Confocal fluorescence spectroscopy of subcutaneous cartilage expressing green fluorescent protein versus cutaneous collagen autofluorescence

Optically monitoring the expression of green fluorescent protein (GFP) in the cartilage underlying the skin of a mouse allows tracking the expression of the chondrocyte phenotype. This paper considers how confocal microscopy with spectral detection can sense GFP fluorescence in the cartilage despite light scattering and collagen autofluorescence from the overlying skin. An in vivo experiment tested the abilities of a topical optical fiber measurement and a confocal microscope measurement to detect GFP in cartilage under the skin versus the collagen autofluorescence. An ex vivo experiment tested the ability of a confocal microscope without and with its pinhole to detect a fluorescent microsphere underneath an ex vivo skin layer versus the collagen autofluorescence. In both systems, spectroscopic detection followed by linear analysis allowed spectral discrimination of collagen autofluorescence (M(C)) and the subdermal green fluorescence (M(G)) due to either GFP or the microsphere. Contrast was defined as M(G)/(M(G)+M(C)). The in vivo contrast for GFP using optical fiber and confocal measurements was 0.16 and 0.92, respectively. The ex vivo contrast for a fluorescent microsphere using a confocal system without and with a pinhole was 0.13 and 0.48, respectively. The study demonstrates that a topical optical fiber measurement is affected by collagen autofluorescence, while a confocal microscope can detect subdermal fluorescence while rejecting collagen autofluorescence.     

Parallel two-channel near- and far-field fluorescence microscopy

We report a new two-channel fluorescence microscopy technique for surface-generated fluorescence. The realized fluorescence microscope allows high resolution imaging of aqueous samples. The core element of the instrument is a parabolic mirror objective that is used to collect the fluorescence at large surface angles above the critical angle of the waterglass interface. An aspheric lens, incorporated into the solid parabolic element, is used for diffraction-limited laser focusing and for collecting fluorescence at low angles with respect to the optical axis. By separated collection of the fluorescence emitted into supercritical and subcritical angles, two detection volumes strongly differing in their axial resolution are generated at the surface of a glass cover slip. The collection of supercritical angle fluorescence (SAF) results in a strict surface confinement of the detection volume, whereas collecting below the critical angle allows gathering the fluorescence emitted several microns deep inside the sample. Consequently, the signals from surface-bound and unbound diffusing fluorescent molecules can be obtained simultaneously.     

Endocytic pathways: combined scanning ion conductance and surface confocal microscopy study

We introduce a novel high resolution scanning surface confocal microscopy technique that enables imaging of endocytic pits in apical membranes of live cells for the first time. The improved topographical resolution of the microscope together with simultaneous fluorescence confocal detection produces pairs of images of cell surfaces sufficient to identify single endocytic pits. Whilst the precise position and size of the pit is detected by the ion conductance microscope, the molecular nature of the pit, e.g. clathrin coated or caveolae, is determined by the corresponding green fluorescent protein fluorescence. Also, for the first time, we showed that flotillin 1 and 2 can be found co-localising with ~200-nm indentations in the cell membrane that supports involvement of this protein in endocytosis. A. Shevchuk and P. Hobson contributed equally to this work.     

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

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.     

Spatially modulated illumination microscopy: online visualization of intensity distribution and prediction of nanometer precision of axial distance measurements by computer simulations

During the last years, measurements considerably beyond the conventional "Abbe-Limit" of optical resolution in far field light microscopy were realized by several light microscopical approaches. Point spread function (PSF) engineering, spectral precision distance microscopy (SPDM), and related methods were used to demonstrate the feasibility of such measurements. SPDM allows the measurement of position and multiple distances between point-like fluorescent objects of different spectral signatures far below the optical resolution criterion as defined by the full width at half maximum of the PSF. Here, we report a software method to obtain online visualization of light distribution in the lateral and axial direction of any object detected in a spatially modulated illumination (SMI) microscope. This strongly facilitates routine application of SMI microscopy. The software was developed using Microsoft Visual C++ running on Windows NT. Furthermore, some aspects of the theoretical limits of the SPDM method were studied by virtual microscopy. For the case of SMI microscopy the precision of axial distance measurements was studied, taking into account photon statistics and image analysis procedures. The results indicate that even under low fluorescence intensity conditions typical for biological structure research, precise distance measurements in the nanometer range can be determined, and that axial distances in the order of 40 nm are detectable with such precision.     

Loss of image quality in photobleaching during microscopic imaging of fluorescent probes bound to chromatin

Prolonged excitation of fluorescent probes leads eventually to loss of their capacity to emit light. A decrease in the number of detected photons reduces subsequently the resolving power of a fluorescence microscope. Adverse effects of fluorescence intensity loss on the quality of microscopic images of biological specimens have been recognized, but not determined quantitatively. We propose three human-independent methods of quality determination. These techniques require no reference images and are based on calculation of the actual resolution distance, information entropy, and signal-to-noise ratio (SNR). We apply the three measures to study the effect of photobleaching in cell nuclei stained with propidium iodide (PI) and chromomycin A3 (CA3) and imaged with fluorescence confocal microscopy. We conclude that the relative loss of image quality is smaller than the corresponding decrease in fluorescence intensity. Furthermore, the extent of quality loss is related to the optical properties of the imaging system and the noise characteristics of the detector. We discuss the importance of these findings for optimal registration and compression of biological images.     

Fluorescence resonance energy transfer determinations using multiphoton fluorescence lifetime imaging microscopy to characterize amyloid-beta plaques

We describe the implementation of a commercial fluorescence lifetime imaging microscopy (FLIM) instrument used in conjunction with a commercial laser scanning multiphoton microscope. The femtosecond-pulsed near-infrared laser is an ideal excitation source for time-domain fluorescence lifetime measurements. With synchronization from the x-y scanners, fluorescence lifetimes can be acquired on a pixel-by-pixel basis, with high spatial resolution. Multiexponential curve fits for each pixel result in two-dimensional fluorescence resonance energy transfer (FRET) measurements that allow the determination of both proximity of fluorescent FRET pairs, as well as the fraction of FRET pairs close enough for FRET to occur. Experiments are described that characterize this system, as well as commonly used reagents valuable for FRET determinations in biological systems. Constructs of CFP and YFP were generated to demonstrate FRET between this pair of green fluorescent protein (GFP) color variants. The lifetime characteristics of the FRET pair fluorescein and rhodamine, commonly used for immunohistochemistry, were also examined. Finally, these fluorophores were used to demonstrate spatially resolved FRET with senile plaques obtained from transgenic mouse brain. Together these results demonstrate that FLIM allows sensitive measurements of protein-protein interactions on a spatial scale less than 10 nm using commercially available components.     

Cell-substrate topology upon ALA-PDT using variable-angle total internal reflection fluorescence microscopy (VA-TIRFM).

Because of the low penetration depth of an evanescent electromagnetic field, total internal reflection fluorescence microscopy (TIRFM) proved to be a powerful technique to examine fluorescent dyes or photosensitizers in close vicinity to the plasma membrane of living cells. In addition, on variation of the angle of incidence of exciting laser light, the penetration depth is varied, so that cell-substrate topology can be examined with nanometer resolution. Using a specific illumination device for TIRFM and a highly sensitive electron multiplying (EM) CCD camera, fluorescence of the photosensitizer protoporphyrin IX (PPIX) was studied in human cancer cells after application of 5-aminolevulinic acid (5-ALA) prior to and after irradiation with sublethal light doses (635 nm, 4 J/cm2). For cells growing on microscope cover slides, cell-substrate distances varied between approximately 20 and 250 nm with a mean distance of approximately 120 nm. On light exposure, these distances generally decreased, and a mean value below 100 nm was attained. Moreover, focal contacts visualized with a fusion protein of yellow fluorescent protein and focal adhesion kinase were maintained on light exposure, i.e., light-induced detachment of cells from their substrate was not likely to occur.     

A fluorescent molecular rotor probes the kinetic process of degranulation of mast cells.

A confocal fluorescence microscope was used to study the exocytotic secretory processes of mast cells in combination with an fluorescent molecular rotor, 9-(dicyanovinyl)julolidine (DCVJ). DCVJ is known to be an unique fluorescent dye which increases its quantum yield with decreasing intramolecular rotation. Here, DCVJ-loaded peritoneal rat mast cells were stimulated with compound 48/80 and their fluorescence images were compared with fluorescence calcium images of fluo-3-loaded mast cells. Subsequent to transient increases in intracellular free calcium ion concentration, DCVJ fluorescence increased dramatically in the cytoplasm and formed a ring-like structure around the nucleus, suggesting the possibility that the dye bound to the proteins composing the cytoskeletal architecture. Furthermore, the increases of DCVJ fluorescence intensities were mostly blocked in the presence of cytochalasin D (10 microM). However, fluo-3 fluorescence intensities still increased after addition of compound 48/80.     

Heuristically optimal path scanning for high-speed multiphoton circuit imaging

Population dynamics of patterned neuronal firing are fundamental to information processing in the brain. Multiphoton microscopy in combination with calcium indicator dyes allows circuit dynamics to be imaged with single-neuron resolution. However, the temporal resolution of fluorescent measures is constrained by the imaging frequency imposed by standard raster scanning techniques. As a result, traditional raster scans limit the ability to detect the relative timing of action potentials in the imaged neuronal population. To maximize the speed of fluorescence measures from large populations of neurons using a standard multiphoton laser scanning microscope (MPLSM) setup, we have developed heuristically optimal path scanning (HOPS). HOPS optimizes the laser travel path length, and thus the temporal resolution of neuronal fluorescent measures, using standard galvanometer scan mirrors. Minimizing the scan path alone is insufficient for prolonged high-speed imaging of neuronal populations. Path stability and the signal-to-noise ratio become increasingly important factors as scan rates increase. HOPS addresses this by characterizing the scan mirror galvanometers to achieve prolonged path stability. In addition, the neuronal dwell time is optimized to sharpen the detection of action potentials while maximizing scan rate. The combination of shortest path calculation and minimization of mirror positioning time allows us to optically monitor a population of neurons in a field of view at high rates with single-spike resolution, ～ 125 Hz for 50 neurons and ～ 8.5 Hz for 1,000 neurons. Our approach introduces an accessible method for rapid imaging of large neuronal populations using traditional MPLSMs, facilitating new insights into neuronal circuit dynamics.     

建立裸鼠皮下荧光子宫内膜异位症模型

背景：将绿色荧光蛋白标记的裸鼠子宫内膜注射于裸鼠皮下,构建子宫内膜异位症动物模型,操作简便,同时可在一定时间内体外动态观察异位病灶的生长,有利于子宫内膜异位症的深入研究。 目的：探讨构建裸鼠皮下子宫内膜异位症荧光模型的方法。 方法：取裸鼠子宫内膜,采用增强型绿色荧光蛋白腺病毒转染后,注射入6只裸鼠皮下(转染荧光组),将未接受增强型绿色荧光蛋白腺病毒转染的子宫内膜组织块注射到另6只裸鼠腹部皮下(对照组)。于注射后第 5,10,15,20,25,30天在体视荧光显微镜下观察皮下绿色荧光强度,记录荧光标记病灶的持续时间,同时行组织学检测两组异位病灶的情况。 结果与结论：成功构建5个内膜异位症活体荧光裸鼠模型及5个内膜异位症活体非荧光裸鼠模型,平均每只裸鼠形成二三个病灶。利用体视荧光显微镜可通过活体动物的皮肤直接观察到体内病灶生长和发展,荧光面积和强度随时间延长逐渐减弱,可持续观察4 周左右。对照组未检测到绿色荧光病灶。获取病灶组织经苏木精-伊红染色,可观察到典型的子宫内膜腺体和基质结构。结果可见这种改良的裸鼠荧光模型可以动态无损伤量化观察病灶生长和发展情况,有助于内膜异位症新药的开发及其发病机制的研究。     

不同细胞培养皿对激光共聚焦显微镜成像效果的影响

目的 探讨3种不同玻片厚度的激光共聚焦专用细胞培养皿制备的细胞样品在激光共聚焦显微镜(CLSM)下采集的荧光染色图像差异.方法通过活细胞及固定后细胞荧光染色实验,采用激光共聚焦显微镜观察细胞形态及荧光亮度,检测不同条件下成像的平均荧光强度,考察3种厚度玻片(0.085～0.13、0.13～0.16、0.16～0.19m...     

A hybrid approach to measuring electrical activity in genetically specified neurons

The development of genetically encoded fluorescent voltage probes is essential to image electrical activity from neuronal populations. Previous green fluorescent protein (GFP)-based probes have had limited success in recording electrical activity of neurons because of their low sensitivity and poor temporal resolution. Here we describe a hybrid approach that combines a genetically encoded fluorescent probe (membrane-anchored enhanced GFP) with dipicrylamine, a synthetic voltage-sensing molecule that partitions into the plasma membrane. The movement of the synthetic voltage sensor is translated via fluorescence resonance energy transfer (FRET) into a large fluorescence signal (up to 34% change per 100 mV) with a fast response and recovery time (0.5 ms). Using this two-component approach, we were able to optically record action potentials from neuronal cell lines and trains of action potentials from primary cultured neurons. This hybrid approach may form the basis for a new generation of protein-based voltage probes.     

Evaluation of a microfluorometer in immunofluorescence assays of individual spores of Bacillus anthracis and Bacillus cereus

A microfluorometer was constructed by modifying a standard fluorescent microscope with a fibre optic eyepiece and a simple photometric system. It was evaluated in direct immunofluorescence assays of Bacillus anthracis and Bacillus cereus spores immobilised on multispot microscope slides. From measurements of stable fluorescent crystals comparable in size to the spores, it was inferred that the fluorescence intensity of a stained bacterium could be measured with good precision. Fluctuation of the exciting light from a mercury vapour lamp did not contribute significantly to the distribution of fluorescence measurements obtained when samples of 20 spores were assessed. Attempts to correlate spore size with fluorescence intensity suggest that spore fluorescence does not increase in a 1 : 1 ratio with surface area; it is therefore possible that the density of antigenic sites on the surface decreases with increasing spore size. It is concluded that differences in the observed fluorescence of individual spores truly reflect differences in fluorescent antibody binding, but the relative contribution of antigenic variability and of artefacts of the staining procedure remain unknown.     

Pure populations of murine macrophages from cultured embryonic stem cells. Application to studies of chemotaxis and apoptotic cell clearance

The use of carboxyfluorescein diacetate succinimidyl ester (CFSE) to measure lymphocyte proliferation by flow cytometry has become one of the most widely utilised assays for assessing lymphocyte responses. The properties of CFSE make it ideal for such a task, covalently labelling cells with a long-lived fluorescence of high intensity and low variance with minimal cell toxicity. No dye in the last 20 years has been capable of replicating CFSE in these respects. However, currently CFSE is limited to following a maximum of 7 cell divisions and is not compatible for use with ubiquitously available fluorescein conjugates or other fluorescent molecules with spectral properties similar to fluorescein, such as EGFP. Here we characterise two new fluorescent dyes for measuring lymphocyte proliferation, Cell Trace Violet (CTV) and Cell Proliferation Dye eFluor 670 (CPD), which have different excitation and emission spectra to CFSE and, consequently, are compatible with fluorescein conjugates. We found that while both CTV and CPD can label cells to a high fluorescence intensity, which is long-lived and has low variability and low toxicity and makes them ideal for long-term tracking of non-dividing lymphocytes in vivo, CTV offers possibly the best available alternative to CFSE in the analysis of cell divisions. We also describe how intercellular dye transfer and cell autofluorescence can affect division resolution with the three different dyes and describe labelling conditions for the three dyes that produce ultra-bright lymphocytes for in vivo tracking studies and allow up to 11 cell divisions to be detected when using CFSE and CTV as the fluorescent dyes.     

Attachment of growth cones on substrate observed by multi-mode light microscopy

Evanescent light illumination was introduced into a multi-mode microscope to construct a new type of total internal reflection fluorescence microscope (TIRFM). This microscope, capable of TIRFM, high resolution video-enhanced differential interference contrast (DIC), epifluorescence, interference reflection (IR) imaging, was combined with an image acquisition system for time-lapse microscopy. Neuronal growth cones of a rat hippocampal neuron were stained with membrane labeling fluorescence dyes (DiI or octadecyl rhodamine B). Dynamic changes of the cell substrate contact of the neuronal growth cone were observed using the multi-imaging capacities of this system. When growth cone regions were stimulated by pressure ejection of a high potassium solution, TIRFM intensity at the basal membrane of the growth cone increased, suggesting that basal membrane of growth cone approaches the glass substrate when excited. The approach of the ventral membrane to the substrate during excitatory stimulation was also observed with IR microscope. The functional importance of cell/substrate contact in growth cones is discussed.     

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

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.     

Confocal fluorescence microscope with dual-axis architecture and biaxial postobjective scanning

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 < or =4.4 microm 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.     

Microfluorometric and fluorometric lipofuscin spectral discrepancies: a concentration-dependent metachromatic effect?

The fluorescent spectral patterns of some lipid peroxidation products and their derivatives have been investigated. A significant concentration-dependent fluorescence shift was found. A variety of suggested age pigment fluorophores, 1,4-dihydropyridines, Schiff base and MDA polymers, all demonstrated a potential for spectral shifts. Along with increased concentration, the fluorescence peaks of these fluorophores shifted from blue (400-490 nm) to golden-yellow or orange-red (500-600 nm). The demonstrated metachromasia is supposed to be an inner-filter effect resulting from molecular polymerization or stacking. Thus, the striking differences between lipofuscin fluorescence spectra obtained by different investigators may be explained as due to large differences in lipofuscin concentration during measurement with different techniques. The pigments are either studied in situ by morphologists and recorded by microscopic fluorometry or by biochemists using spectrofluorometers to measure the extracted and dissolved pigments.     

Functional imaging in bulk tissue specimens using optical emission tomography: fluorescence preservation during optical clearing

Optical emission computed tomography (optical-ECT) is a technique for imaging the three-dimensional (3D) distribution of fluorescent probes in biological tissue specimens with high contrast and spatial resolution. In optical-ECT, functional information can be imaged by (i) systemic application of functional labels (e.g. fluorophore labelled proteins) and/or (ii) endogenous expression of fluorescent reporter proteins (e.g. red fluorescent protein (RFP), green fluorescent protein (GFP)) in vivo. An essential prerequisite for optical-ECT is optical clearing, a procedure where tissue specimens are made transparent to light by sequential perfusion with fixing, dehydrating and clearing agents. In this study, we investigate clearing protocols involving a selection of common fixing (4% buffered paraformaldehyde (PFA), methanol and ethanol), dehydrating (methanol and ethanol) and clearing agents (methyl salicylate and benzyl-alcohol-benzyl-benzoate (BABB)) in order to determine a 'fluorescence friendly' clearing procedure. Cell culture experiments were employed to optimize the sequence of chemical treatments that best preserve fluorescence. Texas red (TxRed), fluorescein isothiocyanate (FITC), RFP and GFP were tested as fluorophores and fluorescent reporter proteins of interest. Fluorescent and control cells were imaged on a microscope using a DSred2 and FITC filter set. The most promising clearing protocols of cell culture experiments were applied to whole xenograft tumour specimens, to test their effectiveness in large unsectioned samples. Fluorescence of TxRed/FITC fluorophores was not found to be significantly affected by any of the test clearing protocols. RFP and GFP fluorescence, however, was found to be significantly greater when cell fixation was in ethanol. Fixation in either PFA or methanol resulted in diminished fluorescence. After ethanol fixation, the RFP and GFP fluorescence proved remarkably robust to subsequent exposure to either methyl salicylate or BABB. The optimized optical clearing procedure of ethanol fixation followed by methyl salicylate clearing preserved the fluorescence of constitutive RFP in whole xenograft tumour specimens, about 1 cc in dimension, indicating successful extension from cell plating experiments to whole tissue samples. Finally, the feasibility of imaging the 3D distribution of viable tumour cells (as indicated by the RFP emission) is demonstrated by optical-ECT imaging of cleared xenograft tumours using an in-house system.