Bi9393 Analytical cytometry Lesson 3 Department of Cytokinetics Institute of Biophysics, CAS, v.v.i. Kralopolska 135 612 65 Brno E-mail: ksoucek@ibp.cz tel.: 541 517 166 Karel Souček, Ph.D. K. Souček Bi9393 Analytical cytometry https://www.bosterbio.com/protocol-and-troubleshooting/flow-cytometry-principle Fluidic systems and hydrodynamics Fluidics Cart Cytometer Fluid system : BD FACSAria II AIR PRESSURE BULK INJECTION Sheath Tank AIR PRESSURE ASPIRATED WASTE (VACUUM) ASPIRATED WASTE (DEGAS) SHEATH FILTER 0” – 9” R1 Sheath Regulator R2 Sample Regulator V17 V20 V5V6 Osborne Reynolds (1842-1912) Re < 2300 Re > 2300 Reynolds number Hydrodynamic focusing in the cuvette Sheath Sample Sheath Sample Sample pressure low, small core stream. Good for DNA analysis High sample pressure, broader core stream. Bad for DNA analysis Laser Beams ANd9GcSG6MDDEyKW2lrEy-4pYcbhalq94g7mws3iCujkWC8szVoIZvRtvw Particle Delivery: Hydrodynamic Focusing Intensities Count Narrow particle focus = Narrow distribution Laser Cross Sectional Area • Sample core is ' pinched ' by fast flowing sheath • Sample volume ratios of 100 – 1000 • Large ratios => low sample inputs • Resolution of particle populations sheath sheath Hydrodynamic core Conventional Instrumentation: Low Flow Rates (12µL/min) Particle Delivery: Hydrodynamic Focusing Conventional Instrumentation: High Flow Rate (60µL/min) Intensities Count Broad particle focus = Broad distribution • Increased sample input = increased core size • Particle distributions broadened, CVs increase • Instrument resolution decreased • Historically, low volumetric sample rates used (25  l/min – 150  l/min) sheath sheath Hydrodynamic core Laser Cross Sectional Area Attune® Acoustic Focusing Cytometer Acoustic Focusing = Better Precision Acoustic focusing module Narrow particle focus = Narrow distribution 12 µL/min 1000 µL/min Acoustic focusing of particles occurs prior to mixing with sheath fluid https://upload.wikimedia.org/wikipedia/commons/3/35/Kundt_tube.png Attune NxT ( 2nd generation ) 100 200 300 400 500 600 700 800 900 1000 Maximum Sample Input Rate (  l/min) Instrument 1 Instrument 2 Instrument 6 Instrument 5 Instrument 4 Instrument 3 Attune® Attune® Throughput Compared to Hydrodynamic Focused Instruments • Attune® can analyze at sample rates from 25µL/min to 1000µL/min without losing accuracy • Traditional Flow Cytometers can only run at most 150µL/min and will sacrifice data quality • Higher sample rates enable dilution of limited samples and analysis of Rare Events Faster Hydrodynamic Focused Instruments • the pressure of the carrier (sheathing) liquid drives the buffer through the cuvette and the higher pressure in the sample tube introduces the sample into the cuvette. • The principle of hydrodynamic focusing aligns the cells in the cuvette "like pearls on a string" before they reach the point where the laser beam intersects. •Hydrodynamic focusing cannot dissociate cell aggregates. Flow cytometry requires a suspension of single cells! Fluidics – summary Principles of flow cytometry and sorting ◼ sorting K. Souček Bi9393 Analytical cytometry Frequency Charge Drop Delay Amplitude SORTING +++++ ----- +++++ ----- +++++ ----- +++++ +++++----- +++++ ++ +++++----- +++++ ++ +++++ + +++++----- +++++ + +++++ ----- +++++----- ++++++++++ ----- +++++----- ++++++++++ ----- - - +++++----- - - - - - - - - - - - - +++++----- - - - - - - - - - - - +++++----- - - - - - - - - - - - +++++----- - - - - -- - - - - +++++----- - - - - - - - - - - http://www.cyto.purdue.edu/cdroms/cyto10a/seminalcontributions/fulwyler.html _ Sorting - Sort Masks Cells are randomized distributed over the stream Sorting - Sort Masks Trailing Interrogated Leading Mask ◼ A region of the stream monitored for the presence of cells ◼ Determines how drops will be deflected if a sorting conflict occurs ◼ Measured in 1/32 drop increments Mask = 0 Mask = 8 Mask = 16 Mask = 32 4 4 8 8 16 16 Conflict Resolution ◼ Precision modes include three types of masks – Yield – Purity – Phase Sorting - Sort Masks Sort decisions are determined by sort masks Target particles in a drop with 1/32-drop resolution Sorting - Yield Mask The yield mask defines how many drops will be sorted Yield mask of 8/32 indicated in blue; target particle shown in green Yield Mask Sorting - Purity Mask Purity mask of 8/32 in blue, 4/32 in each adjacent drop; target particles in green, non-target particles in red Purity Mask Purity Mask Cell sorting - trends ◼ Easy operation ◼ Careful handling – On-chip technology ◼ Size  and security  ◼ Microfluidic-based cell sorting ◼ Spectral cell sorting ◼ Image-based sorting ◼ Buoyancy Activated Cell Sorting (BACS ) – a method that uses low-density particles (microbubbles) for flotation separation. Electronics and data Laser Laser Laser Creation of a Voltage Pulse Tim Voltage TimVoltage Tim Voltage 1 2 3 Height, Area, and Width Time (µs) Voltage Pulse area (A) PulseHeight(H) Pulse Width (W) 0 Signal processing time analogsignalintensity VOLTAGE FSC ~ cell size FL-1 (530/30nm) ~ green fluo. FL-2 (585/42nm) ~ red fluo . Analog/digital conversion Height Width Area ( ∫ ) FL- (H, W, A) FL-1(H) FL-2 (H) dot plot 0 1000 1000 K. Souček Bi9393 Analytical cytometry Voltage In PMT Power Supply Levels 0–1000 Volts Photon In Signal Out Digital data to memory Analog to Digital Conversion Digitization the pulse 16,384 levels Sample the pulse 10 MHz Analog to Digital Converter Parameters • Area: Sum of all height values • Height: Maximum digitized value X 16 • Width: Area/Height X 64K Data is displayed on a 262,144 scale 282 3060 10270 358 4004 9568 14524 AD converters Number of bits # channels distinction 8 256 39.1 mV 10 1024 9.77 mV 12 4096 2.44 mV 14 16384 610  asl 16 65536 153  E 18 262144 38.1  E 20 1048576 9.54  V 22 4194304 2.38  H 24 16777216 596 AD 2 8 = 256 2 10 = 1024 . . . Full scale measurement range = 0 to 10 volts ADC resolution is 12 bits: 212 = 4096 quantization levels ADC voltage resolution is: (10-0)/4096 = 0.00244 volts = 2.44 mV K. Souček Bi9393 Analytical cytometry Logarithmic gain & dynamic range adapted from JPRobinson LIN LOG Data analysis ◼ View data – histogram – dot plot – isometric display – contour plot – chromatic (color) plots – 3D projection ◼ Gating Basic ways to display data K. Souček Bi9393 Analytical cytometry Summary ◼ Fluid systems ◼ Sorting ◼ Signal, data – basic principle At the end of today's lecture you should : 1. Know the basic principles of light scattering 2. and fluorescence; 3. to know what light sources are used in flow cytometry; 4. and how it is detected; 5. know the basic principles of fluid systems and laminar flow. 6. Know the basic principle of data processing and visualization At the end of today's lecture you should : 1. Know the basic principles of light scattering 2. and fluorescence; 3. to know what light sources are used in flow cytometry; 4. and how it is detected; 5. know the basic principles of fluid systems and laminar flow. 6. Know the basic principle of data processing and visualization K. Souček Bi9393 Analytical cytometry