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Gallios – 穩定性絕佳的液流系統

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A New Frontier in Flow Cytometry

FC500 and Gallios Flow Cytometer

美商貝克曼庫爾特有限公司 生命科學部 劉聖德 Tony Liu

sdliu@beckman.com

美商貝克曼庫爾特有限公司 生命科學部 劉聖德 Tony Liu

sdliu@beckman.com

2

ƒ BCI Taiwan Inc.,在台成立29年,西元2000年以來流式細胞分析儀裝機量近100 台

ƒ 台灣MoFlo (4 way sorting) 四年內裝機11台

ƒ Astrios (6 way sorting) 造成全世界一機難求 (中研院分生所)

100000 eps analysis; 70000 eps sorting; purity > 99% at full speed; recovery >90% at full speed 32 bit processing; 100MHz sampling rate

Chromosome sorting; Sperm sorting Single Cell Sorting (CTC 應用) : 99% vs 60% for competitor in each well

完整齊全的產品線

(2)

Gallios – 穩定性絕佳的液流系統

Improved stability for accuracy and reproducibility Improved stability for accuracy and reproducibility

– Onboard sheath reservoir – Filled from external 20 L

sheath container

The isolated onboard sheath reservoir ensures exquisite control of sheath pressure and therefore stream stability

sheath reservoir

Gallios – Optical Design for Stability

Changes in ambient temperature can affect measurements by more than 25%

Customized temperature regulation (TEC) optimizes the internal settings to maintain optical consistency despite ambient temperature changes

Improved stability for accuracy and reproducibility Improved stability for accuracy and reproducibility

• Mean channel and CV

• Reproducibility

• Compensation

(3)

405

488

638

Optic Fibers Forward

Scatter Side

Scatter

Fluorescese Boulevard

Gallios Optical System – 可以關閉沒有使用的雷射

488nm 22 mW FITC PE PE-TR PE-Cy5;PE-Cy5.5 PE-Cy7 638nm 25 mW APC APC-Cy5;APC-A700 APC-Cy7;APC-A750

405nm 40 mW Pacific Blue Pacific Orange Krome Orange

Gallios – Lasers and Dyes

A750A750

Krome Orange

(4)

488 nm Excitation 561 nm Excitation

Gallios – Optional 561nm Laser :

PE Tandem Dye Intensities-488nm or 561nm Excitation

Gallios – Fluorescent Protein Detection

488nm and 561nm Excitation

(5)

Gallios – 三種前向散射光

Customize forward scatter to get more accurate size measurement Customize forward scatter to get more accurate size measurement

A: 3.11%

B: 26.68%

A: 7.20%

B: 13.45%

1-190

1-80

Flow Cell

3 kinds of forward scatter:

1-8o : 8-40 micron 1-19o : 0.5-10 micro

W2 : submicron

RBC Microvesicles WBC

Narrow1-80

Wide 1-190

Lymphocytes與Debris及monocytes分得較清楚 精確計算<1um的micro particles

The Gallios achieves submicron particles resolution with

– An innovative enhanced wide angle forward scatter (W2) – Low noise on the 488nm laser

– Low noise on the scatter electronics

Custom setting to optimize scatter collection of small particles Custom setting to optimize scatter collection of small particles

Forward Scatter

Side Scatter

Forward Scatter

Gallios – W

2

超高解析度可區分0.4um與0.5um微小差異

(6)

血液中Platelet-derived Microparticle 加入已知濃度

Megamix beads以流式細胞儀計數之標準流程, 評估各種機 型之實驗結果

由中心實驗室提供Platelet-Derived Microparticle (小於1um), 3種濃度low, medium及 high concentration, 作盲點測試 (即收到3種樣品, 但是不知道何者為高濃度, 何者為 低濃度)

11台Beckman Coulter : 3種濃度的分佈非常一致

11台BD的流式細胞儀 : 3種濃度分佈非常擴散, 亦即CV值很高

(7)

Beckman Coulter CV %:

Low conc. : 30%; Medium conc. : 15%; High conc. : 17%

BD CV % (內文有提到):

Low conc. : 78%; Medium conc. 87%; High conc. : 91%

Gallios - 優秀的訊號解析度及處理速度

Sensitivity and resolution are also accomplished with superior digital electronics design

Higher bits of information (20-bit)

Baseline restoration (resolve events from noise)

Fast signal processing

Minimize data loss

Electronics optimized for high resolution and fast, accurate processing Electronics optimized for high resolution and fast, accurate processing

(8)

Sensitivity – Discrimination from Noise

Sensitivity : 能偵測出最小螢光訊號的能力 Resolution : Negative及Positive訊號夠不夠集中

Picket Fence現象

Dynamic Range : 最小訊號到最大訊號偵測範圍

30625 eps 19000 eps

10239 eps 5100 eps

Gallios – Sensitivity at High Data Rates

77 eps

High speed without compromise High speed without compromise

Sensitivity is preserved with increasing data rates

Blank

First level bead

(9)

Gallios vs other Flow Cytometer

Resolution – Concentrated Signal No Picket Fence

Sensitivity : 能偵測出最小螢光訊號的能力 Resolution : Negative及Positive訊號夠不夠集中

Picket Fence現象

Dynamic Range : 最小訊號到最大訊號偵測範圍

(10)

Gallos – 高訊號解析度, 低螢光訊號也清楚

bit resolution is analogous to increasing the number of pixels in digital photography

1,048,576channel display 262,144 channel display

4 Megapixel 1 Megapixel

…regard the picket fences in our displays as if they bore the sign ‘beware of the data,’ and not try to play with what is behind them.” Practical Flow Cytometry, 4thedition

More channels of resolution to separate even the dimmest of populations More channels of resolution to separate even the dimmest of populations

Other Systems

Gallios- 20-bit system (20-bit)

(18-bit)

第1及第2個對數格沒有足夠 channels展現低螢光族群, 因此低 log (dim )會產生柵欄效應

No Picket Fence for Gallios

18-bit Systems Gallios

Gallios analysis of CD8+ cells and of CD4+CD25+ regulatory T-cells

Gallios allows use of the entire dynamic range with simultaneous resolution of dim signals even at the low end of the scale Gallios allows use of the entire dynamic range with simultaneous

resolution of dim signals even at the low end of the scale

(11)

Gallios‐20 bit FACSVerse‐18 bit

Dynamic Range – lowest to Highest Signal

Sensitivity : 能偵測出最小螢光訊號的能力 Resolution : Negative及Positive訊號夠不夠集中

Picket Fence現象

Dynamic Range : 最小訊號到最大訊號偵測範圍

(12)

為解決picket fence, 必須將電壓調高

Other System

Gallios

Definition: Fastest sample acquisition rate of the system measured in terms of events per sec

Consequence to Customer: Daily workload can be completed faster

Competitive Overview:

– Gallios: 25,000 events per second, 90% yield (40MHz digital sampling rate)

Max Speed

1. Rare events要花多少時間才能累計足夠細胞分析?

2. 高流速時有多少細胞data會lost?

(13)

High Speed Impact on Rare Events

Long Term HSCs

Long Term HSCs Thy 1.1 TRegs Thy 1.1 TRegs

Data courtesy of John Tigges; Vasilis Toxavidis; and Heidi Mariani (Beth Israel Deaconess Medical Center/Harvard Stem Cell Institute, Boston, MA).

ƒDecreasing signal to noise observed in competitive instrument with increasing data rate

ƒSignificant data loss was observed versus the Gallios

Yield enables you to get accurate results in less time, with fewer cells Yield enables you to get accurate results in less time, with fewer cells

Computer System – User Management

Gallios software enables you to manage users and ensure data integrity Gallios software enables you to manage users and ensure data integrity Multi-user secure log-in

User password management

Administrator control

Billing log for usage fees

(14)

Gallios – Instrument Dashboard

1. 具有儀器狀態燈號,包括樣品流速,sheath fluid不夠及waste滿溢指示燈。

2. Forward / Side light scatter及10色螢光訊 號LED燈號強度顯示,可以快速最佳化

protocol條件設定。此LED訊號顯示對於初 學者之條件設定,幫助尤其巨大。

Gallios – Multi Carousel Loader

1. 標準配備32管multi carousel loader。

2. 具barcode scan功能。

3. 單管auto mixing。

4. Dead volume < 30ul 。

5. 樣品分析完後,自動清洗注射針。

6. Carryover < 0.1% 。

Gallios 可以單管vortex, run到一半還可以轉出來讓user加 buffer或藥, 再轉回去又vortex後再繼續run; 也可以使用 manual mode一次上一管

(15)

Gallios Computer System – Data Display and Analysis (超過60種參數選擇)

簡單化,更方便的操作模式以因應多參數的設定

ƒ 捲軸式調整每個偵測器的訊號放大程度,更簡單也更方便

QuickSET

(16)

525 575 675

FL1 FL2 FL3 FL4 FL5

620 755

Compensation (螢光補償))

FL1 PMT:92=90+2 FL2 PMT:100=85+15

Compensation (螢光補償))

(17)

ƒ 螢光補償系統以及離線螢光補償分析系統使儀器的調整更簡單、更 直觀也可減低樣品重新分析的次數。

QuickComp

簡單化,更方便的操作模式以因應多參數的設定

Gallios – Auto-setup for multi-color setting

Run Standard

Sample

(18)

• 在沒有設定任何螢光補償的情況下,量測每種染劑進入個別螢光通道的強度

• 軟體綜合量測結果,計算出完整的 Compensation Matrix

自動螢光補償設定精靈(Auto-Compensation Setup Wizard)

Gallios TrueView – Accurate Compensation

Classic

Is additional compensation necessary?

TrueView – 6 decades

no additional compensation needed!

(19)

Auto compensation by Beads的謎思

1.Beads無法真實反應每種細胞的實紀情況, 例如細胞外型,背景螢光等等 2.對於tandem dyes例如PE-Cy5.5, PerCP-Cy5.5, PE-Cy7,

APC-Cy5, APC-Cy7, APC-700, APC-750 doesn’t work

Gallios – Auto Setup Wizard by real samples

FITC / PE / APC

PC5.5 / PC7

(20)

Auto compensation by FACSuite CS&T beads

FITC / PE / APC

PC5.5 / PC7

Standardized instrument setup assures reproducible  patterns over time

(stabilized control material)

Auto Setup Wizard by Flow Set Pro – Stability Over Time

(21)

AN26093 AS19123 AT13106 AN26093 AS19123 AT13106

Auto Setup Wizard by Flow Set Pro – Stability across Units

1. 最多可裝配4支雷射,488+638+405+561 lasers 。

2. 專利設計18度Boulevard光學濾片,螢光訊號折損率最低,大幅提升 螢光訊號強度。

3. 內建電子式溫控系統(TEC) ,避免環境溫度變化影響光學系統穩定 性,提升實驗數據再現性,同時延長雷射光源使用壽命。

4. 唯一內建三種前向散射光(1-8o , 1-19o , W2),可依據細胞大小不同進 行分析,超高解析度可區分0.4um與0.5um 極微小差異。

5. 20 bit 訊號解析度為BD系統的四倍,讓弱螢光表現細胞獲得更高圖 形解析度,細胞分群更清晰。

6. 40Mhz訊號處理速度,每秒可分析25,000 events,細胞數據丟失率 少,可在更短時間內獲取微量表現細胞數據。

Beckman Coulter Gallios的特點

(22)

7. 儀器搭配LED指示面板,顯示儀器運作狀態以及訊號強弱程度。

8.內建32管自動進樣轉盤可使用任何材質12 x 75mm試管,上樣前儀 器自動進行排塞以 及單管混勻功能,可避免細胞樣品沉澱。

9.Gallios軟體提供可收取超過60種以上參數 (3種 FSC, 2種SSC, 50 種螢光參數, 飛行時間TOF, Ratio, 以及Time) 。

10.Gallios軟體人性化的操控介面,直觀式拖曳捲軸可幫助初學者輕鬆 根據圖形調整儀器 設定。

11. 簡單和準確的 Auto-Setup Wizard 功能,即使進行多色螢光實驗也 能輕鬆操作。

12. Gallios軟體提供完整的儀器品管以及使用者管理功能,軟體與 Microsoft Office系統相容,實驗數據可直接輸出成PDF, Excel, PowerPoint等格式。

Beckman Coulter Gallios的特點

Cytomics FC500 5 Colors Flow Cytometer

• 488nm blue laser

• Optional 635nm red laser

• FS + SS + 5 FL PMTs

• 2種FS角度 : 1-8o/ 1-19o

• High resolution Digital Signal Processing ( DSP, 220, 1,048,576 channels)

• 5*5 Compensation Matrix

• 32 tubes MCL (standard) or MPL ( optional 96 well plate / 40 tube loader)

• FCS 3.0 file format for list mode data

• Off-line Compensation

• Data with original protocol

• Auto Setup Wizard

• QuickSET (捲軸式電壓調整)

• QuickCOMP (捲軸式螢光補償調整)

(23)

One Laser (488nm) 5 colors: FITC/ PE/ ECD/ PC5/ PC7

Dual Laser (488nm & 635nm) 5 colors: FITC/ PE/ ECD/ APC/ APC-PC7

46

FC500 – 二種前向散射光

Customize forward scatter to get more accurate size measurement Customize forward scatter to get more accurate size measurement

A: 3.11%

B: 26.68%

A: 7.20%

B: 13.45%

1-190

1-80

Flow Cell forward scatter:

1-8o : 8-40 micron 1-19o : 0.5-10 micron

RBC Microvesicles WBC

Narrow1-80

Wide 1-190

Lymphocytes與Debris及monocytes分得較清楚 精確計算<1um的micro particles

(24)

血液中Platelet-derived Microparticle 加入已知濃度

Megamix beads以流式細胞儀計數之標準流程, 評估各種機 型之實驗結果

48

1,048,576 channel display 262,144 channel display

4 Megapixel 1 Megapixel

…regard the picket fences in our displays as if they bore the sign ‘beware of the data,’

and not try to play with what is behind them.” Practical Flow Cytometry, 4thedition

More channels of resolution to separate even the dimmest of populations More channels of resolution to separate even the dimmest of populations

18-bit system

FC500- 20-bit system (20-bit)

(18-bit)

Picket Fence

第1及第2個對數格沒有足夠 channels展現低螢光族群, 因此低 log (dim )會產生柵欄效應

(25)

Friendly design – LED display, 對於初學者有巨大幫助

Forward Scatter Side Scatter

Forward Scatter

Side Scatter

FC500 MCL ( Mutli Carousel Loader )

● MCL Sampler

– FC500 標準配備 – 32 tubes capacity – 內建 Bar-code reader

– 單管混樣,非整盤 / tube rack一起 shaking

– 適用任何品牌12x75 檢體管 – 每個樣品間進行自動清洗 – 可分析 no-wash sample

– Pause / Rotate to allow addition of stimulant or antagonist reagent

(26)

Windows 7 based CXP Software

多重使用者登錄功能:

• 避免使用者間混淆數據與資料

• 利於公用儀器室的管理

拉取式的操控方式

將protocol / data直接拉到此區域

(27)

最簡易的操作介面

ƒ 單一的儀器操作視窗

ƒ Protocol:整合儀器設定與桌面圖示於同一檔案

捲軸式的調整方式

QuickSET:簡化電壓值(HV)的調整

(28)

捲軸式的調整方式

單染FITC與PE,各自上機,扣除掉漏光到其它 偵測器的過程為Compensation

QuickCOMP:簡化螢光補償的流程

FC500 – Auto-setup for multi-color setting

Run Standard

Sample

(29)

57

• 在沒有設定任何螢光補償的情況下,量測每種染劑進入個別螢光通道的強度

• 軟體綜合量測結果,計算出完整的 Compensation Matrix

自動螢光補償設定精靈(Auto-Compensation Wizard)

Off-Line Modified Compensation

(30)

Prism 功能

• 2 色:4 群

• 3 色:8 群

• 4 色:16 群

• 5 色:32 群…….

能在一個同圖形中表示所有細胞分群的百分比

Gating

ƒ多種自動化框選功能可供選擇

ƒAuto Gate 根據細胞分佈密度,軟體可自動框選細胞

(31)

不受限制的疊圖功能

可將統計數據直接輸出至Excel檔中

(32)

可將圖形直接拖引到Word 或PTT檔中,無須轉檔

FlowPAGE (Report)

Available to Save as PDF file

(33)

Kaluza離線分析軟體

65

人性化工作區域設計

Kaluza提供創新直觀化動態工作區域,使用者與分 析途中可任意組織及變動圖形,提供分析數據最佳

彈性及自由度。

放射狀功能選單

Kaluza提供創新的放射狀功能選單,使用者只需 單鍵(one-click)操作即可完成全部圖形以及 region的設定,減少尋找軟體附加功能所浪費的

時間同時提升數據分析效率。

圖形自動排版管理功能

Kaluza獨一無二的自動排版(Auto-Layout)設 計,幫助使用者輕鬆配置工作區域上繁複的分

析圖形,無論分析途中執行任何版面變動,

Kaluza軟體都能即時幫助您執行排版

66

Kaluza離線分析軟體

多參數圖形(樹狀圖、雷達圖)

樹狀圖以最簡易清晰的方式呈現繁複的 數據分析,統計數值可動態隨著圈選區 域的調整進行即時運算。每個樹狀分支

“tree branches”所代表的特定細胞群可 做為進一步數據分析的gate來源。

雷達圖提供多維參數分析,突破傳統單參數 圖或者雙參數圖形分析限制。可更宏觀分析 各種螢光表現族群間相互關聯性,也能夠讓

微量表現細胞更清楚可見。

數據合併功

Kaluza可輕鬆合併多個數據成為單一 檔案進行quality分析,藉由繪製出時 間軸圖形,幫助了解微量表現細胞在 不同時間點或是不同處理條件下的表 現趨勢。對於longitudinal以及time

course研究非常有幫助。

多檔案分析

藉由拖曳功能可將多筆數據同時放入分析清 單中,多檔案分析功能可迅速執行離線螢光

補償以及繪製疊圖。

(34)

1. 最多可裝配2支雷射(488+635 lasers),1支雷射即可分析5色。

2. 內建二種前向散射光(1-8o , 1-19o),可依據細胞大小不同進行分析。

3. 20 bit 訊號解析度為BD系統的四倍,讓弱螢光表現細胞獲得更高圖 形解析度,細胞分群更清晰。

4. 儀器搭配LED指示面板,顯示儀器運作狀態以及訊號強弱程度。

5. 內建32管自動進樣轉盤可使用任何材質12 x 75mm試管,上樣前儀 器自動進行排塞以 及單管混勻功能,可避免細胞樣品沉澱。

6. 軟體人性化的操控介面,直觀式拖曳捲軸可幫助初學者輕鬆根據圖 形調整儀器 設定。

7. 簡單和準確的Auto-Setup Wizard 功能,即使進行多色螢光實驗也 能輕鬆操作。

8. 軟體提供完整的儀器品管以及使用者管理功能,軟體與Microsoft Office系統相容,實驗數據可直接輸出成PDF, Excel及Power Point 等格式。

Beckman Coulter FC500的特點

ƒ Technical Support: 目前 Beckman Coulter台灣分公司擁有3位資深 Flow產品專員,針對客戶各種檢驗項目、儀器操作以及協助條件設定 提供無限次免費服務。

售後服務團隊

20110808-20110810陽明大學貴儀中心FC500 training

(35)

ƒ Service support: Beckman Coulter台灣分公司Flow維修工程師共有9 位負責售後儀器維修及保養,每位工程師擁有至少5年以上的服務年 資,能提供即時而且有效率的維修保養服務。

售後服務團隊

參考文獻

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