Bi9393 Analytická cytometrie Lekce 3 Oddělení cytokinetiky Biofyzikální ústav AV ČR, 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 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 Life Technologies Logo Attune® Acoustic Focusing Cytometer − 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. http://www.humanarray.com/wp-content/uploads/2012/03/LifeTech-Applied-Bio-Attune-6.jpg Life Technologies Logo laser detector fluorochrome 405 VL1 450/50 Pacific Blue, Alexa Fluor 405, Brilliant Violet 421 VL2 522/31 Horizon V500, LIVE/DEAD Aqua VL3 603/48 LIVE/DEAD Yellow, Qdot 605 488 BL1 530/30 FITC, GFP, Alexa Fluor 488, Calcein, LIVE/DEAD Green, ALDEFLUOR, HiLyte 488 BL2 574/26 PE, propidium iodide, Hilyte 555 BL3 640 LP PerCP, Pe-Cy7, PerCP-eFluor 710, LIVE/DEAD Red, 7-AAD Attune (2 lasers, 6 detectors setup) Life Technologies Logo Attune - Key performance features include: •Breakthrough acoustic technology that focuses cells or particles •Highest sample delivery rates commercially available (up to 1,000 μL/minute) •User-selectable collection rates •Equipped with: Blue/Violet: 488 nm (20 mW) and 405 nm (50 mW) lasers •8 parameters—6-color detection plus side scatter and forward scatter •User-changeable bandpass and dichroic filters •Simplified fluorescence compensation •Manual and automated compensation •Adjustable PMT voltage settings •Detection of up to 20,000 events/sec and 20 million events/file •Calibrated delivery volumes for volumetric analysis and absolute cell counts •Electronic resolution of 6 decades •Low fluid consumption (about 1 L/day); self-contained fluids •Countertop instrument—fits on standard lab bench or in laminar • Software: •No software licensing fees •Output file format: FCS 3.0 •Live gating with automatic saving •User and administrator log-in Life Technologies Logo Cancer The Attune® Acoustic Focusing Cytometer, with its fast acquisition times and increased precision, overcomes the technological hurdles involved in analyzing CECs. Stem Cells Side Population Analysis In this study, we demonstrate the ability of the Attune® Acoustic Focusing Cytometer with the blue/ violet laser configuration to quickly analyze a large number of events in search of very rare populations of stem cells. Human Mesenchymal Stem Cells (hMSCs) Adult human mesenchymal stem cells (hMSCs) are rare fibroblast-like cells capable of differentiating into a variety of cell tissues, including bone, cartilage, muscle, ligament, tendon, and adipose. Cell Cycle Analysis Cell cycle analysis is just one example of an application in which precise detection of differences in fluorescence intensity between multiple cell populations is critical... Cell Proliferation Successful proliferation analysis by dye dilution requires sensitive instrumentation and an extremely bright dye to accurately distinguish fluorescently labeled cells from autofluorescence after several cell divisions... Marine Sample Analysis Flow cytometry is a powerful tool for studying the biology, ecology, and biogeochemistry of marine photosynthetic picoplankton... Immunophenotyping The Attune® Acoustic Focusing Cytometer exhibits excellent segregation of populations in immunophenotyping experiments (with up to 6 colors)... Apoptosis The Attune® Acoustic Focusing Cytometer is compatible with a broad offering of reagents and kits for flow cytometric apoptosis testing... GFP & RFP Detection Data for GRP and RFP detection were collected from the Attune® Acoustic Focusing Cytometer using human osteosarcoma cells (U2OS) and BacMam CellLight® reagents... Microbiological Applications In recent years the application of flow cytometry to the study of various microbiological phenomena has increased, finding utility in studies that include detection and quantification... Life Technologies Logo Example: Detecting human circulating endothelial cells using the Attune® Acoustic Focusing Cytometer Circulating endothelial cells (CECs) are mature cells shed from blood vessel walls during the natural process of endothelial cell turnover. Elevated levels of CECs have been reported in a host of pathological conditions including cardiovascular disorders, infectious diseases, immune disorders, post transplantation analysis, and cancer. CECs are reported to be present in very low numbers: 0.01%–0.0001% of all peripheral blood mononuclear cells Life Technologies Logo 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. Life Technologies Logo Attune souhrn Výhody: •rychlost měření •jednoduché ovládání •sw licence bez omezení •snadná výměna emisních filtrů •nízká spotřeba nosné kapaliny (cca 1L denně) • Limitace: •jen dva lasery (6 barev) •pouze originální roztoky •dlouhý (i když automatický) shutdown •sw nedokáže importovat FCS data •nutnost nastavit určitý akviziční objem vzorku • 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 Image Stream & Flowsight Amnis – kombinace průtokové cytometrie a analýzy obrazu Amnis - aplikace Principy průtokové cytometrie a sortrování nsorting nzpracování signálu nanalýza dat nkompenzace signálu 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 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. 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 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 ‹#› 2_color_small baseline 16, 364 Time 340 1985 7650 12420 Analog to Digital Converter ‹#› 2_color_small 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 ‹#› 2_color_small 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 24=16 Lineární a logaritmický obvod nlineární nlogaritmický nkompenzace fluorescenčního signálu 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 Kolik bitů? nPokud konvertujeme analogový signál pomocí 8 bitového ADC – máme 256 kanálů (28=256) odpovídajících rozsahu 0-10 V nRozdíl mezi kanály je 10/256=~40mV 0 50 100 150 200 250 10V 1V 100mV Channels upraveno podle J.P.Robinson Ideální logaritmický zesilovač 0 50 100 150 200 250 10 V 1 V 100 mV 0 50 100 150 200 250 10 V 1 mV Channels Linearní Log 1 V 100 mV 10 mV Log amp upraveno podle J.P.Robinson Logaritmické zesílení & dynamický rozsah upraveno podle J.P.Robinson lin log Kompenzace fluorescenčního signálu n…později n n n n n 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 Histogram distribuce četnosti nHistogram zobrazuje četnost částic pro jeden parametr nJednoduchý výstup nNekorelujeme s dalším parametrem nProblém s identifikací populací K. Souček Bi9393 Analytická cytometrie Dot plot nZobrazuje korelaci dvou libovolných parametrů nJednotlivé tečky představují konkrétní změřené buňky (částice) nHodnoty pro řadu částic mohou ležet ve stejném místě nNemáme informaci o relativní denzitě částic nProblémy s vykreslením v případě velkých objemů dat 4Color TBNK + TruC02 K. Souček Bi9393 Analytická cytometrie Density & contour plot nDensity plot: nZobrazuje dva parametry jako frekvenci četnosti nbarva a nebo její odstín odpovídá četnosti částic n nContour plot: nspojnice spojuje body (částice) se stejnou hodnotou signálu n nV podstatě simulujeme 3D graf – třetí rozměr je frekvence 4Color TBNK + TruC02 4Color TBNK + TruC02 4Color TBNK + TruC02 K. Souček Bi9393 Analytická cytometrie Čas jako jeden z parametrů K. Souček Bi9393 Analytická cytometrie 3D zobrazení n2 parametry + četnost n n n n n n3 parametry společně 4Color TBNK + TruC02 K. Souček Bi9393 Analytická cytometrie 3 Color Combinations Negatives Positives 4+4=8 FITC 4+4+4=12 upraveno podle J.P.Robinson 3 Color Combinations FITC 4+4+4=12 PE-FITC Pattern FITC TR-FITC Pattern FITC TR-PE Pattern Phycoerytherin .1 1 10 100 1000 .1 1 10 100 1000 .1 1 10 100 1000 CD4 CD38 CD45 CD3 Negative Negative CD20 CD8 CD7 CD2 CD8 CD8 (dim) CD2 Negative CD4 CD5 K CD3 CD3 IgG CD3 CD8 CD3 CD20 IgG L CD20 CD20 CD45 upraveno podle J.P.Robinson „Gating“ §Real-time gating vs. softwarový „gating“ §Určení regionů §Strategie „gatingu“ §Analýza kvadrantů §Boolean „gating“ §zpětný „gating“ § Real-Time vs. Software Gating §Real-time (live) gating: -omezuje akceptovaná data během měření § §Software (analysis) gating: -vyřazuje určitá data během následné analýzy upraveno podle J.P.Robinson Určení regionů §Objektivní nebo subjektivní? -školení/schopnosti/trénink § §Možné tvary: -obdelník -elipsa -“free-hand“ (polygon) -kvadrant §Statistika - počet - podíl (%) - průměr, medián, S.D., CV, …. Region vs. gate Region §oblast (plocha) v grafu definovaná uživatelem §mnoho regionů v jednou grafu §ohraničujeme pomocí nich populace našeho zájmu §je možné je barevně odlišit §je definován stejně pro všechny vzorky v analýze § Gate §je definován jako jeden a nebo více regionů zkombinovaných pomocí logických operátorů (AND, OR, NOT; Booleova logika) § § 4Color TBNK + TruC02 K. Souček Bi9393 Analytická cytometrie Region 1 established Gated on Region 1 Using Gates upraveno podle J.P.Robinson Statistika nAritmerický průměr nGeometrický průměr nMedián n– odhad střední hodnoty n– není ovlivněn extrémními hodnotami nSměrodatná odchylka nKoeficient variance nModus – nejčastější hodnota K. Souček Bi9393 Analytická cytometrie Statistika pro histogram K. Souček Bi9393 Analytická cytometrie Analýza kvadrantů (+ +) ( - +) (+ -) (- -) K. Souček Bi9393 Analytická cytometrie Logický „Gating“ (Booleova logika) S překrývajícími se oblastmi máme mnoho možností: upraveno podle J.P.Robinson Boolean Gating Not Region 1: upraveno podle J.P.Robinson Boolean Gating Not Region 2: upraveno podle J.P.Robinson Boolean Gating Region 1 or Region 2: upraveno podle J.P.Robinson Boolean Gating Region 1 and Region 2: upraveno podle J.P.Robinson Not (Region1 and Region 2): Boolean Gating upraveno podle J.P.Robinson 90 Degree Scatter 0 200 400 600 800 1000 8 15 20 30 40 50 100 200 1000 Lymphocytes Monocytes Neutrophils Side Scatter Projection Light Scatter Gating Scale upraveno podle J.P.Robinson Back Gating Region 4 established Back-gating using Region 4 Back gate upraveno podle J.P.Robinson Back Gating 4Color TBNK + TruC02 4Color TBNK + TruC02 K. Souček Bi9393 Analytická cytometrie FITC+ PE+ FITC+ APC+ PE + APC+ 3 Parameter Data Display upraveno podle J.P.Robinson Vícebarevné analýzy generují mnoho dat… 1 2 3 4 5 6 7 8 9 10 3 color 4 color 5 color Log Fluorescence QUADSTATS ++ -- +- -+ Log Fluorescence QUADSTATS ++ -- +- -+ Log Fluorescence QUADSTATS ++ -- +- -+ Log Fluorescence QUADSTATS ++ -- +- -+ Log Fluorescence QUADSTATS ++ -- +- -+ Log Fluorescence QUADSTATS ++ -- +- -+ Log Fluorescence QUADSTATS ++ -- +- -+ Log Fluorescence QUADSTATS ++ -- +- -+ Log Fluorescence QUADSTATS ++ -- +- -+ Log Fluorescence QUADSTATS ++ -- +- -+ Log Fluorescence QUADSTATS ++ -- +- -+ Log Fluorescence QUADSTATS ++ -- +- -+ Log Fluorescence QUADSTATS ++ -- +- -+ Log Fluorescence QUADSTATS ++ -- +- -+ Log Fluorescence QUADSTATS ++ -- +- -+ Log Fluorescence QUADSTATS ++ -- +- -+ Log Fluorescence QUADSTATS ++ -- +- -+ Log Fluorescence QUADSTATS ++ -- +- -+ Log Fluorescence QUADSTATS ++ -- +- -+ Log Fluorescence QUADSTATS ++ -- +- -+ Log Fluorescence QUADSTATS ++ -- +- -+ Log Fluorescence QUADSTATS ++ -- +- -+ Log Fluorescence QUADSTATS ++ -- +- -+ Log Fluorescence QUADSTATS ++ -- +- -+ Log Fluorescence QUADSTATS ++ -- +- -+ Log Fluorescence QUADSTATS ++ -- +- -+ Log Fluorescence QUADSTATS ++ -- +- -+ Log Fluorescence QUADSTATS ++ -- +- -+ Log Fluorescence QUADSTATS ++ -- +- -+ Log Fluorescence QUADSTATS ++ -- +- -+ Mad Scientist Cartoon upraveno podle J.P.Robinson http://www.chromocyte.com/Library/ResizedImages/445/522/Batch_Analysis.png Detection and monitoring of normal and leukemic cell populations with hierarchical clustering of flow cytometry data Cytometry Part A Volume 81A, Issue 1, pages 25-34, 11 OCT 2011 DOI: 10.1002/cyto.a.21148 http://onlinelibrary.wiley.com/doi/10.1002/cyto.a.21148/full#fig1 HCA‐Main hematopoietic populations in normal BM. A: Dendrogram with heatmap‐HCA of 10^4 ungated events acquired from normal BM. Heatmap shows relative levels of all eight parameters (columns) in all 10^4 events (rows) in color coding (blue, low expression; red, high expression). Dendrogram shows the hierarchy of cells based on their similarity in all parameters measured. The x‐axis under the dendrogram represents similarity distance. Colored branches of the dendrogram are selected clusters, as displayed in B. B: The main populations (as identified by HCA) are displayed on a conventional forward versus side scatter dot plot. C: All two‐parameter combination plots of a defined cluster (marked by red rectangle in A). This population is negative for most of the antibodies from the B cell panel and may be difficult to detect by standard gating as it overlaps with other populations in all 28 two‐parameter plots. Nevertheless it is a compact, homogenous population most likely of T cell origin. This red population was drawn on top of the remaining cells (gray). D: Mirror image to C. The gray population was drawn on top of the red population. Fluorescent dyes used were: CD10‐FITC, CD22‐PE, CD117‐PerCP‐Cy5.5, CD38‐PE‐Cy7, CD34‐APC, CD19‐APC‐Cy7, and in all cases pulse height was used. © This slide is made available for non-commercial use only. Please note that permission may be required for re-use of images in which the copyright is owned by a third party. The Flow Cytometry: Critical Assessment of Population Identification Methods (FlowCAP) The goal of FlowCAP is to advance the development of computational methods for the identification of cell populations of interest in flow cytometry data. FlowCAP will provide the means to objectively test these methods, first by comparison to manual analysis by experts using common datasets, and second by prediction of a clinical/biological outcome. Způsoby pomocí kterých lze upravit výsledky: 1. Odstranění „doublets“ 2. Čas jako parametr pro kontrolu kvality Příklad - pro DNA analýzu je třeba: - odstranit „debris“ a shluky - odstranit „doublets“ - udržovat konstantní průtok K. Souček Bi9393 Analytická cytometrie DNA Histogram G0-G1 S G2-M Fluorescence Intensity Co je problém při vícebarevné detekci? K. Souček Bi9393 Analytická cytometrie Emission Spectra–Spectral Overlap EmissionSpectra Kompenzace fluorescenčního signálu při vícebarevné detekci n n n n n n nProces při kterém dochází k eliminaci všech fluorescenčních signálů kromě signálu z fluorochromu který má být na příslušném detektoru detekován nNastavení pomocí mixu mikročástic či buněk označených/neoznačených příslušnými fluorochromy. K. Souček Bi9393 Analytická cytometrie Co je problém při vícebarevné detekci? K. Souček Bi9393 Analytická cytometrie Kompenzace fluorescenčního signálu při vícebarevné detekci n n n n n n K. Souček Bi9393 Analytická cytometrie ‹#› 2_color_small “Bright” = good resolution sensitivity ‹#› 2_color_small Various fluorochromes-stain index ‹#› 2_color_small Choices for 6,- 8,- 10,- and more colors ‹#› 2_color_small Fluorochrome selection considerations ‹#› 2_color_small FITC Spillover 650nm 700nm PerCP-Cy5.5 695/40 500nm 600nm FITC 530/30 Wavelength (nm) 550nm PE 585/42 750nm 800nm ‹#› 2_color_small FITC Compensation 650nm 700nm PerCP-Cy5.5 695/40 500nm 600nm FITC 530/30 Wavelength (nm) 550nm PE 585/42 ‹#› 2_color_small FITC Compensation 650nm 700nm PerCP-Cy5.5 695/40 500nm 600nm FITC 530/30 Wavelength (nm) 550nm PE 585/42 Slide 6 FITC adj FITC comp tab with values ‹#› 2_color_small FITC Compensation 2Fitc comp 1Fitc no comp 3Fitc biexpo Dot plot showing uncompensated FITC data Dot plot showing compensated FITC data Biexponential dot plot showing compensated FITC data ‹#› 2_color_small FITC Spillover 600nm 500nm 550nm FITC 530/30 Wavelength (nm) PE 585/42 Kompenzace fluorescenčního signálu n n n n n n K. Souček Bi9393 Analytická cytometrie FITC positive & negative PE negative beads #2 Kompenzace fluorescenčního signálu n n n n n n K. Souček Bi9393 Analytická cytometrie FITC positive & negative PE negative beads NONE! Kompenzace fluorescenčního signálu n Nastavení kompenzací parametr - detektor amp. FL1 - 544 FL2 - 434 kompenzace FL1 - 1.1%FL2 FL2 - 17.5%FL1 § značené mikročástice – pro běžně konjugované fluorochromy § značené buňky – pro vitální značení ‹#› 2_color_small Effects of Changing PMT Values FITC Voltage Increased by 5 V FITC Voltage Decreased by 5 V Correct Compensation Which marker for compensation? Small errors in compensation of a dim control (A) can result in large compensation errors with bright reagents (B & C). Use bright markers to setup proper compensation. ‹#› 2_color_small BD Comp Beads •Always positive •Bright staining •Save sample (HIV patients) •Use the same antibody for compensation and the real experiment ‹#› 2_color_small BD Comp Beads ‹#› 2_color_small PBMC were stained as shown in a 3-color experiment. Compensation was properly set for all spillovers Courtesy Mario Roederer Fluorescence Minus One Tandemové značky http://www.abcam.com/ps/CMS/Images/Tandem-Dyes_image.jpg Tandemové značky - příklad ‹#› 2_color_small Tandems are light sensitive 0 hours 2 hours 22.5 hours PE (FL2) CD8 CD3 PE-Cy5 PE-Cy7 Time Sample Left in Light Kompenzace - literatura n Mario Roederer - Compensation in Flow Cytometry n Current Protocols in Cytometry (2002) 1.14.1-1.14.20 John Wiley & Sons, Inc. n n M. Loken, D. R. Parks, & L. A. Herzenberg (1977). Two-color immunofluorescence using a fluorescence-activated cell sorter. J. Histochem. Cytochem. 25:899-907. M. Roederer & R. F. Murphy (1986). Cell-by-cell autofluorescence correction for low signal-to-noise systems: application to EGF endocytosis by 3T3 fibroblasts. Cytometry 7:558-565. S. Alberti, D. R. Parks, & L. A. Herzenberg (1987). A single laser method for subtraction of cell autofluorescence in flow cytometry. Cytometry 8:114-119. C. B. Bagwell & E. G. Adams (1993). Fluorescence spectral overlap compensation for any number of flow cytometry parameters. in: Annals of the New York Academy of Sciences, 677:167-184. K. Souček Bi9393 Analytická cytometrie No Data Analysis Technique Can Make Good Data Out of Bad Data! Shapiro’s 7th Law of Flow Cytometry Shrnutí přednášky nsorting nzpracování signálu nvizualizace dat a „gating“ nkompenzace n n Na konci dnešní přednášky byste měli: 1. 1.Znát základní principu sortování, 2.popsat způsob zpracování signálu, 3.rozumět lin / log zesílení signálu, 4.rozeznat jednotlivé způsoby vizualizace dat, 5.chápat základní principy „gatingu“, 6.znát princip kompenzace signálu při vícebarevné detekci. K. Souček Bi9393 Analytická cytometrie