新世代流式細胞儀. Partec GmbH from Münster Germany 派特科技有限公司

Transcription

1 新世代流式細胞儀 Partec GmbH from Münster Germany 派特科技有限公司

2 螢光細胞染色技術 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 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

3 More than 30 years of experience : 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...

4 1971: ICP-11 獲頒卓越產品獎 Award for outstanding product quality the Leipziger Herbstmesse.

5 螢光顯微鏡 vs. 流式細胞儀 判定細胞濃度及強弱螢光細胞的數量 螢光顯微鏡來判讀研究細胞螢光染料的抗體與細胞作用 常因個人眼睛對螢光強弱的靈敏度不同 一顆一顆計算非常費時的 眼睛容易疲勞而誤判 數百顆細胞來分析 統計學上不夠客觀 人為誤差相當的大

6 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 一個光學高度計 一個磅秤 在入口處分發螢光棒 男生拿綠光 女生拿紅光, 或是有人兩種都拿 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, 全部資料也得到了

7 流式細胞儀之數據分析圖形

8 流式細胞儀部分應用 DNA Analysis Cell Cycle Cell Viability: Cell Dying by Apoptosis Tumor Cell Analysis Immunophenotyping Simultaneous DNA content and Cell-Surface Immunofluorescence 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.

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10 儀器操作步驟簡介

11 流式細胞儀工作原理

12 細胞流路與光學照射

13 Hydrodynamic Focusing 細胞之輸送 Sheath fluid Laminar Coaxial Flow The Bernoulli Effect 鞘液流動方向 Velocity Gradient Lower pressure 石英管璧摩擦阻力將細胞往中間低壓區推

14 螢光與光學訊號的意義 SSC: 側面散射光 Side Scatter 大角度散射代表細胞表面顆粒性 (Granularity) 及內部細胞質密度 FSC: 正前方散射 Forward Scatter 細胞顆粒大小 (Size) Fluorescence 螢光散射細胞上螢光物質散射之訊號

15 螢光發光原理及光譜

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18 Flow Cytometer的主要元件

19 各種光源及譜線 : 氣體雷射 Argon 藍光 Argon-Krypton UV 光 HeNe 紅光 HeCd 氣體放電燈 HBO UV lamp Xenon 固態半導體雷射 藍光綠光紅光紫光 UV 波長光 NdYAG 綠光 氣體雷射發光原理

20 氣體雷射工作原理

21 半導體雷射原理

22 雷射發展之趨勢 固態雷射 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 20 Mw, 50mW, 200mW Red 635 nm 25 mw Green NdYAG 532 nm 50 mw, 100mW Violet Laser UV Laser, 8 mw, 20mW Many More to Come

23 光學濾鏡工作原理

24 光電倍增管工作原理

25 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, and processors real potentiometers for PMT acquisition rate cells/s interchangeable filters

26 Stream Sorting

27 Droplet Sorting 488 nm laser FALS Sensor + + Fluorescence detector Charged Plates Single cells sorted into test tubes Purdue University Cytometry Laboratories

28 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

29 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 System UV/Violet, Blue, Green, and Red Solid State Lasers New Level of Price / Performance Ratio

30 CyLab

31 CyFlow in Space - ISS

32 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

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34 CyFlow Space 桌上型設計 三種固態雷射六色螢光系統 : nm nm 20mW UV 365 nm 6 色螢光分析 :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

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36 模組化光學系統

37 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.

38 翡翠水庫水質研究 0.02um 過濾 原水稀釋 50 倍加入 S.B. Green 染劑 0.2um 過濾 中研院地球所環境變遷中心夏復國老師實驗室

39 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)

40 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.

41 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.

42 定體積絕對細胞計數 N c. V = d π r 2 d c = N / V The measurement volume is defined by mechanical measurement, rather than reference beads.

43 定體積絕對細胞計數原理 Computer Controlled Motor Digital Velocity Meter => v Syringe Pump Air c Sample Tube Sample d N Flow Cuvette. V = d π r 2 c = N / V c = N / V Stop Start 不需添加標準濃度粒子試劑 volume:200 ± 10 µl, 流速軟體控制 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

44 德國科技嚴選零件 Partec 流式細胞儀使用與火星計畫 Sujourner 相同的馬達

45 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 < 10 Leukocytes / ml 以單一螢光參數 HBO-Lamp / Excite DAPI Digital counting due to bright CyStain staining 方便快速

46 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.

47 Rare event concentration analysis on CD34+. CD34 Stem Cells / Rare Event

48 Dual laser excitation: Argon and red laser diode. MultiColor gating. 4 color analysis. 多重螢光 4 colors 分析

49 多螢光譜線之重疊

50 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.

51 FloMax 人性化電腦介面

52 FloMax Report

53 FCM Application Workshop