scholarly journals Optimized measurements of separations and angles between intra-molecular fluorescent markers

2015 ◽  
Vol 6 (1) ◽  
Author(s):  
Kim I. Mortensen ◽  
Jongmin Sung ◽  
Henrik Flyvbjerg ◽  
James A. Spudich
Keyword(s):  
Fluorescent Markers

Fading of the diaminobenzidine reaction product in the confocal scanning laser microscope

1989 ◽  
Vol 47 ◽  
pp. 146-147
Author(s):  
JS Deitch ◽  
KL Smith ◽  
JW Swann ◽  
JN Turner
Keyword(s):  
Pyramidal Neurons ◽  
Light And Electron Microscopy ◽  
Scanning Laser ◽  
Laser Exposure ◽  
Confocal Scanning Laser Microscope ◽  
Fluorescent Markers ◽  
Before And After ◽  
Staining Protocol ◽  
Confocal Scanning ◽  
Confocal Scanning Laser

Neurons labeled with horseradish peroxidase and reacted with diaminobenzidine (DAB) can be imaged using a confocal scanning laser microscope (CSLM) in the reflection mode. In contrast to fluorescent markers, the DAB reaction product is thought to be stable and can be observed by both light and electron microscopy. We have investigated the sensitivity of the DAB reaction product to laser irradiation, and present the spectrophotometric properties of DAB before and after exposure in the CSLM.Pyramidal neurons in slices of rat hippocampus were injected with biocytin (a biotin-lysine complex), fixed overnight in 4% paraformaldehyde, and vibratome sectioned at 75 μm. Biocytin was detected with avidin-HRP (1:200) in 0.5% Triton X-100, incubated in DAB (0.5 mg/ml) with or without 0.04% nickel ammonium sulfate (Ni), dehydrated, and imaged in a Bio Rad MRC-500 CSLM with an argon ion laser (488 and 514 nm). Spectrophotometric measurements of the soma were made on a Zeiss microspectrophotometer, as a function of laser exposure (100-1000 scans) and staining protocol.

A Fused Thiophene-S,S-Dioxide-Based Super-Photostable Fluorescent Marker for Lipid Droplets

2020 ◽  
Author(s):  
Masayasu Taki ◽  
Keiji Kajiwara ◽  
Eriko Yamaguchi ◽  
Yoshikatsu Sato ◽  
Shigehiro Yamaguchi
Keyword(s):  
Small Molecules ◽  
Lipid Droplets ◽  
Trajectory Analysis ◽  
Super Resolution ◽  
Fluorescent Marker ◽  
Selective Staining ◽  
Fluorescent Markers ◽  
Biological Studies ◽  
Particle Level

Lipid droplets (LDs) are essential organelle in most eukaryotes, and tracking intracellular LDs dynamics using synthetic small molecules is crucial for biological studies. However, only a limited number of fluorescent markers that satisfy all requirements, such as the selective staining of LDs, high photostability, and sufficient biocompatibility, have been developed. Herein, we report a series of donor-p-acceptor dyes based on the thiophene-containing fused polycyclic scaffold [1]benzothieno[3,2-<i>b</i>][1]benzothiophene (BTBT), in which either or both thiophene rings are oxidized into thiophene-<i>S</i>,<i>S</i>-dioxide to form an electron-accepting building block. Among these dyes, LAQ1 satisfied all the aforementioned requirements, and allowed us capturing ultra-small LDs on the endoplasmic reticulum (ER) membrane by stimulation emission depletion (STED) microscopy with a super-resolution below the diffraction limit of light. Moreover, the extremely high photostability of LAQ1 enabled recording the lipolysis of LDs and the concomitant lipogenesis as well as long-term trajectory analysis of micro LDs at the single particle level in living cells.

scholarly journals A novel high-content screening approach for the elucidation of C. jejuni biofilm composition and integrity

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Matthew V. X. Whelan ◽  
Jeremy C. Simpson ◽  
Tadhg Ó Cróinín
Keyword(s):  
Atmospheric Oxygen ◽  
Chicken Meat ◽  
Live Cells ◽  
High Throughput Analysis ◽  
Aerobic Conditions ◽  
Poultry Products ◽  
Source Of Infection ◽  
Fluorescent Markers ◽  
Oxygen Conditions ◽  
Bacterial Gastroenteritis

Abstract Background Campylobacter jejuni is the leading cause of bacterial gastroenteritis worldwide and the main source of infection is contaminated chicken meat. Although this important human pathogen is an obligate microaerophile, it must survive atmospheric oxygen conditions to allow transmission from contaminated chicken meat to humans. It is becoming increasingly evident that formation of biofilm plays a key role in the survival of this organism for extended periods on poultry products. We have recently demonstrated a novel inducible model for the study of adherent C. jejuni biofilm formation under aerobic conditions. By taking advantage of supercoiling mediated gene regulation, incubation of C. jejuni with subinhibitory concentrations of the Gyrase B inhibitor novobiocin was shown to promote the consistent formation of metabolically active adherent biofilm. Results In this study, we implement this model in conjunction with the fluorescent markers: TAMRA (live cells) and SytoX (dead cells, eDNA) to develop a novel systematic high-content imaging approach and describe how it can be implemented to gain quantifiable information about the integrity and extracellular polymeric substance (EPS) composition of adherent C. jejuni biofilm in aerobic conditions. We show that this produces a model with a consistent, homogenous biofilm that can be induced and used to screen a range of inhibitors of biofilm adherence and matrix formation. Conclusions This model allows for the first time a high throughput analysis of C. jejuni biofilms which will be invaluable in enabling researchers to develop mechanisms to disrupt these biofilms and reduce the viability of these bacteria under aerobic conditions.

Microbial bioburden of inpatient and outpatient areas beyond patient hospital rooms

2021 ◽  
pp. 1-5
Author(s):  
Jennifer L. Cadnum ◽  
Basya S. Pearlmutter ◽  
Annette L. Jencson ◽  
Hanan Haydar ◽  
Michelle T. Hecker ◽  
...  
Keyword(s):  
Bacterial Pathogens ◽  
Outpatient Clinics ◽  
Acinetobacter Baumanii ◽  
Healthcare Facilities ◽  
Fluorescent Markers ◽  
Clostridioides Difficile ◽  
Vancomycin Resistant ◽  
Healthcare Associated ◽  
Cleaning And Disinfection ◽  
Surgery Center

Abstract Objective: To investigate the frequency of environmental contamination in hospital areas outside patient rooms and in outpatient healthcare facilities. Design: Culture survey. Setting: This study was conducted across 4 hospitals, 4 outpatient clinics, and 1 surgery center. Methods: We conducted 3 point-prevalence culture surveys for methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, Clostridioides difficile, Candida spp, and gram-negative bacilli including Enterobacteriaceae, Pseudomonas aeruginosa, Acinetobacter baumanii, and Stenotrophomonas maltophilia in each facility. In hospitals, high-touch surfaces were sampled from radiology, physical therapy, and mobile equipment and in emergency departments, waiting rooms, clinics, and endoscopy facilities. In outpatient facilities, surfaces were sampled in exam rooms including patient and provider areas, patient bathrooms, and waiting rooms and from portable equipment. Fluorescent markers were placed on high-touch surfaces and removal was assessed 1 day later. Results: In the hospitals, 110 (9.4%) of 1,195 sites were positive for 1 or more bacterial pathogens (range, 5.3%–13.7% for the 4 hospitals) and 70 (5.9%) were positive for Candida spp (range, 3.7%–5.9%). In outpatient facilities, 31 of 485 (6.4%) sites were positive for 1 or more bacterial pathogens (range, 2% to 14.4% for the 5 outpatient facilities) and 50 (10.3%) were positive for Candida spp (range, 3.9%–23.3%). Fluorescent markers had been removed from 33% of sites in hospitals (range, 28.4%–39.7%) and 46.3% of sites in outpatient clinics (range, 7.4%–82.8%). Conclusions: Surfaces in hospitals outside patient rooms and in outpatient facilities are frequently contaminated with healthcare-associated pathogens. Improvements in cleaning and disinfection practices are needed to reduce contamination.

Vacuole Partitioning During Meiotic Division in Yeast

Genetics ◽  
1996 ◽  
Vol 144 (2) ◽  
pp. 445-458 ◽  
Author(s):  
Amy D Roeder ◽  
Janet M Shaw
Keyword(s):  
Alkaline Phosphatase ◽  
Meiotic Division ◽  
Cell Walls ◽  
Vacuolar Membrane ◽  
Carboxypeptidase Y ◽  
Fluorescent Markers ◽  
Immunofluorescence Studies ◽  
Membrane Bound ◽  
Subsequent Staining ◽  
Spore Cell

Abstract We have examined the partitioning of the yeast vacuole during meiotic division. In pulse-chase experiments, vacuoles labeled with the lumenal ade2 fluorophore or the membrane-specific dye FM 4-64 were not inherited by haploid spores. Instead, these fluorescent markers were excluded from spores and trapped between the spore cell walls and the ascus. Serial optical sections using a confocal microscope confirmed that spores did not inherit detectable amounts of fluorescently labeled vacuoles. Moreover, indirect immunofluorescence studies established that an endogenous vacuolar membrane protein, alkaline phosphatase, and a soluable vacuolar protease, carboxypeptidase Y, were also detected outside spores after meiotic division. Spores that did not inherit ade2- or FM 4-64-labeled vacuoles did generate an organelle that could be visualized by subsequent staining with vacuole-specific fluorophores. These data contrast with genetic evidence that a soluble vacuolar protease is inherited by spores. When the partitioning of both types of markers was examined in sporulating cultures, the vacuolar protease activity was inherited by spores while fluorescently labeled vacuoles were largely excluded from spores. Our results indicate that the majority of the diploid vacuole, both soluble contents and membrane-bound components, are excluded from spores formed during meiotic division.

Using conventional fluorescent markers for far-field fluorescence localization nanoscopy allows resolution in the 10-nm range

2009 ◽  
Vol 235 (2) ◽  
pp. 163-171 ◽  
Author(s):  
P. LEMMER ◽  
M. GUNKEL ◽  
Y. WEILAND ◽  
P. MÜLLER ◽  
D. BADDELEY ◽  
...  
Keyword(s):  
Far Field ◽  
Fluorescent Markers ◽  
Nm Range

scholarly journals Site Specific Three-dimensional Structural Analysis in Tissues and Cells Using Automated DualBeam Slice &View

2007 ◽  
Vol 15 (2) ◽  
pp. 26-31 ◽  
Author(s):  
Ben Lich
Keyword(s):  
Electron Microscopy ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Three Dimensional ◽  
Surface Region ◽  
Dimensional Structure ◽  
Near Surface ◽  
Imaging Capability ◽  
Fluorescent Markers ◽  
Confocal And Electron Microscopy

DualBeam instruments that combine the imaging capability of scanning electron microscopy (SEM) with the cutting and deposition capability of a focused ion beam (FIB) provide biologists with a powerful tool for investigating three-dimensional structure with nanoscale (1 nm-100 nm) resolution. Ever since Van Leeuwenhoek used the first microscope to describe bacteria more than 300 years ago, microscopy has played a central role in scientists' efforts to understand biological systems. Light microscopy is generally limited to a useful resolution of about a micrometer. More recently the use of confocal and electron microscopy has enabled investigations at higher resolution. Used with fluorescent markers, confocal microscopy can detect and localize molecular scale features, but its imaging resolution is still limited. SEM is capable of nanometer resolution, but is limited to the near surface region of the sample.

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