Life technologies Invitrogen Tali User manual

user guide
Tali™ Image-Based Cytometer
Catalog Number T10796
Publication Part Number MAN0003766
Revision Date 28 April 2011

2
Contents
Product Contents....................................................................................................................................3
Introduction ..................................................................................................................4
Description of Tali™Image-Based Cytometer........................................................................................4
Specifications..........................................................................................................................................9
Methods ...................................................................................................................... 11
Getting Started......................................................................................................................................11
Guidelines for Performing Tali™Assays...............................................................................................14
Select a Tali™Assay ..............................................................................................................................15
Load Sample .........................................................................................................................................16
Focus Image..........................................................................................................................................18
Measure the Background (optional).....................................................................................................19
Run Sample...........................................................................................................................................20
Analyze Data .........................................................................................................................................25
Export Data ...........................................................................................................................................28
Maintenance ...............................................................................................................31
Update the Tali™Image-Based Cytometer Firmware..........................................................................31
Adjust the Camera Alignment ..............................................................................................................32
Calibrate the Tali™Image-Based Cytometer .......................................................................................34
Clean the Tali™Image-Based Cytometer .............................................................................................36
Appendix.....................................................................................................................37
Safety Information ................................................................................................................................37
Accessory Products ..............................................................................................................................39
Technical Support.................................................................................................................................40
Purchaser Notification..........................................................................................................................41

3
Product Contents
Tali™Image-Based
Cytometer
The Tali™Image-Based Cytometer (Cat. no. T10796) is shipped with the
components listed below:
Component Quantity
Tali™Image-Based Cytometer 1 each
Tali™Cellular Analysis Slides, box of 50 1 box
Tali™Image-Based Cytometer power cords, pack of 4 cords
(for U.S./E.U./U.K./Australia)
1 each
Tali™Image-Based Cytometer USB Drive
(includes the user manual)
1 each
Tali™Image-Based Cytometer Quick Reference Card (QRC) 1 each
Certificate of Analysis (CoA) 1 each
Tali™Calibration Beads* 1 kit
* Tali™Calibration Beads are shipped separately and include 1 tube each of Tali™
Green Calibration Beads, Tali™Red Calibration Beads, and Tali™Alignment
Beads.
Upon receiving the
instrument
Examine the instrument carefully for damage incurred during transit. Ensure that all
parts of the instrument, including accessories listed above, are included with the
product. Damage claims must be filed with the carrier; the warranty does not cover
in-transit damage.
See page 11 for instructions on installing the Tali™Image-Based Cytometer.
Registering your
instrument
Visit www.invitrogen.com/tali to register your instrument. You will be asked to
supply the serial number, your name, and your contact details. Registering your
instrument ensures that you will receive notifications of software upgrades and
information on new assays for use with the Tali™Image-Based Cytometer.
Intended use For research use only. Not for human or animal therapeutic or diagnostic use
Purpose of this
manual
The Tali™Image-Based Cytometer User Manual is designed to help you learn how
to use the instrument. The manual includes step-by-step instructions for instrument
set-up, data collection, data handling, and instrument maintenance; it does not
contain assay specific protocols.
For information on specific Tali™Assays and detailed protocols, refer to the product
information sheets (PIS) supplied the individual assay kits. For your convenience,
these PISs are included in the Tali™Image-Based Cytometer USB Drive; they can
also be downloaded at www.invitrogen.com/tali.

4
Introduction
Description of Tali™Image-Based Cytometer
Tali™Image-Based
Cytometer
The Tali™Image-Based Cytometer is a 3-channel (bright field, green fluorescence,
red fluorescence) benchtop assay platform that uses state-of-the-art optics and
image analysis to perform assays for cells in suspension, including GFP and RFP
expression, apoptosis, cell viability (live, dead, and total cells), and cell counting
assays. It is compatible with a wide variety of eukaryotic cells.
Using only 25 µL of sample volume, the Tali™Image-Based Cytometer takes
10 seconds to 2 minutes for a typical assay, depending on complexity of the assay
and number of fields captured.
In addition to the bright field channel, the Tali™Image-Based Cytometer features
two fluorescent channels (green and red), enabling it to simultaneously count green
or red fluorescent stains, as well as cells expressing GFP and RFP.
The Tali™Image-Based Cytometer offers an intuitive user interface, and provides the
option to save data and generate a report, which can then be transferred to a PC
using the USB drive supplied with the instrument or available separately.
The Tali™Image-Based Cytometer is supplied with disposable Tali™Cellular
Analysis Slides (see page 8), which are also available separately (see page 39 for
ordering information).
See next page for details on various parts of the Tali™Image-Based Cytometer.
Features Important features of the Tali™Image-Based Cytometer are:
Provides a user-friendly, benchtop design for simple, fast, and highly accurate
three-parameter population analysis.
Uses the Tali™assays optimized for the Tali™Image-Based Cytometer.
Uses disposable Tali™Cellular Analysis Slides that eliminate washing steps and
cross contamination between samples.
Presents comprehensive data with graphic reports and allows the export of data
as .csv (comma separated value), .jpg, and .pdf files for archiving and sample
comparisons.
Continued on next page

5
Description of Tali™Image-Based Cytometer, continued
How Tali™Image-
Based Cytometer
counts cells
The Tali™Image-Based Cytometer simultaneously captures a series of bright field
and fluorescent images (i.e., fields of view) of the sample in the Tali™Cellular
Analysis Slide and uses sophisticated digital image analysis algorithms to determine
total and fluorescent cell counts and calculate their concentrations. All Tali™Assays
share the same basic workflow and user interface, but differ in which channels the
algorithm is used for analysis.
Using the touch screen user interface of the Tali™Image-Based Cytometer, you can
select the number of images (i.e., fields of view) to capture for each experiment and
gate the counts by cell size and/or fluorescence intensity (where applicable). After
capturing, you can select each field of view for review, zoom in and out of the
selected field of view, display channel-specific layers (i.e., images captured through
bright field, green-fluorescent and red-fluorescent channels or any combination of
them), and identify the cells counted through each channel.
Overview of Tali™
Assays
The Tali™Image-Based Cytometer incorporates image-based cell counting and
fluorescence detection algorithms to perform the assays listed below for cells in
suspension. The instrument captures up to 20 fields of view per sample with each
field of view covering 0.233 µL of the sample, and presents the relevant data (e.g.,
cell count vs. cell size, cell count vs. fluorescence) in tables and histograms. The data
from the analysis, including the image files, can be downloaded to a USB flash drive
immediately after the assay and transferred to a computer for sample comparisons.
Viability – determines the number and proportion of viable and dead cells
using the Tali™Viability Kit – Dead Cell Red (Cat. no. A10786), which stains the
dead cells red.
Apoptosis – distinguishes between apoptotic, dead, and live cell populations
using the Tali™Apoptosis Kit (Cat. no. A10788), counts the cells in each
population, and calculates relative amount of each population in the sample.
GFP/RFP – counts and calculates the relative amounts in the sample of cells
expressing GFP or RFP (or stained with any green- or red-fluorescent stain), as
well as any unstained or non-fluorescent cells. The GFP/RFP assay menu also
includes combined GFP/RFP and viability assays. Use the Tali™Viability Kit –
Dead Cell Red (Cat. no. A10786) with cells expressing green fluorescent proteins
and use the Tali™Viability Kit – Dead Cell Green (Cat. no. A10787) with cells
expressing red fluorescent proteins.
Quick count – provides quick and accurate cell counts without the need for
staining your cells and determines the concentration of your sample and the
average cell size.
For more information on Tali™Assays and detailed Tali™Assay-specific protocols,
refer to the product information sheets (PIS) supplied the individual assay kits. The
product information sheets can also be downloaded at www.invitrogen.com/tali.
Continued on next page

6
Description of Tali™Image-Based Cytometer, continued
Front view of Tali™
Image-Based
Cytometer
The front view of the Tali™Image-Based Cytometer illustrating the various parts of
the instrument is shown below.
Power Button
The power button is used to turn the instrument on and off. The blue status light
indicates that the instrument is on.
Touch screen Display
The touch screen display, located in the front of the instrument, contains buttons for
all the functions needed and displays data from the cell count.
Slide Port
The slide port is used to insert the Tali™Cellular Analysis Slide containing the
sample into the instrument for analysis.
USB Port
The USB port allows you to transfer and save the cell count data and images to your
computer for record keeping and printing purposes. The USB drive supplied with
the instrument or any other standard USB drive is inserted into the USB port for
data transfer. See page 29 for instructions on exporting data files.
Image Adjustment (Focus) Knob
The image adjustment (focus) knob is used to adjust the image quality to obtain
better contrast between the cells and the background. Correctly focusing the image
is important to obtain accurate cell counts and fluorescence measurements for
various Tali™Image-Based Cytometer assays.
Continued on next page

7
Description of Tali™Image-Based Cytometer, continued
Rear and side view
of Tali™Image-
Based Cytometer
The rear and side view of the Tali™Image-Based Cytometer illustrating the various
parts of the instrument is shown below.
Power Inlet
The power inlet connects the instrument to an electrical outlet through the supplied
power cord and the appropriate plug, based on the electrical outlet configuration in
your country.
On/Off Switch
The on/off switch is located above the power inlet and is as the main power switch.
It is not necessary to use the on/off switch for day-to-day operation of the
instrument.
Continued on next page

8
Description of Tali™Image-Based Cytometer, continued
User interface The touch screen user interface of the Tali™Image-Based Cytometer is used to
operate the instrument and consists of:
The touch screen buttons to operate the instrument
The digital display to show the image of cells and data from the sample
The example below shows the Measure screen of the Apoptosis assay.
Tali™Cellular
Analysis Slides
The Tali™Cellular Analysis Slides are disposable slides composed of polylactic acid
(PLA), a low-fluorescence plastic. Each slide holds the sample in two separate,
enclosed chambers (A and B), allowing the analysis of two different samples or
replicates of the same sample. Each chamber has a 25-µL sample capacity. The Tali™
Image-Based Cytometer captures a series of images (i.e., fields of view) of the
sample in the chamber and then analyzes them using algorithms specific for the
Tali™Assay selected. See page 16 for guidelines on loading the Tali™Cellular
Analysis Slides.

9
Specifications
Physical
characteristics
Instrument type: Benchtop cell counter and suspension cell-based
assay platform
Instrument dimensions: 11 ½” (W) 11 ½” (H) 17 ½” (D)
Weight: 19.4 lbs
Operating power: 100–240 VAC, 2.5 A, 120 V
Frequency: 50/60 Hz
Electrical input: 12 VDC, 13 A
Installation site: Indoor use only
Operating temperature: 15–29°C
Operating humidity: < 80% (non-condensing)
Technical
specifications
Processing time: 10 seconds to 2 minutes, depending on the number
of fields captured
Sample concentration range: 1 × 105–1 × 107cells/mL
Recommended sample
diameter range: 5–60 µm
Required sample volume: 25 µL
Firmware: Tali™Image-Based Cytometer Firmware
(visit www.invitrogen.com/tali for updates)
USB Drive : 4 Gigabyte
Optics Optics: 3 channels (bright field, green fluorescence, red
fluorescence)
Excitation: Green channel LED: 458 20 nm
Red channel LED: 530 20 nm
Filters: Green channel: 466/40 EX, 495 LP Di, 525/50 EM
Red channel: 543/22 EX, 580 LP Di, 585 LP EM
Camera: 1.3 Mega pixels, 4X objective, 4X or 16X digital
zoom
Tali™Cellular
Analysis Slide
Material: Polylactic acid (PLA)
Dimensions: 110 mm (w) × 24 mm (d) × 1.9 mm (h)
Chamber volume: 25 µL

10
Using the Tali™Image-Based Cytometer
Workflow The Tali™Image-Based Cytometer captures a series of images (i.e., fields of view) of
the sample in Tali™Cellular Analysis Slides and uses sophisticated digital image
analysis algorithms to perform assays for cells in suspension. All Tali™Assays share
the same basic workflow and user interface, but differ in which channels the images
are captured and which algorithm is used for analysis. The table below describes the
major steps for using the Tali™Image-Based Cytometer.
Step Action Page
1 Optional: Adjust global counting settings 13
2 Select the Tali™Assay you wish to perform 15
3 Load your sample. 16
4 Focus the field of view 18
5 Optional: Perform a new background measurement or select a
previously recorded background (not applicable to the Quick
Count Assay)
19
6 Capture images of the sample 20
7 Review captured images 21
8 Set gates and thresholds 23
9 Annotate and export your data 25
Before using the Tali™Image-Based Cytometer for the first time, you need to align
the cameras and calibrate the instrument using the calibration beads provided with
the instrument. See page 32 for more information.
Continued on next page

11
Methods
Getting Started
Introduction This section provides information on installing your Tali™Image-Based Cytometer,
setting the date and time, and updating the firmware. It also provides information on
adjusting the counting settings to optimize the analysis of different sample types.
Install the Tali™
Image-Based
Cytometer
1. After unpacking the instrument, place the instrument on a flat, level, dry
surface.
2. Plug one end of the power cord appropriate for your region into the Tali™
Image-Based Cytometer.
3. Plug the power cord into the electrical outlet. Be sure to use only the power
cord supplied with your instrument. Powering the instrument with an
unapproved power cord may damage the instrument.
4. Turn on the main power switch (i.e., On/Off switch) located above the power
inlet port (see image on page 7).
5. When you are ready to use, turn on the Tali™Image-Based Cytometer by
pressing the Power button on the front of the instrument.
Home screen When the Tali™Image-Based Cytometer is turned on, the instrument initializes and
displays the Home screen. From the Home screen, you can proceed immediately to
Tali™Assays (Quick count, Viability, Apoptosis, or GFP/RFP), or use the navigation
bars to access saved data (Data screen) or adjust instrument settings (Settings
screen).
Continued on next page

12
Getting Started, continued
Settings screen Press Settings on the navigation bar to display the Settings screen.
The Settings screen allows you to:
Calibrate and align the instrument to ensure optimal performance (page 32)
Update to install new firmware versions as they become available (page 31)
Set up date and time (see below)
Adjust counting settings (sensitivity and circularity) (page 13)
Set up date and
time
The date and time is already preset when you receive the instrument. To reset the
date and time, use the Date and Time roller wheels that respond to the movement
of a finger across the screen as if they were wheels.
1. Press Settings on the touch screen; the Settings
screen is displayed.
2. Select the date and time by bringing the
desired value to the center position on the
roller wheel located under Screen settings.
The updated date/time is displayed on the top
right corner of the screen.
Once the date/time is set, there is no need to
set it each time the instrument is turned on.
Continued on next page

13
Getting Started, continued
Adjust counting
settings
The Tali™Image-Based Cytometer comes with pre-set parameters to match the
majority of cultured cell types which can be accurately counted without changing
any of the default parameters. Counting settings of the Tali™Image-Based
Cytometer allows you to adjust the global image analysis parameters for specific or
mixed cell types; you must be determine these settings empirically.
From the Settings screen, you can adjust the following global parameters for
counting:
Sensitivity refers to the contrast of the objects from the background. Increasing
the sensitivity makes the instrument more sensitive to objects in the bright field,
while decreasing the sensitivity makes the instrument less sensitive and is
useful if there is more debris in the cell sample.
Circularity is used to determine which objects to include in the measurement
based on their roundness. Increasing the value for circularity requires objects to
be rounder for inclusion in the measurement and may be useful if you need to
distinguish perfectly round cells from more oddly-shaped cells. Decreasing the
circularity allows objects to be less round for inclusion in the measurement and
may be useful if the cell type is not particularly circular or perhaps oddly
shaped.
The default settings for sensitivity and circularity
are 6 and 8, respectively. To adjust the Counting
settings:
1. Press Settings on the touch screen.
2. The Settings screen is displayed. Move the
slider bar located under Sensitivity to adjust
the sensitivity setting to the desired level.
3. Move the slider bar located under Circularity
to adjust the circularity setting to the desired
level.
In addition to sensitivity and circularity, you can also adjust the parameters for the
cell size and GFP and/or RFP-fluorescence, which are specific to the assay in use.
For more information, see Set gates and thresholds, page 23.

14
Guidelines for Performing Tali™Assays
General guidelines To obtain the best results, follow these recommendations:
Wear gloves during sample handling.
Do not touch the optical surfaces of the Tali™Cellular Analysis Slides. Hold the
slides by the edges. See page 16 for guidelines on loading the Tali™Cellular
Analysis Slides.
Use the Tali™Image-Based Cytometer at room temperature only (10–35ºC).
The Tali™Image-Based Cytometer contains delicate optics. Place the instrument
on a flat, dry surface that is free from excessive vibration. Do not spill liquids on
the surface of the instrument or introduce liquids into its interior.
The Tali™Image-Based Cytometer can accurately count cells 5 µm to 60 m in
diameter.
The recommended sample concentration range for the Tali™Image-Based
Cytometer is 1 × 105to 1 × 107cells/mL; however, the sample concentration
does not need to be exact to perform an assay.
For accurate viability count results, ensure the counting area is covered with cell
suspension and count cells immediately after staining per the assay protocol.
Re-calibrate the Tali™Image-Based Cytometer after updating the firmware or
when using assay reagents not formulated for the instrument. To recalibrate the
Tali™Image-Based Cytometer, see page 32.
The internal working memory of the Tali™Image-Based Cytometer is
145 Gigabytes, sufficient for storing comprehensive data from 1000 sample runs.
However, we recommend that you save your data to the USB drive after
completing each experiment. Using the USB drive, you may then transfer the
data to your PC as described in Export Data (page 25).
After using Tali™Cellular Analysis Slides, appropriately dispose of them as
biohazardous waste. Do not reuse the Tali™Cellular Analysis Slides.
Turn off the Tali™Image-Based Cytometer at the end of the day.
Accuracy vs. assay
speed
The Tali™Image-Based Cytometer performs counts by simultaneously capturing
bright field and fluorescent images of the sample in the Tali™Cellular Analysis Slide
and then analyzing the captured fields of view using assay-specific algorithms.
You can obtain higher accuracy by capturing more images, and thus analyzing
more cells.
You can obtain faster assay speeds by analyzing fewer fields of view.
You can calculate the theoretical CV using the following equation:


nn
where nis the number of cells counted.

15
Select a Tali™Assay
Select a Tali™Assay 1. To select a Tali™Assay, press the appropriate assay button (Quick count,
Viability, Apoptosis, or GFP/RFP) on the Home screen. In this example, the
Apoptosis Assay is selected.
Note: See page 5 for an overview of Tali™Assays.
2. To name the sample before performing the assay, press Name now. The alpha-
numeric keyboard pop-up screen is displayed.
3. Using the keyboard, type the name of the sample series using up to 40 alpha-
numeric characters, and then press Save. Each sample run in the series will be
appended with a number to reflect the order in which it was run.
To return to the previous screen without assigning a name to the sample series,
press Close.
Note: If you select Name later, the instrument automatically assigns a name for each
sample series by date and time. You can later rename the individual samples from the
Data screen.

16
Load Sample
Load the Tali™
Cellular Analysis
Slide
The Tali™Cellular Analysis Slides are plastic, disposable slides that hold the sample
in two separate, enclosed chambers (A and B) that allow the analysis two different
samples or replicates of the same sample. Each chamber has a 25-µL sample
capacity. Follow the guidelines below to load your samples into the Tali™Cellular
Analysis Slides.
Do not touch the optical surfaces of the Tali™Cellular Analysis Slides. Hold the
slides by the edges.
Use 25 µL of sample volume per slide chamber. Do not overfill or underfill the
slide chambers.
Pipet the sample at an angle of approximately 80° into the half moon-shaped
sample loading area (see figure below). The sample is loaded into the chamber
through capillary action.
Take care to avoid forming bubbles in the sample or to cause back splatter.
The Tali™Image-Based Cytometer counts the cells in the chamber opposite the
loading direction.
For example, to count the sample in Chamber A, you need to insert the Tali™
Cellular Analysis Slide into the instrument with the Chamber B-side first (see
figure below). For inserting the Tali™Cellular Analysis Slide into the
instrument, see page 17.
Important
Tali™Cellular Analysis Slides are specially designed for use with the Tali™Image-
Based Cytometer exclusively. Use of other slides will result in inaccurate cell counts
and can damage the Tali™Image-Based Cytometer. See page 39 for ordering
information.
The slide port button, located on the top right side of the touch screen, is
used only for opening the slide port to eject the Tali™Cellular Analysis
Slide in case of an error. Pressing this button will not insert the slide into
the instrument or eject it after a run is completed.
Continued on next page

17
Load Sample, continued
Insert the Tali™
Cellular Analysis
Slide into the
instrument
1. Load 25 µL of your sample (per slide chamber) into the Tali™Cellular Analysis
Slide as described on page 16.
2. The Tali™Image-Based Cytometer counts the cells in the chamber opposite the
loading direction. For example, to count the sample in Chamber A, insert the
Tali™Cellular Analysis Slide into the slide port of the instrument with the
Chamber B-side first until it stops (see figure below). Do not forcefully push the
slide any further.
3. Touch Press to insert new sample; the slide will automatically be pulled into
the instrument.
Note: If you are performing a background measurement, the button for inserting the
sample will read “Press to insert unstained control” (see page 19).

18
Focus Image
Focus the field of
view
1. Before running your sample, focus your cells using the image adjustment
(focus) knob on the right side of the instrument.
2. Touch Zoom to review the image.
Correctly focused images have uniformly dark-colored cells surrounded by
bright halos.
Cells may be undercounted when the transition between the background
and the edges of the cells are fuzzy and the cells have undefined
boundaries.
Cells maybe overcounted when they have bright centers and dark
perimeters.

19
Measure the Background (optional)
Measure the
background
(optional)
Measuring the background fluorescence using unstained samples allows you to
obtain more accurate sample counts. Note that the Background feature is not
available for the Quick count assay, because this assay does not use fluorescent
reagents. To measure the background for Tali™Viability, Apoptosis, and GFP/RFP
assays, follow the instructions below.
1. Touch the Background tab. The Background screen opens.
If there are no background measurements for the assay saved on the instrument,
the In use dropdown menu will display “No Background” and the run button
will be labeled “Press to insert new unstained cell control” (see image above). If
you choose not to run a background control, the instrument will assign a
fluorescence threshold for you, which you can change manually after
performing your assay.
If you have already measured the background, the dropdown menu will
display the latest background measurement on file.
2. To use a previously obtained background measurement, select the file name of
the measurement from the In use drop-down menu.
To perform a new background measurement, load the Tali™Cellular Analysis
Slide with the unstained cell control, insert the slide into the Tali™Image-Based
Cytometer (see page 16), and touch Press to insert new unstained cell control.
To replace a previously obtained background measurement, select the file you
want to replace from the In use drop-down menu, insert the Tali™Cellular
Analysis Slide containing the new unstained cell control into the instrument,
and touch Press to insert new unstained cell control.
3. After the unstained control sample is automatically pulled into the instrument,
touch Press to run unstained cell control.

20
Run Sample
1. To specify the number of fields of
view to capture, touch the arrow
button on the # of images to capture
drop-down menu and select from
the available options.
Capture images
2. To capture the specified number of fields of view, touch Press to run sample.
While the image capture is in progress, an ongoing update is provided by the
progress bar.
3. After capturing the specified number of fields of view, the Tali™Image-Based
Cytometer automatically ejects the slide and provides the data from the analysis
of the captured images in the Sample tab. The analysis includes information
about the average cell size, the number of cells counted, total cell concentration,
as well assay-specific data and histogram plots.
Note: In this example, the Tali™Apoptosis
Assay-specific data include the
concentration, percentage, and number of
live, dead, and apoptotic cells, as well as
the histograms for cell size and
fluorescence intensity for Annexin V and
propidium iodide (PI).
4. To run the next sample, insert the new slide, and repeat the procedure.
Continued on next page