Microsoft PowerPoint - FCM seminor at Utsunomiya university pptx

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Principle of Flow

Cytometry and Outline of

EC800 Cell Analyzer

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What is Flow Cytometry

You can get the characteristics of cells by

measurement various light in the flow

Flow

Cells in Motion

Cyto

Cell

Metry

Measure

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• Cell Analyzer

– Data acquisition and analysis

– Auto sample loader

• Cell Sorter

– Data acquisition and analysis

– Cell sorting (collection)

Cell analyzer EC800 Cell Sorter SH800Z Cell analyzer SP6800Z

Flow Cytometer

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6/9/2015 4

Basics of Flow Cytometry

•cells in suspension

•flow in single-file through

•an illuminated volume where they

scatter light and emit fluorescence

•that is collected, filtered and

converted to digital values

•that are stored on a computer

Fluidics:

Optics

Electronics

Original Slide from Bob Murphy, CMU

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Principle of Flow Cytometry

• If laminar flow made in flow cell, cells are made to pass the center of sheath stream forcibly by using the

hydrodynamic focusing

• Cells are detected one by one with laser beam

• Various emitted light are efficiently collected, separated by optical filters and detected with PMTs

• Detectors output voltage signals related to brightness

• Signals are converted to digital value

Fluidics

Bandpass Filters Dichroic Mirrors Optics Detectors Electronics Computing

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Conceptual scheme of Flow Cytometer

Fluidics

Computing

Electronics

Optics and Detectors

From : http://en.wikipedia.org/wiki/Flow_cytometry

Flow Cell

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Fluidics System

Fluidics

Sheath

Sample

Sheath

Cell Laser Difference of the pressure Sample and Sheath makes Hydrodynamic Focusing in the Flow cell. Sample flow is very narrow, so cells line up and flow at constant velocity.

Flow Cell

Flow Cell

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Optical System

Band pass filter (BP)

Flow Cell Dichroic long pass _{filters （DLP）}

Separate fluorescence based on wavelength When a cell passes a laser beam in flow cells, the cell emit scatter and fluorescence from the fluorescent dye. Forward Scatter (FSC), Side Scatter (SSC) and fluorescence (FL) are separated based on their wavelength by optics filters and lead to detectors (PMT).

FSC

SSC

Green

Yellow

Red

Pass only limited wavelength light

From: http://flow.csc.mrc.ac.uk/?page_id=5

Detectors for Fluorescence （PMT)

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Separation of Fluorescence by Optical Filters

For Example : Red fluorescence(660nm)

a b c d e

a.

639nm DLP pass it (∵ 660nm > 639nm)

b.

752nm DLP reflect it (∵ 660nm < 752nm)

c.

685nm DLP reflect it (∵ 660nm < 685nm)

d.

665/30nm BP(665±15nm) pass

(∵ 650nm <660nm < 680nm)

e.

FL4(PMT) detect it

FL4

PMT

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Multi‐Laser / Multi‐Color

Multi‐Laser gives you flexibility choice of Flourochromes.

405nm 488nm 561nm 638nm

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Can provide you various information of cells at once

Multi-Laser / Multi-Color

Laser

Flourochromes

EmissionMax.

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Electronics / ADC / Plot

• The scatter and the fluorescence occur when a cell passes a laser beam. Detectors make out voltage pulse caused from each intensity. • Pulse Height are directly proportional to intensity. However, they change by detector's sensitivity control. Histogram Cytogram Time Voltage pulse Area • Integrated value (Area) shows total amount of light from the cell. • These analog value are converted to digital value by ADC. Height

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Histogram

X axis ： Intensity of light

Make plots, identify the cell populations

Visualization of the cell poplations

Cytogram

Show correlation of two parameters (light).

Dot density = population density.

Parameter (Log axis)

Number of cells Number of cells Parameter (Lin axis) Parameter (Log a xis)

Parameter (Lin axis)

Parameter (Log axis)

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Cells (micro particle suspension)

• Size :

0.5 μm ～40 μm

• Concentration :

1x10

6

～5x10

6

個/mL

Blood cells

Cell line

Pant

Bacteria

Marline life

Cytokine etc.

Marker （cell surface, intra culler）

DNA content （Cell cycle, Diploidy）

Cell kinetics （Active oxygen, etc.）

Cell death （Apoptosis）

Gene expression （Fluorescence protein）

Intra culler stain, Capture beads

（Cytometric Bead Array : CBA）

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What are the markers for lymphocyte subsets?

Granulocytes

Lymphocytes

Monocytes

WBC

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Parameters on FCM

For Individual cell

1. Scatter

• Forward Scatter （FSC） ⇒ Cell size

• Side Scatter （SSC） ⇒ Internal structure of the cell

2. Fluorescence

•

Dyes

3. Time

4. others (Ratio, EV, etc.)

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Forward Scatter : FSC

= relevant to cell size

*

Side Scatter : ＳＳC

= relevant to Internal structure of the cell

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単球顆粒球リンパ球

Scatter plot (FSC vs SSC)

WBC sample

Size

Large

small

In

te

rnal

st

ru

ct

u

re

Simple

Complex

FSC Lin SSC Lin

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“cell surface marker”

• There are many protein molecules on cell surface

• Dye conjugated Monoclonal Antibody bind to them

specifically.

membrane

(21)

More ～Fluorescence

Dye conjugated antibody

Excitation Emission

Immunophenotyping

Cell surface

marker (antigen)

CD No.

(22)

Negative

Dim

Bright

Positive

Many antibody bind to the cell that express

many antigen.

Dye conjugated antibody

Emit fluorescence ＝ AB bind to cell ＝ Positive

(23)

Negative

Dim

Bright

Cell numbers

dim ←

intensity

→ bright

Posotive

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P. 24

PE

FITC

+/‐

‐/+

+/+

‐/‐

2 FL Cytogram

Single positive

(FITC)

Single positive (PE)

Double negative

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There are many many fluorescence dyes ：

• For DNA ： Cell Cycle analysis, Apoptosis, etc. (DAPI, PI, etc.)

• Activated Oxygen, pH, Calcium con. Etc. ： Kinetics analysis (Flou-3, Flou-4, etc.)

• Gene expression : Fluorescence protein : GFP,CFP, YFP, mCherry, etc.

Many fluorescence dyes

GFP

G0 G1 S G2 M

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フローサイト

メトリー

Immuno‐ phenotyping DNA/RNA analysis Death, Apoptosis sorting Kinetics analysis

FCM applications

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Immunophenotyping～

Lymphocytes subset analysis

Lymphocytes

Monocytes

リンパ球は以前に増して機能的な多数のサブセットに分類されるが形態学的にこれらのサブセットを識別することは困難であり、フローサイトメトリーにより機能的な各サブセットの目印(マーカー)となるものを指標として, サブセットの分類や解析を行う CD：Cluster of differentiation T helper T Cytoxic 代表的マーカー CD2, CD3, CD5, CD7,

CD4

CD2, CD3, CD5, CD7,

CD8

CD5, CD19, CD20 , CD21 CD2, CD7, CD16, CD56 CD14 T Cell

Cell type B Cell NK Cell Monocytes

Typical

marker

(28)

Positive region made refer to Negative control

% of positive region

Immunophenotyping～

Lymphocytes subset analysis

Multi-Color analysis ： setting of positive region

(29)

FS

L

M

G

Gating

全体

Make a gate region around

lymphocytes population

Immunophenotyping～

Lymphocytes subset analysis

(30)

（incorrect gate）

（No gate = all events）

（Only in the gate）

30

(31)

FITC

PE

PE‐Cy5

T 細胞

CD3‐FITC

CD56‐PE‐Cy5

Lym subset

Immunophenotyping～

Lymphocytes subset analysis

Multi-Color analysis

CD19

CD20

CD56

CD16

CD3

CD2 CD5 CD7

CD4

Th

CD8‐FITC

CD3‐PE‐Cy5

T細胞subset

CD3

CD8

CD2 CD5 CD7

Tc

B 細胞

NK 細胞

CD3

CD2 CD5 CD7 CD4 CD8

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Immunophenotyping～

Lymphocytes subset analysis

Fluorescence emission is very broad. The part of it overlap on other detectors. It is necessary to

set the compensation for view the data plot correctly.

Multi-Color analysis ： Compensation

PE/Cy7

BV421 FITC PE APC PerCP/

Cy5.5

Pacific Blue

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FITC’s emission

PE’s emission

Band pass

Detection by FL2 (PE)

Detection by FL1 (FITC)

No compensation

FITC overlap to PE detector, PE overlap to FITC detector.

FL1 FL2 FL1 FL2

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Compensation are applied.

subtraction

Compensation Matrix

Band pass filter

FL1 FL2

FITC’s emission

PE’s emission

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FITC Single positive control sample

Under compensation

Over compensation

Good compensation

Immunophenotyping～

Lymphocytes subset analysis

(36)

Point

1. Reactivity

2. Antibody reagent

※

Antibody clone, maker, product lot.

3. Conjugated Dye

4. Check the overlaps.

Negative

Performance

definition

Positive

CD4/FITC

Vendor Median values Resolution

Positive Negative M(pos) / M(neg)

A 217,4 3,4 64 B 105,6 3,18 33 C 162,8 3,29 50

Immunophenotyping～

Lymphocytes subset analysis

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CD3-FITC CD3-PE CD3-APC

Emission intensity are different between each dyes.

Stain Index = D/W

D =陰性ピークと陽性ピークの中央値(Median)の差 W = 2 x rSD (robust standard deviation)

Holden Maecker & Joe Trotter, Nature Methods 5, (2008)

Immunophenotyping～

Lymphocytes subset analysis

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Immunophenotyping～：T

_Reg

identify

CD3‐FITC

CD4‐APC

CD25‐PE‐Cy7

CD127‐PE

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DNA analysis～Cell cycle

Stain with DNA binding dye (PI, etc.)

⇒ Fluorescence Intensity ∝ DNA Content

（intensity of G2 phase cells are 2 times G0/G1 phase cells）

Tumor cells often aneuploidy.

Cell Cycle＝increase of the cell

G0～G1～S～G2～M～G1～S～G2～M～G1～・・・・

Classic and important application

G0 G1

S G2

(40)

Only in Gate

DNA analysis ～ Cell cycle analysis

Technical Tip1: Doublet discrimination

All events

Area Height Width Heig ht

(41)

From : http://www.bio‐protocol.org/e973

蛍光パラメーターのパルスAreaおよびwidthを用いる電

気的ダブレット除去により、2つの細胞が結合しているダ

ブレットや細胞凝集物によるデータのアーティファクトを

除くことができる。

1. 細胞間のわずかな差(DNA等）を定量的測定の精度

2. 解析データの直線性および正確さ

3. ソーティング時の純度

の向上につながります。

DNA analysis ～ Cell cycle

Technical Tip 1: Doublet discrimiation

Width Ar ea

(42)

sample/sheath

Laser Laser

FL Lin

event

CV（％）= 100×（Peakの標準偏差）/（Peakの平均値）るアプリケション

High Flow Rate > CV large

適用：細胞マーカー解析（免疫蛍光法、蛍光抗体法）、蛍光タンパク質発現解析など、蛍光データを Log 表示で解析するアプリケーション

FL Lin

event

Low Flow Rate > CV small

適用： Cell Cycle Analysis（細胞周期解析）など、蛍光データ を Lin 表示で解析するアプリケーション

DNA analysis ～ Cell Cycle analysis

Technical Tip 2: sample pressure v.s. CV

Sample

pressure :

High

Sample

pressure :

Low

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目的の遺伝子に蛍光タンパク遺伝子を連結し、細胞に導入

→目的遺伝子が発現すると、蛍光タンパク遺伝子も発現

→蛍光タンパクがある細胞＝遺伝子発現細胞

遺伝子発現細胞の蛍光標識

・目的遺伝子を発現する細胞を解析、ソーティング

レーザー：

405nm

,

488nm

,

561nm

Gene expression ～ Fluorescence protein

CFP GFP CFP DsR e d DsRed HcR e d

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6/9/2015 44 BSC-A FSC-A BSC-A FSC-A

E. coli

Micro biology ～ cell count

例：細菌計数アッセイ：細菌の同定に使用する複数の蛍光核酸染色剤の混合液と正確なサン

プル量の測定を可能にする校正済みビーズ懸濁液が含まれている

bacteria/Yeast

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Intra celluler protein ～Cytokine assay

Intra celluler Cytokine Staining Assay

特長：

• 刺激による細胞のサイトカイン産生能力を検出できる

• 細胞表面マーカーと組み合わせることで、ヘテロなサンプル中の特定のポピュレーションを絞っ

て観察することも、異なるポピュレーションを比較することも可能

• 同時に複数のサイトカインを検出することができる

http://www.bdbiosciences.com/jp/research/ics/techniques/

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フローサイトメトリーによる実験の流れ

準備段階

•使用する抗体・蛍光色素の割り当てを計画

•細胞の準備・校正用サンプルの準備

フローメーターの最適化 •シース流の安定化 •ソーティングの場合は液滴形成・偏向電圧の調整 •PMT検出器電圧の最適化 •蛍光漏れ込み補正 •標的目標の解析・分取用ゲートを決定サンプルの解析・ソーティング

•ソーティング時の細胞濃度に注意

•液滴の安定性をモニタリング

•実験目的にあわせて回収容器を選別

(47)

フローサイトメトリーの特徴

☆高速測定：最大数万細胞/秒での客観的測定が可能

☆マルチパラメータ解析：複数蛍光色素（ラベル）の組合せに

より多重パラメータデータを取得＆解析できる

☆高再現性：同一条件での測定が可能

☆高速分取：測定結果に従い、特定の細胞集団を

生きたまま分取可能

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6/9/2015 50

Blu-ray

Zoom up of Disc

Hi-Speed flow

Cell

Lens

Laser

Lens

Laser

Hi-Speed spin

Flow Cytometer

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Conventional cell sorters require adjustment of optical axis, droplet formation, side stream adjustment, decision of delay time by a dedicated operator. However,

SH800Z eliminates the need for these tasks. Also, the adoption of a plastic chip sorting, washing operation is no longer time-consuming. In addition, we have achieved a small 1/3 compared to conventional products by taking advantage of the integrated technology and compact mechanical design technology cultivated in the Blu-ray optical techniques.

As "personal" cell sorter, the SH800Z will contribute to improvement of the workflow efficiency, and cost reduction for everyone including customers who heavily use the sorter for their research.

Cell Sorter SH800Z

Next Generation Cell Sorter

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１．Full automatic setup

• about 10 min.

• no user’s adjustment

• unnecessary the FCM specialist

２．Easy maintenance

• disposable sort chip

• quick change of sample line

• no carriover

３．Compact body and 4 lasers

・ W ５５ｃｍ, D 55cm

(1/3 of conventional sorter)

・ 488,405,561,638nm

(53)

• One to four selectable and upgradable laser excitation options

405 nm, 488 nm, 561 nm, 642 nm

• Four to six PMT’s, field upgradeable, with user changeable filters

Any Fluorochrome Anywhere

• Flexible autoloader for 12x75 mm, PCR, or Eppendorf tubes, and more than 10 plate formats Flexibility at your fingertips, no extra charge

convenient affordable flow cytometry analysis

Cell Analyzer EC800

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１．Hybrid Flow cell (Laser and Electronics)

• Laser : Scatter(FS, SS), Fluorescence

(FL1-FL5)

• ＥＶ (Electronic Volume) : Impedance

54

EV

FS

SS

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P. 55

55

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P. 56

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Separation : Cells – Debris (EV v. s. FS)

Adhesion cell line sample data

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P. 57

24bit Wide dynamic ADC

２．Adjustment of gains is available after acquisition

Digital gain adjustment

Touch screen

57

Drag and Drop

1000 times than conventional

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P. 58

58

Compensation : No affected by Digital gain adjustment

Compensation

PMT’s Voltage Adjustment

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P. 59

59

３．Full automatic sampler

• Syringe sampling system

• Auto probe cleaning

• Tube and Well plate are available

• Side holder for Reagents

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P. 60

60

Tube and well plate

Well plate (24, 96, 384ウェル） are available

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The SP6800 Spectral Analyzer is Sony's newest innovative life science system fundamentally expanding the way cell and biomarker analysis can be performed. This system incorporates a unique optical bench, Blu-ray™ disc technology, and advanced algorithms to deliver some of the most accurate and precise data available. To improve accuracy of data, this system also provides unique functions to display and analyze cellular auto fluorescence and allows the user to easily automatically remove.

世界初 “スペクトル型” フローサイトメーター

(62)

c

SP6800

Revolutionary Optics System:

32ch array PMT

Unique 32 micro lens array to improving optical efficiency

High transparency Prisms 4ch PDA detects each event’s location

Unique Flow cell Chip

Micro Lens Array Auto Alignment

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6/9/2015 63 Sample flow Laser FL signal 蛍光波⻑ 個々の細胞の蛍光スペクトル

SP6800 measure FL intensity with 32 PMTs Wavelength Intensit y Wavelength 450nm – 800nm Spectrum Plot

Overlay Spectrum curves of many cells

Spectrum Plot

Wavelength (nm) Intensity In ta ns it y 32ch PMT FL Spectrum High Frequency. Low Frequency.