Flowcytometry Analysis of Cell Characteristics
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Diagnostic immunology Definition. Uses of Flowcytometery. Instrument Overview. Principles of flow cytometry. Light scatter. Fluorescence. Gating. FACS cell sorter
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Flowcytometry Analysis of Cell Characteristics
- 1. Flowcytometery By Ihab Muhammad Mahmoud Medical Research Institute - Alexandria
- 2. • Definition. • Uses of Flowcytometery. • Instrument Overview. • Principles of flow cytometry. • Light scatter. • Fluorescence. • Gating.
- 3. Definition • An analytical technique that quantifies the frequencies of cells binding to fluorescent antibodies and scattering light in characteristic ways. • Powerful technique for the analysis of multiple parameters of individual cells within heterogenous population.
- 4. Uses of Flowcytometery • Flowcytometry is the sine qua non. • Used in range of applications: – Immunophenotyping. – Ploidy anaysis. – Cell counting. – GPF expression analysis.
- 5. Clinical Applications of Flow Cytometry • Cell cycle analysis. • Apoptosis(Bcl2,p53 and sub G1,……..) • Evaluation of DNA Histogram. • Tumor marker • Immunophenotyping Applications • HIV monitoring. • Analysis of leukemias and lymphomas • Sperm haploidy and chromatin analysis • RNA and DNA analysis for down syndrome. • And another many markers.
- 9. Fluidics system: Present the samples to the interrogation point.
- 10. Lasers: Light source for scatter and fluorescence.
- 11. Optics: Gather and direct the lights.
- 13. The electronics and computer system: Convert the signals from the detectors into digital data and perform the analysis.
- 14. Principles of flow cytometry
- 16. Hydrodynamic focusing Particles or cells are passed through the laser beam one at a time.
- 17. Light Scattering
- 18. Forward Scatter The magnitude of forward scatter is roughly proportional to the size of the cell.
- 19. The voltage pulse generated by a cell passing in front of the laser beam
- 20. Size comparison
- 21. Forward Scatter The magnitude of each pulse record by cell is proportional to the cell size.
- 22. Forward scatter histogram Is a graphical representation of the size distribution within the cell population within the sample. This histogram only represent one distribution data.
- 23. Forward scatter histogram The single population in the forward scatter histogram in reality is multiple populations…… Only distinct by looking at the data in second dimension.
- 24. Overview – Side scatter A second light scattering detector is placed at approximately 90° to the path of the laser beam.
- 25. Side scatter
- 26. Side scatter Side scatter is caused by granularity and the structural complexity inside the cell.
- 27. Side scatter
- 29. 2D Dot blot
- 30. 2D Dot blot The peaks from the forward and side scatter histograms correlate with color in the two dimension dot blot
- 31. 2D Scatter Plot of Blood Multipara metric analysis is the real power of flow cytometery.
- 32. Using fluorescence in flow cytometery Fluorophore-labelled antibody.
- 33. Energy state diagram (fluorescence)
- 34. Fluorescence detection Labeled cells travel the same path as side-scatter and directed to series of filters and mirrors. The particular wavelength is delivered to particular detector.
- 35. Fluorescence one color histogram The fluorescence light is detected to an appropriate detector to where translated to a voltage pulse proportional to the amount of fluorescence emitted .
- 36. Fluorescence one color histogram
- 37. Two color experiment, spectra compatible
- 38. Filters collect two colors
- 39. Gatting • Gates are used to isolate subsets of cells or “populations” on a plot • Allows the ability to look at parameters specific to only that subset
- 40. Gatting
- 41. 2 Parameter Histogram FITC FL PE FL Negative Population Single Positive FITC Population Single Positive PI Population Double Positive Population
- 42. Cell Sorting • When the cells meet the criteria, they are broken into droplets containing the cells • An electrical charge is applied to the stream just before the cell of interest breaks off the stream. • When the drops fall, they fall between two metal plates, one charged positively, the other negatively. • The charged cell will be attracted to the plate of opposite charge . • Droplets can be collect for further examination.
- 44. Referances • https://www.youtube.com/watch?v=sfWWxF BltpQ • Judy Owen, Jenni Punt, Sharon Stranford Kuby Immunology 2013. • http://www.motifolio.com/6111179.html
Editor's Notes
- sine qua non (without which, nothing) Immunophenotyping is the analysis of heterogeneous populations of cells for the purpose of identifying the presence and proportions of the various populations of interest. Antibodies are used to identify cells by detecting specific antigens expressed by these cells, which are known as markers. ploidy analysis A flow cytometry technique which evaluates a cell’s chromosome content—a parameter of aggressiveness in cancer. In general, diploidy (i.e., the presence of 2 haploid sets of chromosomes) is a normal or near-normal state; in contrast, anaplastic and aggressive tumours are more often aneuploid or hyperdiploid. Ploidy analysis is used to prognosticate malignancies in bone (osteosarcoma), as well as in bladder, breast, colon, endometrial and ovarian carcinomas, and lymphoma. The green fluorescent protein (GFP) is a protein composed of 238 amino acid residues (26.9 kDa) that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. ... In cell and molecular biology, the GFP gene is frequently used as a reporter of expression. Green fluorescent protein (GFP) is a protein that causes the Aequorea victoria jellyfish to glow. The protein is coded for by a single gene. ... If the GFP gene is inserted correctly, it can be expressed in organisms other than jellyfish. The GFP gene can be used as a visual tag for the expression of other genes. In cells where the gene is expressed, and the tagged proteins are produced, GFP is produced at the same time. Thus, only those cells in which the tagged gene is expressed, or the target proteins are produced, will fluoresce when observed under fluorescence microscopy.
- Principles of flow cytometry. Cells introduced into the sample injection port are focused within a stream of sheath fluid and pass one by one in front of the laser beam. Forward scattered light is detected by a photodiode. Side-scattered light and emitted fluorescence of various wavelengths is detected by photomultiplier tubes, after passage through a series of dichroic mirrors and light filters.
- Laser and sample intersect and optics collect the resulting scattering and fluorescence
- Cells in suspension are hydrodynamically focused into a narrow stream by being introduced inside a rapidly moving column of sheath fluid. Every time a cell passes in front of the laser beam, light is scattered, and this interruption of the laser signal is recorded.
- One detector, called a photodiode, picks up light scattered in the forward direction (i.e., within a few degrees of the original path of the laser beam). High forward light scattering (within 5 to 10 degrees of the light path of the laser beam) provides an indication that the cells responsible for this light scattering are large. The more forward light scatter, the larger the cell, and so the amount of light scattered in the forward direction can be used as a rough measure of the range of sizes of the cells in the stream.
- As cells pass in front of the laser beam, they emit or scatter light that is detected by a series of photomultiplier tubes (PMTs). The PMT receives the photons and converts them into a voltage pulse whose height, width and area is digitized and represented by the cytometer software
- The scattered light is received by the detector is translated into a voltage pulse . Small cell produces small amount of forward scatter Large cells produce large amount of forward scatter The magnitude of each pulse record by each cell is proportional to the cell size.
- A second light scattering detector is placed at approximately 90° to the path of the laser beam. The amount of side scattered light offers an indication of the extent of intracellular complexity of the scattering cells. The more side-angle light scatter, the more intracellular membranous structures, such as endoplasmic reticulum (ER) and mitochondria, are present in the scattering cell.
- A second light scattering detector is placed at approximately 90 to the path of the laser beam. The amount of side scattered light offers an indication of the extent of intracellular complexity of the scattering cells. The more side-angle light scatter, the more intracellular membranous structures, such as endoplasmic reticulum (ER) and mitochondria, are present in the scattering cell.
- The signals collected by side-scatter detector can be blotted on one dimension histogram like with forward scatter.
- The one dimension histogram do not necessarily show the complexity of the cell populations. Only distinguished by looking in the data in the second dimension This comes to be used as two dimension dot blot The peaks from the forward and side scatter histograms correlate with color in the two dimension dot blot
- Lymphocytes: too small cells showing low internal complexity Monocytes: medium size cells but slightly more complexity Neutrophils and other granulocytes: large cells having a lot of internal complexity
- Fluorescently tagged antibodies bound to the surface antigens of particular cells can be excited by the laser. Monoclonal antibody and FACS technologies were developed at around the same time, and the two technological breakthroughs proved synergistic: the more antibodies that were available for cell typing and sorting, the more informative flowcytometric experiments became.
- After excitation, they emit light that is recorded by a series of photomultiplier tubes located at a right angle to the laser beam.
- Each photomultiplier tube fluorescence detector is placed behind a series of dichroic filters and mirrors, so that it only receives and detects light within a particular range of wavelengths. Flow cytometers count every cell as it passes the laser beam and record the level of emitted fluorescence at a number of different wavelengths, as well as the amounts of forward- and side-scattered light for each cell; an attached computer stores all the data for each cell, which can then be called up by the analysis soft ware as needed. This technology is advancing very quickly, and flow cytometers capable of detecting 8 to 12 fluorescence and light-scattering parameters are routinely used in clinical and research laboratories.
- Fluorescence data is collected in generally the same way the forward and side scatter data in a population of labeled cells some will be brighter than the other The fluorescence light is detected to an appropriate detector to where translated to a voltage pulse proportional to the amount of fluorescence emitted . All the voltage pulses are recorded and presented graphically.
- On the left is a scatter plot of forward scatter (abscissa) versus side scatter (ordinate) of a sample of human white blood cells. Lymphocytes are gated and displayed in red. On the right is a plot of lymphocytes stained with anti-CD4 (ordinate) or anti-CD8 (abscissa) antibodies. On the left of Figure 20-19b, we show an example of a typical forward and side scatter plot from human white blood cells. On the right of figure is a plot of the fluorescence intensity of cells derived from the region shown gated in red in the scatter plot, which represents small, lowlight- scattering cells, or lymphocytes. These cells have been stained with a fluorescein-conjugated antibody to CD8; therefore, CD8-bearing T cells will fluoresce in the green part of the spectrum. Green fluorescence is detected in the FL1, or green channel of the detector. Similarly, CD4 T cells have been stained with an antibody conjugated with a fluorochrome (e.g., phycoerythrin, or PE) that is detected in the FL2, or red channel. This plot therefore shows us that, of the cells falling into the lymphocyte gate, 44% were red-staining CD4-bearing cells and 23% were green-staining CD8-bearing cells.