This is it; the real magic starts here. With the CytoSMART® Lux3 BR properly placed inside the incubator, a culture vessel placed on the device and the software active, you can begin with imaging an experiment.
Live View of the Cells
To get the right images with the Lux3 BR, you need to configure the settings first.
In case you have multiple devices connected to the same laptop (also see section “Use Multiple Devices (Lux3 BR Duo Kit/Multi Lux3 BR)”) you can rename the tab of your device (Fig. 4.1A) to make it easy to identify which device is used.
Fig. 4.1. Live view of cells. A) Rename the device tab. B) Mini map to see the zoom level and position on the live view. C) Adjust Zoom level. D) Adjust focus level. E) Adjust exposure level. F) Start online experiment. G) Capture raw image.
Start with selecting the right focus level. You can use the “Slider bar” (Fig. 4.1D) to adjust the focus. To make more precise adjustments, you can either use the plus and minus buttons, or the arrow keys on your keyboard.
To make finding the correct settings easier, you can scroll and move the live view of the cells, in a similar manner that you would do this on an online map. To keep track of where you currently are and reset to the full view, you can use the mini map (Fig. 4.1B).
You can add 2x digital zoom to the 10x optical zoom of the Lux3 BR by ticking the box “Zoom in” (Fig. 4.1C). Please note that the number of pixels in an image remains the same, so this will make your field of view smaller.
Exposure represents the amount of time the cells get illuminated (Fig. 4.1E). You can make the image brighter by increasing the exposure time. If you decrease the exposure time, the image becomes darker. You can adjust the exposure time using the slider bar or by typing the desired value.
After you have finished the configuration, then you can “Start online experiment” (Fig. 4.1F), or quickly capture the raw image (Fig. 4.1G). This raw image will be stored locally on your computer, and the file location will be automatically opened in Windows Explorer.
Create Project:
A project is used to hold multiple experiments and it is necessary when creating an experiment. First, when creating a project (Fig. 4.2), you need to provide a project name (Fig. 4.2A).
By default, you will be assigned as the project owner. Optionally, you can give additional users access to a project by adding their email address (Fig. 4.2B). These additional users will receive an invitation email to join the project.
Fig. 4.2. Create project menu. A) Define name of the project. B) Add other users access to your project. C) Add an optional description. D) Change the color of the project icon. E) Create a new project.
Next, you can add an optional project description (Fig. 4.2C) and customize the color of the project icon (Fig. 4.2D). Here you can provide additional information regarding the project, for all users with project access.
After entering all details, select “Create my project” to create a new project (Fig. 4.2E).
Start Experiment:
In the “Start new experiment” page (Fig. 4.3) you first have to add the experiment to a project, either by selecting an existing project from the dropdown menu (Fig. 4.3A) or by creating a new project (Fig. 4.3B). This last option will take you to the “Create project” view (Fig. 4.2).
An experiment name is required to continue (Fig. 4.3C). Additionally, an experiment specific description can be added (Fig. 4.3D).
You can next select algorithms that will be used to analyze the images (Fig. 4.3E), where you can choose for the confluence algorithm or scratch assay algorithm.
Important: It is not possible to select analysis algorithms after starting the experiment, so always make sure to include the required algorithm(s) in this step.
Next, you have the option to take a single snapshot of your experiment or perform a time-lapse experiment (Fig. 4.3F). When selecting time-lapse, set the desired imaging frequency by customizing “Snapshot interval” in hours and minutes (Fig. 4.3G). You can keep the experiment running until you decide to stop it by selecting the “Open ended” option, or you can define a “Custom end date” (Fig. 4.3H).
Important: Since biological cells can be susceptible to light, it is advised not to use a higher recording frequency than needed.
Fig. 4.3. Start new experiment. A) Select an existing project to store your experiment. B) Create a new project for your experiment. C) Define a name for your experiment. D) Add an optional description. E) Select algorithms for your experiment. F) Choose between a single snapshot or time-lapse experiment. G) Configure interval for time-lapse experiment. H) Choose between a defined end date or an open-ended experiment. I) Go back to the live view of the cells. J) Start the experiment.
Once you are satisfied with the settings of your experiment, select “Start my experiment” (Fig. 4.3J). At this point, the imaging will begin and a confirmation email with a link to the experiment will be sent to the project owner and optional additional users.
When the imaging has started, the system will go into sleep mode (Fig. 4.4). In case you would like to check your cells or adjust the focus level between scans, tap or click on the screen to wake up from sleep mode. The camera and LED will turn on so you can adjust the focus level if necessary.
The button to quickly capture a raw image (Fig. 4.1G) will be disabled during a running experiment.
Fig. 4.4. Sleep view.
End Experiment:
Once an experiment is running, you can end it at any time. To end an experiment, select the red “Stop experiment” button (Fig. 4.5A) from the sleep mode or live screen and select “OK” in the dialog box (Fig. 4.5B) to confirm. A confirmation email indicating that the project has ended will be sent.
Fig. 4.5. End experiment dialog. A) Stop the experiment button. B) Confirm or cancel the end experiment dialog.