Unit 4_TU_FC.pdf

FLOW CYTOMETRY OPTICS (FSC) Forward Angle Light Scatter Laser FALS Sensor Optics Side Scatter Channel (SSC)  The amount of light scattered at right angle to the incident light beam depends on the internal complexity of th...

FLOW CYTOMETRY OPTICS (FSC) Forward Angle Light Scatter Laser FALS Sensor Optics Side Scatter Channel (SSC)  The amount of light scattered at right angle to the incident light beam depends on the internal complexity of the particle, this known as wide angle or Side Scatter (SSC) , side scatter detected at 90, to the laser beam. SSC) ( Optics 90 Degree Light Scatter Laser FALS Sensor 90LS Sensor OPTICS PROPERTIES OF FSC& SSC PROPERTIES OF FSC& SSC  As the cell passes through the laser beam, light is scattered in all directions and that scattered in the forward direction is proportional to the square of the radius of a sphere, and so to the size of the cell or particle.  The cells may be labeled with fluorochrome-linked antibodies or stained with fluorescent membrane, cytoplasmic or nuclear dye. What can a Flow Cytometer (FCM) tell us about a cell?  Its relative size (Forward Scatter—FSC)  Its relative granularity or internal complexity (Side Scatter—SSC)  Its relative fluorescence intensity (FL1, FL2, FL3, FL4) Optics - Fluorescence Channels  Any fluorescent molecule present in or on the particle will absorb energy from the laser light and release the absorbed energy at longer wave length, the emitted light is collected by lenses and detectors, emitted fluorescence intensity is proportional to the amount of fluorescent compound on the particle. Optical Design PMT 5 PMT 4 Sample PMT 3 Dichroic Flow cell Filters PMT 2 Scatter PMT 1 Laser Sensor Band pass Filters Electronics  The scattered light from particles passing the laser light is converted to digital values that stored in the computer for analysis. Electronics FSC vs SSC Dot Plot Fluorescence And Antibodies To The Rescue Fluorescence and Fluorochrome.  The coupling of monoclonal antibodies with fluorescent dyes is necessary for recognition and enumeration by flow cytometer.  Each fluorochrome possesses a distinctive spectral pattern of absorption (excitation) and emission.  The property of fluorescence is that, the fluorochromes present on the cell absorb the laser light and re-emit the light at a lower energy and longer wave length.  The most popular fluorochrome used in immuno-fluorescent is fluorescein isothiocyanate (FITC), which has an absorption maximum between 450 and 550 nm.  Another example of an excellent combination is Phycoerythrin (PE) as a second fluorochrome since it can absorb light at higher wave lengths than the FITC. APPLICATION Applications in Clinical Laboratories  Immunophenotyping (HIV)  CD4 absolute counts  Leukemia and lymphoma immunophenotyping  Cell cycle and ploidy analysis of tumors  Reticulocyte enumeration  Flow cross-matching (organ transplantation)  Stem cell enumeration  Residual white blood cell detection  (QC platelet, red blood cells) APPLICATION Research Laboratories  Immune function studies  Hematopoietic stem cells  Multi-drug resistance studies (cancer)  Kinetics studies (cell function) Platelet analysis (coronary disease)  Environmental sample analysis  Flow and FISH TYPES OF MEASUREMENTS  Intrinsic parameters: Cell size & Granularity.  Extrinsic parameters: Surface Antigens  Single or multiple surface membrane antigens are detected with fluorescinated monoclonal antibodies examples, CD45, CD4, CD8, etc). Fluorescent Dyes And Antibodies Fluorochromes Are Molecules That Emit  Fluorescence Upon Excitation With Light Ex. FITC (Fluorescein Isothiocyanate) PE (Phycoerythrin) PerCP (Peridinin Chlorophyll Protein) APC (Allophycocyanin) Some Fluorochromes Are Proteins, Some  Are Small Organic Compounds Ex. PE (Phycoerythrin)-Protein Ex. FITC (Fluorescein Isothiocyanate) Principles Of Fluorescence E= h f =f Excitation Spectra Of Fluorochromes Emission Spectra