IncuCyte Zoom Video Tutorial and User Guide

Posted on Updated on

Here is a guide that was written by Rachael McCloy from our lab on how to use
the IncuCyte Zoom Kinetic Imaging System.
We recently published a paper using this machine in Cell Cycle 

Part 1- How to set up an experiment

Part 2- How to analyse an experiment

Incucyte ZOOM User Guide

NB: Click here to download a PDF version

Considerations before setting up experiment

-Most vessels can be used; multi-well plates, dishes or flasks. Adapters can be found in cupboard

-We have a scratch wound maker for use with ImageLock plates (24well)

-Even seeding is crucial for good results

-Do not write on lids where the scan will take place

-If you are measuring in the green channel use phenol-free media to reduce background fluorescence

-If you want accurate cell counts, you must have fluorescent marker in your cells e.g. H2B-mCherry

-Incucyte is set to a 2-hour scan pattern for everybody, you cannot change this.

– Have your plate in the instrument at least 15 mins before starting first scan to allow condensation to settle

Downloading and connecting to software

-IncucyteZOOM software can be downloaded from Garvan software server. The user manual is also in this folder

-Software is only compatible with PC, so you need to run a parallels program if you are on a mac

1) Open software >connect to a device>zoom40061>connect device

2) Enter user name and password (see Rachael or Rob for a login)

Adding a plate to instrument

1) Check scan status on instrument before loading your vessel. If it is scanning, wait until the scan has finished.

2) Press the eject button to open drawer

3) Insert your vessel into the silver frame and ensure it is sitting square. Push the drawer closed.

Schedule a scan

-Front of drawer=bottom row

1) From the Task List choose Schedule Scans>click on the position you wish to add your plate to>Add vessel>Choose your vessel type

2) Scan Setup tab

>Channel selection, Default acquisition times are usually ok, but you can change if you have had issues in the past. If you are using both Green+Red channels together you will need to do Spectral Unmixing (like compensation). You can do this before you run the scans, or after on the resulting images.

>Scan Pattern>choose your scan pattern from the list, or to make a new one click the ‘edit scan patterns’ tab at bottom right.

>Job Type>Basic Analyser

>Processing Definition>Choose one that best fits your assay. It is a requirement of the machine that one is selected to run the assay, but you will be creating a new one later on anyway, so it is not crucial that you select the exact one here.

>Name>give your experiment a name, including your initials and date

>Notes>add any notes you wish to.

3) Properties Tab

>Label>put your experiment name in here again

>Cell Type>note your cell type

>Plate Map>If you have created a plate map with the ‘Plate Map Editor’ you can attach it here.

4) APPLY.

Note: Make sure you never touch the ‘Reload’ button, this will clear everybody’s experiments that are running

5) Make sure you EXIT the program when you are finished.

6) When your experiment has finished running, remove the plate from the instrument. You also need to remove it from the software>click on your vessel>Remove vessel>APPLY

DATA ANALYSIS

1) Task List>Search

>Scanned vessels-these are your raw images

>Analysis jobs-raw images with a PD mask over them and resulting metrics

>Processing Definitions

>Image Collections

Creating an Image Collection

-An Image Collection is a group of images that can be used to create or test a Processing Definition. Image collections will typically contain 3-8 images that represent the range in your data. Too many images in an Image Collection should be avoided as it takes too long to process

1) Task List>Search>Scanned Vessels>choose your experiment

2) Choose the image you want to add to collection>Add to Image Collection>New>Give it a name>Choose required channels

3) Choose the image you want to add the same collection>Add to Image Collection>Existing. Repeat this until you have 3-8 images in your collection

Creating a Processing Definition (PD)

-PDs are what define the parameters used to analyse all images within an experiment. Each new experiment will likely require a new PD, but you can often re-use a PD across similar experiments. You should ALWAYS check in detail that your PD is correct for each new experiment, do not assume it will be.

1) Task List>Search>Scanned vessels>choose your experiment

2) New processing definition>select your image collection

3) Preview>Adjust the parameters to give the mask you require. Each time you change a parameter, click Preview again to see the changes

4) When you are happy with your PD >File>Save As>name your PD

Launch Analysis Job

-An analysis job will analyse images and produce metrics based on the parameters in the PD you have created.

1) Task List>Search>Scanned vessels>choose your experiment

2) Launch new analysis job

>Job type>basic analyser

>Processing definition>select yours

>Name>Name your results output

>Define the time-points you wish to use

>Select the wells you wish to analyse

>OK

Exporting Results

1) Task List>Search>Analysis Jobs>choose your analysis result

2) Check a few wells to see that the PD has created the correct mask

3) Graph/Export>Graph/Export

>Choose Metric you wish to export, the time-frame and the wells

>Group>None

>Microplate graph>a summary of all the wells

>Graph>creates a graph that you can print

>Export>will export a .txt file that you can open in excel

(tick -Break data down into individual images)

4) Utilities

>Export Current Image>Choose image type>Export

>Customise with Image Designer to add scale bar and details

>Export Movie or Image Set>Choose time range and wells>Export

Advertisements

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out / Change )

Twitter picture

You are commenting using your Twitter account. Log Out / Change )

Facebook photo

You are commenting using your Facebook account. Log Out / Change )

Google+ photo

You are commenting using your Google+ account. Log Out / Change )

Connecting to %s