These particles have something in common.pptVIP

These particles have something in common Parts of a flow cytometer Fluidics Provide a constant stream of sheath Transport the sample to the interrogation point Arrange and focus the cells to the laser intercept Optics Focus the excitation light Collect the emitted light Electronics Convert the optical signals into electronic signals Send the signals to the analysis computer Computer Display data graphically Control instrument settings What a flowcytometer is Very basically, a flow cytometer is an automated fluorescence microscope (in fact, that is how the first prototype instruments looked like). Like a microscope, some adjustments have to be made to optimally illuminate and collect the light. The basic microscope The automated Microscope Basic fluidics of the FACSAria Hydrodynamic focussing in the cuvette Basic Optics Optical filters Octagon Detection System Summary Excitation light is steered with prisms and lenses to the interception point Emitted light is collected using lenses and is split up with dichroic mirrors and filters Tasks for the electronical system Convert the optical signals into electonic signals (voltage pulses) Digitise the data Analyse Height (H), Width (W) and Area (A) of the pulse Send the data to the analysis computer How a voltage pulse from the PMT is generated Height, Area, and Width Threshold Summary Instrument settings Workstation Data saving Visualization of data Example: Determine the percentage of CD3, CD4, and CD8 populations from whole blood Prepare the instrument Proper adjustment of FSC and SSC voltage Parameters (I) Parameters Defining the population of interest (often just named ?gating“) About ?Gating“ Adjusting the fluorescence settings Theory of quadrant analysis Real life:FITC-fluorescence overspill FITC Compensation FITC Compensation PE-fluorescence overspill Automatic Multicolour Compensation Summary What we have seen: the emission spectra of common fluorochromes (FITC, PE) the spectral overlap of fluorochro