SP6800
Spectral Analyzer

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SP6800 Spectral Analyzer

The Sony SP6800 Spectral Analyzer uses spectral technology to optimize sensitivity and enhance dim signal detection by collecting photons from 420nm to 800nm. Spectral technology also simplifies multicolor panel design, by eliminating bandpass filters and conventional compensation matrices while delivering better data and simplifying visualization for the study of heterogeneous populations.Novel global standardization mode automatically sets the system to a master specification with a single click. This capability eliminates instrument variability from day to day and across multiple instruments for greater reliability.
Advanced electronics and patented optical technologies bring simplicity to Spectral Analysis workflows. Sony’s patented Flowpoint™ core stability and tracking system and automated QC ensure the highest resolution possible of target populations.

The system features easy to use software that automates alignment and laser delay with set up wizards and simplified voltage settings. Each system includes FCS Express™ software in addition to Sony analysis packages to offer the highest flexibility in analysis.

The SP6800 is for non-clinical research use only and is a Class 1 laser product.

SP6800 System Analyzer

The SP6800 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 to achieve greater flexibility for panel design and more accurate visualization of results.

SP6800 Software

The SP6800 software is easy to learn and use. It guides researchers from set-up to panel design, acquisition, analysis, and shutdown. User preferences allow users and administrators set up options for overall instrument operation and experiment set up to facilitate use and unattended operation procedures.

System Start Up

At start up Align Check and Performance QC wizards check instrument calibrations, using beads to ensure the instrument is operating optimally. On screen instructions guide the user through procedures, then display progress and report results. The performance report displays MESF, Q, and B values to describe real-time fluorescence detection performance. If desired, Align Check and Performance QC reports can be displayed in historical context.

Standardization

Standardization mode sets the system to a master specification to eliminate variability in a single instrument or among instruments located across sites. This unique capability allows experiments performed on any Sony cell analyzer to produce highly reliable, accurate, and reproducible results. This function also eliminates setup subjectivity for collaborative or long term studies with different operators or experience levels across sites.

When engaged, standardization mode sets the SP6800 to predetermined global settings that eliminate instrument variability.

Spectral Library

The Spectral Library lets users create a personal reagent library that simplifies experiment creation and saves time. An Acquisition Wizard assists the users with step by step instructions to acquire and analyze single positive controls for your spectral library. Once acquired the spectral reference for that reagent including the spectral index are available for future use. Information from the spectral library is available to users with a simple click improving accuracy and streamline panel design.

Experiment Creation

Experiments can be created using a template, an existing experiment, a single stained, or multicolor assay, in the Create Experiment window. Users can point and click to choose (or edit) an existing experiment and can easily select templates for wells or plates when creating a new experiment. A setup Assay Wizard guides users through the creation of a singlestained or multicolor assay simplifying experiment creation.

Acquisition Functions and Analysis

All acquisition functions, including instrument settings are controlled from the Acquisition Window. Worksheet tools let users choose how the data is displayed- (such as plot types), and customize for their analysis needs. Plots and statistics provide real-time information during acquisition. To increase sample acquisition to the cuvette, a variable booster lets users set acquisition speed from low (33ul/min) normal or high (250ul/min) offering flexibility.

FCS Express from De Novo Software

Spectral Analyzers from Sony include FCS Express, from De Novo Software. FCS Express offers a range of new analysis tools from live gating to batch analysis. Native support for Sony Spectral data files to enable sophisticated data transformations and visualizations such as spectral overlays, tSNE, Spade, and heat maps.

Live Gating

Dot plots and spectral plots can be set up with live gating. Live gating allows users to find positive and negative populations quickly and easily for added assurance about target populations.

SP-image-8

Flexible Analysis

Flexible data analysis can include integrated spreadsheets, custom calculations, charting and regression analysis.
SP-image-9

Batch and Report

Batch analysis lets you process any number of samples with one click. To support presentation, data can be exported directly to PowerPoint, PDF, and Excel.

Spectral Analysis Technology

Spectral analysis technology is the foundation of the SP6800 system. Spectral flow cytometry streamlines workflow and yields better data by keeping all the light collected. In conventional systems, overlapping fluorescence is subtracted using color compensation, so less light is collected. Instead, spectral flow cytometers sum the fluorescence together and then use unmixing to mathematically separate the colors. This powerful capability also simplifies workflow including panel design, and improves visualization of autofluorescence. A unique prism collection system delivers emitted light to a 32 channel PMT. This produces 66 data points of signal detection for fluorescence and bright auto fluorescence to achieve accurate visualizations of fluorescent populations. This lets researchers see the complete spectral fingerprint of each fluorochrome from 420nm to 800nm.

Visualization of fluorescent cell populations using the analyzer’s unique prism collection system.

Spectral Unmixing

A powerful capability of spectral technology is Unmixing. This allows researchers to separate fluorophores into pure signals that measure the quantity of each fluorophore at each pixel to more accurately measure data for analysis.

Spectral Unmixing separates each spectral fingerprint for complete and optimal visualization of fluorochromes.

Spectral unmixing

Uniform measurement of Fluorescent Emissions

A correction system adjusts the offset and sensitivity of each channel of the 32 channel PMT to ensure a uniform and accurate measurement of fluorescent emission from 420nm to 800nm. The corrective system brings a standardization to all 32 PMTs ensuring the user is getting the most reliable data with the ease of adjusting only one voltage. This saves time over conventional flow cytometry operation where users must calibrate each individual PMT.

Pre and Post Correction Profile. Each graph illustrates pre and post correction in the SP6800 32 channel PMT. The correction improves accuracy of spectral visualization.
Spectral emission of PE Cy5 pre and post correction. Corrections support accurate unmixing of closely overlapping fluorescence spectra.

Subtracting Auto-Fluorescence Improves Visualization

In conventional flow cytometry cellular auto-fluorescence produced by pyridine (NAD/NADH), flavin (FMN, FAD), and other intracellular oxidative reactions can cause fluorescent signal contamination of other fluorescent markers. Other common sources of auto-fluorescence include cell fixation and permeabilization. Using spectral technology, auto-fluorescent spectral fingerprints can be subtracted to allow researchers to see the true fluorescent population.

Unstained mouse splenocytes were analyzed with the SP6800 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.
Auto-fluorescence can result in the appearance of additional cell populations leading to erroneous data interpretation. A. T cells expressing GFP and stained with an antibody conjugated to Alexa Fluor® 700 were analyzed with the SP6800. A 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.

Fluorochrome Panel Guide

Application data dye options and combinations. Select one from each group.

SP6800 Applications

16 color panel on Human Peripheral Blood


16 color stained sample of Human Peripheral Blood


Application Data: Brilliant Violet Dyes

Spectral Analysis allows multi-laser excited fluorochromes to be run without using a complicated compensation matrix.

human-peripheral-2L-405nm

BV421BV605
BV510BV650
BV570BV711
BV785

brilliant-human-pbmc-graphs

Sample data from Human PBMCs stained with seven markers conjugated to Brilliant Violet dyes offered by Sony Biotechnology. D. All populations were clearly resolved including fluorochomes with significant spectral overlap such as BV605 and BV650 (D).


12-color Staining of Human Peripheral Blood Leukocytes

12-color-leukocytes-table
12-color-leukocytes


Application 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 SP6800 is capable of analyzing all of these by looking at all photons from 420nm to 800nm and unmixing each unique spectral fingerprint. 

Instrument Specifications

SP6800 instrument specification
Instrument
Configuration2 laser model, 488/6382 laser model, 405/4883 laser model, 405/488/638
Model NumberLE-SP6800ZBLE-SP6800ZCLE-SP6800ZE
Optics
Laser Specification
LasersSemiconductor 2 laser model
488nm, 638nm
Semiconductor 2 laser model
405/488
Semiconductor 3 laser model
405/488/638
Maximum power (at flow cell)488nm 40mW
638nm 60mW
405nm 60mW
488nm 40mW
405nm 60mW, 488nm 40mW
638nm 60mW
Detection Optics
Scatter signalsForward scatter (FSC), Side scatter (SSC)
Spectroscopic methodPrism array spectroscopy
Fluorescent channels32 channel PMT (wavelength: 500-800nm)32 channel PMT (wavelength: 500-800nm) PMT x 2(420-440nm, 450-470nm)
System
Signal ResolutionHeight 20 bit, Area 32 bit Sampling frequency: 50MHz
Measurement parameters64 channels, FSC, SSC,
cell position XY
66 channels, FSC, SSC, cell position XY
Pulse shape parametersHeight, Area, Width
Optical alignmentAutomated alignment with Corefinder™ technology
Event Rate10,000eps (standard)/20,000eps (maximum)
Fluidics
Sample loader typeSingle auto-loader
Supporting sample tube5ml polystyrene round tubes
Sheath Flow SpeedLow (approx. 3m/s), Mid (approx. 5m/s), High (approx. 10m/s)
Detection channel dimensions200μm x 200μm
Performance
Fluorescence sensitivityFITC: 120 MESF / PE: 70 MESF
LinearityFITC R2 ≧0.995 / PE R2 ≧0.995
Detection size range0.5~40μm (beads)
Fluorescence detection resolution488nm Laser/CH16, 638nm Laser/CH27* : ≧2.5% (HPCV)
Software
Unmixing algorithmsSpectrum method (LSM, WLSM, PSA and Constraint option), Reverse matrix method (Conventional)
AutofluorescenceAutofluorescence spectral detection
Virtual FilterPossible to change wavelength region for each fluorochromes in analysis
Export Data FormatFlow Cytometry Standard (FCS) 3.0, 3.1
Main Unit
DimensionsMain unit: Width 60.0cm x Depth 63.5cm x Height 71.3cm
Fluidics cart: Width 78.6cm x Depth 52.1cm x Height 58.0cm
WeightMain unit: approx. 99kg (dried weight)/Fluidics cart: approx. 32kg (dried weight)
Air pressure supply350kPa~450kPa (51psi~65psi)
AC Power supplyAC100V 50/60Hz, AC120V 60Hz
Power consumption220W (max)
Operating temperature16-29 degrees Celsius, Room temperature variation: within 5 degrees Celsius
Operating humidity20% to 80% (condensation free)
Recommended PCSP6800 workstation

*Except for LE-SP6800ZC (This HPCV value is calculated for red laser)

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