The rapid analysis of single marine cells by flow cytometry

1990 ◽  
Vol 333 (1628) ◽  
pp. 99-112 ◽  
Keyword(s):  
Flow Cytometry ◽  
Single Cells ◽  
Rapid Analysis ◽  
Light Scatter ◽  
Fluorescent Labelling ◽  
Photosynthetic Organisms ◽  
Novel Technique ◽  
Multiple Measurements ◽  
Fluorescence Light ◽  
Near Future

Analytical flow cytometry (AFC) is a novel technique for the rapid (more than 103 s-1) analysis and sorting of single cells based upon simultaneous, multiple measurements of laser-induced particle fluorescence, light scatter and impedance. Originally developed for biomedical use, AFC is now being adapted to analyse single-celled organisms such as phytoplankton and bacteria which are present as trace but functionally important components in seawater. Marine AFC has been used to analytically differentiate and sort these organisms from the heterogeneous assemblage of particles present in seawater. Chlorophyll autofluorescence is an unique biomarker for photosynthetic organisms and has been used to analyse phytoplankton cytometrically both in the laboratory and at sea. A theoretical and practical framework for the cytometric quantitation of cellular chlorophyll in phytoplankton based on autofluorescence is presented. Other subcellular constituents such as enzymes, lipids, nucleic acids and toxins in phytoplankton have recently been analysed by AFC using immuno-, induced or applied fluorescent labelling techniques. Examples are presented together with novel developments in fringe areas of cytometry that are likely to influence AFC of single marine cells in the near future.

An optofluidic system with integrated microlens arrays for parallel imaging flow cytometry

Lab on a Chip ◽  
2018 ◽  
Vol 18 (23) ◽  
pp. 3631-3637 ◽  
Author(s):  
Gregor Holzner ◽  
Ying Du ◽  
Xiaobao Cao ◽  
Jaebum Choo ◽  
Andrew J. deMello ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
High Speed ◽  
Parallel Imaging ◽  
Single Cells ◽  
Rapid Analysis ◽  
High Speed Imaging ◽  
Microlens Arrays ◽  
Imaging Flow Cytometry

In recent years, high-speed imaging has become increasingly effective for the rapid analysis of single cells in flowing environments.

scholarly journals Rugosity in Grimontia hollisae

2006 ◽  
Vol 73 (4) ◽  
pp. 1215-1224 ◽  
Author(s):  
S. K. Curtis ◽  
M. H. Kothary ◽  
R. J. Blodgett ◽  
R. B. Raybourne ◽  
G. C. Ziobro ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Wheat Germ ◽  
Cellular Differentiation ◽  
Lectin Binding ◽  
Single Cells ◽  
Light Scatter ◽  
Agar Surface ◽  
Galanthus Nivalis ◽  
Vibrio Hollisae

ABSTRACT Grimontia hollisae, formerly Vibrio hollisae, produces both smooth and rugose colonial variants. The rugose colony phenotype is characterized by wrinkled colonies producing copious amounts of exopolysaccharide. Cells from a rugose colony grown at 30�C form rugose colonies, while the same cells grown at 37�C form smooth colonies, which are characterized by a nonwrinkled, uncrannied appearance. Stress response studies revealed that after exposure to bleach for 30 min, rugose survivors outnumbered smooth survivors. Light scatter information obtained by flow cytometry indicated that rugose cells clumped into clusters of three or more cells (average, five cells) and formed two major clusters, while smooth cells formed only one cluster of single cells or doublets. Fluorescent lectin-binding flow cytometry studies revealed that the percentages of rugose cells that bound either wheat germ agglutinin (WGA) or Galanthus nivalis lectin (GNL) were greater than the percentages of smooth cells that bound the same lectins (WGA, 35% versus 3.5%; GNL, 67% versus 0.21%). These results indicate that the rugose exopolysaccharide consists partially of N-acetylglucosamine and mannose. Rugose colonies produced significantly more biofilm material than did smooth colonies, and rugose colonies grown at 30�C produced more biofilm material than rugose colonies grown at 37�C. Ultrastructurally, rugose colonies show regional cellular differentiation, with apical and lateral colonial regions containing cells embedded in a matrix stained by Alcian Blue. The cells touching the agar surface are packed tightly together in a palisade-like manner. The central region of the colony contains irregularly arranged, fluid-filled spaces and loosely packed chains or arrays of coccoid and vibrioid cells. Smooth colonies, in contrast, are flattened, composed of vibrioid cells, and lack distinct regional cellular differences. Results from suckling mouse studies showed that both orally fed rugose and smooth variants elicited significant, but similar, amounts of fluid accumulated in the stomach and intestines. These observations comprise the first report of expression and characterization of rugosity by G. hollisae and raise the possibility that expression of rugose exopolysaccharide in this organism is regulated at least in part by growth temperature.

Fast intracellular pH determination in single cells by flow-cytometry

1981 ◽  
Vol 68 (5) ◽  
pp. 265-266 ◽  
Author(s):  
G. Valet ◽  
A. Raffael ◽  
L. Moroder ◽  
E. W�nsch ◽  
G. Ruhenstroth-Bauer
Keyword(s):  
Flow Cytometry ◽  
Intracellular Ph ◽  
Single Cells

scholarly journals Applications of Flow Cytometry to Clinical Microbiology

2000 ◽  
Vol 13 (2) ◽  
pp. 167-195 ◽  
Author(s):  
Alberto Álvarez-Barrientos ◽  
Javier Arroyo ◽  
Rafael Cantón ◽  
César Nombela ◽  
Miguel Sánchez-Pérez
Keyword(s):  
Flow Cytometry ◽  
Clinical Microbiology ◽  
Ex Vivo ◽  
Analytical Techniques ◽  
Clinical Samples ◽  
Clinical Microbiology Laboratory ◽  
Commercial Kits ◽  
Antimicrobial Treatments ◽  
Near Future

SUMMARY Classical microbiology techniques are relatively slow in comparison to other analytical techniques, in many cases due to the need to culture the microorganisms. Furthermore, classical approaches are difficult with unculturable microorganisms. More recently, the emergence of molecular biology techniques, particularly those on antibodies and nucleic acid probes combined with amplification techniques, has provided speediness and specificity to microbiological diagnosis. Flow cytometry (FCM) allows single- or multiple-microbe detection in clinical samples in an easy, reliable, and fast way. Microbes can be identified on the basis of their peculiar cytometric parameters or by means of certain fluorochromes that can be used either independently or bound to specific antibodies or oligonucleotides. FCM has permitted the development of quantitative procedures to assess antimicrobial susceptibility and drug cytotoxicity in a rapid, accurate, and highly reproducible way. Furthermore, this technique allows the monitoring of in vitro antimicrobial activity and of antimicrobial treatments ex vivo. The most outstanding contribution of FCM is the possibility of detecting the presence of heterogeneous populations with different responses to antimicrobial treatments. Despite these advantages, the application of FCM in clinical microbiology is not yet widespread, probably due to the lack of access to flow cytometers or the lack of knowledge about the potential of this technique. One of the goals of this review is to attempt to mitigate this latter circumstance. We are convinced that in the near future, the availability of commercial kits should increase the use of this technique in the clinical microbiology laboratory.

scholarly journals Glyoxal fixation facilitates transcriptome analysis after antigen staining and cell sorting by flow cytometry

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0240769
Author(s):  
Prasanna Channathodiyil ◽  
Jonathan Houseley
Keyword(s):  
Flow Cytometry ◽  
Transcriptome Analysis ◽  
Mammalian Cells ◽  
Single Cells ◽  
Rna Extraction ◽  
Cyclin B1 ◽  
Immunofluorescent Staining ◽  
Rna Seq ◽  
Simple Method ◽  
Staining Methods

A simple method for extraction of high quality RNA from cells that have been fixed, stained and sorted by flow cytometry would allow routine transcriptome analysis of highly purified cell populations and single cells. However, formaldehyde fixation impairs RNA extraction and inhibits RNA amplification. Here we show that good quality RNA can be readily extracted from stained and sorted mammalian cells if formaldehyde is replaced by glyoxal—a well-characterised fixative that is widely compatible with immunofluorescent staining methods. Although both formaldehyde and glyoxal efficiently form protein-protein crosslinks, glyoxal does not crosslink RNA to proteins nor form stable RNA adducts, ensuring that RNA remains accessible and amenable to enzymatic manipulation after glyoxal fixation. We find that RNA integrity is maintained through glyoxal fixation, permeabilisation with methanol or saponin, indirect immunofluorescent staining and flow sorting. RNA can then be extracted by standard methods and processed into RNA-seq libraries using commercial kits; mRNA abundances measured by poly(A)+ RNA-seq correlate well between freshly harvested cells and fixed, stained and sorted cells. We validate the applicability of this approach to flow cytometry by staining MCF-7 cells for the intracellular G2/M-specific antigen cyclin B1 (CCNB1), and show strong enrichment for G2/M-phase cells based on transcriptomic data. Switching to glyoxal fixation with RNA-compatible staining methods requires only minor adjustments of most existing staining and sorting protocols, and should facilitate routine transcriptomic analysis of sorted cells.

Comparison of turbidimetric and light-scattering measurements of immunoglobulins by use of a centrifugal analyzer with absorbance and fluorescence/light-scattering optics.

1980 ◽  
Vol 26 (10) ◽  
pp. 1459-1466 ◽  
Author(s):  
L P Hills ◽  
T O Tiffany
Keyword(s):  
Light Scattering ◽  
Kinetic Curve ◽  
Nonlinear Least Squares ◽  
Light Scatter ◽  
Assay Sensitivity ◽  
Human Immunoglobulins ◽  
Least Squares Fit ◽  
Scattering Measurements ◽  
Fluorescence Light ◽  
Antigen Antibody

Abstract We have developed a centrifugal analyzer with both fluorescence/light-scatter and conventional absorbance optics. The instrument is used in this investigation to study the formation of antigen-antibody complexes by light scattering and turbidimetric measurements, and to develop assays for human immunoglobulins G, A, and M. Concentrations are calculated from a nonlinear least-squares fit of calibrators, and antigen excess is automatically detected from kinetic curve characteristics. Precisions and patients’ results are presented, and assay sensitivity and reliability in the detection of antigen excess are compared. We also investigated the effects of centrifugal force on complex formation. Both nephelometry and turbidimetry can be very satisfactorily adapted to centrifugal analyzers. We present a model to describe the observed differences between the light-scatter and the turbidity data.

Sign in / Sign up

Export Citation Format

Share Document