Two-photon autofluorescence microscopy and spectroscopy of Antarctic fungus: new approach for studying effects of UV-B irradiation

We combined two-photon fluorescence microscopy and spectroscopy to provide functional images of UV-B (280-315 nm) induced stress on an Antarctic fungus. Two-photon excitation microscopy was used to characterize the distribution of autofluorescence inside the spore and the hyphae of the fungus. The imaging analysis clearly shows that the autofluorescence response of spores is higher than that of hyphae. The imaging analysis at different depths shows that, strikingly enough, the spore autofluorescence originates from the cell wall and membrane fluorophores. The spectroscopic results show moreover that the fluorescence spectra of spores are redshifted upon UV-B irradiation. Tentative identification of the chromophores involved in the autofluorescence response and their biological relevance are also discussed on the basis of a previous steady-state fluorescence spectroscopic study performed on both whole spore suspension and organic-soluble extracts.     

Monitoring of specific methanogenic activity of granular sludge by confocal laser scanning microscopy during start-up of thermophilic upflow anaerobic sludge blanket reactor

Confocal, laser-scanning microscopy was applied to acquire coenzyme F₄₂₀-based autofluorescence images of middle sections of sludge granules during start-up of a thermophilic reactor that were seeded with mesophilically-grown microorganisms of granular sludge. Digital images were analyzed to calculate weighted averages of autofluorescence. The values were related (r ²=0.97) to specific methanogenic activities of granular sludge as the granules developed to steady state.     

Improved in vivo whole-animal detection limits of green fluorescent protein-expressing tumor lines by spectral fluorescence imaging

Green fluorescent protein (GFP) has been used for cell tracking and imaging gene expression in superficial or surgically exposed structures. However, in vivo murine imaging is often limited by several factors, including scatter and attenuation with depth and overlapping autofluorescence. The autofluorescence signals have spectral profiles that are markedly different from the GFP emission spectral profile. The use of spectral imaging allows separation and quantitation of these contributions to the total fluorescence signal seen in vivo by weighting known pure component profiles. Separation of relative GFP and autofluorescence signals is not readily possible using epifluorescent continuous-wave single excitation and emission bandpass imaging (EFI). To evaluate detection thresholds using these two methods, nude mice were subcutaneously injected with a series of GFP-expressing cells. For EFI, optimized excitation and emission bandpass filters were used. Owing to the ability to separate autofluorescence contributions from the emission signal using spectral imaging compared with the mixed contributions of GFP and autofluorescence in the emission signal recorded by the EFI system, we achieved a 300-fold improvement in the cellular detection limit. The detection limit was 3 x 10(3) cells for spectral imaging versus 1 x 10(6) cells for EFI. Despite contributions to image stacks from autofluorescence, a 100-fold dynamic range of cell number in the same image was readily visualized. Finally, spectral imaging was able to separate signal interference of red fluorescent protein from GFP images and vice versa. These findings demonstrate the utility of the approach in detecting low levels of multiple fluorescent markers for whole-animal in vivo applications.     

Use of confocal laser scanning microscopy in systematics of insects with a comparison of fluorescence from different stains

Abstract Confocal laser scanning microscopy has become a valuable tool for a wide range of investigations in the biological sciences, but its use in insect systematics has been neglected. Confocal microscopy depends on the degree of fluorescence of the examined specimens, which is aided either by fluorescent dyes or autofluorescence of the specimen. This study provides methods for using a combination of fluorescent dyes and autofluorescence to provide images that document the value of confocal microscopy for systematic research with insects. Fluorescence was compared from Lepidoptera genitalia dissections that were unstained or stained with merbromin (mercurochrome), safranine, chlorazol black E, eosin Y, eosin Y + chlorazol black E, and orange‐G. The unstained specimen showed that chitin autofluorescences to a small degree. The comparison of stains showed that use of eosin Y provides the best images, followed by safranine and mercurochrome. Orange‐G and chlorazol black are the least fluorescent and provide poor images, even when chlorazol black is combined with eosin.     

Time-gated in vivo autofluorescence imaging of dental caries.

Laser-induced time-resolved autofluorescence from carious lesions of human teeth was studied by means of ultrashort pulsed laser systems, time-correlated single photon counting and time-gated imaging. Carious regions exhibited a slower fluorescence decay with a main 17 ns fluorescence lifetime than healthy hard dental tissue. The long-lived fluorophore present in carious lesions only emits in the red spectral region. Fluorescence decay time and spectral characteristics are typical of fluorescent metal-free porphyrin monomers. The spatial distribution of the long-lived endogenous porphyrin fluorophore within the tooth material was detected by time-gated nanosecond autofluorescence imaging. In particular, high contrast video images were obtained with an appropriate time delay of 15 ns to 25 ns between excitation and detection due to the suppression of short-lived autofluorescence of healthy tissue. First in vivo applications are reported indicating the potential of time-resolved fluorescence diagnostics for early caries- and dental plaque detection.     

Phasor analysis of multiphoton spectral images distinguishes autofluorescence components of in vivo human skin

Skin contains many autofluorescent components that can be studied using spectral imaging. We employed a spectral phasor method to analyse two photon excited autofluorescence and second harmonic generation images of in vivo human skin. This method allows segmentation of images based on spectral features. Various structures in the skin could be distinguished, including Stratum Corneum, epidermal cells and dermis. The spectral phasor analysis allowed investigation of their fluorescence composition and identification of signals from NADH, keratin, FAD, melanin, collagen and elastin. Interestingly, two populations of epidermal cells could be distinguished with different melanin content. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Detecting and quantifying colocalization of cell surface molecules by single particle fluorescence imaging

Single particle fluorescence imaging (SPFI) uses the high sensitivity of fluorescence to visualize individual molecules that have been selectively labeled with small fluorescent particles. The positions of particles are determined by fitting the intensity profile of their images to a 2-D Gaussian function. We have exploited the positional information obtained from SPFI to develop a method for detecting colocalization of cell surface molecules. This involves labeling two different molecules with different colored fluorophores and determining their positions separately by dual wavelength imaging. The images are analyzed to quantify the overlap of the particle images and hence determine the extent of colocalization of the labeled molecules. Simulated images and experiments with a model system are used to investigate the extent to which colocalization occurs from chance proximity of randomly distributed molecules. A method of correcting for positional shifts that result from chromatic aberration is presented. The technique provides quantification of the extent of colocalization and can detect whether colocalized molecules occur singly or in clusters. We have obtained preliminary data for colocalization of molecules on intact cells. Cells often exhibit particulate autofluorescence that can interfere with the measurements; a method for overcoming this problem by triple wavelength imaging is described.     

真菌的自发荧光现象研究

本试验用激光共聚焦扫描显微镜对三株不同真菌的菌丝、分子孢子以及子实体的自发荧光现象进行了观察,结果发现：处于相同培养条件的三株真菌的荧光分布特点不一,并且同一真菌在不同培养基条件下所表现的荧光现象亦有差异,说明真菌的自发荧光具有种属特异性并受到培养基质等因素的影响,而培养时间对荧光强度及类别的影响不显著。化学试剂的处理试验表明,弱碱性物质对真菌的荧光具有减弱效应,而强碱性物质对真菌的荧光则表现为增强,提示不同真菌细胞内的自发荧光物质结构上和组分上的差异,其变化可导致荧光性质的改变。     

Microspectrofluorometry of autofluorescence emission from human leukemic living cells under oxidative stress.

Image cytometry was applied to study the intracellular localization of autofluorescence and the influence of an oxidative stress on this emission. K562 erythroleukemia cancer cells were analyzed with a microspectrofluorometer, coupled with a Argon laser (Ar+) (363 nm). From each cell, 15 x 15 emission spectra were recorded in the 400-600 nm spectral range to generate a spectral image of autofluorescence. The intracellular locations of the autofluorescence emission and of the specific mitochondrial probe rhodamine 123 (R123) were matched. Under a 363 nm excitation, all spectra from K562 cells show equivalent profiles with a 455 nm maximum emission, near of reduced nicotinamide adenine dinucleotide-(Phosphate) solution (NAD(P)H) (465 nm maximum emission). The spatial distribution of autofluorescence is homogeneous and different from the one of R123. Hydrogen peroxide (H2O2) (200 microM) and menadione (Men) (5 microM) induce a weak spectral change and a decrease in autofluorescence intensity, down to 40% of the initial emission. Doxorubicin (Dox) induces a dose-dependent decrease in autofluorescence emission and a release of intracellular free radicals. When cells were pre-treated 1 h with 1 mM glutathione (GSH), Dox induces a lower free radicals release, no significant variation of autofluorescence intensity and a lower growth inhibitory effect. Images cytometry of autofluorescence suggest that the intracellular NAD(P)H would not be restricted to mitochondrial compartments. The release of free radicals was associated with a decrease in autofluorescence intensity, mainly attributed to NAD(P)H oxidation both inside and outside mitochondria.     

Nicotinamide adenine dinucleotide fluorescence spectroscopy and imaging of isolated cardiac myocytes.

Nicotinamide adenine dinucleotide (NADH) plays a critical role in oxidative phosphorylation as the primary source of reducing equivalents to the respiratory chain. Using a modified fluorescence microscope, we have obtained spectra and images of the blue autofluorescence from single rat cardiac myocytes. The optical setup permitted rapid acquisition of fluorescence emission spectra (390-595 nm) or intensified digital video images of individual myocytes. The spectra showed a broad fluorescence centered at 447 +/- 0.2 nm, consistent with mitochondrial NADH. Addition of cyanide resulted in a 100 +/- 10% increase in fluorescence, while the uncoupler FCCP resulted in a 82 +/- 4% decrease. These two transitions were consistent with mitochondrial NADH and implied that the myocytes were 44 +/- 6% reduced under the resting control conditions. Intracellular fluorescent structures were observed that correlated with the distribution of a mitochondrial selective fluorescent probe (DASPMI), the mitochondrial distribution seen in published electron micrographs, and a metabolic digital subtraction image of the cyanide fluorescence transition. These data are consistent with the notion that the blue autofluorescence of rat cardiac myocytes originates from mitochondrial NADH.     

Intensity-based energy transfer measurements in digital imaging microscopy

Investigation of protein-protein associations is important in understanding structure and function relationships in living cells. Using Förster-type resonance energy transfer between donor and acceptor labeled monoclonal antibodies we can assess the cell surface topology of membrane proteins against which the antibodies were raised. In our current work we elaborated a quantitative image microscopic technique based on the measurement of fluorescence intensities to calculate the energy transfer efficiency on a pixel-by-pixel basis. We made use of the broad excitation and emission spectrum of cellular autofluorescence for background correction of images. In addition to the reference autofluorescence images (UV background) we recorded three fluorescent images (donor, acceptor and energy transfer signal) of donor-acceptor double labeled samples, and corrected for spectral spillage of the directly excited donor and acceptor fluorescence into the energy transfer image. After careful image registration we were able to calculate the energy transfer efficiency on a pixel-by-pixel basis. In this paper, we also present a critical comparison between results obtained with this method and other approaches (photobleaching and flow cytometric energy transfer measurements).     

Optimization of Single- and Dual-Color Immunofluorescence Protocols for Formalin-Fixed,Paraffin-Embedded Archival Tissues

Performance of immunofluorescence staining on archival formalin-fixed paraffin-embedded human tissues is generally not considered to be feasible, primarily due to problems with tissue quality and autofluorescence. We report the development and application of procedures that allowed for the study of a unique archive of thymus tissues derived from autopsies of individuals exposed to atomic bomb radiation in Hiroshima, Japan in 1945. Multiple independent treatments were used to minimize autofluorescence and maximize fluorescent antibody signals. Treatments with NH₃/EtOH and Sudan Black B were particularly useful in decreasing autofluorescent moieties present in the tissue. Deconvolution microscopy was used to further enhance the signal-to-noise ratios. Together, these techniques provide high-quality single- and dual-color fluorescent images with low background and high contrast from paraffin blocks of thymus tissue that were prepared up to 60 years ago. The resulting high-quality images allow the application of a variety of image analyses to thymus tissues that previously were not accessible. Whereas the procedures presented remain to be tested for other tissue types and archival conditions, the approach described may facilitate greater utilization of older paraffin block archives for modern immunofluorescence studies.     

Detecting fluorescent protein expression and co-localisation on single secretory vesicles with linear spectral unmixing

Many questions in cell biology and biophysics involve the quantitation of co-localisation and the interaction of proteins tagged with different fluorophores. However, the incomplete separation of the different colour channels due to the presence of autofluorescence, along with cross-excitation and emission "bleed-through" of one colour channel into the other, all combine to render the interpretation of multi-band images ambiguous. Here we introduce a new live-cell epifluorescence spectral imaging and linear unmixing technique for classifying resolution-limited point objects containing multiple fluorophores. We demonstrate the performance of our technique by detecting, at the single-vesicle level, the co-expression of the vesicle-associated membrane protein, VAMP-2 (also called synaptobrevin-2), linked to either enhanced green fluorescent protein (EGFP) or citrine [a less pH-sensitive variant of enhanced yellow fluorescent protein (EYFP)], in mouse cortical astrocytes. In contrast, the co-expression of VAMP-2-citrine and the lysosomal transporter sialine fused to EGFP resulted in little overlap. Spectral imaging and linear unmixing permit us to fingerprint the expression of spectrally overlapping fluorescent proteins on single secretory organelles in the presence of a spectrally broad autofluorescence. Our technique provides a robust alternative to error-prone dual- or triple colour co-localisation studies.     

Two-photon confocal microscopy: a nondestructive method for studying wound healing

Two-photon confocal microscopy is a new technology useful in nondestructive analysis of tissue. The pattern generated from laser-excited autofluorescence and second harmonic signals can be analyzed to construct a three-dimensional, microanatomical, structural image. The healing of full-thickness guinea pig skin wounds was studied over a period of 28 days using two-photon confocal microscopy. Three-dimensional data were rendered from two-dimensional images and compared with conventional, en face, histologic sections. Two-photon confocal microscopy images show resolution of muscle, fascia fibers, collagen fibers, inflammatory cells, blood vessels, and hair. Although these images do not currently have the resolution of standard histology, the ability to noninvasively acquire three-dimensional images of skin promises to be an important tool in wound-healing studies.     

Quantitative comparison of the sensitivity of detection of fluorescent and bioluminescent reporters in animal models

Bioluminescent and fluorescent reporters are finding increased use in optical molecular imaging in small animals. In the work presented here, issues related to the sensitivity of in vivo detection are examined for standard reporters. A high-sensitivity imaging system that can detect steady-state emission from both bioluminescent and fluorescent reporters is described. The instrument is absolutely calibrated so that animal images can be analyzed in physical units of radiance allowing more quantitative comparisons to be performed. Background emission from mouse tissue, called autoluminescence and autofluorescence, is measured and found to be an important limitation to detection sensitivity of reporters. Measurements of dual-labeled (bioluminescent/fluorescent) reporter systems, including PC-3M-luc/DsRed2-1 and HeLa-luc/PKH26, are shown. The results indicate that although fluorescent signals are generally brighter than bioluminescent signals, the very low autoluminescent levels usually results in superior signal to background ratios for bioluminescent imaging, particularly compared with fluorescent imaging in the green to red part of the spectrum. Fluorescence detection sensitivity improves in the far-red to near-infrared, provided the animals are fed a low-chlorophyll diet to reduce autofluorescence in the intestinal region. The use of blue-shifted excitation filters is explored as a method to subtract out tissue autofluorescence and improve the sensitivity of fluorescent imaging.     

盘基网柄菌野生型KAx-3发育过程中自发荧光的观察

社会性变形虫盘基网柄菌(Dictyostelium discoideum)是研究发育和致病机理的重要模式生物之一.发育过程中自发荧光的研究有利于流式细胞仪检测时确定基础荧光阈值、细胞分选和判定荧光标记实验的准确性.本文用荧光显微镜对盘基网柄菌野生型KAx-3不同发育阶段在4种不同波长的激发光(蓝光、绿光、黄光和紫外线)...     

Variability of spectra of laser-induced fluorescence of colonic mucosa: its significance for fluorescence detection of colonic neoplasia

To determine the extent of a natural variability of the spectra of the autofluorescence and its significance for a reproducibility of different approaches typically used in studies on fluorescence detection of colonic lesions. Two independent series of experiments have been conducted during three years in the same laboratory. Macroscopic tissue specimens obtained during operations of patients with colonic cancers were studied in vitro. The tissues were excited using UV lines of c.w. He-Cd laser and pulsed nitrogen laser and the autofluorescence spectra were recorded for areas visually diagnosed as normal or pathologically changed mucosa. Natural variability of the autofluorescence spectra of colonic tissues seems to be most important factor limiting sensitivity and specificity of the diagnostic algorithms. The mean fluorescence spectra obtained for normal mucosa and its neoplastic lesions differ significantly but the differences are difficult to observe because of the high natural variability among the individual spectra. Further studies of biological basis of the colonic autofluorescence are necessary for a progress in the field of fluorescence detection of colonic neoplastic lesions.     

Photobleaching and Fluorescence Recovery of RPE Bisretinoids

The autofluorescence of the retina that originates primarily from lipofuscin fluorophores in retinal pigment epithelial cells, is observed to undergo photobleaching during the acquisition of fundus autofluorescence images. Bisretinoid fluorophores isolated from retinal pigment epithelial cells have the spectral characteristics consistent with their being the source of fundus autofluorescence. Clinically relevant experiments were designed to better understand conditions in the micromilieu of bisretinoid fluorophores that can influence fluorescence efficiencies, photobleaching, and subsequent fluorescence recovery of this fluorophore. The consumption of the bisretinoid A2E due to photooxidation-induced degradation was quantified in solvent systems of variable relative permittivity (formerly called dielectric constant), in micelles, and in phospholipid vesicles of varying composition. Reorganization within biphasic systems was also examined. A2E content was measured by high performance liquid chromatography (HPLC) and fluorescence intensity was quantified spectroscopically. As solvent polarity was increased, A2E fluorescent spectra exhibited red-shifted maxima and reduced intensity. A2E was depleted by light irradiation and the loss was more pronounced in less polar solvents, lower concentrations of anionic surfactant, and in gel- versus fluid-ordered phospholipid liposomes. Conditions that permit A2E aggregation promoted photooxidation/photodegradation, while movement of A2E between bisphasic systems was associated with fluorescence recovery after photobleaching. The fluorescence characteristics of A2E are subject to environmental modulation. Photooxidation and photodegradation of bisretinoid can account for fundus autofluorescence photobleaching. Return of fluorescence intensity after photobleaching likely occurs due to redistribution of A2E fractions amongst co-existing heterogeneous microdomains of the lysosomal compartment.     

Real-time detection of breast cancer at the cellular level

Novel optoelectronic instrumentation has been developed for the multispectral imaging of autofluorescence emitted by metabolic fluorophores. The images resolve individual cells while spectra are collected for each pixel in the images. These datacubes are generated at a rate of 10 per second—fast enough for surgical guidance. The data is processed in real time to provide a single color-coded image to the surgeon. To date, the system has been applied to fresh, ex vivo, human surgical specimens and has distinguished breast cancer from benign tissue. The approach is applicable to in vivo measurements of surgical margins and needle-based optical biopsies. Ongoing work demonstrates that the system has great potential for translation to a hand-held probe with high sensitivity and specificity.