Green fluorescent cells on a black background.

Fluorescence Capture Protocol

Three green cells and four blue cells in a circle.

This protocol helps to set up a Fluorescence Capture using HoloMonitor® M4FL and the HoloMonitor® App Suite software. The HoloMonitor® Fluorescence Capture provides holography time-lapse images for further analysis by any other HoloMonitor® App Suite application and accompanying fluorescence images for Single Cell Tracking and Cell Morphology analysis.



  • Time-lapse images
  • Time-lapse videos
  • Fluorescence intensity values
  • Selected application data analysis




Seed the cells with about 5 % confluence (ca. 6000 – 11000 cells/cm2).
►Please note that too few cells may lead to inadequate results due to auto-focus failure.
►Please note that a positive fluorescent control that
is representative of your exmeriment set up is essential.

Place the vessel in the incubator and let cells attach for 2-24 hours.

Start the software and wait for complete instrument initialization.

Run an auto-calibration. With successful calibration (as shown in the picture), the instrument is ready to use.

Vessel Vendor cat.
HoloLid™ Final working
Growth area,
Vessel cut in
a holder
Sarstedt TC-dish 3583.3900711103.0 mL/well8.00NA
Sarstedt TC 6-well plate83.3920.005711203.0 mL/well8.80top left
Sarstedt lumox® 24-multiwell plate94.6000.014711301.9 mL/well1.90top left
Sarstedt lumox® 96-multiwell plate94.6000.02471140170 µL/well0.34top left
ibidi® μ-dish 35 mm, high81156711112.5 mL/well3.50NA
ibidi® μ-plate 24 Well Black80241711312.5 mL/well1.90NA
Eppendorf CCCadvanced® FN1 – 6 well0038110010711503.0 mL/well9.40bottom right

Sterilize the HoloLids™ according to the specific HoloLid™ protocol.

Add the treatment to your cells. The final working volumes per well, essential for using HoloLids™, are shown in the table above.

Slide the cell culture vessel onto the Vessel holder, its grips facing towards you. Ensure that the vessel is parallel to the holder. There is a spring that holds the vessel in place.
►When using multi-well plates, place them with the cut-off corner to the left.

Replace the standard lids with the HoloLid™.

Put the vessel holder with the sample on the HoloMonitor® M4 stage.


Focus Holography image

Go to Live View Tab on the left side panel.

Select the correct vessel map from the drop-down list.

Find a position with cells. Preferably, the position should be as centered in the vessel as possible.

In Controls, make sure that the holography image of the cells is in focus by adjusting the Z-position of the stage using the up/down arrows. Ensure that Autofocus is ticked and then adjust the Z-position until the Software focus bar shows 1300±5.
► The Z-position will be saved with the calibration and used as the default Z-position during Capture Setup.

Live View window, Controls tab

Calibrate Fluorescence Focus Plane

Go to the Fluorescence tab.

Turn on Fluorescence live capture by pressing the Play button (or take a Single image repeatedly) while manually adjusting the Fluorescence focus until you get a fluorescence signal.
► You can use the arrow keys on the keyboard.

Check that the view is set to mixed channel.

Set Exposure time and Gain. For more information see HoloMonitor® M4FL Setup and Operation Manual.
► Increasing gain value might be beneficial when the fluorescence signal is weak, and App Suite struggles to find focus automatically.

Press Autofocus to automatically set best focus offset.

If the software fails to find optimal focus, adjust it manually by adjusting the fluorescence focus slider.

Press Calibrate.

Use the Position, Scale and Rotation controls to adjust and align the fluorescence overlay to fit the holographic image.
► This is an iterative step. Thus, position, scale, and rotation adjustments do not need to be performed sequentially.
► If needed, you can decrease the Step size to make finer adjustments. You can also change the opacity of the fluorescence overlay to compare it with the holographic image better.

Turn Off the Fluorescence Live Capture.

Press Save calibration when the fluorescence focus and alignment are as good as possible.

Go to the Apps tab and select the Fluorescence Capture Application and proceed by clicking the Setup button.

Live View window, Fluorescence tab

Fluorescence signal intensity can be evaluated using the measure tool (or histogram), which are found to the right of the Live View image.

To reduce the fluorescent exposure during calibration, turn off fluorescence live capture. Instead, take a single fluorescence image every time you need to confirm a change during the calibration.

If the arrows in Controls for Microscope Stage were used in
step 1.3 to navigate to the location used for calibration, the
holographic position might need to be corrected:

Go to Controls.

Turn OFF the Holographic Phase channel by p ressing the Stop button .

Press Correct Pos.

Turn ON the Holographic Phase channel by pressing the Play button.

Turn OFF the Holographic Phase channel by pressing the Stop button.

Take Single Fluorescence Capture.

Check if Holographic Channel and Fluorescence Channel images are aligned. Repeat steps if needed.

App Suite main window with selected Fluorescence Capture


Basic setup: Describe the experiment and assign treatments to the wells

Enter the experiment name, optional experiment description, and cell types.

Select the correct vessel map from the drop-down list.

Map treatments on the vessel map. Select wells by
marking them with the left mouse button while moving
the cursor over the relevant well(s).

Add the treatment name(s) in the text box below the vessel map and click Add / press Enter. It is possible to
add wells as individual treatments. Marked well(s) are light blue, selected wells will appear dark blue.

Proceed to Capture setup.

Basic Setup window

Capture setup: Set experiment time settings, choose and validate capture positions

Adjust the default settings for duration and interval.

Set Fluorescence capture interval.
► Captures set too often will induce phototoxicity.

Setup capture positions.

Adjust the fluorescence exposure time and gain if needed in the Fluorescence tab.

Ensure that the storage requirement for the experiment does not exceed the computer capacity.

When satisfied with the experiment setup, click Proceed
to Capture

Capture Setup window with opened steup tabs

For the best Fluorescence imaging result, the Holographic Image Software focus value should be as close as possible to 1300±5. Adjusting the Actual Z-value for each position might be necessary.

Capture setup: Manually choose and validate capture position

Setting capture locations:

Setting Holographic focus:

Setting Fluorescence focus:

Capture Setup window with opened steup tabs

Capture: Review the experiment in real-time during the time-lapse

Click Start Capture.

To stop the experiment ahead of time, click the Stop Capture button.
► It is not possible to restart the experiment once it has been stopped.

To pause the experiment, click the Pause button. Press Resume button to continue the experiment.
► Missed frames can be appended to the end of the experiment or skipped.

Click on Preview Results to preview the captured images during the run.
►Wait for the experiment to fully finish before starting data analysis.

When the Experiment capture finishes, click the Show Result button to go directly to the Experiment Overview tab.

Capture window


Experiments tab

Click Experiments to see a list of the experiments.

Click on the experiment title to open an experiment summary.

Click Open or double-click left mouse button to open the results page.

Experiments tab

Experiment overview tab

See the experiment summary, view all images and go to the experiment setup.

Generate In-depth analysis data (fluorescence green area (μm2), fluorescence green average, fluorescence green max, fluorescence green min, fluorescence green std, and fluorescence green sum) from the captured images by clicking on the Cell Morphology or Single Cell Tracking icon. A new window for the In-depth analysis will open.

Create New Guided Assay Results from this experiment by clicking the button.
► Note that only the DHM images will be analysed using the guided assay analysis.

A screenshot from App Suite showing the Experiment overview of a fluorescent experiment.
Experiment overview tab

In-depth analysis

Identify cells window
  • The first step for Cell Morphology analysis and Single Cell Tracking is to identify the cells.
  • The software automatically calculates cell count and confluence for each image.
  • Review the image quality and include/ exclude images from results analysis. See the Image guide for more information.
  • In the Identify cells under Fluorescence tab, adjust Holographic and Fluorescence Image overlay automatically or manually for individual frames, selected frames, or all frames.
  • Follow corresponding Cell Morphology or Single Cell Tracking application protocols for data analysis.

One experiment — multiple results

Generating Guided Assay results

In the Experiment overview page under Guided Assay Results select Create New Result.

Choose the type of analysis in the pop-up window and name the new result.

If there are previous analyses it is possible to tick copy image analysis from and select the experiment to copy from. This will copy the image analysis settings from the selected result including all changes.
► For further data analysis steps, please see the respective assay protocol.

Press create.

Screenshot from App Suite showing the re-analysis promt.
Experiment overview tab

Generating In-depth Assay results

In the Experiment overview page select the In-depth assay icon for the wanted result.

Follow the respective assay protocol.

Cell proliferation assay protocol reanalysis