Basics of a Flow Cytometer
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1 Basics of a Flow Cytometer Deborah Michel OCT
2 What is Flow Cytometry flow cytometry is the measurement of cells/particles in a flow system, which delivers the cells/particles (0.2 to 150 µm) singly past a point of measurement. Points to consider Flow Light Detection detector
3 What can a Flow Cytometer Tell Us About a Cell/Particle? Relative size by Forward Scatter Right angle light detector Relative granularity or internal complexity by Side Scatter Incident Research question by Relative fluorescence intensity (ie marker)
4 Examples of application of forward and side scattering high internal complexity and large size
5 Examples of application of forward and side scattering Nanodiamond and titanium oxide particles in cellular uptake studies using HeLa cells increases the side scatter with little effect on forward scattering S. Alwani and Dr. Badea research
6 What is Fluorescent Light? The fluorochrome absorbs energy from the laser The fluorochrome releases the absorbed energy by emission of photons of a longer wavelength excitation emission
7 Fluorescence
8 FACSCalibur Fluidics Introduces and focuses the cells for interrogation Optics Generates and collects the light signals Electronics Converts the optical signal to digital signal, processes the signal and communicate with the computer
9 Fluidics
10 Hydrodynamic focusing Slower moving sample stream is injected into a faster moving sheath stream Surface tension and laminar flow causes the sample to be wicked off into a narrower faster moving stream within the sheath stream (stream within a stream) Alignment of cells within this stream are controlled by velocity of the two streams Flow cell Higher resolution (Quantitative) Lower resolution (Qualitative)
11 Optics Excitation/Emission 2 lasers (488 and 635nm) 4 filters BP and LP (FL1 to 4) Photomultiplier tubes 488 laser FL1 530/30nm Pre- Amplifier Levels: E-1 E00 E01 E02 E03
12 Electronics Pulse height (H), area (A) and width (W) Converts analog signals to proportional digital signals A H W Practical Flow Cytometry Haematology Diagnostics
13 Electronics Volt Histogram (one parameter)
14 Plotting Data Channel values
15 Plot Stats (Cell Quest Pro) Peak Channel 286 Peak to 1000 MMMM = x i n GGG MMMM = 11 lll x i n M. Poorghorban and Dr. Badea research
16 Types of Plots Histogram Dot plot Density plot Contour plot 3D plot Dr. Badea research
17 Flow Cytometer Setup Set the forward and side scatter detectors for your untreated, unstained cell population of interest Set fluorescence detectors sensitivities Using more than one fluorescence marker? Do you need to correct spectral overlap with compensation? Collect data from your samples
18 Setting FSC and SSC Set forward and side scatter detectors to untreated cells. HeLa cells Beads
19 Setting Fluorescence Detectors Set FL1 and FL2 detectors so that the auto fluorescence from unstained cells are set within the first log decade (10 0 to 10 1 ) PE (FL2) FITC (FL1)
20 Compensation When analysing more than one color be careful of spectral overlap FITC PE Fl2-%Fl1 Digital FACS have software for this. Flowcyt.salk.edu
21 Compensation Cells + FITC PE (FL2) FL2 % FL1 PE (FL2) Q1 Q2 Quadrant Statistics Quad Events X Mean Y mean Q1 0 *** *** Q2 0 *** *** Fl2-%FL1 Q Q FITC (FL1) Q3 FITC (FL1) Q4 Y mean
22 Compensation FITC PE Fl1-%Fl2 Flowcyt.salk.edu
23 Compensation Cells + FITC PE (FL2) FL2 % FL1 PE (FL2) Q1 Q2 Quadrant Statistics Quad Events X Mean Y mean Q1 0 *** *** Q2 0 *** *** Fl2-%FL1 Q Q FITC (FL1) Q3 FITC (FL1) Q4 Y mean Cells + PE PE (FL2) FL1 % FL2 PE (FL2) Fl1-%FL2 Q1 Q3 Q2 Q4 Quadrant Statistics Quad Events X Mean Y mean Q Q2 0 *** *** Q Q4 0 *** *** FITC (FL1) X mean FITC (FL1)
24 Quiz Over compensated Under compensated Compensated A. Doig et al. Practical Flow Cytometry in Haematology Diagnosis
25 Gating Isolate populations of interest Gating an area will make your analysis more specific Can remove dead cells and debris Cannot discriminate between cells with the same scattering properties
26 Gating Example Isolating populations of interest
27 Gating Example Cannot discriminate between cells with the same scattering properties A. Doig et al. Practical Flow Cytometry in Haematology Diagnosis
28 Back Gating Example Which population is Lymphocytes? Leucocytes CD45+ Monocytes CD14+ Lymphocytes CD14-, CD45+Bright Back gating of P1 Fluorescence of P2 A. Doig et al. Practical Flow Cytometry in Haematology Diagnosis
29 Applications Immunophenotyping / Intracellular antigens measurement DNA/RNA: cell cycle, aneuploidy, endoreduplication, kinetics DNA base ratios Chromatin structure Apoptosis (DNA degradation, mitochondrial membrane potential, permeability changes, caspase activity) Membrane potential Membrane fluidity Membrane fusion/runover Intracellular calcium (ions) flux Intracellular ph Sulfhydryl groups/glutathione Cell viability Cell tracking and proliferation Intracellular reactive oxygen species (Oxidative burst) Cell proliferation Cell enumeration Cell volume and morphological complexity Cell pigments (f.ex. chlorophyll or phycoerythrin) Drug delivery Multidrug resistance (MDR) Phagocytosis Pathogen-host cell adherence Differentiation Identification of stem cells Reticulocyte, platelet etc analysis Microparticles analysis Assessing infection/transfection levels Monitoring of the electropermeabilization of cells Cytotoxicity assay Enzymatic activity Cell activation Protein-protein interactions (FRET, split-gfp) Protein modifications, phospho-proteins Activation of signalling pathways Cytokine Secretion Sorting (f.ex sperm sorting for sex preselection) Karyotyping Telomere length
30 Build Your Own Flow Cytometer 36 th Annual Course in Flow Cytometry, Brunswick Maine, 2012
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Copyright 1999 2010 by Mark Brandt, Ph.D. 12
Introduction to Absorbance Spectroscopy A single beam spectrophotometer is comprised of a light source, a monochromator, a sample holder, and a detector. An ideal instrument has a light source that emits