Attune NxT: Acoustic Focusing Flow Cytometercore.nhri.org.tw/userfiles/file/Attune_NxT_basic_training_NHRI(1).pdf · Attune NxT: Acoustic Focusing Flow Cytometer Experience the Ultimate

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Attune NxT: Acoustic Focusing Flow Cytometer

Experience the Ultimate Speed without Compromising Performance

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Introduction to Flow Cytometry

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What is Flow Cytometry?

CYTOMETRY is the measurement of physical or chemical characteristics of cells or particles

FLOW CYTOMETRY measurements are made as individual cells or particles in flowing stream pass through a flow cytometry instrument

• Performed on single cell suspensions

• Provides discrete measurements from each cell in the sample

• Provides a distribution of the measured characteristics in the sample

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The Flow Cytometer – “A Different kind of Microscope”

Light

Sample

Laser

Data Analysis

Flow cytometry is similar to a microscope.

Microscope produces an image of a cell

A flow cytometer collects and quantifies scattered light and fluorescence. The output produced is numbers.

5

What makes a Flow Cytometer?

Abbreviated : FCM

Flow Cytometer is made up of 3 subsystems:

• Fluidics

To introduce and focus the cells for interrogation

• Optics

To generate and collect the light signals

• Electronics

To convert the optical signals to proportional electronic signals for computer analysis

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1. Cells in a single profile pass through the flow cell

� Cells Focusing

2. Laser hits individual cell passing through the narrow tube called flow cell

� Interrogation Point

3. Deflected light hits a series of detectors (PMTs)

4. The signal from detectors are interpreted by a computer

Flow cell figure taken from http://www.med.umich.edu/flowcytometry/training/lessons/lesson1/index.htmSchematic Diagram of cytometer taken from Immunology: The Immune System in Health and Disease, 3rd ed. By: C. Janeway and P. Travers

Principle of Flow Cytometry

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What happens to light when it hits a cell?

Laser Light Scatter

� When laser light interacts with a cell, light is scattered in all directions

� The light scatter depends on size and internal complexity of the cell

� We look at Forward Light Scatter and Side Light Scatter.

8

What are Forward and Side Scatter?

Laser Light Scatter

� Forward Scattered light (FSC) is proportional to cell-surface area or size

� Side-scattered light (SSC) is proportional to cell granularity/internal complexity of the cell. SSC is usually collected at 90 degrees to the laser beam

.

..

....

.

. .

.Laser

FS

C detector

SSC detector

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Laser Light Scatter

Measurements of FS and SS

- allow for differentiation of cell types in a heterogeneous cell population,

- look for changes in cell health.

Ammonium chloride lysed whole bloodAged culture of Jurkat T cells: green are live cells & red are dead cells

FSS

S

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Fluorescence 3-step process

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Fluorescent Light – Common Definitions

• Fluorescence : is the result of a three stage process in molecules called fluorophores, or fluorescent dyes.

• Absorption spectrum : The wavelength range over which a fluorescent compound can be excited

• Emission spectrum : The range of emitted wavelengths of a fluorescent compound, it is a longer wavelength than the absorption wavelength

• Auto-Fluorescence : Otherwise known as background fluorescence that originates from endogenous sample constituents or from unbound or nonspecifically bound probes

What happens to light when it hits a fluorescently labeled cell?

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Fluorescence Spectrum

Stokes

Shift

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Three Color Immunophenotyping Experiment

Sample: PBMCs from Human Blood

Measure: % T-lymphocytes CD4+% T-lymphocytes CD8+

T-lymphocytes specific

Antibody Fluorescent Probe

1 Anti-CD3 Alexa Fluor® 488

2 Anti-CD4 R-PE

3 Anti-CD8R-PE Alexa Fluor®

700 dye tandem

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Alexa Fluor® 488 fluorescence (CD3)

Three Color Experiment – Data presentation

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R-P

E A

lexa

Flu

or®

700

fluo

resc

ence

(C

D8)

Three Color Experiment – Data representation

Alexa Fluor® 488 fluorescence (CD3)

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Attune ® NxT SystemsFluidicsOpticsElectronics

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The purpose of a fluidics system is to transport pa rticles in a fluid stream to the laser beam for interrogation

For optimal illumination, the stream transporting the particles should be in the center of the laser beam

Only one particle should move through the laser beam at a time

Fluidics system needs to be free of air bubbles & debris

Fluidics System

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Focused laser

shea

th

shea

th

Hydrodynamic core

Focused laser

shea

th

shea

th

High sample flow rate (e.g., 200 µL/min)

Low sample flow rate (e.g., 12 µL/min)

IntensityC

ount

Broad particle focus = Broad distribution

Intensity

Cou

nt

Narrow particle focus = Narrow distribution

Particle positioning in laser is important

Traditional hydrodynamic focusing

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Acoustic focusing

Prior to wrapping in sheath

IntensityC

ount


Intensity

Cou

nt


1,000 µL/min12.5 µL/min

High sample input flow rates allow for more sample flexibility

Acoustic focusing

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Comparable Results at Fast Detection Speed

Traditional Cytometers

µl/min

CV= 4.83 CV=6.12 CV=7.76

12 µL/min 35 µL/min 60 µL/min

Hydrodynamic Focusing Only

Attune NxT

µl/min

12.5 µL/min 25 µL/min 100 µL/min 200 µL/min 500 µL/min 1,000 µL/min

CV=2.99

CV=3.03

CV=2.76

CV=2.94

CV=2.70

CV=2.96

Up to10x Faster than Traditional Cytometers

Acoustically Enhanced Hydrodynamic Focusing

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Problems with Concentrated Sample

• Aggregation (Clogging)

• Coincidence

• Antibody non-specific binding (Higher background)

• Significant cell loss in tube dead volume and sample line

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Status indicator lights

Attune® NxT Fluidics System

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Focusing Fluid Reservoir

1ml SampleSyringe

Attune® NxT Fluidics System

Valve

Fluid LinesSensor Connections

Waste FocusingFluid

WashFluid

ShutdownFluid

Focusing Fluid Filters

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Determining Sample Concentration

• The Concentration Statistic can be selected from the Statistics Ribbon

• Values are displayed as Events/µL

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Attune ® NxT Optical System

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Four Laser Optical Configuration

16 Detection Channels

• Violet 405 nm• 4 Colors• Optional SSC

• Blue 488 nm• 3 Colors• FSC/SSC

• Yellow 561 nm• 4 Colors

• Red 637 nm• 3 Colors

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Spatially Separated Lasers

• Spatially Separated Lasers

• All lasers spatially separated

• Co-linear lasers limit the combination of colors with similar emission

• Improved compensation for multi-color panels

• More choices for colors

• 6 color experiments with no compensation with 4 laser instrument

• Minimal compensation for popular dyes

• Example: FITC vs. PE

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561nm Yellow Laser

• Closest wavelength to PE excitation peak, provides better sensitivity to PE and PE tandems

• Optimal excitation for DsRed, mCherry, RFP, and other fruit proteins

• No spillover from FITC

561nm is close to the PE max. excitation peak

488nm488nm48

8nm

561n

m

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Filters

Bandpass Filter Longpass Filter

Shortpass Filter Dichroic Mirror

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Violet Blue Yellow Red - Standard Configuration

417LP

Blank

577LP

BlankBlank

654LP

780/60710/50

503LP

780/60

Blank

695/40

603/

48

Bla

nk

440/

5051

2/25

670/

1472

0/30

Bla

nk

585/

1662

0/15

695/

40

488/

10+O

D2

530/

3059

0/40

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Violet SSC Configuration

417LP

Blank

440/

50

710/50

603/

4851

2/25

Blank

405/

10+O

D2

417LP

710/50

603/

4851

2/25

415 dichroic LP405/10 BP - OD2 filterBlank 25 mm holder

Violet Side Scatter Kit

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No-Lyse No -Wash Applications

Sid

e S

catte

r

CD45 FITC fluorescence

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Dilute your samples, not your data quality

Immunophenotyping mouse whole blood presents a challenge due to the limited sample volume available (≤100 µL/day/animal) particularly in longitudinal studies.

Small volumes limit the ability to perform multicolor phenotypingexperiments with the required compensation and fluorescence-minus-one (FMO) controls

The sample dilution required in no-lyse, no-wash methods (to achieve low coincidence with red blood cells and platelets) generally dilutes the cell sample to such an extent that the time required to acquire sufficient events at the flow rates available in traditional instruments is extremely long.

No-Lyse No-Wash Applications

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Simplified sample preparation workflowsCollect Sample

Process toSingle cell Suspension

5-10 min

Lyse red blood cells

or enrich forwhite blood

cells 30-60min

Stain15-30 min

Wash 5-10 min Wash 5-10 min Wash 5-10 minAcquire Samples

Total sample prep time ~1.5-2.5hrs

Add 1-2 hours for intracellular markers

Collect Sample

Lyse red blood cells

or enrich forwhite blood

cells 30-60min

Stain15-30 min

Wash 5-10 min Wash 5-10 minAcquire Samples

XX X

dilute

Generic sample preparation workflow

No Lyse/No Wash sample preparation workflow

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NLNW using 405 and 488 nm SSC

Data courtesy Jordi Petriz, Josep Carreras Leukemia Research Institute, Barcelona, Spain

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1. Dual SSC – Blue Laser SSC/Violet Laser SSC

• Utilization of Violet Scatter to differentiate white from red blood cells

2. Fluorochrome-conjugated CD45 and Glycophorin-A Abs can be used to differentiate white and red blood cells

No Lyse/No Wash assays on the Attune® NxT

FSC (Blue 488 nm) (x106)

SS

C (

Vio

let 4

05 n

m)

(x10

6 )

SSC (Blue 488 nm)

SS

C (

Vio

let 4

05 n

m)

CD45 Pacific OrangeTM

Gly

coph

orin

A F

ITC

FSC (Blue 488 nm) (x103)S

SC

(B

lue

488

nm)

(x10

3 )

SSC (Blue 488 nm)

SS

C (

Vio

let 4

05 n

m)

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Attune® NxT Optical Configurations

Laser Configuration - #

detectors Pa

c B

lue

, C

FP

Pa

c G

ree

n

Pa

c O

ran

ge

Qd

ot

70

5

FIT

C,

GFP

, A

F48

8,

YFP

PE

, A

F56

8,

PI

PI,

PE

-TxR

ed

,

PE

-AF6

10

Pe

rCP

, P

E-C

y5

.5,

Pe

rCP

-Cy

5.5

PE

-Cy

7,

QD

80

0

PE

, A

F56

8,

PI,

DsR

ed

PE

-Tx

Re

d,

mC

he

rry

PE

-Cy

5.5

, P

E-

AF7

00

, P

erC

P-C

y5

.5

PE

-Cy

7,

QD

80

0

AP

C

AF7

00

AP

C-A

F75

0 (

Cy

7)

filters →

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0/5

0

51

2/2

5

60

3/4

8

71

0/5

0

53

0/3

0

57

4/2

6

59

0/4

0

69

5/4

0

78

0/6

0

58

5/1

6

62

0/1

5

69

5/4

0

78

0/6

0

67

0/1

4

72

0/3

0

78

0/6

0

Blue - 4

Blue-Red -7

Blue-Violet -8

Blue Yellow -7

Blue Red Violet -11

Blue Violet Yellow -11

Blue Red Violet Yellow - 14

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Attune ® NxT Electronics System

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Electronics

Function of Electronic System:

• Converts detected light signals into proportional electronic signals (voltage pulses)

• Electronic signals are processed by the on-board processor

• Converts electronic signals from the detectors into digital data used for analysis

• Interface with the computer for data transfer

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Sample Presentation: Voltage Pulse

Time

Vol

tage

Time

Vol

tage

TimeV

olta

ge

Laser

Laser

SampleFlow

Laser

Voltage Pulse in PMT

Peak:HeightWidthArea

Laser

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Time(µ Seconds)

Vol

ts

Pulse Area

Pul

se H

eigh

t

Pulse Width

0

Sample Presentation: Voltage Pulse

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Pulse to Parameters

Attune default: All parameters (A-H-W) on all channels

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In most cases, data analysis should include gating on single cells.

Linear Relationship of Pulse Height to Pulse Area

Pulse width double pulse height Pulse width double pulse area

Doublet discrimination

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• Every fluorescent molecule emits light with a particular spectrum unique to that molecule

• These emission spectra overlap and is in some cases very significant• Compensation is the process by which we correct for "spillover“

Compensation

AF488 R-PE PerCP-Cy5.5530/30 574/26 695/40 780/60

Laser 488nm

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Three Color Experiment

Compensation controls

1. Cells only (optional)2. Cells + Anti-CD3 only3. Cells + Anti-CD4 only4. Cells + Anti-CD8 only

Which Controls are needed to compensate?

Antibody Fluorescent Probe

1 Anti-CD3 Alexa Fluor® 488

2 Anti-CD4 R-PE

3 Anti-CD8 PerCP-Cy5.5

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Why do we need to compensate?

Uncompensated Compensated

Single stained control for PerCP-Cy5.5

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Compensation: Background Fluorescence Modes

• Auto Fluorescence Correction Choices:

• Negative Gate

• Unstained Control

• None

Background Mode When to Use?

Negative GateWith a “mixed bag” of controls such as cells and beads; or using different cell populations (lymphs and monos).

Unstained Control When all controls are of the same type (beads, all lymphs)

NoneRarely used but in cases where background is negligible or cannot be ascertained.

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Spillover Matrix

• At the end of Auto-compensation:

• Spillover Matrix is automatically calculated

• Compensation is applied on all samples

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On Plot Compensation Adjustment Tools

• Plot Compensation adjustment tools displayed

• Tool allows manual adjustment of a selected plot

• % spillover

• Quadrant statistics

• Dragging populations

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Thank you!

Attune NxT: Acoustic Focusing Flow Cytometercore.nhri.org.tw/userfiles/file/Attune_NxT_basic_training_NHRI(1).pdf · Attune NxT: Acoustic Focusing Flow Cytometer Experience the Ultimate

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