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新世代流式細胞儀. Partec GmbH from Münster Germany. 派特科技有限公司. Fluorescence Based Flow Cytometry. Invented in 1968 in the University of M ün ster, Germany at the Institute of Radiobiology by Professor Dr.Wolfgang Göhde.
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新世代流式細胞儀 Partec GmbH from Münster Germany 派特科技有限公司
Fluorescence Based Flow Cytometry • Invented in 1968 in the University of Münster, Germany at the Institute of Radiobiology by Professor Dr.Wolfgang Göhde. • First Published paper in Jan. 1969 “Dittrich W., Göhde W. "Impulsfluorometrie bei Einzelzellen in Suspensionen", Zeitschrift für Naturforschung (1969), Tübingen, Band 24 b, Vol. 3” • “Impulse Fluorometry” was the name used in the paper, “Impulse Cyto Photometry Technology” was called and still can be found in the Internet. • Hertzenberg started flow cytometry together with Bernhard Shaw (at that time V. P. of Becton Dickenson) in 1973, when the NIH had decided to develop an own American Flow Cytometer
More than 30 years of experience... 1969: The ICP 11. The first commercial flow Cytometer is born -the legendary ICP-11, developed in Münster and distributed by Phywe AG, Göttingen. Electronics size and fashion changed since that time...
1971: ICP-11獲頒卓越產品獎 Award for outstanding product quality of theon the Leipziger Herbstmesse.
需要判定細胞濃度及強弱螢光細胞的數量 一個光學高度計 一個磅秤 在入口處分發螢光棒 裝設一個紅光與綠光偵測及計數器 讓來客漁貫進入… 最後一個客人進入時資料也得到了 螢光顯微鏡 vs. 流式細胞儀 • 主辦單位想由演奏會台上 數觀眾中男生及女生人數
Sheath fluid Laminar Coaxial Flow The Bernoulli Effect 鞘液流動方向 Lower pressure Velocity Gradient 細胞之輸送 Hydrodynamic Focusing Viscous drag along walls. Particles move to low pressure area
Laser Light 雷射與螢光訊號 High angle scatter : SSC 大角度散射代表細胞內部 細胞質密度及表面顆粒性 Granularity Low angle scatter : FSC 細胞顆粒大小 螢光散射 Fluorescence at longer wavelengths. Intrinsic內部螢光 (自體螢光 auto fluorescence) and extrinsic.
DNA Analysis Cell Cycle Cell Viability: Cell Dying by Apoptosis Tumor Cell Analysis Immunophenotyping Simultaneous DNA content and Cell-Surface Immuno- fluorescence Analysis Matching for Solid Organ Transplantation Cell Membrane Potential Analysis Sperm Chromatin Structure Assay Intracellular pH Intracellular Ionized Ca Lysosomal Proton Pump Activity “No-Wash” procedures for Nuclei Antigen Detection Simultaneous Analysis of Cellular RNA & DNA content PRINS & FISH Enzyme Kinetics Reticulocyte Analysis & Maturity Index Cell Sorting …. 流式細胞儀部分應用
Light Source Various light sources: • Argon • NdYAG • HeNe • Red Laser Diode • HBO UV lamp • HeCd
Sorting 細胞分選原理 • 1979: • The PAS-II. • mercury arc lamp (UV) • best DNA measurement precision (CV< 1%) • 3 optical parameters + electrical cell size • first piezo activated cell sorting (patented), real time acquisition • absolute cell counting • Motorola 6809, 68000 and 68020 processors • real potentiometers for PMT high voltage • acquisition rate 10000 cells/s • interchangeable filters
Cytopeia InFlux • High Performance Sorter • High Speed Sorting up to 50,000/sec • Easy to Operate • Multiple Lasers • Blue 200mw 488 • Green, Red • Solid State UV Laser • Highly Hygiene for Cell Culture by Disposal Sorting device
體積小 低耗能源 輸出功率高 啟動時間短不需暖機 使用壽命長 目前現有發展雷射 : Blue 488 nm 200 mW Red 635 nm 25 mW Green NdYAG 532 nm 50 mW Violet Laser UV Laser Many More to Come … 雷射發展之趨勢 – 固態雷射
CyFlow Compact Highest Stability and Highest Precision Power Connection: Regular 100/240 VAC or Car Battery (12 V) Single Platform Volumetric Absolute Counting Up to 5 Parameters (FSC, SSC, FL1, FL2, FL3) Single Laser SystemUV/Violet, Blue, Green, and Red Solid State Lasers New Level of Price / Performance Ratio
CyFlow ML • 桌上型設計 • 可裝 3 組雷射及 UV 燈 (red, green, blue, violet lasers) • FloMax® Software • 16 參數 (FSC, 2 x SSC, FL-1 - FL-13) • 體積小 (size: ca. 56 cm x 65 cm x30cm) • Windows™ XP 作業系統 • New 16 bit 數位處理系統 • 定體積絕對細胞計數功能True Volumetric Absolute Counting • 德國光學系統 超強螢光訊號靈敏度低於 100 MESF • FCS, SSC 可偵測粒子直徑 < 0.2 um
CyFlow® Space • 桌上型設計 • 雙固態雷射六色螢光系統: 20 mW @ 488 nm 25 mW @ 635 nm option • 6 色 Immunophenotyping: FITC + PE + PE-Cy5,PE-Cy5.5, PerCP, PI, ECD + PE-Cy7 + APC, APC-Cy5.5 + APC-Cy7 • 德國光學系統 超強螢光訊號靈敏度低於 100 MESF • FCS, SSC 可偵測粒子直徑 < 0.2 um • 定體積絕對細胞計數功能True Volumetric Absolute Counting • 細胞分選系統 PPCS Particle and Cell Sorter option
偵測微細粒子之靈敏度 Supported by the use of a PMT for the FSC parameter, the small white blood cell platelets (PLT) can safely be discriminated from red blood cells (RBC) in the scatter plot.
Yeast Analysis DNA and Lipids with Double Excitation DAPI (DNA, FL4) and red staining (neutral lipids, FL3) of brewery yeasts after double excitation with HBO-lamp (UV) and argon ion laser (488 nm, 20 mW)
Bacteria Analysis RFP in E. coli Measurement of RFP-expression (FL2: orange-red) of an E. coli population after excitation with an YAG-laser (523 nm, 50 mW): 40% of the E. coli population show the red fluorescence due to RFP-expression.
Microbiology Analysis in Food Bacteria in chocolate drink. left: control, right: contaminated sample. The discrimination between “chocolate” particles and bacteria is based on spectral analysis in the two fluorescence channels.
定體積絕對細胞計數 The measurement volume is defined by mechanical measurement, rather than reference beads.
定體積絕對細胞計數原理 • 不需添加標準濃度粒子試劑 • volume:200 ± 10 µl, 流速軟體控制 0 - 120 uL/sec • coincidence losses: < 1% at analysis rates < 2000/s • 分析後可以任意圈選而得到不同區域細胞群之濃度
德國科技堅持 嚴選零件 Partec流式細胞儀使用與火星計畫 Sujourner 相同的馬達
減白血液製品的Leukocytes數目 • 機械式體積測量精確度高 • 不需添加標準濃度粒子試劑 • 可偵測濃度低至 < 0,01 Leukocytes / µl (= 10 / ml) • 以單一螢光參數 HBO-Lamp / ExciteDAPI • “Digital” counting due to bright CyStain staining • 方便快速
CD4/CD8 單位體積之細胞數目 Routine concentration analysis on CD4/CD8 for HIV monitoring. The concentration of subpopulations can as well be analyzed “offline”, after analysis.
CD34 Stem Cells / Rare Event Rare event concentration analysis on CD34+.
多重螢光 4 colors 分析 • Dual laser excitation: Argon and red laser diode. • MultiColor gating. • 4 color analysis.
FloMax – 螢光校正補償 • N-color cross-talk compensation. • Matrix-based: Compensation of any parameters, e.g. FL1-FL3. • Correction of compensation at any time: No need to rerun samples.
