primary oleic acid-induced changes in the transport properties of the hydrophilic probe sulforhodamine B included increases in the vehicle to skin partition coefficient and the skin diffusion coefficient. These findings utilizing the two-photon scanning fluorescence microscopy methodology and data analysis described here demonstrate that, in addition to providing three-dimensional images that clearly delineate probe distributions in the direction of increasing skin depth, the subsequent quantification of these images provides additional important insight into the mechanistic changes in transdermal transport underlying the visualized changes in probe distributions across the skin.

Intracellular location and nuclear targeting of the Spi-1, Spi-2 and Spi-3 gene-derived serine protease inhibitors in non-secretory cells

Proteases and their inhibitors are indispensable for the regulated activation and/or degradation of structural and functional proteins involved in basic cellular processes, e.g. in cell cycle control, cell growth, differentiation and apoptosis. In this context the serine protease inhibitors derived from the murine Spi-1, Spi-2 and Spi-3 genes, and their human homologs, deserve reconsideration. Microsequencing data indicate that a fraction of the three serpins has the capability to constitute a well characterized proteinase K, high salt and SDS-stable complex which coisolates with DNA under salting out conditions from various cell and tissue types. This tight association with DNA isolated under conditions designed to deproteinize DNA efficiently points to an in situ preformed chromatin complex. Accordingly, in addition to their well known functions as 'serum protease inhibitors' the Spi-1 and Spi-2 gene-derived proteins appear to have intracellular functions as well. The involvement of the three serpins in chromatin complexes requires their nuclear translocation. Application of (enhanced) green fluorescent protein technology and optical section microscopy reveals that truncation of the N-terminal signal sequences of the Spi-1 and Spi-2 gene-encoded proteins is a prerequisite for their nuclear translocation while non-truncated fusion proteins are enriched at the nuclear indentation which is the site of the Golgi apparatus and the centrosome. The identification of new species of intracellular serpins is of potential interest with respect to accumulating evidence for serine protease inhibitor-dependent inhibition or prevention of apoptosis.