attune™ acoustic focusing cytometer designed for...

Attune™ Acoustic Focusing Cytometer

Designed for Performance

2

Particle Delivery: Hydrodynamic FocusingConventional Instrumentation

Intensity

Count

Narrow particle focus = Narrow distribution

Laser Cross

Sectional Area

• Sample core is ‘pinched’ by fast flowing sheath

• Sample volume ratios of 100 – 1000

• Large ratios => low sample inputs

• Resolution of particle populations

sheath

sheath

Hydrodynamic core

3

sheath

sheath

Intensity

Count

Broad particle focus = Broad distribution

• Increased sample input = increase core size

• Particle distributions broadened

• Instrument resolution decreased

• Historically, low volumetric sample rates

used (25 µl/min – 150 µl/min)

Laser

Cross Sectional Area

Particle Delivery: Hydrodynamic FocusingConventional Instrumentation

4

Attune™: The Acoustic Idea

19891989 19981998 20052005 20062006 20072007 20082008 20092009 20102010

Kaduchak builds acoustic levitation device

Applies catastrophe theory to small droplets

No acoustic force With acoustic force

~ 1872 Dust observed acoustically levitating in organ pipes

5

Attune™ Idea to Market Time Line

19891989 19981998 20052005 20062006 20072007 20082008 20092009 20102010

Kaduchak builds acoustic levitation device

Applies catastrophe theory to small droplets

Gary Salzman suggests working w/Kaduchak to apply acoustic levitation/focusing to flow cytometry

Funding is non-existent in early years; project is boot-legged

Greg Goddard begins working on acoustic focusing capillaries with Kaduchak

Now a Life Employee

Mike Ward begins acoustic focusing postdoc in Kaduchak’slab

Now a Life Employee

First ‘stable’ and ‘repeatable’acoustic focusing device achieved

Kaduchak PI of NIH National Flow CytometryResource

Acoustic CytometrySystems (ACS) is born –G. Kaduchak and M. Ward

Mike Olszowy witnesses acoustic flow cytometry in Los Alamos

ACS collaborations begin with outside companies

Invitrogen acquires ACS

Greg & Mike Ward on-site in Eugene

6

Line Driven Tubes Containing Fluids

Particles/cells

Background fluid

Capillary

Line source

Potential minimum

Force potential experienced by

an erythrocyte in PBS

-100

-90

-80

-70

-60

-50

-40

-30

50 100 150 200

20

40

60

80

100

120

140

160

180

200

Key Points:• Tight particle focus at low linear

velocities• Particle concentration effect

(dilute samples)

7

Acoustic Focusing in Action

10 µm fluorescently-labeled beads

8

Acoustic Focusing = Better Precision

Acoustic focusing

sheath

sheath

Acoustic focusing

sheath

sheath

Acoustically focused sample stream

Laser


Laser


9

Acoustically Focused Sample

Acoustic focusing

sheath

sheath

• Volume Ratio of Sheath : Sample is 1 – 100

Intensity

Count

Narrow particle focus = Narrow distribution

Highest volumetric sample throughput

Sample velocity can be controlled

with adjustable dwell times

Laser Cross Sectional

Area

10

Hydrodynamic Focusing

Acoustic Focusing

Sheath

Sheath

Samplea) b) c)

Hydrodynamic focusing

Acoustic Focusing

• Wrap sample with a slower flowing sheath

• Optics stay clean

• Sheath:Sample ratios of 1:1 to 100:1 allowing transition from

hydrodynamically focused samples to acoustically focused samples

• Capable of running concentrated AND dilute samples

The Meaning of Precision

11

Applications Where the Attune™ Cytometer Delivers Critical Performance Value to Scientists:

• Cell Cycle Analysis and Proliferation

• Phospho Protein / Intracellular Analysis

• Rare Event Detection

• Immunophenotyping

• Apoptosis

Applications – Cell Cycle Analysis and Proliferation

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G0G1: 41.73% Mean: 50.34

− CV: 4.83%

G2M: 20.44% Mean: 98.67

− G2/G1: 1.96

S-Phase: 37.83% Mean:73.46

G0G1: 40.16% Mean: 50.00

− CV: 6.12%

G2M: 20.89% Mean: 98.00

− G2/G1: 1.96

S-Phase: 38.95% Mean:73.19

G0G1: 44.60% Mean: 49.97

− CV: 7.76%

G2M: 29.23% Mean: 95.13

− G2/G1: 1.90

S-Phase: 26.17% Mean:69.12

Hydrodynamic Focusing Instrument Cell Cycle Data

Low - 12 µl/min

Medium - 35 µl/min

High - 60 µl/min

As Sample Rates

Increase

Note overt

degradation of CV

values and changes in

data

13

G0G1: 42.81 % Mean: 53.65

− CV: 3.17 %

G2M: 18.47 % Mean: 104.32

− G2/G1: 1.94

S-Phase: 38.72 % Mean: 77.70

G0G1: 45.20 % Mean: 52.13

− CV: 3.22 %

G2M: 14.51 % Mean: 101.25

− G2/G1: 1.94

S-Phase: 40.29 % Mean: 74.27

G0G1: 41.97 % Mean: 53.51

− CV: 3.16 %

G2M: 19.86 % Mean: 104.04

− G2/G1: 1.94

S-Phase: 38.18 % Mean: 77.78

G0G1: 41.54 % Mean: 52.88

− CV: 4.16 %

G2M: 20.79 % Mean: 102.76

− G2/G1: 1.94

S-Phase: 37.67 % Mean: 75.74

G0G1: 40.25 % Mean: 50.21

− CV: 4.21 %

G2M: 21.20 % Mean: 97.41

− G2/G1: 1.94

S-Phase: 38.55 % Mean: 72.32

Attune™ Cytometer Cell Cycle Data Acoustic Focusing

25 µl/min

100 µl/min

200 µl/min

500 µl/min

1000 µl/min

As Sample Rates

Increase, little to no

change in CV and

data quality even at

sample rates >16x

faster than a

hydrodynamic

focusing cytometer

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Application:Hydrodynamic Focusing vs. Attune™ Acoustic Focusing -Cell Cycle

For Hydrodynamic Focusing, note that the CV climbs significantly as sample rate increases.

Whereas for Attune™ Cytometer, CV remains quite stable, though slightly higher at highest flow rate.

60 µl/min

1000 µl/min

Coefficient of Variation (CV)

= Standard Deviation/ Mean

CV

Hyd

ro F

ocus

_60u

l/min

Hyd

ro F

ocus

_35u

l/min

Hyd

ro F

ocus

_12

ul/m

in

15






• Apoptosis

Applications – Phospho Protein / Intracellular Analysis

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1:40 Ab dilution

Hydrodynamic Focusing Instrument

Phospho Specific Ab-PE

Attune™Normal Sensitivity

1:80 Ab dilution1:60 Ab dilution

Phospho Antibody Staining Hydrodynamic Focusing Instrument vs. Attune™

Note Dramatic Differences in CV and

% Positive

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• Apoptosis

Applications – Rare Even Detection

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Acoustic Sample Throughput Comparisons

100 200 300 400 500 600 700 800 900 1000

Maximum Sample Input Rate (µl/min)

Instrument 1

Instrument 2

Instrument 6

Instrument 5

Instrument 4

Instrument 3

Attune™

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Rare Event Analysis and Cell Counting Attune™

Instrument Acquisition

TimeCD34+ Cells/µl Derived Count

using Beads

CD34+ Cells/µl

Direct Measurement

Hydro Focus13 min 49

sec8.01

Hydro Focus – No

Direct Measurement

Attune™ 1 min 17 sec 7.91 8

Data gate on live cells

CD45 Pacific Blue™ Fluorescence

Counting Beads

CD45+Cells

CD34 PE Fluorescence

Counting Beads

Data gated on CD45 positive cells

CD34+ Cells

−S

SC

Data gated on CD45 positive cells

Counting Beads

CD34 PE Fluorescence

CD34+ Cells

−S

SC

Data gated on live cells

Counting Beads

CD45 Pacific Blue™ Fluorescence

CD45+ Cells

Hydro Focus

CytometerAttune™

20






• Apoptosis


21

Immunophenotyping:CD4-Pacific Blue™ vs CD8-Pacific Orange™

Lymphocyte gate

11 1

1

22

Sample % Positive % Negative Signal: Noise

CD8 26.619 69.775 88.690

CD4 51.721 48.262 14.515

CD3 75.952 19.136 142.020

CD56 18.254 81.734 39.563

CD19 6.557 93.417 180.480

Attune™ 6-color Staining –

Common Lymphocyte Subsets

Cytotoxic T Cells

T Helper Cells

T Cells

NK Cells

B Cells

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Attune™ 6-color Staining – Dual Plots

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• Apoptosis


25

F2N12S Membrane Asymmetry Probe

Ratiometric dye is self calibrating

Independent of cell size, cell

concentration and instrument variation

5 minute labeling time, no wash

No special buffer required

Use with adherent cells also

a

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Dual labeling of F2N12S vs SYTOX® AADvanced™ dead stain ratio of 405ex with 530/30 and 585/42 ratio emission

control

apoptotic

1 1

1

1

Attune™

User Centered Innovation

28

UCI Approach - Hardware

Observation

Rapid Prototyping

New Prototypes

Major Pain Points:

• Sample introduction a “cave” on most instruments.

• High rates of fluid consumption.

• Fear of sample loss due to long set-up times.

29

Design Footprint

• Footprint designed for right- or left-handed operation

• Fits on standard lab bench and into standard tissue culture hood

• Plug into standard outlet (no additional infrastructure requirements)

Sample

Sample

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Tube Lifter/Multiple tube sizes

• Spring-load carriage accepts multiple tube sizes

• Dash-pot damping for gently lowering/reduced carry over

• 12x75, 1.5 and 2 ml centrifuge, 17 x 100 tubes

31

Multi-Function Indicator Lights

Aerosol Shield

Quick Release Drip Tray

UCI Approach - Hardware

“On-Board” Fluidics:

• > full day use without refill

• Alternating Male/female connectors prevent incorrect bottle placement

• Back lighting / attention cycling

Sealed under-tray:

• Holds full liter to capture leaks, spills

• Spans entire floor

• Sloped forward to bring drips forward – away from electronics

Optical Filter Storage Under Lid

32

UCI Approach – Graphic User Interface / Software

Observation

Rapid Prototyping

Story Telling / Synthesis

Major Pain Points:

• Engineering logic in most software.

• Non-intuitive, lacks modern software GUI approaches.

• Fear of lost data from incorrect instrument settings.

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Software / Reagents to operate

34

Daily Performance Report

35

Tracking of Daily Performance / Levey-Jennings Charts

36

Attune™ Acoustic Focusing Cytometer

Ergonomics:

� ~60 lbs. (27 Kg.) vs. >300 lbs. (136 Kg.) comp.

� Fluidics cart—not needed, fluidics on-board vs. >100 lb. (45 Kg.) comp.

� Fluidics consumption—estimate ~1 L per day full use vs. up to 7 L per day comp.

� Reduced footprint, 22”x17”x16”

� Fits on all standard lab benches

� Intuitive, easy-to-use, full-featured software

� Fits in standard tissue culture hood

� Whisper quiet

� Includes 24” monitor, tower computer mouse and keyboard

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Acoustic Technology Web Support

● Web-based Attune™ Technology videos etc.:

− www.appliedbiosytems.com/attune> ‘virtual’ walk-through of instrument with Mike

● Flow Cytometry Reagents from Invitrogen:− http://www.invitrogen.com/site/us/en/home/Pr

oducts-and-Services/Applications/Cell-and-Tissue-Analysis/Flow-Cytometry.html

● Violet Laser Reagent Resource Guide:− http://www.invitrogen.com/etc/medialib/en/file

library/cell_tissue_analysis/pdfs.Par.55940.File.dat/B-081478_Flow_Cytometry_Violet_Laser_Resource_Guide.pdf

● Tutorials on Flow and Data Analysis:− http://www.invitrogen.com/site/us/en/home/s

upport/Tutorials.html

38

Discover Life with Attune™

Attune™ - What new life will you discover?

• Flexibility to define Prochlorococcus and multiple heterotrophs

• Industry exclusive high sensitivity variable flow rate

• High power violet laser

• Direct cell concentration measurements

Detect and determine concentration of Prochlorococcus populations from ALOHA site Surf (5 m) marine water samples

Marine Biology

39

Be a Pioneer in the Detection of Circulating Endothelial Cells

Attune™ - How low can you go?

• Statistically significant rare event analysis in <1/10th the time

• Absolute counts of all populations

• Multi-parametric analysis

• No sample concentration required

Cancer Biology

40

Find the Drugs others Miss

Attune™ - What new drugs will you discover?

• Tighter CVs yield unprecedented detail

• Industry exclusive high sensitivity variable flow rate

• Unmatched intracellular antigen detection

• Discover the lower affinity compounds

Drug Screening

Hydrodynamic Focusing Instrument

Attune™Normal Sensitivity

Note Dramatic Differences

in CV and % Positive

41

Cell Proliferation Analysis by Dye Dilution

● Cell division results in equal partitioning of dye between daughter cells.

● Fluorescence of daughter cells is half that of parent cell

First

Generation

Second

Generation

Third

Generation

Fourth

Generation

Brightness

Nu

mb

er

of

Cell

s

CellTrace™ Violet stain

42

CellTrace™ Violet stain

Human peripheral blood mononuclear cells were stained with CellTrace™ Violet, and stimulated with CD3 and IL-2 for 7 days in culture. Cells were stained with CD4 Alexa Fluor® 488 and SYTOX® AADvanced™ dead cell stains prior to analysis on the Attune™ Acoustic Focusing Cytometer. Live CD4 positive cells were gated for CellTrace Violet plot, and using the algorithms in proliferation modeling software (ModFit LT™, Verity Software House) to provide statistics about each generation of cells in a population.

43

Multiplexed Analysis CellTrace™ Violet on the Attune™ Acoustic Focusing Cytometer

Several generations of live CD4+ T Cells as visualized in the Attune™ Acoustic Focusing Cytometer

CellTrace™ Violet Fluorescence

Num

ber

of cells

counte

d

Several generations of live CD4+ and CD4-T Cells as visualized in the Attune™Acoustic Focusing Cytometer.

CellTrace™ Violet FluorescenceC

D4-A

lexa F

luor®

488

Flu

ore

scence

attune™ acoustic focusing cytometer designed for...

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