scholarly journals Characterization and Quenching of Autofluorescence in Piglet Testis Tissue and Cells

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Yanfei Yang ◽  
Ali Honaramooz
Keyword(s):  
Laser Scanning ◽  
Intrinsic Fluorescence ◽  
Laser Scanning Microscopy ◽  
Epifluorescence Microscopy ◽  
Fluorescence Labeling ◽  
Confocal Laser ◽  
Target Cells ◽  
Tissue Sections ◽  
Sudan Black B ◽  
Testis Tissue

Significant intrinsic fluorescence in tissues and in disassociated cells can interfere with fluorescence identification of target cells. The objectives of the present study were (1) to examine an intrinsic fluorescence we observed in both the piglet testis tissue and cells and (2) to test an effective method to block the autofluorescence. We observed that a number of granules within the testis interstitial cells were inherently fluorescent, detectable using epifluorescence microscopy, confocal laser scanning microscopy, and flow cytometry. The emission wavelength of the autofluorescent substance ranged from 425 to 700 nm, a range sufficiently broad that could potentially interfere with fluorescence techniques. When we treated the samples with Sudan Black B for different incubation times, the intrinsic fluorescence was completely masked after treatment for 10–15 min of the testis tissue sections or for 8 min of the testis cells, without compromising specific fluorescence labeling of gonocytes with lectin Dolichos biflorus agglutinin (DBA). We speculate that the lipofuscin or lipofuscin-like pigments within Leydig cell granules were mainly responsible for the observed intrinsic fluorescence in piglet testes. The method described in the present study can facilitate the identification and characterization of piglet gonocytes using fluorescence microscopy.

scholarly journals Implementation of Accurate and Fast DNA Cytometry by Confocal Microscopy in 3D

2005 ◽  
Vol 27 (4) ◽  
pp. 225-230
Author(s):  
Lennert S. Ploeger ◽  
André Huisman ◽  
Jurryt van der Gugten ◽  
Dionne M. van der Giezen ◽  
Jeroen A. M. Beliën ◽  
...  
Keyword(s):  
Genomic Instability ◽  
Confocal Laser Scanning Microscopy ◽  
Dna Content ◽  
Laser Scanning ◽  
Clinical Applications ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
Dna Cytometry ◽  
Tissue Sections

Background: DNA cytometry is a powerful method for measuring genomic instability. Standard approaches that measure DNA content of isolated cells may induce selection bias and do not allow interpretation of genomic instability in the context of the tissue. Confocal Laser Scanning Microscopy (CLSM) provides the opportunity to perform 3D DNA content measurements on intact cells in thick histological sections. Because the technique is technically challenging and time consuming, only a small number of usually manually selected nuclei were analyzed in different studies, not allowing wide clinical evaluation. The aim of this study was to describe the conditions for accurate and fast 3D CLSM cytometry with a minimum of user interaction to arrive at sufficient throughput for pilot clinical applications. Methods: Nuclear DNA was stained in 14 μm thick tissue sections of normal liver and adrenal stained with either YOYO-1 iodide or TO-PRO-3 iodide. Different pre-treatment strategies were evaluated: boiling in citrate buffer (pH 6.0) followed by RNase application for 1 or 18 hours, or hydrolysis. The image stacks obtained with CLSM at microscope magnifications of ×40 or ×100 were analyzed off-line using in-house developed software for semi-automated 3D fluorescence quantitation. To avoid sectioned nuclei, the top and bottom of the stacks were identified from ZX and YZ projections. As a measure of histogram quality, the coefficient of variation (CV) of the diploid peak was assessed. Results: The lowest CV (10.3%) was achieved with a protocol without boiling, with 1 hour RNase treatment and TO-PRO-3 iodide staining, and a final image recording at ×60 or ×100 magnifications. A sample size of 300 nuclei was generally achievable. By filtering the set of automatically segmented nuclei based on volume, size and shape, followed by interactive removal of the few remaining faulty objects, a single measurement was completely analyzed in approximately 3 hours. Conclusions: The described methodology allows to obtain a largely unbiased sample of nuclei in thick tissue sections using 3D DNA cytometry by confocal laser scanning microscopy within an acceptable time frame for pilot clinical applications, and with a CV small enough to resolve smaller near diploid stemlines. This provides a suitable method for 3D DNA ploidy assessment of selected rare cells based on morphologic characteristics and of clinical samples that are too small to prepare adequate cell suspensions.

Sudan Black B Reduces Autofluorescence in Murine Renal Tissue

2011 ◽  
Vol 135 (10) ◽  
pp. 1335-1342 ◽  
Author(s):  
Yan Sun ◽  
Hong Yu ◽  
Dong Zheng ◽  
Qi Cao ◽  
Ya Wang ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Kidney Tissue ◽  
Fluorescein Isothiocyanate ◽  
Renal Tissue ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Frozen Sections ◽  
Paraffin Sections ◽  
Biopsy Material ◽  
Sudan Black B

Context.—Renal tissue emits intense autofluorescence, making it difficult to differentiate specific immunofluorescence signals and thus limiting its application to clinical biopsy material. Objective.—To identify and minimize autofluorescence of renal tissue and demonstrate a simple, efficient method to reduce autofluorescence using Sudan black B. Design.—In this study, the sources and features of autofluorescence emitted from kidney tissue were examined. Broad autofluorescence was visualized in both frozen and paraffin kidney sections of normal mice and mice with Adriamycin-induced nephropathy using confocal laser scanning microscopy. Autofluorescence appeared in commonly used 4′,6-diamidino-2-phenylindole, fluorescein isothiocyanate, and Texas Red channels but not in far-red channel, and emitted extensively from red cells, injured tubulointersitial cells, and protein casts in diseased kidney. To eliminate autofluorescence, Sudan black B was used on formaldehyde-fixed paraffin sections and frozen sections of mouse kidney. The effects of Sudan black B in various concentrations were tested on kidney tissue. Results.—The 0.1% Sudan black B effectively blocked autofluorescence from both paraffin and frozen sections without adversely affecting specific fluorescence signals. Interestingly, the solvent for Sudan black B, 70% ethanol, was also shown to reduce autofluorescence on frozen sections, but not on paraffin sections. Conclusions.—This study demonstrates a simple, efficient, and cost-effective method to reduce autofluorescence using Sudan black B, and also provides a comprehensive approach to identify and minimize autofluorescence of renal tissue.

Gradients in the taxonomic composition of different microbial systems: comparison between biofilms for advanced waste treatment and lake sediments

1998 ◽  
Vol 37 (4-5) ◽  
pp. 159-166 ◽  
Author(s):  
I. Röske ◽  
K. Röske ◽  
D. Uhlmann
Keyword(s):  
Laser Scanning ◽  
Flow Reactor ◽  
Treatment Plant ◽  
Water Reservoir ◽  
Laser Scanning Microscopy ◽  
Microbial Colonization ◽  
Epifluorescence Microscopy ◽  
Confocal Laser ◽  
Sediment Layer ◽  
Glass Slides

The application of in situ hybridization with group specific oligonucleotide probes detected by epifluorescence microscopy and confocal laser scanning microscopy was tested to identify spatial gradients in the distribution of bacteria in biofilms of plug flow reactors and in the bottom sediment layer of a drinking water reservoir. The two tubular biofilm reactors were fed with the effluent from a full scale biological wastewater treatment plant to which were added the chlorophenols whole degradation was being investigated. One was operated as a continuous-flow reactor and the other as a sequencing batch reactor. The vertical gradients in the microbial colonization of the sediment were analyzed by means of glass slides exposed to the sediment. In the biofilms of both reactors the beta-Proteobacteria dominated. The Cytophaga-Flavobacterium group and the Gram-positive bacteria were also abundant. Only small amounts of gamma-bacteria could be detected. This is contrary to findings using traditional cultivation methods. Unlike the biofilms in the reactor, the bacterial Aufwuchs on the glass slides in the sediment presented a surprising diversity of morphological types and size classes of bacteria.

scholarly journals Possible Overestimation of Surface Disinfection Efficiency by Assessment Methods Based on Liquid Sampling Procedures as Demonstrated byIn SituQuantification of Spore Viability

2011 ◽  
Vol 77 (17) ◽  
pp. 6208-6214 ◽  
Author(s):  
I. Grand ◽  
M.-N. Bellon-Fontaine ◽  
J.-M. Herry ◽  
D. Hilaire ◽  
F.-X. Moriconi ◽  
...  
Keyword(s):  
Peracetic Acid ◽  
Laser Scanning ◽  
Sampling Procedure ◽  
Standard Test ◽  
Laser Scanning Microscopy ◽  
Fluorescence Labeling ◽  
Confocal Laser ◽  
Content Type ◽  
Surface Disinfection ◽  
Sampling Procedures

ABSTRACTThe standard test methods used to assess the efficiency of a disinfectant applied to surfaces are often based on counting the microbial survivors sampled in a liquid, but total cell removal from surfaces is seldom achieved. One might therefore wonder whether evaluations of microbial survivors in liquid-sampled cells are representative of the levels of survivors in whole populations. The present study was thus designed to determine the “damaged/undamaged” status induced by a peracetic acid disinfection forBacillus atrophaeusspores deposited on glass coupons directly on this substrate and to compare it to the status of spores collected in liquid by a sampling procedure. The method utilized to assess the viability of both surface-associated and liquid-sampled spores included fluorescence labeling with a combination of Syto 61 and Chemchrome V6 dyes and quantifications by analyzing the images acquired by confocal laser scanning microscopy. The principal result of the study was that the viability of spores sampled in the liquid was found to be poorer than that of surface-associated spores. For example, after 2 min of peracetic acid disinfection, less than 17% ± 5% of viable cells were detected among liquid-sampled cells compared to 79% ± 5% or 47% ± 4%, respectively, when the viability was evaluated on the surface after or without the sampling procedure. Moreover, assessments of the survivors collected in the liquid phase, evaluated using the microscopic method and standard plate counts, were well correlated. Evaluations based on the determination of survivors among the liquid-sampled cells can thus overestimate the efficiency of surface disinfection procedures.

scholarly journals CXCL12-Abundant Reticular (CAR) Cells Direct Megakaryocyte Protrusions Across the Bone Marrow Sinusoid Wall

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 722
Author(s):  
Nicole Wagner ◽  
Kristina Mott ◽  
Berin Upcin ◽  
David Stegner ◽  
Harald Schulze ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Blood Circulation ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Tissue Sections ◽  
Complex Process ◽  
Block Face ◽  
Platelet Release ◽  
Cell Retraction

Megakaryocytes (MKs) release platelets into the lumen of bone marrow (BM) sinusoids while remaining to reside within the BM. The morphogenetic events of this complex process are still not fully understood. We combined confocal laser scanning microscopy with transmission and serial block-face scanning electron microscopy followed by 3D-reconstruction on mouse BM tissue sections. These analyses revealed that MKs in close vicinity to BM sinusoid (BMS) wall first induce the lateral retraction of CXCL12-abundant reticular (CAR) cells (CAR), followed by basal lamina (BL) degradation enabling direct MK-sinusoidal endothelial cells (SECs) interaction. Subsequently, an endothelial engulfment starts that contains a large MK protrusion. Then, MK protrusions penetrate the SEC, transmigrate into the BMS lumen and form proplatelets that are in direct contact to the SEC surface. Furthermore, such processes are induced on several sites, as observed by 3D reconstructions. Our data demonstrate that MKs in interaction with CAR-cells actively induce BMS wall alterations, including CAR-cell retraction, BL degradation, and SEC engulfment containing a large MK protrusion. This results in SEC penetration enabling the migration of MK protrusion into the BMS lumen where proplatelets that are adherent to the luminal SEC surface are formed and contribute to platelet release into the blood circulation.

Culture and Detection of Campylobacter jejuni within Mixed Microbial Populations of Biofilms on Stainless Steel

2007 ◽  
Vol 70 (6) ◽  
pp. 1379-1385 ◽  
Author(s):  
SHERIASE Q. SANDERS ◽  
DOROTHY H. BOOTHE ◽  
JOSEPH F. FRANK ◽  
JUDY W. ARNOLD
Keyword(s):  
Stainless Steel ◽  
Campylobacter Jejuni ◽  
Laser Scanning ◽  
The United States ◽  
Laser Scanning Microscopy ◽  
Epifluorescence Microscopy ◽  
Confocal Laser ◽  
Atmospheric Conditions ◽  
Culture Methods ◽  
Bacterial Populations

Campylobacter jejuni is the most frequently reported cause of foodborne illness in the United States, but its survival outside the host is poor. The objective of this research was to examine the formation and composition of biofilms by C. jejuni alone and within mixed bacterial populations from the poultry-processing environment. C. jejuni growth was assessed with four media, two temperatures, and two atmospheric conditions to develop culture methods for liquid media that would allow growth within the biofilms. Growth kinetics was followed at four cell densities to determine temporal compatibility within biofilm mixtures. Analysis of the biofilms by confocal laser scanning microscopy showed that C. jejuni formed a biofilm when incubated without other bacteria. The average surface area of stainless steel covered by C. jejuni increased by 50% from 24 to 48 h, remained level to 96 h, and then decreased by 88% by 168 h. C. jejuni and mixed bacterial populations formed biofilms during incubation periods of up to 7 days. The area of the mixture was significantly greater than for C. jejuni alone at 24 h, was approximately the same at 48 h, and was significantly less by 168 h. When incubated with either of two initial inoculum densities of other bacteria, the number of C. jejuni was enhanced after 24 h. The intensity of fluorescence and cell viability were monitored by epifluorescence microscopy. This study provides the basis for studying interactions of Campylobacter spp. with other bacteria in the environment, which will aid in the design of effective intervention strategies.

Population dynamics of rumen microbes using modern techniques in rumen enhanced solid incubation

2003 ◽  
Vol 48 (4) ◽  
pp. 113-119 ◽  
Author(s):  
N. Raizada ◽  
V. Sonakya ◽  
R. Dalhoff ◽  
M. Hausner ◽  
P.A. Wilderer
Keyword(s):  
Population Dynamics ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Epifluorescence Microscopy ◽  
Rumen Content ◽  
Confocal Laser ◽  
Rumen Microbes ◽  
Microbial Population Dynamics ◽  
The Stability

The microbial ecology of the rumen is very complex. Different species of bacteria, protozoa, and fungi are involved in digestion of plant material in ruminants. In spite of complicated interrelationships among the various groups of microorganisms in the rumen ecosystem, Bacteria and Archaea are believed to play a major role because of their numerical predominance and metabolic diversity. In this work we are presenting the results for microbial population dynamics of rumen microbes during two-stage anaerobic digestion of grass. The reactors were inoculated with fresh rumen content. Fluorescent in situ hybridization, confocal laser scanning microscopy and epifluorescence microscopy were employed for microbial investigation. It was observed that Bacteria dominated in the hydrolytic reactor (1st stage) whereas Archaea were predominant in the methanogenic reactor (2nd stage). The stability of the methanogenic reactor was result of the dominance of Methanosaeta species (mainly the filamentous type).

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