attune acoustic focusing cytometer training -...

AttuneTM Acoustic Focusing Cytometer Training

Manik Punj

Attune Training

Attune Training Agenda

Section 1 An Introduction to Flow Cytometry

Section 2 An Introduction to Acoustic Focusing

Hydrodynamic Focusing vs. Acoustic Focusing

Section 3 Instrument Systems

Optics, Fluidics, Electronics

2 | Life Technologies Proprietary & Confidential | 11/7/2011

Break

Section 4 Performance Tracking

Section 5 Software Overview

Ribbons and Menus

What is Flow Cytometry?

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

FLOW CYTOMETRY measurements are made as cells or particles in suspension pass individually through a flow cytometer instrument


cytometer 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

Flow Cytometry: What Can I do?

• Flow cytometric measurement records data from single cells

• Rapid statistical analysis on large numbers of cells are obtained

• Identifying subpopulations within a heterogeneous population

• Ability to identify cell populations on multiple characteristics


• Ability to identify cell populations on multiple characteristics

• Ideally suited for blood and other cells in suspension

• Ability to archive data

• Data format allows post acquisition analysis

Flow Cytometry Basics

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

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


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.htm

Particle Delivery: Hydrodynamic Focusing

Laser Cross Sectional Area

Hydrodynamic core

−narrow particle focus = narrow


−sh

eath

−sh

eath

−Intensity

−C

ount

−narrow particle focus = narrow distribution


Low Flow Rate High Flow Rate



−sh

eath

−sh

eath

−sh

eath

−sh

eath


−C

ount

broad particle focus = broad distribution



−sh

eath

−sh

eath

−Intensity

• Increase sample input volume = increase flow rate = decrease in pressure difference = increase core diameter

• Particle distributions broadened

• Instrument resolution decreased

• Volumetric sample rates = 25 ul/min –150 ul/min

Acoustic Focusing:

How does Attune™ Cytometer Differ from Conventional Flow Cytometers?


What is Acoustic Focusing? A century old phenomenon


No acoustic force With acoustic force

Acoustic Focusing Capillary

FocusedParticles/cellsCapillary

Piezo-electric device

~20cm acoustic waves – similar to ultrasound used to visualize a fetus in utero.

Laser


Particles/cellsCapillaryLaser

(~10µm high)

~ ~~~~ ~ ~ ~~ ~

Flow

•Sheath is not required to focus cells•Flow rate can also be increased while maintaining resolution•Flow rate past laser can be precisely controlled to very slow rates,

allowing more fluorescence and scatter signal per particle

Acoustic Focusing High Flow Rate

Laser Cross Section

Low Flow Rate


Acoustically focused

−sh

eath

−sh

eath

−sh

eath

−sh

eath

PiezoelectricTransducer

Acoustic Focusing Cytometry: Practical Considerations

• Acoustic forces cause cells to line up in the center of the capillary. No sheath flow is necessary for particle alignment.

• Flow rate past laser can be precisely controlled to

Piezoelectric ultrasonic device

CapillaryCells


• Flow rate past laser can be precisely controlled to very slow rates, allowing more fluorescence and scatter signal per particle (better sensitivity)

• Faster sample flow rates and speedy analysis of dilute samples facilitates rare event analysis

device

Laser(~10µm high)

Detector

Acoustic Focusing


Acoustically Focused SampleC

ount

−narrow particle focus = narrow distribution

Laser Cross Section


Acoustic focusing

−sh

eath

−sh

eath

−Intensity

−C

ount

Focus Cells Long Before They Get ThereVariable Laser Interrogation Times


High sensitivity mode/High sample rate Low sensitivity mode/High sample rate mode

Variable flow speeds

Standard

Laser Interrogation Point Laser Interrogation Point

High SensitivityTransit Time = 40 usec

Total Volume = 600ul/min

Velocity = ~0.5m/s

Standard SensitivityTransit Time = 10 usec

Total Volume = 2400 ul/min

Velocity =~2m/s

Controlling Cell Speed Without Hydrodynamic Focus

High sensitivity Standard sensitivity100 ul/min 100 ul/min0.5 meters/sec 2 meters/sec

FocusingSolution Manifold

Laser

Detector


Manifold

Cells

Piezoelectric ultrasonic device

Transit Modes and Times

• Standard (2400 ul/min total volume)

Pre-set Sample Input Rate (in ul/min)*

Focusing Fluid Input Rate (in ul/min)

Focusing Fluid to Sample Ratio

25 2375 95:1

100 2300 23:1

200 2200 11:1


• High Sensitivity (600 ul/min total volume)

500 1900 3.8:1

1000 1400 1.4:1

*note that the particle velocity and interrogation time remains constant regardless of the sample input rate since the total volume (sample and focusing fluid) is constant at this transit mode

Pre-set Sample Input Rate (in ul/min)

Focusing Fluid Input Rate (in ul/min)

Focusing Fluid to Sample Ratio

25 575 23:1

100 500 5:1

Questions ?


Questions ?

• Flow Cytometer is comprised of 3 subsystems:

• Fluidics - To introduce and focus the cells for interrogation

Section 2 - Instrument Systems


• Optics - To generate and collect the light signals

• Electronics - To convert the optical signals to proportional electronic signals for computer analysis

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

Three conditions needed for optimal interrogation:

• The stream transporting the particles should pass through the

Fluidics


• The stream transporting the particles should pass through the focal point of the laser beam.

• Optimally one particle should move through the laser beam at one time.

• Fluidics system needs to be free of air bubbles & debris.

Fluidics


Attune Focusing Fluid – is an isotonic, buffered, azide-free support/carrier reagent for transporting particles through the capillary assembly to the flow cell for laser interrogation. It contains a preservative and detergent designed to minimize bubble formation. Prevents sample from coming into contact with the walls of the flow cell (optical cuvette).

Attune™ Wash Solution – is a ready-to-use solution for removing cellular debris and dyes from the fluidics system of the instrument.

Fluids


Attune™ Shutdown Solution – is a 10X solution that prevents bubbleformation in the fluidics system of the instrument. Prepare a 1:10 dilution ofthe shutdown solution in deionized water.

10% Household Bleach Solution in deionized water (0 .5% Sodium hypochlorite) –decontaminates the fluidics lines. Prepare this solution fresh daily and use during the shutdown procedure.

Deionized water – used for diluting Attune™ Shutdown Solution and bleach,as well as for long-term storage of the instrument.

Collection Panel

Run Status indicated the event rate as events per second and the progression of sample collection

Collection criteria allows users to define the endpoint of collection


Collection controls allows users to run, record, stop sample or clear data

Collection mode allows users to run in high sensitivity mode or standard mode as well as choose sample input rate

Fluidics Functions

• De-bubble is a user-initiated function for clearing bubbles in the fluidics lines of the cytometer.

• Wash is a user-initiated system cleaning between sticky samples. This function requires user supplied 10% bleach solution.

• Unclog function is a user-initiated back flush operation to remove clogs from the sample probe and flow cell.

• Rinse flushes system between samples to minimize carryover. This


• Rinse flushes system between samples to minimize carryover. This function is run automatically between samples, but it can also be user initiated.

• Shutdown is an automated function that decontaminates, cleans, rinses and powers off the cytometer. This mode requires user supplied bleach, Attune™ Wash Solution, and Attune™ Shutdown Fluid.

• Startup primes the instrument fluidics with Attune™ Focusing Fluid.

• Stop is used to end any running script.

• Clear Error is used to erase any error prompts.

Status Lights


Routine Maintenance

Procedure Frequency

• Shutdown Daily

• Visual inspection of sample injection port Daily

• Visual inspection of fluidics tanks and connections Daily

• Visual inspection of syringe pumps Daily

• Computer maintenance Weekly


• Optical filter cleaning Monthly

• Fluidics maintenance As needed*

• Replacing syringes As needed*

• Changing focusing fluid filter As needed*

* The frequency of maintenance depends on how often you run the cytometer. If the Attune is to be un-used for a period of time exceeding two weeks, perform the shutdown function using distilled water.

Daily maintenance

29 11/7/2011 | Life Technologies™ Proprietary and confidential

Daily maintenance

Daily operation - visual inspections

• Fluidics compartment: make sure there is no fluid or salt residue on the floor of the

compartment, around the connectors, or tube junctions.

• Check the fluid levels. Fill/empty as needed

focusing fluid

wash solution

shutdown solution

waste


• Syringe compartment - make sure there is no fluid or salt residue on the floor of the

compartment

focusing fluid filter – change if there are any signs of debris/dirt or if the

focusing pump stays on for too long. If working correctly, the pump should only be

on seconds/less than a minute.

syringes – change if they leak or have salt residue build-up

Daily operation – startup / shutdown

• Startup Function

• Starts the fluidics, optics and

electronics

• Warms up the lasers (give 15

minutes before running samples)

• Initializes the syringe pumps

• Primes the instrument fluidics

• Shutdown Function

• Turns off lasers

• Cleans and rinses the fluid lines

• Refills fluid lines with shutdown

solution


• Ensures that:• all fluidic lines are clean

• Syringe pumps are filled with fresh

focusing fluid

• Lasers are warmed up to operating

temperature

• Ensures that:

• Fluid lines are filled with a solution

that prevents crystal formation and

bubbles

• Back up your experiments on a regular basis to a secondary storage device

• Defragment the hard drive of the computer weekly

Regular Computer Maintenance Procedures


• Minimize memory usage: -When planning the experiments remember to delete parameters that you do not need i.e. only collect the parameters needed

Questions?


Questions?

Optics

Band Pass FiltersDichroic Mirrors

50mW 405nm Violet Laser

20mW 488 Blue Laser


Laser Light ScatterForward Scattered light (FS) is proportional to cell-surface area or size.

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


.

..

....

.

. .

.Laser

FS detector

SS detector

Fluorescence- Common Definitions

Fluorescence - is the result of a three stage process in certain 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 know as background fluorescence that originates from endogenous sample constituents or from unbound or nonspecifically bound probes.

This Box is to cover up the word summary and arrow


Optical Filters


There are five types of optical filters used in flow cytometry:• Bandpass filter (BP)• Longpass filter (LP)• Shortpass filter (SP)• Dichroic mirror (DM)• Neutral density filter (ND)

Band Pass Filters

Long Pass

Short Pass


Dichroic Mirror

Laser Channel FilterWavelength

Range

488 nm Blue 20 mW laser

BL1 530/30 551-545 (FITC)BL2 574/26 561-587 (PE)BL3 690/50 665-715BL4 780/60 750-810FSC 488/10 483-493

638 nm Red 50 mW laser

RL1 660/20 650-670RL2 780/60 750-810SSC 638/10 633-643

Product Overview: Attune ® Laser Configuration


Laser Channel FilterWavelength

Range

488 nm Blue 20 mW laser

BL1 530/30 515-545BL2 574/26 561-588BL3 640LP >640

405 nm Violet 50 mW laser

VL1 450/40 430-470VL2 522/31 507-537VL3 603/48 579-627FSC 405/10 400-410SSC 405/10 400-410

•Compensation is the process by which we correct for "spillover“

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

•These emission spectra overlap, in some cases very significantly

Compensation

530/3046.8% FITC


46.8% FITC0.4% R-PE0% PerCP

575/2610.1% FITC53.9% R-PE0% PerCP

648 LP0.1% FITC8.8% R-PE99.7% PerCP

Uncompensated vs. Compensated Data


Questions?


Questions?

Electronics

Functions of Electronics:

• Converts detected light signals into electronic signals

• Electronic signals are process by the computer system

• Converts signals from detectors into digital data used for analysis


Electronics and Computers

Time

Vol

tage

SampleFlow

Laser

Voltage Pulse in PMT


Time

Vol

tage

Time

Vol

tage

Laser

Laser

PeakHeightWidthArea

Electronic Pulse

Vol

ts

Pulse Area

Pul

se H

eigh

t


Time(µ Seconds)

Pul

se H

eigh

t

Pulse Width

0

Threshold (“trigger”)

Electronics: Overview

FL2

Time

TimeFSC

FL1

Data

SS

C

FSC


Time

Time

FL1

SSC

Time

FL3

Data Processor

FL2

Doublets

Doublet Discrimination


Singlets

Questions?


• Allows you to monitor performance of the instrument

• Critical to ensure accuracy and sensitivity of instrument

• Includes:

•Running the same bead particle set (AttuneTM Performance Beads)

•Monitoring changes in the CV

Instrument Performance Tracking


•Monitoring changes in mean fluorescent intensity

•Tracking linearity of instrument

•Evaluating quantum efficiency and background

•Sets laser delay

•Sets the instrument’s performance baseline

• Provides information about all the lasers and detection channels

Attune Performance Tracking Beads• A mixture of beads of four fluorescence emission intensities in

equal concentration− Blank− Dim− Medium− Bright


• 3mL vial

PN: 4449754

Running Baseline

• Uses performance tracking beads

• CSV file obtained from Applied Biosystems® website

• Performed any time a new lot of AttuneTM Performance beads are used

• Performed after any major maintenance on the instrument

• The percent half-peak coefficient of variation (%HPCV) of the brightest


• The percent half-peak coefficient of variation (%HPCV) of the brightest bead is recorded

• PMT is adjusted to place the brightest bead at target MFI values, and voltage values for each channel are recorded

• Relative quantum efficiency (rQ) and relative Background (rB) is calculated for each channel

• Linear regression is calculated and recorded

• Laser delay setting is also automatically calculated

Running Daily Performance Check

• Performed after baseline values have been defined

• AttuneTM Performance Beads are used to run daily performance measurements to track the performance of the cytometer

• Run the performance test at least once per day that the instrument is used

• Determines the voltage required to place the brightest bead in the target MFI, and calculates the delta PMT voltages as compared to the baseline.


• %HPCV of the bright bead is recorded

• Relative quantum efficiency (rQ) and relative Background (rB) is calculated for each channel

• Linear regression is calculated and recorded

• Laser delay setting is also automatically calculated

• Levy-Jennings charts provides a record of %HPCV and PMT voltage to check for shifts and trends

Daily Performance Check


Levey-Jennings Reports



A calibration curve plot showing limit of detection (LOD), limit of

quantification (LOQ), dynamic range, and limit of linearity (LOL).

PMT Voltage

What to do if performance check fails

1. Repeat performance check

2. Check the Performance test troubleshooting section in the users

guide.

3. If additional help is required, don’t hesitate to call .

4. Call: 800-831-6844 or 1-800-327-3002, option 4 (5 am-5 pm PST), or

email: [email protected]

5. The AB Call Center will open a call or simply transferring the call to

the TAC group if assistance is needed.

6. Provide the serial number and contact information


6. Provide the serial number and contact information

www.appliedbiosystems.com/support

Request instrument repair

Other maintenance

59 11/7/2011 | Life Technologies™ Proprietary and confidential

Other maintenance

Other maintenance - Optics

Filter holders


• Check for dust or scratches

• Remove any dust from the surface with a blower (compressed gas) or a soft brush.

• If necessary, gently clean using a clean lens cloth and lens cleaning solution or MeOH

• Frequency – monthly or less

Other maintenance optics• Important notes:

− Always allow at least 10 min for the lasers to reach operating temp

− Powering the instrument on/off within 30 minutes can decrease the laser lifetime.

− The “Shutdown” and “Wash scripts” powers off the lasers automatically, allow at

least 10 min for the laser to reach operating temperature

− If you cancel shutdown, allow at least 10 min for the lasers to reach operating

temperature


Other maintenance – Fluidics decontamination

• Potential problem - contamination− Check for cloudiness or debris

− Discolored solution or brown marks on sensor

• Cleaning instructions: User bulletin # 4468798

Jun 2011

• Frequency: monthly

• Perform ‘system flush’ if the instrument will not


• Perform ‘system flush’ if the instrument will not

be used for ≥ 2 weeks

• Replacement part #s

- 1L waste # 90039272

- 1L focusing fluid # 90039273

- 250 ml wash soln # 90032053

- 250 ml shutdown soln # 90039274

Other maintenance - focusing fluid filter and syringes

Focusing Fluid Filter


Syringes/Pumps door10ml Syringe 1ml Syringe

Other maintenance – Focusing fluid filter

Crack

Focusing fluid filter PN 4456564


� The filters will grow some contamination over time. If you can see brownish red

spot, and if the filter is intact, it should still keep this growth out of the bottle.

� The focusing fluid filter air gap needs to point down ( )

� Replacing focusing fluid filter every 6 months reduce the risk of any potential

contamination in the lines.

� Don’t hesitate to replace focusing fluid filter !

Other maintenance - Syringes

• Potential problem:

− Check for leaks

− Erratic fluid draws from fluidics tanks or SIP

− No fluid draws up from the fluidics tanks or SIP

• Part numbers:

- 1 ml syringe 4452079


- 1 ml syringe 4452079

- 10 ml syringe 4452819

• Frequency: as needed

Syringe replacement – directions in manual (V 1.2.5)• Run “Shutdown script”. The plunger will be lowered and instrument will turn off

• Open the Pump door and remove the plunger lock screw

• Unscrew the syringe from the valve by rotating it counter-clockwise

• To install a new syringe, pull the plunger down

• Align the syringe with valve and syringe plunger holder assembly

• Rotate clockwise until the syringe end cap seal hits the bottom of the valve

• After bottoming out, rotate clockwise ¼ turn to ensure complete seal


• After bottoming out, rotate clockwise ¼ turn to ensure complete seal

• Align the hole in the plunger with the hole in the plunger holder assembly

• Insert the plunger lock screw and tighten

Notes:

• No tools should be used to tighten the syringe to the valve.

• Over tightening the syringe beyond the above recommendation could result in

damage to the syringe and/or valve.

• Proper syringe-to-valve seal is crucial for the operation when fluids are cycling

through the system.

Other maintenace: System flush / long term shutdown

• Perform system flush if the Attune will be shutdown for > 2 weeks

− 1. Replace all fluidics container (focusing fluid, wash and shutdown tanks) with

de-ionized water

− 2. Run startup

− 3. Run shutdown function using deionized waster on the SIP instead of bleach

− 4. When shutdown is complete, empty all fluidics tanks and allow to air dry

• This process will prevent crystals from clogging the fluids system.


• This process will prevent crystals from clogging the fluids system.

Other maintenance: Software • Set up user accounts with operator privileges

• Browser

− Minimize the number of experiments displayed in the browser

− Close all experiments except the one that is currently active (by clicking on the

arrow to minimize experiments)

− Remove experiments from browser but keep data on hard drive

Right click on the experiment name,


Right click on the experiment name,

Click on delete experiment – the following options are displayed

> Yes – will delete the experiment and associated files

> No – will delete the experiment from the browser and keep the associated files (on

the hard drive)

> Cancel – will make no changes

• Back up your experiments on a regular basis to a secondary storage device

• Check thumb drives for viruses, run Antivirus software to remove threats

• Defragment Hard Drive and run Disk Cleaner (Monthly)

• Minimize memory usage:

Instrument settings - delete parameters not needed i.e. only collect

Other maintenance - regular computer maintenance procedures


Instrument settings - delete parameters not needed i.e. only collect

the parameters needed

Summary Maintenance

Procedure Frequency

• Start up / Shutdown Daily

• Visual inspection of sample injection port Daily

• Visual inspection of fluidics tanks and connections Daily

• Visual inspection of syringe pumps Daily

• Computer maintenance Weekly




• Fluidics maintenance Monthly

• Replacing syringes As needed*

• Changing focusing fluid filter As needed*

• The frequency of maintenance depends on how often you run the cytometer.

• If the Attune is to be un-used for a period of time exceeding two weeks, perform the shutdown function

using distilled water.

Software Overview

Main Features:

Tracking and Performance

Experiment Explorer


Collection Panel

Ribbons

Menus

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.© 2010 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners.

NOTICE TO PURCHASER: Limited Use Label License

The products shown in this presentation may be covered by one or more Limited Use Label License(s). Please refer to the respective product documentation or the Applied Biosystems website under www.appliedbiosystems.com for the


Applied Biosystems website under www.appliedbiosystems.com for the comprehensive license information. By use of these products, the purchaser accepts the terms and conditions of all applicable Limited Use Label Licenses. These products are sold for research use only, and are not intended for human or animal diagnostic or therapeutic uses unless otherwise specifically indicated in the applicable product documentation or the respective Limited Use Label License(s).

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