Bi9393 Analytická cytometrie Lekce 3 Oddělení cytokinetiky Biofyzikální ústav AVČR, v.v.i Královopolská 135 612 65 Brno e-mail: ksoucek@ibp.cz tel.: 541 517 166 Karel Souček, Ph.D. K. Souček Bi9393 Analytická cytometrie https://www.bosterbio.com/protocol-and-troubleshooting/flow-cytometry-principle Fluidics Cart Cytometer Fluidní systém: 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 Hydrodynamic focussing 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 Particle Delivery: Hydrodynamic Focusing Intensity 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) Intensity Count Broad particle focus = Broad distribution • Increased sample input = increase core size • Particle distributions broadened, CVs increase • Instrument resolution decreased • Historically, low volumetric sample rates used (25 ml/min – 150 ml/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 Attune NxT (2nd generation) 100 200 300 400 500 600 700 800 900 1000 Maximum Sample Input Rate (ml/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 • tlak nosné (oplašťující) kapaliny vede pufr kyvetou a vyšší tlak ve zkumavce se vzorkem zavádí vzorek do kyvety. • Princip hydrodynamického zaostření zarovná buňky v kyvetě „jako perly na šňůrce“ předtím než dojdou do bodu kde protnout paprsek laseru. •Hydrodynamické zaostření nemůže oddělit buněčné agregáty. Průtoková cytometrie vyžaduje suspenzi jednotlivých buněk! Fluidika – shrnutí 2 Principy průtokové cytometrie a sortrování ◼ sorting K. Souček Bi9393 Analytická cytometrie +++++ ----- +++++ ----- +++++ ----- +++++ +++++----- +++++ ++ +++++----- +++++ ++ +++++ + +++++----- +++++ + +++++ ----- +++++----- ++++++++++ ----- +++++----- ++++++++++ ----- - - +++++----- - - - - - - - - - - - - +++++----- - - - - - - - - - - - +++++----- - - - - - - - - - - - +++++----- - - - - -- - - - - +++++----- - - - - - - - - - - http://www.cyto.purdue.edu/cdroms/cyto10a/seminalcontributions/fulwyler.html Frequency Charge Drop Delay Amplitude SORTING SORTING Sheath Pressure Nozzle SizeFrequency Amplitude SORTING Drop delay Interrogation point Breakoff SORTING Each sort setup includes: Sheath pressure Breakoff window values Side Stream window values Instrument window > Laser tab values SORTING - Streams Good Bad SORTING – Setup Side Streams Drop Delay interrogation point drop delay breakoff Waste BD FACS™ Accudrop technology ◼ Accudrop beads ◼ Diode laser ◼ Camera ◼ Optical filter Sorting - Sort Masks Cells are randomized distributed over the stream 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 - trendy ◼ Snadná obsluha ◼ Šetrná manipulace – On-chip technologie ◼ Velikost  a bezpečnost  ◼ Microfluidic-based cell sorting ◼ Spectral cell sorting ◼ Image-based sorting ◼ Buoyancy Activated Cell Sorting (BACS™) – metoda, která používá částice s nízkou hustotou (mikrobubliny) pro flotační separaci. Flow Cytometry Standard data file format. FCS 3.1 http://www.isac-net.org/images/stories/documents/Standards/fcs3.1_normativespecification_20090813.pdf Spidlen, J. et al. Cytometry. Part A : the journal of the International Society for Analytical Cytology 77, 97-100, (2010). Laser Laser Laser Creation of a Voltage Pulse Time Voltage TimeVoltage Time Voltage 1 2 3 Height, Area, and Width Time (µs) Voltage Pulse area(A) PulseHeight(H) Pulse Width (W) 0 Signal processing time analogsignalintesity 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 Zesílení signálu (!) lin nebo log K. Souček Bi9393 Analytická cytometrie Voltage In PMT Power Supply Levels 0–1000 Volts Photon In Signal Out Digital data to memory Analog to Digital Conversion Digitize 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 262,144 scale 282 3060 10270 358 4004 9568 14524 AD převodníky Počet bitů # kanálů rozlišení 8 256 39.1 mV 10 1024 9.77 mV 12 4096 2.44 mV 14 16384 610 mV 16 65536 153 mV 18 262144 38.1 mV 20 1048576 9.54 mV 22 4194304 2.38 mV 24 16777216 596 nV 28 = 256 210 = 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 Analytická cytometrie Logaritmické zesílení & dynamický rozsah upraveno podle J.P.Robinson lin log Analýza dat ◼ Zobrazení dat – histogram – dot plot – isometric display – contour plot – chromatic (color) plots – 3 D projection ◼ Gating Jednoduché způsoby pro zobrazení dat K. Souček Bi9393 Analytická cytometrie Shrnutí ◼ Světlo, fluorescence ◼ Optické systémy ◼ Fluidní systémy ◼ Sorting ◼ Signál, data – základní princip Na konci dnešní přednášky byste měli: 1. znát základní principy rozptylu světla a 2. fluorescence; 3. vědět jaké zdroje světla se využívají v průtokové cytometrii; 4. a jakým způsobem je detekováno; 5. znát základní principy fluidních systémů a laminárního proudění. 6. Znát základní princip zpracování a vizualizace dat K. Souček Bi9393 Analytická cytometrie