Bi9393 Analytical cytometry Department of Cytokinetics Institute of Biophysics AVČR, vvi Královopolska 135 612 65 Brno e-mail: ksoucek @ ibp.cz phone: 541 517 166 Karel Souček, Ph.D. K. Souček Bi9393 Analytical cytometry https://www.bosterbio.com/protocol-and-troubleshooting/flow-cytometry-principle 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 K. Souček Bi9393 Analytická cytometrie Ways to view data K. Souček Bi9393 Analytická cytometrie Frequency distribution histogram ◼ Histogram shows particle frequency for a single parameter ◼ Simple Output ◼ We don't correlate with the next parameter ◼ The Problem of Identifying Populations K. Souček Bi9393 Analytická cytometrie Dot plot ◼ Displays the correlation of two arbitrary parameters ◼ The individual dots represent specific measured cells (particles) ◼ Values for a number of particles can lie in the same location ◼ We don't have information about the relative density of particles ◼ Rendering issues with large amounts of data K. Souček Bi9393 Analytická cytometrie Density & contour plot Density plot: ◼ It displays two parameters as frequency frequency ◼ the colour or shade corresponds to the particle frequency Contour plot: ◼ It connects points (particles) with the same signal value ◼ Basically, we're simulating a 3D graph – the third dimension is frequency K. Souček Bi9393 Analytická cytometrie Time as one of the parameters R1 K. Souček Bi9393 Analytická cytometrie Statistics ◼ Arithmeric mean ◼ Geometric mean ◼ Median – estimation of the mean value – is not affected by extreme values ◼ Standard deviation ◼ Coefficient of variance ◼ Mode – the most common value K. Souček Bi9393 Analytická cytometrie Statistics for histogram K. Souček Bi9393 Analytická cytometrie Quadrant Analysis (+ +)( - +) (+ -)(- -) K. Souček Bi9393 Analytická cytometrie Logical "Gating" (Boolean logic) With overlapping areas, we have many options: 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 Back Gating Region 4 established Back-gating using Region 4 logPE Back gate upraveno podle J.P.Robinson Back Gating K. Souček Bi9393 Analytická cytometrie Data analysis tools HW Manufacturers • Beckman Coulter • Kaluza • Becton Dickinson • FACSDiva • FACSSuite • FlowJo • BioRad • Sony • Milteney • … Universal Platforms • Commercial • FlowJo • FCS Express Freeware • Flowing Software • Cyflogic • BioConductor - Flowcore Specifying Regions/Gates Objective or subjective? ▪Training/Skills/Training Possible shapes: ▪rectangle ▪ellipse ▪"free-hand" (polygon) ▪quadrant Statistics - count - Share (%) - mean, median, S.D., CV, .... Gating – data reduction technique ◼ Flow stability gating — to capture events once the flow stream has stabilized, eliminating effects of clogging, back-pressure, and other instrument issues. ◼ Pulse geometry gating — to remove doublets from the dataset. ◼ Forward and side scatter gating — to remove debris and other events of non-interest while preserving cells based on size and or complexity. ◼ Subsetting gating — to rely on expression of markers and what they identify. Using viability dyes and dump channels further narrow to the cells of interest. This is where Fluorescence Minus One (FMO) controls become critical in defining the populations of interest. ◼ Backgating — to provide visualization of cells in final gate at higher level. Gating & Quality Control 1)Singlets 2)Time 3)viability http://expertcytometry.com/3-flow-cytometry-gates-that-will-improve-the-accuracy-of-your-facs-data-analysis/ http://expertcytometry.com/6-flow-cytometry-gating-tips-that-most- scientists-forget/ Gating & Quality Control https://www.flowjo.com/learn/flowjo-university/flowjo/tutorial/33 Scatter gating Subset gating http://expertcytometry.com/6-flow-cytometry-gating-tips-that-most-scientists-forget/ Gating and checking settings FMO ~ Fluorescence Mines One Back gating Data visualization and data interpretation Herzenberg LA, Tung J, Moore WA, Herzenberg LA, Parks DR (2006) Interpreting flow cytometry data: a guide for the perplexed. Nat Immunol 7: 681-685 Herzenberg LA, Tung J, Moore WA, Herzenberg LA, Parks DR (2006) Interpreting flow cytometry data: a guide for the perplexed. Nat Immunol 7: 681-685 Multi-color analyses generate a lot of data... 1 2 3 4 5 6 7 8 9 10 3 color 4 color 5 color Log Fluorescence QUADSTATS LogFluorescence ++ -- +- -+ Log Fluorescence QUADSTATS LogFluorescence ++ -- +- -+ Log Fluorescence QUADSTATS LogFluorescence ++ -- +- -+ Log Fluorescence QUADSTATS LogFluorescence ++ -- +- -+ Log Fluorescence QUADSTATS LogFluorescence ++ -- +- -+ Log Fluorescence QUADSTATS LogFluorescence ++ -- +- -+ Log Fluorescence QUADSTATS LogFluorescence ++ -- +- -+ Log Fluorescence QUADSTATS LogFluorescence ++ -- +- -+ Log Fluorescence QUADSTATS LogFluorescence ++ -- +- -+ Log Fluorescence QUADSTATS LogFluorescence ++ -- +- -+ Log Fluorescence QUADSTATS LogFluorescence ++ -- +- -+ Log Fluorescence QUADSTATS LogFluorescence ++ -- +- -+ Log Fluorescence QUADSTATS LogFluorescence ++ -- +- -+ Log Fluorescence QUADSTATS LogFluorescence ++ -- +- -+ Log Fluorescence QUADSTATS LogFluorescence ++ -- +- -+ Log Fluorescence QUADSTATS LogFluorescence ++ -- +- -+ Log Fluorescence QUADSTATS LogFluorescence ++ -- +- -+ Log Fluorescence QUADSTATS LogFluorescence ++ -- +- -+ Log Fluorescence QUADSTATS LogFluorescence ++ -- +- -+ Log Fluorescence QUADSTATS LogFluorescence ++ -- +- -+ Log Fluorescence QUADSTATS LogFluorescence ++ -- +- -+ Log Fluorescence QUADSTATS LogFluorescence ++ -- +- -+ Log Fluorescence QUADSTATS LogFluorescence ++ -- +- -+ Log Fluorescence QUADSTATS LogFluorescence ++ -- +- -+ Log Fluorescence QUADSTATS LogFluorescence ++ -- +- -+ Log Fluorescence QUADSTATS LogFluorescence ++ -- +- -+ Log Fluorescence QUADSTATS LogFluorescence ++ -- +- -+ Log Fluorescence QUADSTATS LogFluorescence ++ -- +- -+ Log Fluorescence QUADSTATS LogFluorescence ++ -- +- -+ Log Fluorescence QUADSTATS LogFluorescence ++ -- +- -+ upraveno podle J.P.Robinson 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. Other ways to visualize multidimensional data ◼ t-SNE, viSNE – t-Distributed Stochastic Neighbor Embedding – viSNE is a tool for reducing high-parameter data down to two dimensions – visually identify interesting and rare biological subsets – allow to gate single cell events across different samples. ◼ SPADE – Spanning-tree Progression Analysis of Density-normalized Events – way to automatically identify populations in multidimensional flow cytometry data files – clusters cells into populations and then projects them into a tree ◼ https://docs.flowjo.com/flowjo/advanced-features/dimensionality-reduction/tsne/ Dimensions reduction AMID, Ehsan; WARMUTH, Manfred K. TriMap: Large-scale dimensionality reduction using triplets. arXiv preprint arXiv:1910.00204, 2019. Dimensions reduction „… to create a map.“ t-SNE t-Stochastic Neighborhood Embedding Opt-SNE/FIt-SNE Fast Fourier Transform-accelerated Interpolation-based t-SNE Local structure 10x faster than t-SNE UMAP Uniform Manifold Approximation and Projection Average in Local and Global structure Transition of cells EmbedSOM Efficiently embed FlowSOM maps into 2D Poor in Local and Global structure Fast algorithm TriMap Dimensionality reduction technique based on triplet constraints Excellent in Global structure Transition of cells Preprint Courtesy of B. Kvokačková, R. Fedr Emission Spectra–Spectral Overlap What is the problem with multicolor detection? K. Souček Bi9393 Analytical cytometry Fluorescence signal compensation in multicolor detection ◼ A process in which all fluorescence signals are eliminated except for the fluorochrome signal to be detected on the detector ◼ Adjustment using a mix of microparticles or cells labeled/unlabeled with the appropriate fluorochromes. K. Souček Bi9393 Analytická cytometrie Choices for 6,- 8,- 10,- and more colors Fluorochrome selection considerations Various fluorochromes-stain index Kompenzace fluorescenčního signálu K. Souček Bi9393 Analytická cytometrie FITC positive & negative PE negative beads#2 Kompenzace fluorescenčního signálu K. Souček Bi9393 Analytická cytometrie FITC positive & negative PE negative beadsNONE! Kompenzace fluorescenčního signálu 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 set up proper compensation . Tandem Dyes Tandem Dyes Tandem Dyes - examples Tandems are light sensitive 0 hours 2 hours 22.5 hours PE (FL2) CD8 CD3 PE-Cy5PE-Cy7 Time Sample Left in Light https://www.bdbiosciences.com/en-us/resources/bd-spectrum-viewer Factors that Effect Compensation ◼ Reagent Lot-to-Lot Variation ◼ Fluorochrome Stability ◼ Sample-to-Sample Variation ◼ Assay Staining Conditions ◼ Another solution ? #1 The Idea (1931) " Simplicissimus Karl Arnold Mobile Telephony" by Source (WP:NFCC#4). Licensed under Fair use via Wikipedia Invention (1973) Martin Cooper , Motorola Innovation (2007) Steve Jobs , Apple Spectral flow cytometry JP Robinson, Purdue University Spectral flow cytometry Conventional vs. spectral analysis http://www.sonybiotechnology.com/images/sp6800/Fluorescent_Protei ns_and_Fluorochromes.jpg ◼ Another solution ? #2 Single Cell Mass Cytometry Cells were covalently labeled with a bifunctional compound, maleimido-mono-amide-DOTA (mDOTA). This compound can be loaded with a lanthanide(III) isotope ion, and reacts covalently with cellular thiol groups through the maleimide moiety. Single Cell Mass Cytometry Seven unique lanthanide isotopes were used to generate 128 combinations, enough to barcode each sample in a 96-well plate. The seven lanthanide isotopes, their masses and their locations on the 96well plate are shown. Single Cell Mass Cytometry Single Cell Mass Cytometry Imaging Mass Cytometry Compensation - literature Mario Roederer - Compensation in Flow Cytometry Current Protocols in Cytometry (2002) 1.14.1-1.14.20 John Wiley & Sons, Inc. 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. Maciorowski, Z., Chattopadhyay, P.K., & Jain, P. (2017). Basic multicolor flow cytometry. Current Protocols in Immunology, 117, 5.4.1–5.4.38. doi: 10.1002/cpim.26 K. Souček Bi9393 Analytická cytometrie No Data Analysis Technique Can Make Good Data Out of Bad Data! Shapiro’s 7th Law of Flow Cytometry Summary of the lecture ◼ Data analysis ◼ Compensation ◼ Quality control, principles At the end of today's lecture, you should know: 1. Pronciples of gating and data analysis 2. Principles of compensations 3. What are the basic principles of multispectral and mass cytometry At the end of today's lecture, you should know: 1. Pronciples of gating and data analysis 2. Principles of compensations 3. What are the basic principles of multispectral and mass cytometry K. Souček Bi9393 Analytical cytometry