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 Flow Cytometry Fundamental Principle 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 V5 V6 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 ANd9GcSG6MDDEyKW2lrEy-4pYcbhalq94g7mws3iCujkWC8szVoIZvRtvw − Particle Delivery: Hydrodynamic Focusing Intensity 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 • − − − − − − − − − Hydrodynamic core − Conventional Instrumentation: Low Flow Rates (12µL/min) > To really understand what is different and unique about acoustic focusing, one first needs to understand the current most commonly used method for focusing, called hydrodynamic focusing. In this method, the sample is injected into a stream of sheath fluid where it is completely surrounded by sheath, the sample core is then centered in the sheath fluid where the laser beam will then interact with the particles. Based on principles relating to laminar flow, the sample core remains separate but coaxial within the sheath fluid. The flow of sheath fluid accelerates the particles and restricts them to the center of the sample core. This process is known as hydrodynamic focusing. Where the sample core is narrow, the particles interact with the laser beam optimally and a narrow distribution results as seen in the histogram above. Particle Delivery: Hydrodynamic Focusing − Conventional Instrumentation: High Flow Rate (60µL/min) − Intensity 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) − − − − − − − − − − − − − − Hydrodynamic core Laser Cross Sectional Area > Acoustic Focusing Technology Overview http://www.invitrogen.com/etc/medialib/images/Cell-Analysis/other.Par.17809.Image.320.200.1.attune- jpg.gif Attune® Acoustic Focusing Cytometer https://www.thermofisher.com/content/dam/LifeTech/global/life-sciences/cellanalysis/Images/0715/aco ustic-focusing-animation.gif Thermo Fisher Scientific Logo − Acoustic Focusing = Better Precision Acoustic focusing Module − − − − − − − − − − − − − − − − − − − − − − − − Narrow particle focus = Narrow distribution manifolds.jpeg acousticcapillary.jpeg 12 µL/min 1000 µL/min Acoustic focusing of particles occurs prior to mixing with sheath fluid > Fundamentals of acoustic focusing in flow cytometry: Picture in the middle is a comparison of a manifold taken from a traditional hydrodynamic focusing cytometer (left) compared to a manifold taken from the Attune Cytometer and represented “in action” by the cartoons directly to the left and right of the photograph. The picture on the far right shows a full acoustic device as well as the manifold at the top. 1. Here, regardless of the sample volume that is moved through the flow cell, cells are focused by the acoustic device prior to entering the manifold and, ultimately, the flow cell. 2. So, at low sample volumes (left) and high sample volumes (right), focus is maintained. 3. This also means that very little sheath is needed. It is only used to allow for varying the amount of sample while maintaining the correct flow rate; and, is used to prevent reagent from staining the inside of the cuvette. https://upload.wikimedia.org/wikipedia/commons/3/35/Kundt_tube.png AugustKundt.jpg http://www.humanarray.com/wp-content/uploads/2012/03/LifeTech-Applied-Bio-Attune-6.jpg Life Technologies Logo 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 With Precious and Rare samples, the problem is that certain cells may not have enough volume of the original sample or cells may be fragile and can not withstand the centrifugation required to concentrate enough to run at low sample input rates. With the Attune® cytometer, being able to increase the sample rate up to 10x that of a traditional flow cytometer enables these customers to run rare or dilute samples. Also, because you can dilute your sample prior to running it on the Attune, customers with limited sample can save some of their sample for other analyses i.e. PCR, western, imaging etc. • 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 nsorting K. Souček Bi9393 Analytická cytometrie +++++ ----- Blue tissue paper +++++ ----- +++++ ----- +++++ +++++ ----- +++++ ++ +++++ ----- +++++ ++ +++++ Blue tissue paper + +++++ ----- +++++ + +++++ Blue tissue paper Blue tissue paper ----- +++++ ----- +++++ +++++ Blue tissue paper Blue tissue paper ----- +++++ ----- +++++ +++++ ----- - - +++++ ----- - - - - - - - - - - - - - +++++ ----- - - - - - - - - - - - Blue tissue paper - +++++ ----- - - - - - - - - - - Blue tissue paper - +++++ ----- - - - - - - - - - - Blue tissue paper - +++++ ----- - - - - - - - - - - http://www.cyto.purdue.edu/cdroms/cyto10a/seminalcontributions/fulwyler.html n n SORTING Frequency Charge Drop Delay Amplitude SORTING SORTING Sheath Pressure Nozzle Size Frequency 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 Aria FINAL picturestest_Page_062_Image_0001 Aria FINAL picturestest_Page_142_Image_0002 Good Bad SORTING – Setup Side Streams Aria FINAL picturestest_Page_062_Image_0001 Aria_II_UG_draft5_Page_165_Image_0002 Drop Delay interrogation point drop delay breakoff Waste BD FACS™ Accudrop technology nAccudrop beads nDiode laser nCamera nOptical filter n Before sorting, you need to make sure that you have an accurate drop delay setting. The Aria has integrated Accudrop technology which provides a simple way to adjust the drop delay. The components used for Accudrop: Diode laser: A low-power red laser which illuminates the lower portion of the stream when the sort block door is closed (there’s a safety interlock). Camera: Provides an image of the side streams visible in this window. (located behind the round window in the sort block) Optical filter: Used for viewing the fluorescence from the droplets containing Accudrop beads. (point out the button on this image that moves the filter into place) Accudrop beads: Beads that are excited by the diode laser and emit within the range of the optical filter. The drop delay is the distance between the laser intercept and the last connected drop, measured in time. The system needs to know when to charge the stream so that the intended droplet is charged before it is detached from the stream. This diagram shows the diode laser and the lower camera window. When setting the drop delay, the waste aspirator drawer is blocking the collection tubes, so all beads will go to waste. Essentially, Accudrop allows us to view the accuracy of our sort in real-time, without having to do a post-sort analysis. Sorting - Sort Masks Cells are randomized distributed over the stream Sorting - Sort Masks aspirator Trailing Interrogated Leading Mask nA region of the stream monitored for the presence of cells nDetermines how drops will be deflected if a sorting conflict occurs nMeasured in 1/32 drop increments – Mask = 0 Mask = 8 Mask = 16 Mask = 32 4 4 8 8 16 16 At the laser intercept particles are defined as wanted or not based on the gates you created in the software. However, while the particle is traveling down the stream another decision is made on whether or not the particle will be sorted based upon the Sort Precision mode. A sort precision mode is made up of a combination of masks. A mask is a region of the stream that will be considered to make a decision if there’s a sorting conflict. Each mask is measured in 1/32 drop increments. The next few slides will discuss each individual mask, then we’ll look at how those three masks are combined into precision modes. Note to instructor: Encourage customers to hold questions about putting the masks together until after you’ve discuss the basic definitions of all three. Conflict Resolution nPrecision modes include three types of masks –Yield –Purity –Phase n 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 nSnadná obsluha nŠetrná manipulace –On-chip technologie nVelikost i a bezpečnost h nMicrofluidic-based cell sorting nSpectral cell sorting nImage-based sorting n nBuoyancy Activated Cell Sorting (BACS™) –metoda, která používá částice s nízkou hustotou (mikrobubliny) pro flotační separaci. n Flow Cytometry Standard data file format. FCS 3.1 http://www.isac-net.org/images/stories/documents/Standards/fcs3.1_normativespecification_20090813.p df Spidlen, J. et al. Cytometry. Part A : the journal of the International Society for Analytical Cytology 77, 97-100, (2010). D:\Kaja\Desktop\Výstřižek.PNG Laser Laser Laser Creation of a Voltage Pulse Time Time Time 1 2 3 Height, Area, and Width Time (µs) Pulse area(A) Pulse Width (W) 0 Signal processing time 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-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 nZobrazení dat –histogram –dot plot –isometric display –contour plot –chromatic (color) plots –3 D projection nGating Způsoby pro zobrazení dat 4Color TBNK + TruC02 4Color TBNK + TruC02 4Color TBNK + TruC02 4Color TBNK + TruC02 K. Souček Bi9393 Analytická cytometrie Shrnutí nFluidní systémy nSorting nSignál, data – základní princip n 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 7. K. Souček Bi9393 Analytická cytometrie