SP6800 Spectral Analyzer

Transcription

1 SP l nalyzer Sony Biotechnology Inc.

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3 SP l nalyzer The Sony SP l nalyzer uses spectral technology to optimize sensitivity and enhances dim signal detection by collecting photons from nm to nm. l technology also simplifies multicolor panel design, by eliminating bandpass filters and conventional compensation matrices to allow greater flexibility. This revolutionary approach uses spectral unmixing to expand the way cellular and microbiological samples are analyzed delivering the most accurate visualization of fluorescent populations available to scientists using flow cytometry. l unmixing makes analysis simpler and easier by separating individual spectral fingerprints to allow scientists to better visualize their data. This delivers a more comprehensive picture to see rare populations and decreases the complexities associated with working with fluorescent proteins and multi laser excited fluorochromes. dvanced electronics and patented optical technologies bring simplicity to l nalysis workflow. Sony s patented FlowPoint core stability and tracking system and automated QC ensure the highest resolution possible of target populations. Uses spectral analysis technology that optimizes sensitivity while simplifying application design and workflow. Enhances dim signal detection for better visualization of rare populations, fluorescent proteins and fluorochromes excited by multiple lasers. Incorporates advanced electronics and patented optical technologies for greater stability, and automated quality control that deliver very high resolution of target populations. Easy to use software features automated alignment and laser delay via set up wizards, easy acquisition with simplified voltage settings and flexible analysis that enables populations to be gated or seen spectrally to ensure accuracy.

4 Microfluidics flow cell chip maximizes signal with auto positioning to guarantee high sensitivity. Made of durable plastic with an embedded quartz cuvette, the chip is easy to replace when needed. The nm, 88nm and 8nm excitation lasers are positioned to reduce fluorescent noise. They enable the system to support or more fluorescent parameters. The FlowPoint detection system precisely tracks the core stream shape and position in the flow cell as well as the cross-sectional position of each passing particle to provide highly reliable measurements. This patented technology ensures core stability and the highest resolution. Cells Y X Scatter nalysis Forward and Side Scatter parameters to allow relative size and complexity measurements. x, ll events SSC_ x, FSC_ unique prism collection system Delivers light through consecutive prisms allowing optimal signal with minimal loss of light. Emitted light passes through a Channel PMT that produces data points of signal detection to analyze emitted photons from nm to nm to ensure accurate visualization. Positive Positive LD/8_H LD/8_H LD88_H LD88_H Positive Positive

5 SP System Overview The SP spectral flow analyzer improves sensitivity and simplifies application design, workflow and analysis over conventional flow cytometers. This is achieved using spectral analysis technology, advanced electronics and patented optical technologies. These capabilities, unique to Sony Biotechnology systems, allow experienced and novice flow cytometrists achieve greater flexibility for panel design and more accurate visualization of results. Software The SP software is designed to be simple and intuitive. QC wizard simplifies set up with step-by-step and automated procedures to ensure optimal alignment, and laser delay. Figure. SP QC wizard simplifies set up. Flexibility in the software allows voltage to be set during acquisition, using one voltage setting to adjust all PMTs. l unmixing can occur during acquisition or analysis, saving time. Figure. cquisition is simplified requiring only one voltage adjustment to set all PMTs. Sample acquisition is simplified using the SP. Figure. Flexible analysis lets populations be gated or visualized spectrally for greater accuracy. To ensure accuracy populations can be gated and visualized spectrally to ensure they are accurately defined as single positive, double positive or negative.

6 l nalysis Technology l analysis technology, the foundation of the SP system lets researchers see the complete spectral fingerprint of each fluorochrome from nm to nm. unique prism collection system delivers emitted light to a channel PMT. This produces data points of signal detection for fluorescence and bright auto fluorescence to achieve the most accurate visualizations of fluorescent populations (Figure ). LD/8_ LD88_ 8 Figure. Visualization of fluorescent cell populations using the analyzer s unique prism collection system. cquisition l technology simplifies multicolor flow cytometry by running a negative control, to define the spectral fingerprint for auto-fluorescence, and single positive controls to define the reference spectra. Controls are then matched against the sample to mathematically unmix each spectral fingerprint to easily produce dot plot results (Figure ). Sample Sample Sample Sample Figure Sample Sample Sample LD8_H LD8_H LD8_H LD8_H CPS CPS CPS CD_-Cy7_ CD_-Cy7_ - - PC PC PC PC PC PC PC PC CDRO_-Cy_ CDRO_-Cy_ - CD_-Cy7_ F F F F F F F F PC-Cy7 PC-Cy7 PC-Cy7 PC-Cy7 PC-Cy7 PC-Cy7 PC-Cy7 PC-Cy7 CD_CD_- F F CD_-Cy7_ CD_-Cy7_ CD_-Cy7_ CD_-Cy7_ CD_-Cy7_ CD_-Cy7_ PC PC CD_CD_- -Cy7 -Cy7 PerCP-Cy. PerCP-Cy. - CD_-Cy7_ CD_-Cy7_ - - CD_-Cy7_ Cy -Cy CD_-Cy7_ CD_-Cy7_ CD_-Cy7_ CD_-Cy7_ CD_-Cy7_ CD_-Cy7_ -Cy -Cy -Cy PerCP-Cy. PerCP-Cy. PerCP-Cy. -Cy7 -Cy7 -Cy7 -Cy -Cy -Cy PerCP-Cy. PerCP-Cy. PerCP-Cy. -Cy7 -Cy7 -Cy7 -Cy -Cy7 -Cy PerCP-Cy. PerCP-Cy. -Cy7 -TR -TR CD_-Cy7_ - - CPS CPS CPSCPS CPS CPS CD_-Cy7_ CD_-Cy7_ CD_-Cy7_ CD_-Cy7_ CD_-Cy7_ CD_-Cy7_ CDRO_-Cy_ CDRO_-Cy_ CDRO_-Cy_ CDRO_-Cy_ CDRO_-Cy_ CPS CPSCPS CD_-Cy7_ CD_-Cy7_ CD_-Cy7_ CD_-Cy7_ CD_-Cy7_ CD_-Cy7_ CPS CPS CPS CPS CPSCPS CD_-Cy7_ CD_-Cy7_ CD_-Cy7_ CD_-Cy7_ CD_-Cy7_ CD_-Cy7_ - - CPS CPSCPS CPS CPSCPS CPS CPSCPS CD_-Cy7_ CD_-Cy7_ CPS CD_-Cy7_ CPS CD_-Cy7_ CD_-Cy7_ CD_-Cy7_ CPS CPS CPS CPS CPS - -TR -TR F F F F F FF -TR -TR -TR F FF -TR -TR -TR F F F FF F FF F F nm/8nm Lasers CD_-Cy7_ nm Laser CD_-Cy7_ CD_-Cy7_ CD_-Cy7_ CD_-Cy7_ CD_-Cy7_ CD_-Cy7_ LD8_H CPS CPS CPSCPS HLDR_PerCP-Cy._ HLDR_PerCP-Cy._ LD8_H LD8_H LD8_H LD8_H l lgorithm lgorithm lgorithm lgorithm lgorithm lgorithm Unmixing lgorithm lgorithm lgorithm CPS HLDR_PerCP-Cy._ HLDR_PerCP-Cy._ CPS HLDR_PerCP-Cy._ HLDR_PerCP-Cy._ HLDR_PerCP-Cy._ HLDR_PerCP-Cy._ HLDR_PerCP-Cy._ CD8_lexa CD8_lexa Fluor Fluor Cd/_PC-Cy7_ Cd/_PC-Cy7_ Cd/_PC-Cy7_ Cd/_PC-Cy7_ Cd/_PC-Cy7_ CPS CDR_lexa CDR_lexa Fluor Fluor 88_ 88_ CPS CPSCPS CPS CPSCPS Cd/_PC-Cy7_ Cd/_PC-Cy7_ LD88_H CD8_lexa CD8_lexa Fluor _ Fluor _ CD8_lexa CD8_lexa Fluor _ Fluor _ CD8_lexa Fluor _ CD8_lexa CD8_lexa Fluor _ Fluor _ CD_-TR_ CD_-TR_ CD_-TR_ CD_-TR_ CD_-TR_ CD_-TR_ CD_-TR_ LD88_H LD88_H LD88_H LD88_H LD88_H LD88_H CDR_lexa CDR_lexa Fluor 88_ Fluor 88_ CDRO_-Cy_ CDRO_-Cy_ CPS CPSCPS CDR_lexa CDR_lexa Fluor 88_ Fluor 88_ CDR_lexa Fluor 88_ CDR_lexa CDR_lexa Fluor 88_ Fluor 88_ Flow Cytometry Data CD_-TR_ CD_-TR_ LD88_H LD88_H Cd/_PC-Cy7_ Cd/_PC-Cy7_ Flow Flow Flow Cytometry Cytometry Cytometry Data Data Data Flow Cytometry Data Unmixing Results Flow Flow Flow Cytometry Cytometry Cytometry Data Data Data Sample Sample

7 Reliability and Quality Data The FlowPoint detection system precisely tracks the core stream shape and position in the flow cell as well as the cross-sectional position of each passing particle. This ensures core stability and delivers highly reliable measurements. This patented technology ensures the system achieves the highest resolution possible of the targeted population by showing real-time and saved data of sample core stream (Figure ). Y +um um Count -um um +um X K K 88ch9- K Figure. FlowPoint patented technology ensures core stability, and delivers the highest resolution possible of targeted population by showing core stream data for both real-time and saved data. Samples that are not in the center of the core can be easily excluded preventing artifacts from bubbles or skewed particles. uto-fluorescence In conventional flow cytometry cellular auto-fluorescence produced by pyridine (ND/NDH), flavin (FMN, FD), and other intracellular oxidative reactions can cause fluorescent signal contamination such as FITC, and Pacific Blue channels. Other common sources of autofluorescence include cell fixation and permeabilization. Using spectral technology, auto-fluorescent spectral fingerprints can be subtracted to allow researchers to see the true fluorescent population (Figure 7, 8). lexa Fluor _ - -. With uto-fluorescent ll events H-Q: 88.% H-Q: 8.% H-Q:.% H-Q:.% lexa Fluor _ B. Without uto-fluorescent - - ll events H-Q: 9.% H-Q: 8.% H-Q:.% H-Q:.7% ctivated T Cells with GFP and lexa Fluor - GFP_ - GFP_ O T:.9% S V Y C. With uto-fluorescent D. Without uto-fluorescent B_BV_ Y:.7% S:.% - - R: 9.% - - F/8 LD/8_ - - LD/8_ - 8 LD/8_ SSC_ ll events GFP Pos:.9% GFP neg: 98.7% SSC_ ll events GFP Pos:.% GFP neg: 99.% Liposomes with GFP Figure 7. mouse splenocytes were analyzed with the SP revealing three distinct auto-fluorescent populations. Using the spectral fingerprints obtained in analysis, the appropriate auto-fluorescence can be removed, increasing the precision and quality of results. - GFP_ - GFP_ Figure 8. uto-fluorescence can result in the appearance of additional cell populations leading to erroneous data interpretation.. T cells expressing GFP and stained with an antibody conjugated to lexa Fluor were analyzed with the SP. double positive population is present in the uncorrected density plot. B. This double positive population disappears when the auto-fluorescent spectral fingerprint is subtracted. C. Liposomes from cells expressing GFP were analyzed. D. This uncovered a small positive GFP population after auto-fluorescence was subtracted. 7

8 l Library l technology eliminates the need for bandpass filters and conventional compensation matrices to allow for greater flexibility in multicolor application panel design. This lets researchers save single positive fluorochromes in a l Library to easily import them into current and future experiments. Cy /Dazzle 9 PerCP Cy. Pacific Blue FITC lexa Fluor efluor9 lexa Fluor 9 efluor NC Cy PerCP lexa Fluor Cy7 PI 7-D BD Horizon V mcyan Brilliant Violet Brilliant Violet Brilliant Violet 7 Brilliant Violet Brilliant Violet Brilliant Violet 7 Brilliant Violet 78 Qdot Qdot Qdot Qdot 8 Qdot Qdot Qdot 7 lexa Fluor 7 PC lexa Fluor PC Cy7 l Unmixing l unmixing separates each spectral fingerprint to better visualize each fluorochrome marker. In doing so it lets researchers gain insights by delivering a more comprehensive picture of rare populations, fluorescent proteins and multi laser excited fluorochromes. Figure 9. l Unmixing separates each spectral fingerprint for complete and optimal visualization of fluorochomes. G: 8.% CD_PC_ - E: 9.8% G: 8% F: 8% Unmixing CD_PC_ - E: 9.% F: % - IL B - IL cells High uto-fluorescence Positive cells E G F LD-88_ LD-88_ LD-88_ s s s Figure. l analysis eliminates false-positives and delivers more accurate analysis over conventional flow cytometry. Mouse splenocytes were stained with CD PC and anti-il- antibody conjugated to.. In this conventional density plot it is unclear if the light-blue region is a dim, weak double positive, or non-specific binding. B. Using l analysis the spectral data of each region is compared against the l Library to unmix the sample. This reveals the light blue region is high auto-fluorescence. 8

9 pplications pplication data dye options and combinations using the SP. Fluorochromes laser BV Pac Blue BV Qdot Qdot BV7 BV Qdot LD/8_H LD/8_H BV Pac Blue BV Qdot Qdot 8 BV7 BV Qdot BV 8 LD/8_H LD/8_H 8 8 Qdot Qdot7 BV7 BV78 LD/8_H LD/8_H 8 LD/8_H 8 8 LD/8_H LD/8_H 8 8 Figure. SP is capable of running more fluorochromes with fewer lasers saving costs and greater flexibility. This allows the SP to handle more dye options and combinations that are not possible with conventional flow cytometers. On the left are a compilation of individual fluorochromes run on the SP. Qdot BV LD/8_H LD/8_H LD/8_H LD/8_H Qdot Qdot 7 BV7 BV78 Fluorochromes 88 laser Cy F88 F F Cy LD88_H LD88_H LD88_H LD88_H LD88_H F88 F F Cy Dazzle 9 F Cy Cy. LD88_H LD88_H LD88_H LD88_H LD88_H Cy Dazzle 9 PerCP F Cy7 F PerCP LD88_H LD88_H PerCP Cy. PerCP F Cy7 Fluorochromes 8 laser PC F PC Cy7 PC LD/8_H LD8_H LD/8_H F Zombie NIR PC Cy7 Zombie NIR Zombie NIR 8 8 LD/8_H 8 9

10 color panel on Human Peripheral Blood pplication data using lasers 88nm Laser nm Laser CD lexa Fluor 88 HL-DR Pacific Blue CD8 CD8 BV CD Dazzle 9 CD lexa Fluor CDR CD BV BV7 CD9 CD Cy Cy. (nm) CD CDb BV BV (nm) CD PerCP efluor 7 CDc BV7 CD Cy7 CD BV78 B C D E x, SSC_ ll events Granulocytes Monocytes CD_PerCP eflour7_ - - Granulocytes CD-CD CD_BV_ - CD-CD CD-CD.7% CD_BV_ - CD-CD Natural Killer Cells CD_BV_ - CD-CD:.7% - - CD_-Cy7_ F CD+ G - CD_-F_ CDc-CD- H - CD_lexa Fluor 8_ CD9-CD I - - CD_lexa Fluor 8_ PLSM B Cells - - CD_lexa Fluor 8_ CD9c-CD PLSM B Cells CD_BV78_ - - CDc-CD- CD_-Cy._ - CD_BV_ - - CDb_BV_ - - Memory B Cells Naive B Cells - J - - CDc_BV7_ Monocytes K CD - CD9_-Cy_ L - HL-DR_PacificBlue_ ll events CDR_BV_ CD-CD CD positive CD_BV78_ - - CD_BV78_ - - CD_BV78_ - CD-CD - - CDc_BV7_ - CD_PerCP eflour7_ - - CDc_BV7_ Figure. Examples of the resolution of several important cell populations using only two lasers with the SP. Clear population resolution is obtained with highly overlapping fluorochomes such as -Cy/-Cy. (G) and Pacific Blue/BV (H).

11 color stained sample of Human Peripheral Blood pplication data using lasers 88nm Laser nm Laser CD lexa Fluor 88 HL-DR Pacific Blue CD8 CD BV CD Dazzle 9 CD lexa Flour CD CD BV BV7 CD9 CD Cy Cy. CD BV (nm) (nm) CD PerCP eflour 7 8nm Laser CD Cy7 CD8 PC CDc lexa Flour CDR PC-Cy7 (nm) ll events Granulocytes CD+ x, SSC_ Granulocytes Monocytes CD_PerCP eflour7_ CD+CD+ Grans CD_BV_ - NK Cells CD_-Cy._ - - CD9+CD+ - - CD_-Cy7_ CD_-F_ - - CD_lexa Fluor 88_ - - CD9_-Cy_ CD9+CD+ Cytotoxic T Cells Monocytes CD8_PC_ Plasma B Cells: 99.7% CD8 - Cytotoxic T Cells CDR_PC-Cy7_ - Cytotoxic Naive T Cells Cytotoxic Effector T Cells CD_BV_ - CD positive - - HL-DR_PacificBlue_ - - CD_lexa Fluor 88_ - CD8_PC_ - - CD_lexa Fluor 88_ Monocytes Granulocytes Helper T Cells:.8% Helper Naive T Cells CD+CD+ Monocytes CD_Dazzle 9_ - - CDR_PC-Cy7_ - Helper Effector T Cells CD_-F_ - - CD+CD+ B Cells CD_BV7_ - CD8_PC_ - - CD+CD8- Granulocytes - - CD_lexa Fluor 88_ - CD8_PC_ - - CD_BV_ - - CD_PerCP eflour7_ CD_-F_ Figure. Example of the detection of several important cell populations using sixteen fluorochromes excited by three lasers. Unlike conventional flow cytometers that can detect up to five off the blue, in this example we demonstrate that with the SP eight fluorochromes can be excited off the blue with clear sample resolution. Even highly overlapping fluorchromes such as -Cy and -Cy. can be resolved.

12 pplication Data: Brilliant Violet Dyes l nalysis allows multi-laser excited fluorochromes to be run without using a complicated compensation matrix. BV BV BV7 BV BV BV7 BV78 (nm) B C D E H CD+ CD+ CD+ CD+ x, CD+ 8 SSC_ CD8_BV7_ - - CD_BV_ - - CD/_BV_ - CD_BV_ - CD_BV7_H - CD_BV_ - CD_BV_ - - CD9_BV_ - - CD_BV78_ Figure. Sample data from Human PBMCs Stained with seven markers conjugated to Brilliant Violet dyes. offered by Sony Biotechnology. D. ll populations were clearly resolved including those fluorochomes with significant spectral overlap such as BV and BV (D). pplication Data: Sony lexa Dyes Sony lexa Fluor and lexa Fluor with lexa Fluor88 and on Human Peripheral Blood CD8 lexa Fluor 88 CD lexa Fluor CD lexa Fluor CD (nm) x, SSC_ Cells CD - CD8_lexa Fluor 88_ CD_lexa Fluor _ - CD_lexa Fluor _ - CD_lexa Fluor _ Figure. Human peripheral blood was stained with the fluorochrome conjugated antibodies and analyzed with the SP using the blue laser for excitation. Due to their spectral overlap fluorochromes, lexa Fluor 88,, and cannot be used together on conventional flow cytometers. However the SP can distinguish these fluorophores.

13 pplication Data: Brilliant Violet and Q Dots Unlike conventional flow cytometers, l nalysis enables use of Brilliant Violet and Qdot fluorochromes using spectral unmixing. BV and Qdot D D D LD88_ LD/8_ CD_Qdot _ 9% E: 9.8% (nm) 8 CD_BV_ BV and Qdot D D D LD88_ LD/8_ LD/8_ 8% F:.8% (nm) 8 CD8_Qdot _ BV7 and Qdot 7 D D D LD88_ LD/8_ CD78_Qdot 7_ E:.8% (nm) 8 CDRO_BV7_

14 pplication Data: Dyes with issues Common problems in flow cytometry occur when running fluorochromes with emission peaks that are too close to one another, multi-laser excitations, fluorescent proteins, and unstable tandems. The SP is capable of analyzing all of these by looking at all photons from nm to nm and unmixing each unique spectral fingerprint. CD8_BV_ Cells - (nm) - CD_PacificBlue_ Example : Pacific Blue (7nm emission) and Brilliant Violet (nm emission) PC excitation PC emission Cy excitation Cy emission CD8_-Cy_ E - - (nm) - - CD_PC_ Example : Cy (excited with 88nm and 8nm lasers) and PC (excited with 8nm laser) ll events LD88_H GFP_ Example : Fluorescent GFP and YFP need no special bandpass filter set with the SP Normal Cy7 C LD88_ Cy7 falling apart C F LD88_ Unmixing _ Cy7_ Example : Cy7 tandem is prone to falling apart causing issues with resolution when using conventional flow cytometers. Since l nalysis unmixes the spectral fingerprint, the SP produces excellent resolution.

15 Specifications Configuration laser model, 88/8 laser model, /88 laser model,/88/8 Model Number LE-SPZB LE-SPZC LE-SPZE Laser specification Lasers Semiconductor laser model 88nm, 8nm Semiconductor laser model /88 Semiconductor laser model, /88/8 Maximum power (at flow cell) 88nm mw 8nm mw nm mw 88nm mw nm mw, 88nm mw 8nm mw Optics Irradiation form Detection optics Scatter signals Spectroscopic method -spot dual-axis irradiation Forward scatter (FSC), Side scatter (SSC) Prism array spectroscopy Fluorescent channels channel PMT (wavelength: -nm) channel PMT (wavelength: -nm) PMT x (-nm, -7nm) Signal resolution Height bit, rea bit Sampling frequency: MHz System Measurement parameters Pulse shape parameters channels, FSC, SSC, cell position XY Height, rea, Width channels, FSC, SSC, cell position XY Fluidics Optical alignment Event Rate Sample loader type Supporting sample tube Sheath Flow Speed Detection channel dimensions Fluorescence sensitivity utomated alignment with Corefinder TM technology,eps (standard)/,eps (maximum) Single auto-loader ml polystyrene round tubes Low (approx. m/s), Mid (approx. m/s), High (approx. m/s) μm x μm FITC: MESF / : 7 MESF Performance Linearity FITC R.99 / R.99 Detection size range.~µm (beads) Software Fluorescence detection resolution Unmixing algorithms utofluorescence Virtual Filter 88nm Laser/CH, 8nm Laser/CH7* :.% (HPCV) Spectrum method (LSM,, PS and Constraint option), Reverse matrix method (Conventional) utofluorescence spectral detection Possible to change wavelength region for each fluorochromes in analysis Export Data Format Flow Cytometry Standard (FCS).,. Dimensions Main unit: Width.cm x Depth.cm x Height 7.cm Fluidics cart: Width 78.cm x Depth.cm x Height 8.cm Main Unit Weight ir pressure supply C Power supply Power consumption Operating temperature Operating humidity Recommended PC Main unit: approx. 99kg (dried weight)/fluidics cart: approx. kg (dried weight) kpa~kpa (psi~psi) CV /Hz, CV Hz W (max) -9 degrees Celsius, Room temperature variation: within degrees Celsius % to 8% (condensation free) SP workstation * Except for LE-SPZC (This HPCV value is calculated for red laser. )

16 North merica/international Japan Europe 7 North First Street San Jose, C 9 U.S.. Voice: FX: sales@sonybiotechnology.com -7-, Konan, Minato-Ku, Tokyo, 8-7 Japan Tel: Fax:+-88- sales_japan@sonybiotechnology.com The Heights, Brooklands. Weybridge, Surrey, KT XW, UK Tel: + () sales_eu@sonybiotechnology.com Sony Biotechnology Inc. ll rights reserved. Sony, the Sony logo, Sony Biotechnology Inc. and logo, are trademarks of Sony Corporation. lexa Fluor and Pacific Blue are registered trademarks of and licensed under patents assigned to Life Technologies Corporation. Brilliant Violet, Brilliant Violet, BV, Brilliant Violet 7, BV7, Brilliant Violet, BV, Brilliant Violet, and BV, Brilliant Violet, BV, Brilliant Violet 7, BV7, Brilliant Violet 78 and BV78 are trademarks of Sirigen Group Ltd. -Dazzle 9 and Zombie NIR are a trademarks of Biolegend. Cy and CyDye are trademarks of GE Healthcare Limited. icyt products containing Cy, Cy. or Cy7 are manufactured under license from GE Healthcare under U.S. Patent Numbers,9,87;,7,7 and patents or pending applications that are continuations, continuations-in-part, re-examinations, divisionals, reissues or foreign equivalents thereof. Qdot is a registered trademark of Invitrogen Corporation. PerCP efluor is a registered trademark of ebioscience Corporation. ll other trademarks are property of their respective owners. For Research Use Only. Not for use in diagnostic or therapeutic procedures. The SP l nalyzer is classified as a Class laser product....