introduction to the amnis imaging flow cytometry

Flow Cytometry Plus High

Content Imaging on the

ImageStream® MKII

Amal El-Mabhouh, PhD

Field applications Scientist

Introduction to the Amnis

Imaging Flow Cytometry

complexity simplified. | © Copyright 2019 Luminex Corporation 2

Now Part of LuminexAmnis® and Guava® Flow Cytometry Systems


Amnis History & Timeline• Founded in Seattle in 1999, by David Basiji and Bill Oryton

• 1st generation ImageStream®100 launched in 2005.

• 2nd generation ImageStream®X launched in 2009.

• Lower cost FlowSight® launched in 2011

• Amnis acquired by Merck/Millipore late 2011

• 3rd generation ImageStream®X MkII launched in 2012

• Non-imaging, CellStream® flow cytometer launched in 2018

• Acquired by Luminex corporation January 2019


Imaging Cytometry Allows for Probe Quantification

and Location

4


Standard Cytometry with Visual Confirmation

Human PBMC

60X Magnification

CD45-AF488

Nucleus-DRAQ5

Darkfield

10,000 cells imaged

CD45 vs SSC

basophils

eosinophils

monocytes

lymphocytes

neutrophils

Sid

e s

catt

er

Inte

nsity

CD45 AF488


Quantitative Morphological Analysis

Human PBMC

60X Magnification

CD45-AF488

Nucleus-DRAQ5

Darkfield

10,000 cells imaged

nuclear lobe count

basophils

eosinophils

monocytes

lymphocytes

neutrophils

Sid

e s

catt

er

Inte

nsity

CD45 AF488

0 1 2 3 4 5

0

500

1e3

1.5e3

2e3

2.5e3

Lobe Count_Morphology(M6, Ch6)_Ch60 1 2 3 4 5

Fre

quency

0

500

1e3

1.5e3

2e3

2.5e3

Lobe Count_Morphology(M6, Ch6)_Ch6

Fre

qu

en

cy

Neutrophils

0 1 2 3 4 5

0

20

40

60

80

100

120


Fre

quency

0

20

40

60

80

100

120


Fre

qu

en

cy

Basophils

0 1 2 3 4 5

0

500

1e3

1.5e3

2e3

2.5e3

3e3


Fre

quency

0

500

1e3

1.5e3

2e3

2.5e3

3e3

Kidney nuclei


Fre

qu

en

cy

Lymphocytes

0 1 2 3 4 5

0

50

100

150

200

250

300


Fre

quency

0

50

100

150

200

250

300


Fre

qu

en

cy

Monocytes

0 1 2 3 4 5

0

30

60

90

120


Fre

quency

0

30

60

90

120


Fre

qu

en

cy

Eosinophils


Flow Cytometry with ImagesImaging Flow Cytometry= Best of Both Worlds

Combines the speed, sensitivity and phenotyping abilities of flow cytometry

7

Single, focused

R3R3R3

R4R4R4

R3R3R3

R4R4R4

1e70 1e3 1e61e51e4-1e3

Intensity_Ch04

1e3

0

1e4

-1e3

1e5

1e7

1e6

Inte

nsity

_Ch0

5 Population Count %Gated Mean Ch04 Mean Ch05

R3-R4- 1846 62.5 642 50

R3 507 17.2 986 264836

R4 439 14.9 219405 419

R2

......

1e4 1e7-1e3 1e61e50 1e3

Intensity_Ch04

0.5

2

0

1

1.5

Nor

mal

ized

Fre

quen

cy

…with the detailed imagery and functional insights of microscopy.

Imaging flow cytometry enables applications that can’t be performed by

flow cytometry or microscopy alone.


Amnis Imaging Flow Cytometry

8


Principles of Operation: ImageStream Layout

9


ImageStream® Key Compnents

Single load or

96-well plate autosampler

Camera 1Camera 2

Spectral

Decomposition

Element 1

Spectral Decomposition

Element 2

Auto Focus

Flow Speed

“Pinhole” Splitter

Retro-reflectors

20X

40X

60X

785nm 642nm 592nm 561nm 488nm 405nm830nm

Mag.

EDF

Element 1

EDF

Element 2Flow Cell

5 Laser System

Full Color

Brightfield


Objectives

11


12 Channels 2 Camera System

12


TDI CCD technology provides superior sensitivity

Laser Core CCD Image Database

Time Delayed Integration (TDI)

• Amnis core technology

• Increased photon collection time provides superior sensitivity

• Object synchronization yields in-focus imagery


Objectives

14


Data From Amnis Imaging Flow Cytometers

15

Ease of Use

16


Daily ASSIST Self-Calibration17

Laser Alignment

Brightfield Illumination

Darkfield Illumination

Fluidic Performance

Focus Quality

Spectral Characteristics

Synchronization

Image Resolution

• All results logged with date and time stamp

• Uses a stable, low cost QC reagent run each day

• Takes about 40 minutes

Automatically calibrate/tests of critical parameters:


Simple Instrument Operation

18


IDEAS Image Analysis Software

1

• Wizards for simplified

image analysis.

Including Feature Finder.

• Automated

compensation for

complex color

combinations.

• Building blocks for

assisted analysis.

• 85 features per channel,

14 function masks, and

user defined features

help advanced users

with quantitative imaging

applications.

Tabular DataImage Gallery

Workspace

Quantitative Image Cytometry

20


Amnis Cytometry• The ImageStream statistical microscopy platform advances research…

But How??

• Science-driven conclusions require

Collection of critical data sets

Objective analysis backed by statistics

• The value of Amnis image cytometry is:

• The benefit that statistics brings to microscopy

• The benefit that imagery brings to cytometry

• The benefit of multiplexed single cell measurements

21


The Benefit Statistics Brings to Microscopy

22


The Benefit Imagery Brings to Cytometry

23

Cytometry typically measures intensity.

Focus

1e70 1e3 1e61e51e4-1e3

Intensity_MC_Ch07 DAPI

1e3

0

1e4

-1e3

1e5

1e7

1e6

Inte

nsity

_M

C_C

h11 A

ctn

647

Total

Intensity

Max

Intensity

Mean

Intensity

244881 889 335

244881280 135

244881856 359

These three cells have the same intensity.


The Benefit Imagery Brings to Cytometry

24

Total

Intensity

Max

Intensity

Mean

Intensity

244881 889 335

244881280 135

244881856 359

Cytometry typically measure intensity.

These three cells have the same red intensity.

Measuring not only total intensity but the location of the intensity can

characterize unique subsets of cells.

The cells differ greatly in the size, shape and location of that intensity.


APC/T Call Immune Synapse Analysis


Step 1: Identify Candidate Conjugates


Step 2: Identify CD3+/CD19+ Conjugates

Doublets

......

1e51e4-1e3 0 1e3

Intensity_CD19_AF488

-1e4

1e5

1e4

1e3

0

-1e3

Inte

nsity

_CD

3_P

E-T

R

Double Positive

Use CD3-TxRed and CD19-AF488 intensity on putative conjugates to identify double positives

FS


Step 3: Identify Conjugates with One APC

Double Positive

......

0 1e3500 1.5e3

Area_CD19_AF488

0.6

0.9

0.3

Aspect

Ratio_C

D19_A

F488

CD19+

Use Aspect Ratio and Area of CD19-AF488 imagery of double positives to identify conjugates with one APC


CD19+

......

1000 300200 400

Area_CD3_PE-TR

0.8

0.2

0.4

0

1

0.6

Asp

ect

Rat

io_C

D3_

PE

-TR CD3+

Use Aspect Ratio and Area of CD3-PE/TxRed imagery of single APC conjugates to ID T-Cell/APC conjugates

Step 4: Identify Conjugates with One T-Cell


Step 5A: Analyze Synapse Using Actin Image

• Recruitment of actin at the synapse results in a high local pixel intensity

• “Valley Mask” on DAPI nuclear image isolates synapse region

• Increased intensity of actin within Valley mask indicates synapse formation

Cell conjugates with no organized immunological synapse

Cell conjugates with an organized immunological synapse

Low synapse intensity

High synapse

intensity


Automatic identification of the immune synapse by masking


Step 6: Assess Conjugate Fraction of Sample


Internalization and Co-localization in Primary Plasmacytoid Dendritic Cells

33



Cy5-CpGB Internalization on y-axis

Similarity Bright Detail (x-axis) used to

measure organelle colocalization

Co-localization score In

tern

ali

za

tio

n s

co

re



35

24.6

CD71 (endosomes)

1.5 18.8

CD107a (lysosomes)

1.6 26.9 66.8


Autophagy LC3 Clustering Using the Feature Finder


Feature Finder Wizard


Feature Finder Wizard: Autophagy28


Feature Finder Wizard: Autophagy


41

Cellular Exosomes Analysis


42 Exosomes Uptake-Cellular derived exosomes (SW780) can be identified using the ImageStream

TEM

Western

ImageStream

PKH26 BF


43

Exosomes Uptake- Uptake of SW780 Derived Exosomes with SW780 Cells

43

4hr. Incubation with exosomes


44

Exosomes Uptake-Uptake of SW780 derived exosomes with SW780 cells


45

Pre-treat cells or exosomes with Heparin for 30min. Incubate for 4hr.

Exosomes Uptake-Uptake of SW780 derived exosomes with SW780 cells


NFkB Translocation in THP-1 and PBMCs

46

Cell with full

Translocation

Cell without

translocation

Cell with some

translocation

Nuclear Extracts

Western Result:

Dark Band No Band Dim Band 2Dim Band 1

• Quantifying translocation cell-by-cell is essential with heterogeneous

samples and / or incomplete translocation


NFKB Translocation in Whole Blood Circulating pDC

47

Analyze nuclear

localization in pDC using

the ImageStreamx

BF CD123 BDCA2 DAPI NF-kB

Incubate whole blood

with R848

Immunophenotype for

pDC (CD123-PE /

BDCA2-PETxRed) and

stain for NF-kB and DAPI


NFKB Translocation in Whole Blood Circulating pDC

48

Intensity_CD123

Inte

nsit

y_B

DC

A2

CD123+BDCA-

pDC (CD123+BDCA+)

Eosinophil

BF SSC DAPI CD123 BDCA2

17139

17684

22815

25063


16739

22664

23366

23556


11419

23944

37536

53381


Translocation Using Similarity

49

S = -2.07 S = +2.82

7-AAD Pixel Intensity

NF

-kB

Pix

el In

ten

sity

7-AAD Pixel Intensity

NF

-kB

Pix

el In

ten

sity

Untranslocated Translocated

7-AAD

image


Translocation Using Similarity

50

-2.2

-1.9

-0.9

-1.4

-1.5

-0.5 +0.1 +1.4 +1.8

+2.6

+0.9+0.5

+4.0

+3.3

+3.1

+2.8

“The benefit of multiplexed single cell measurements”


NFKB Translocation in Whole Blood Circulating

pDC

51

3-1 1 20

Similarity_NFkB/DAPI

0

4

5

2

3

1

No

r ma

liz

ed

Fre

qu

en

cy 0.05

Untreated

3-1 1 20

Similarity_NFkB/DAPI

0

2

3

4

1

5

No

r ma

liz

ed

Fre

qu

en

cy 0.66R848

BF BDCA2 DAPI NF-kB DAPI/NF-kB

256

2552

5268

6782

BF BDCA2 DAPI NF-kB DAPI/NF-kB

2226

3095

3509

3686


NFKB Translocation in Whole Blood Circulating

pDC

52

R848 - pDC activation

0.0

0.4

0.8

1.2

5 15 60

Time (min)

Sim

_N

FkB

/DA

PI,

Med

R848 - pDC activation

-0.4

0.0

0.4

0.8

1.2

0.00001 0.0001 0.001 0.01 0.1 1 10

[R848] (mg/mL)

Sim

_N

FkB

/DA

PI,

Med


Summary

• The ISX Mark II exceeds the fluorescence sensitivity of conventional

cytometers for easier experiment design and better data quality

• Imagery of every cell facilitates debris/doublet ID, improves gating, and

validates rare event data

• Quantitative image analysis of large populations enables a wide range of

functional assays that cannot be performed using flow cytometry or

microscopy

53

Thank you For Your Attention


Data Acquisition Form

55


Sample Preparation• Select an appropriate cell type for the magnification. Typically 2um to 100um

diameter.

• Select fluorochromes appropriate for your lasers.

• Fluorescently label cells and resuspend in 50ul running buffer in 1.5ml tube.

• Prepare 10^6 cells per sample (this is 2x10^7th cells/ml).

• Label single color compensation controls.

• Label good biological controls so you know the experiment worked.

• To start, experiments should be limited to around 10 samples.

• Most FACS staining procedures should work. See “Current Protocols in Cytometry” and the protocols and tips in the Amnis knowledgbase.

56


Sample Quality

57

introduction to the amnis imaging flow cytometry

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