Selective fluorescence of zymogen granules of pancreatic acinar cells stained with hematoxylin and eosin.

Following staining with hematoxylin and eosin Y, paraffin sections of mouse pancreas were examined by transmitted light, epifluorescence and confocal laser scanning microscopy. Light microscopy revealed that the nuclei of pancreatic acinar cells were located basally, while the apices of the cells appeared eosinophilic, although the secretory granules were difficult to visualize. Under violet-blue light excitation, the zymogen granules at the apices of the acinar cells showed strong yellowish fluorescence; the other part of the cytoplasm was only faintly fluorescent and the nuclei and the supporting tissues were nonfluorescent. Confocal laser scanning microscopy resulted in clear pictures of the zymogen granules and their distribution within the cell. The fluorescent emission of zymogen granules was certainly the result of eosin Y staining, because hematoxylin is not a fluorochrome and the zymogen granules are not autofluorescent. Staining with eosin Y alone, however, did not result in clear fluorescent images of zymogen granules or any other cellular structures. Our observation shows that the fluorescence emission of eosin Y allows easy and precise recognition of zymogen granules of pancreatic cells.     

Selective fluorescence of zymogen granules of pancreatic acinar cells stained with hematoxylin and eosin

Following staining with hematoxylin and eosin Y, paraffin sections of mouse pancreas were examined by transmitted light, epifluorescence and confocal laser scanning microscopy. Light microscopy revealed that the nuclei of pancreatic acinar cells were located basally, while the apices of the cells appeared eosinophilic, although the secretory granules were difficult to visualize. Under violet-blue light excitation, the zymogen granules at the apices of the acinar cells showed strong yellowish fluorescence; the other part of the cytoplasm was only faintly fluorescent and the nuclei and the supporting tissues were nonfluorescent. Confocal laser scanning microscopy resulted in clear pictures of the zymogen granules and their distribution within the cell. The fluorescent emission of zymogen granules was certainly the result of eosin Y staining, because hematoxylin is not a fluorochrome and the zymogen granules are not autofluorescent. Staining with eosin Y alone, however, did not result in clear fluorescent images of zymogen granules or any other cellular structures. Our observation shows that the fluorescence emission of eosin Y allows easy and precise recognition of zymogen granules of pancreatic cells.     

Selective fluorescence of zymogen granules of pancreatic acinar cells stained with hematoxylin and eosin

Following staining with hematoxylin and eosin Y, paraffin sections of mouse pancreas were examined by transmitted light, epifluorescence and confocal laser scanning microscopy. Light microscopy revealed that the nuclei of pancreatic acinar cells were located basally, while the apices of the cells appeared eosinophilic, although the secretory granules were difficult to visualize. Under violet-blue light excitation, the zymogen granules at the apices of the acinar cells showed strong yellowish fluorescence; the other part of the cytoplasm was only faintly fluorescent and the nuclei and the supporting tissues were nonfluorescent. Confocal laser scanning microscopy resulted in clear pictures of the zymogen granules and their distribution within the cell. The fluorescent emission of zymogen granules was certainly the result of eosin Y staining, because hematoxylin is not a fluorochrome and the zymogen granules are not autofluorescent. Staining with eosin Y alone, however, did not result in clear fluorescent images of zymogen granules or any other cellular structures. Our observation shows that the fluorescence emission of eosin Y allows easy and precise recognition of zymogen granules of pancreatic cells.     

In Vivo Staining by Dis and Trisazo Dyes, Especially of Bone and Elastic Tissue

The localization and retention of dis and trisazo dyes in connective tissues and bone was studied in rats and rabbits. Chlorazol fast pink, 5% in 0.9% NaCl. was injected intraperitoneally, 25 mg/kg each day for 2 days in newborn, growing, mature and 17-18 day pregnant rats, and up to 5 days in young rabbits. The dye was also injected at different time intervals during the development of strontium-induced rickets in growing rats, and in animals with abscess walls following subcutaneous injection of 0.1 ml turpentine. Animals were killed at-intervals thereafter, and comparison of in vivo staining of 5% solutions of chlorazol fast pink, chlorantine fast red, chlorazol black E, chlorazol sky blue, chlorazol sky pink, chlorazol green, chlorazol violet, pontamine green and pontamine sky blue was made by intraperitoneal injection in rats. Soft and hard tissue specimens were embedded in polyester resin or in paraffin wax and sectioned at 5-7 μ. Chlorazol fast pink stained some connective tissues and growing bones. The main intensity of staining occurred within 24 hr and gradually decreased but was still detectable after 6 mo in elastic tissues. In thin plastic sections, colouration was brilliant, not in osteoid tissue, but at calcifying bone margins and in elastic fibres. Dye localized at calcifying bone margins was incorporated within calcified tissues and then subsequently lost through remodelling. Such staining was not seen in paraffin-embedded material. Dye uptake was greatly reduced in rachitic rats, and wide osteoid seams were coloured faint pink, but where calcification was still occurring, colouration was brilliant. Similarly collagenous tissue in abscess walls was only lightly stained, in contrast to brilliant colouration of elastic tissues and macrophages. Of the 9 dyes tested, only chlorazol fast pink and chlorazol sky blue stained bone and elastic tissue in vivo. This prolonged retention and staining by these 2 dyes, unlike the others, was associated with their presence in the proximal convoluted tubules of the kidney.     

The Staining of Radulae

A review of the various methods of staining and mounting radulae is given. Normally the radula should be extracted with 0.5 to 1% sodium hydroxide solution, and the associated tissues removed before staining. Two staining methods are recommended for facilitating the interpretation of radulae. Newly formed teeth and the bases of older ones are well stained by saturated aqueous chlorazol black E (up to 10 minutes). A more uniformly stained specimen) in which the cusps of all but the young teeth are alone stained, may be obtained by using the “oxidation-dahlia technic”. The radula is oxidized in N/10 potassium permanganate solution until black and subsequently decolorized in saturated aqueous oxalic acid. It is then stained in 0.1% aqueous dahlia (Hofmann's violet), the staining time varying from 10 to 30 minutes, according to the material. It is then dehydrated and passed through xylene and clove oil into Canada balsam. Other mountants may be employed but glycerin jelly is only recommended for the rapid examination of unstained radulae. Several other staining methods are mentioned, and general precautions to be observed while mounting are discussed.     

Cytochemical characterization of the modified Guard procedure a regressive staining method for demonstrating chromosomal basic proteins

Summary A number of acidic dyes, including various fluorochromes, were substituted for biebrich scarlet in the modified Guard (1959) procedure, a regressive staining method which appears to demonstrate basic chromosomal proteins. These substitutions were made to test the possibility that dyes other than biebrich scarlet might provide advantages in sensitivity and/or contrast, or that more control could be exerted over the differentiation step in which solutions of phosphotungstic and phosphomolybdic acids (polyacids) are used.Of the dyes tested in this investigation, six were found to be especially suitable: procion brilliant blue, procion yellow, geranine G, brilliant sulfoflavine, eosin Y, and eosin B. While procion brilliant blue could be used as an absorption dye only, the other dyes were used more profitably as fluorochromes. The various dyes displayed considerable variability in the ease with which they could be displayed from substrates with polyacid solutions during the differentiation step. Procion brilliant blue, procion yellow, and geranine G were displaced gradually and thus resembled biebrich scarlet. In contrast, eosin B, eosin Y, and brilliant sulfoflavine were displaced more easily from all but the most highly condensed chromatin in substrates. Brilliant sulfoflavine yielded exceptionally bright and nearly selective fluorescence of consensed chromosomes in division, X chromosomes of grasshopper spermatocytes, and sperm heads.Weak, but selective fluorescence was observed when monazo sulfonated dyes, including ponta chrome violet SW, eriochrome black, diamond red, and ponta chrome blue black, were substituted in the modified Guard procedure. Similar results were obtained with solochrome cyanin R. As expected, these dyes seemed to function as weakly acidic dyes.     

Fluorescent staining of elastic tissue with Rhodamine B and related xanthene dyes

Summary Frozen sections, cut at 8 from various fresh tissues, were dried for 5 minutes over sulfuric acid, and then stained for 4 minutes in a 0.1% aqueous solution of Rhodamine B (Chroma) at pH 8.O. After soaking twice in butanol for 2 minutes each, the sections were kept on a warmer at 60° C for 2 minutes and then mounted in oil of cedar. Under ultraviolet microscopy, elastic fibers selectively stained yellow-orange against a pale blue background. No autofluorescence of elastic fibers was observed in guinea pig, rat, hamster or rabbit tissue in contrast to the autofluorescence of elastic fibers typically seen in human tissue.Similar fluorescent staining of the elastic tissue could be achieved by using a number of related xanthene dyes including pyronine, eosin B, acridine orange, pholoxin, phloxin B. More distantly related auranine O and thiazol yellow G were likewise used with success as fluorochromes for elastic fibers.Supported by a John A. Hartford Foundation Grant.     

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.     

Selective Staining of Protein and Starch in Wheat Flour and its Products

The main constituents of wheat flour and many wheat flour products are wheat protein (gluten) and starch granules. The specific staining of the protein present was effected by 10 min in 0.1% aqueous ponceau 2R (C.I. No. 16150) acidified with 3—4 drops of 1 N H₂SO₄ per 50 ml of staining solution, followed by rinsing in 2 changes of distilled water, dehydrating, clearing and mounting in a resinous medium in the normal way. Staining of starch was as follows: sections or flour smears were brought to water, treated for 10 min in a protein-blocking reagent (Taninol ADR—Imperial Chemical Industries—used in 1% aqueous solution) rinsed, then stained for 3 mins in 0.5% aqueous chlorazol violet R (C.I. No. 32445) or for 10 min in either 0.5% aqueous chlorazol violet N (C.I. No. 22570), or chlorazol black E (C.I. No. 30235). Staining was followed by thorough rinsing, normal dehydration and clearing and mounting in a medium of R.I. about 1.49 to enhance visibility of unstained starch grains. The methods are applicable to flour smears, cryostat and wax sections.     

Standardization of the Romanowsky staining procedure: an overview

Commerically available Romanowsky blood stains are variable mixtures of thiazein dyes and brominated fluorescein derivatives with varying degrees of metallic salt contamination in a number of different solvent systems. There is a need for standardized Romanowsky stains of constant composition, which, when used in conjunction with a carefully controlled specimen preparation technique, should give consistent performance. Such a preparation system would be of great value to hematologists in general and would be essential to the validity of data obtained by the digital processing of blood cell images. It is possible to prepare standardized Romanowsky stains as mixtures of two or three dye components, namely, eosin Y, azure B and methylene blue, although azure B has only recently become commercially available at an acceptable degree of purity. The logistic problems of stain standardization are discussed.     

3-d image analysis of fluorescent drug binding

Fluorescent ligands provide the means of studying receptors in whole tissues using confocal laser scanning microscopy and have advantages over antibody- or non-fluorescence-based method. Confocal microscopy provides large volumes of images to be measured. Histogram analysis of 3-D image volumes is proposed as a method of graphically displaying large amounts of volumetric image data to be quickly analyzed and compared. The fluorescent ligand BODIFY FL-prazosin (QAPB) was used in mouse aorta. Histogram analysis reports the amount of ligand-receptor binding under different conditions and the technique is sensitive enough to detect changes in receptor availability after antagonist incubation or generic manipulations. QAPB binding was concentration dependent, causing concentration-related rightward shifts in histogram. In the presence of 10 microM phenoxybenzamine (blocking agent), the QAPB (50 nM) histogram overlaps the autofluorescence curve. The histogram obtained for the 1D knockout aorta lay to the left of that control and 1B knockout aorta, indicating a reduction in 1D receptors. We have shown, for the first time, that it is possible to graphically display binding of a fluorescent drug to a biological tissue. Although our application is specific to adrenergic receptors, the general method could be applied to any volumetric, fluorescence-image-based assay.     

Efficient Blind Spectral Unmixing of Fluorescently Labeled Samples Using Multi-Layer Non-Negative Matrix Factorization

The ample variety of labeling dyes and staining methods available in fluorescence microscopy has enabled biologists to advance in the understanding of living organisms at cellular and molecular level. When two or more fluorescent dyes are used in the same preparation, or one dye is used in the presence of autofluorescence, the separation of the fluorescent emissions can become problematic. Various approaches have been recently proposed to solve this problem. Among them, blind non-negative matrix factorization is gaining interest since it requires little assumptions about the spectra and concentration of the fluorochromes. In this paper, we propose a novel algorithm for blind spectral separation that addresses some of the shortcomings of existing solutions: namely, their dependency on the initialization and their slow convergence. We apply this new algorithm to two relevant problems in fluorescence microscopy: autofluorescence elimination and spectral unmixing of multi-labeled samples. Our results show that our new algorithm performs well when compared with the state-of-the-art approaches for a much faster implementation.     

Confocal microscopy: principles and applications to the field of reproductive biology

Confocal microscopy allows analysis of fluorescent labeled thick specimens without physical sectioning. Optical sections are generated by eliminating out-of-focus fluorescence and displayed as digitalized images. It allows 3-dimensional reconstruction (XYZ) and time-analysis (XYT), thus providing unique chance to link morphology with cell function. Since images are obtained by scanning, excess illumination of the specimen and quick decrease of the fluorescent signal are avoided. Resolution obtained with a Laser Scanning Confocal Microscopy (LSCM) is theoretically better than that of a conventional microscope. The preparation of the specimen may be based on standard techniques, such as immunocytochemistry applied to fixed cells, or on staining of living cells, following the use of different fluorescent probes at the same time (colocalization). In our laboratory, we use the LSCM system Fluoview version 2.1 (Olympus) to study reproductive biology of animals and humans. We work on stainings of oocytes and blastocysts (mouse, bovine, human), and human ovarian tissues. We study mitochondrial distribution, cortical granule migration, calcium oscillations and spindle quality to link culture conditions and oocyte quality. Staining of F-actin is used to check transzonal projections (in zona pellucida) or to detect abnormalities following experimental treatment. Blastocyst quality is analyzed in sequential optical sections for microfilament organization and counting of total cell number (staining with phalloidin (actin) and picogreen (DNA). Trophectoderm and inner cell mass distribution (differential staining), apoptotic cells (TUNEL method) and viable cells (live/dead test) are also evaluated. Confocal imaging can be helpful for rapid determination of follicle density (staining with AM Calcein) and follicle morphology (picogreen) in ovarian cortical biopsies. The current review describes the principles of confocal microscopy and illustrates its applications to the field of reproductive biology by a large collection of pictures.     

Hyperspectral imaging microscopy for identification and quantitative analysis of fluorescently‐labeled cells in highly autofluorescent tissue

Standard fluorescence microscopy approaches rely on measurements at single excitation and emission bands to identify specific fluorophores and the setting of thresholds to quantify fluorophore intensity. This is often insufficient to reliably resolve and quantify fluorescent labels in tissues due to high autofluorescence. Here we describe the use of hyperspectral analysis techniques to resolve and quantify fluorescently labeled cells in highly autofluorescent lung tissue. This approach allowed accurate detection of green fluorescent protein (GFP) emission spectra, even when GFP intensity was as little as 15% of the autofluorescence intensity. GFP‐expressing cells were readily quantified with zero false positives detected. In contrast, when the same images were analyzed using standard (single‐band) thresholding approaches, either few GFP cells (high thresholds) or substantial false positives (intermediate and low thresholds) were detected. These results demonstrate that hyperspectral analysis approaches uniquely offer accurate and precise detection and quantification of fluorescence signals in highly autofluorescent tissues. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Clearing, cuticle removal, and staining for the fertile bracts (lemmas and paleas) of grass anthoecia.

The epidermis of the bracts enclosing the flower of grasses contains epidermal cell patterns which are indicative of phylogenetic and systematic relationships among taxa. Treating the heavily cutinized anthoecial bracts (fertile lemma and palea) with 10% NaOH results in the removal of sufficient cuticle to allow examination of the cells of the epidermis. After clearing and removal of the cuticle, the bracts are bleached, washed, dehydrated, and if studied by light microscopy, stained in 2% chlorazol black E and mounted in Diaphane; or, if studied by scanning electron microscopy, dried by the critical-point method and either left uncoated or coated with a film of various conductive metals.     

Two-color Fluorescence Labeling in Acrolein-fixed Brain Tissue

Acrolein is a potent fixative that provides both excellent preservation of ultrastructural morphology and retention of antigenicity, thus it is frequently used for immunocytochemical detection of antigens at the electron microscopic level. However, acrolein is not commonly used for fluorescence microscopy because of concerns about possible autofluorescence and destruction of the luminosity of fluorescent dyes. Here we describe a simple protocol that allows fine visualization of two fluorescent markers in 40-μm sections from acrolein-perfused rat brain. Autofluorescence was removed by pretreatment with 1% sodium borohydride for 30 min, and subsequent incubation in a 50% ethanol solution containing 0.3% hydrogen peroxide enhanced fluorescence labeling. Thus, fluorescence labeling can be used for high-quality detection of markers in tissue perfused with acrolein. Furthermore, adjacent acrolein-fixed sections from a single experiment can be processed to produce high-quality results for electron microscopy or fluorescence labeling. (J Histochem Cytochem 58:359–368, 2010)     

Functional Imaging of Mitochondria in Saponin-permeabilized Mice Muscle Fibers

Confocal laser-scanning and digital fluorescence imaging microscopy were used to quantify the mitochondrial autofluorescence changes of NAD(P)H and flavoproteins in unfixed saponin-permeabilized myofibers from mice quadriceps muscle tissue. Addition of mitochondrial substrates, ADP, or cyanide led to redox state changes of the mitochondrial NAD system. These changes were detected by ratio imaging of the autofluorescence intensities of fluorescent flavoproteins and NAD(P)H, showing inverse fluorescence behavior. The flavoprotein signal was colocalized with the potentiometric mitochondria-specific dye dimethylaminostyryl pyridyl methyl iodide (DASPMI), or with MitoTracker™ Green FM, a constitutive marker for mitochondria. Within individual myofibers we detected topological mitochondrial subsets with distinct flavoprotein autofluorescence levels, equally responding to induced rate changes of the oxidative phosphorylation. The flavoprotein autofluorescence levels of these subsets differed by a factor of four. This heterogeneity was substantiated by flow-cytometric analysis of flavoprotein and DASPMI fluorescence changes of individual mitochondria isolated from mice skeletal muscle. Our data provide direct evidence that mitochondria in single myofibers are distinct subsets at the level of an intrinsic fluorescent marker of the mitochondrial NAD–redox system. Under the present experimental conditions these subsets show similar functional responses.     

Clearing, Cuticle Removal, And Staining For The Fertile Bracts (Lemmas And Paleas) Of Grass Anthoecia

The epidermis of the bracts enclosing the flower of grasses contains epidermal cell patterns which are indicative of phylogenetic and systematic relationships among taxa. Treating the heavily cutinized anthoecial bracts (fertile lemma and palea) with 10% NaOH results in the removal of sufficient cuticle to allow examination of the cells of the epidermis. After clearing and removal of the cuticle, the bracts are bleached, washed, dehydrated, and if studied by light microscopy, stained in 2 % chlorazol black E and mounted in Diaphane; or, if studied by scanning electron microscopy, dried by the critical-point method and either left uncoated or coated with a film of various conductive metals.     

A single laser method for subtraction of cell autofluorescence in flow cytometry

In flow cytometry cell autofluorescence often interferes with efforts to measure low levels of bound fluorescent antibody. We have developed a way to correct for autofluorescence on a cell-by-cell basis. This results in improved estimates of real staining and better separation of the fluorescence histograms of stained and non-stained cells. Using a single laser, two-color fluorescence measurement system and two-color compensation electronics, autofluorescence and one fluorescent reagent are measured (rather than two fluorescent reagents). With fluorescein-conjugated antibodies the signal in the 515 to 555 nm range (green fluorescence) includes both fluorescein emission and part of the cellular autofluorescence. In the cases we have investigated, autofluorescence collected at wavelengths above 580 nm ("red") is well correlated with the green autofluorescence of the cells. A fraction of this red fluorescence is subtracted from the green fluorescence to produce an adjusted fluorescein output on which unstained cells have zero average signal. Use of this method facilitates the selection of rare cells transfected with surface antigen genes. Culture conditions affect the level of autofluorescence and the balance between red and green autofluorescence. When applied with fluorescein-conjugated reagents, the technique is compatible with the use of propidium iodide for live/dead cell discrimination.