White and grey cells on a black background.
L929 cells imaged by HoloMonitor

Image Quality Guide

The objective of HoloMonitor reflecting the illuminating red laser light

This image guide intends to assist HoloMonitor® M4 users when evaluating image quality during experiment setup and result analysis. Please contact support@phiab.com if you need further assistance assessing image quality.

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EXPERIMENT SETUP

Before you start

Check the status of your cells: image a maximum of 80% confluency in a field of view to allow the software algorithm to identify individual cells from the background. HoloMonitor® M4 is designed to visualize and quantify adherent monolayer cell cultures, therefore your cells should not grow in multiple layers or in suspension cell culture.

Always perform Auto-calibration before every experiment. The calibration minimizes background noise.

calibration
For a susscesful auto-calibration, all values (indicated by black arrow
heads) should be in the green range.

Reasons for failed auto-calibration:

  • If all values are in the red region – the laser does not produce enough light. Reasons: dirty optics, laser being not properly connected or dirty, laser requires periodical recalibration. For resolving issues, consult the Setup and Operation Manual.
  • If Hologram noise is in the red range – HoloMonitor® M4 might have dirt on the optics or dirt inside the instrument. Clean the objective, if the problem persists, contact technical support (by writing email to support@phiab.com) for further assistance.

During Capture Setup

Inspect the images when setting up the capture for the experiment. Run a full or quick validation to ensure good image quality. If the image quality is poor, a warning sign (yellow exclamation mark) appears.

Having cells grow too sparsely may introduce image analysis warnings. Make sure there are at least a few cells in the field of view. Adjust the capture position location if necessary.

Reasons for poor image quality could be: incorrect focus, condensation, dirt and particles, air bubbles, smears and scratches, or the vessel’s optical properties.

Below are examples of poor quality images prompted due to various reasons.

Cell are growing in multilayers or in suspension

Black and white image showing a grey multilayer of indistinguishable cells.
Cell growing in multiple layers cannot be identified or quantified.
White, unfocused and blurry suspension cells on a black background.
Cells growing in suspension cannot be focused, tracked or quantified.

High background noise

Black and white image showing a green and an orange circle around grey, blurry cells.
Thin cells (green circle) and strong background noise (orange circle).
A minimal background noise is especially important when working with
very thin cells to distinguish cells from a strong background noise.

Incorrect focus

Holographic image showing grey cells on a black background with a green checkmark in the upper right corner.
Correct focus
Unfocused, blurry grey cells on a black background with an orange exclamation mark in the upper, right corner.
Incorrect focus
Black and white image showing unfocused cells with an orange exclamation mark in the upper right corner.
Incorrect focus
Unfocused, blurry grey cells on a black background with an orange exclamation mark in the upper, right corner.
Incorrect focus
Screenshot from App Suite showing the manuan focus adjustment controls.

MANUAL FOCUS ADJUSTMENT: The focusing tool is located in the Controls tab. Move the black square or click on the Arrow buttons to move the stage up and down. Save an adjusted focus setting to the selected position by using the Apply Current Focus button. For details, consult the Setup and Operation Manual.

Condensation

Black and white holographic image showing condensation.
Some condensation obstructing field of view
Black and white holographic image showing condensation.
Field of view fully obstructed by condensation

Tips to reduce condensation:

  • Use pre-warmed cell culture media, as the cold one introduces a high probablity of condensation forming outside of the vessel. The condensation disappears once the media in a well is warm (1-2 hours after addition).
  • Use HoloLids™ to eliminate image disturbances caused by condensation and surface vibrations inside the cell culture vessel. Details and user instructions are available in the HoloLids™ protocol.

Dirt and particles

Black and white holographic image with two blue arrows pointing at particles.
Particles
Holographic image showing grey cells on black background, with two blue arrows pointing at dust particles.
Dirt/dust creates “swirly patterns“ in the image

Tips to avoid dirt and particles:

  • Never use paper or fabric to remove liquid drops or condensation. Fibers will stick onto the cell culture vessel.
  • Always work in a clean environment – use LAF-bench to prevent dirt particles getting into the cell culturing media.
  • Clean the HoloMonitor® M4 outer surfaces according to the Setup and Operation Manual.
  • It is recommended to service HoloMonitor M4 yearly. For more information contact technical support (by writing an email to support@phiab.com) or your local distributor .

Airbubbles, smears and scratches

Black and white image showing an airbubble.
Airbubble at the top right corner
White cells on a grey background. The image is blurry due to smear on the vessel.
Smear
Black and white image showing a scratch in the plastic vessel and two blue arrows pointing at floating, blurry cells.
Thin scratch and floating cells (blue arrows)
Black and white image showing a thick scratch in the plastic vessel.
Thick scratch

Tips to prevent airbubbles, smears and scratches:

  • To remove airbubbles use clean air. This can be created by using an ethanol dispensing bottle with inner tube removed, and which contains on a small volume (100-150 mL) of 70% ethanol. Press the bottle carefully while directing the air stream towards the bubble. Do not touch the cell media.
  • To prevent smears – always wear laboratory gloves.
  • To prevent smears and scratches – only hold the cell culture vessel at its sides and do not touch the vessel bottom or lids.

Low optical quality plastics of cell culture vessels

Grey brightfield picture showing cells, with two blue arrows pointing at grey lines in the background indicating interference from the plastics used.
Plastic interference lines visible in bright field
Holographic image with grey cells on a black background, with blue arrows pointing att lines due to plastic interference.
Plastic interference lines visible in holographic image
Holographic image with grey cells on a dark grey background, with blue arrows pointing at lines due to plastic interference.
Plastic interference lines visible in holographic image
Holographic image with grey cells on a black background, with blue arrows pointing at lines due to plastic interference.
Plastic interference lines visible in holographic image

Tip to avoid low quality plastic:

Result analysis

Guided Assays

HoloMonitor cell count assay icon

Cell Counter

HoloMonitor cell QC assay icon

Cell Quality Control

HoloMonitor cell proliferation assay icon

Kinetic Proliferation Assay

HoloMonitor cell motility assay icon

Kinetic Motility Assay

HoloMonitor kinetic dose response assay icon

Kinetic Dose Response Assay

When the capture finishes, click the Show Result button to get directly to the Experiment Results page.

The assay results are generated during image capturing. The software automatically analyzes quantitative data generated from each image.

Image analysis tab

  • An image analysis algorithm filters out images of poor quality and excludes them from results data.

The captured images from the experiment are displayed in a tree-view list. Clicking an item in the tree will show a list of all images contained in the selected level/category.

Checked check boxes — all images are included.
Empty check boxes — all images are excluded.
Check boxes with a dash — some images are included.
Symbols in the list show cell analysis (yellow exclamation mark) or image quality (red exclamation mark) warning.

It is possible to adjust the Sensitivity of image(s) and
view the image threshold and markers.

Screenshot from App Suite showing the Image analysis tab.
Image analysis tab
  • It is users responsibility to verify that the software algorithm was correctly applied during image analysis and that results are accurate.
  • User can choose to adjust sensitivity of the image threshold, and to include or exclude images from results analysis.
  • For more information, see the Image Analysis Section in the Setup and Operation Manual for HoloMonitor® M4.

Below are some examples on how to improve image analysis by adjusting the sensitivity of the image and therefore the threshold of cell detection. Additionally cases, when the frames should be excluded from the analysis by unchecking the check box, are shown.

Adjust cell detection sensitivity

Two images. The left one showing red dots on a blue background, indicating a too sentitive mask. A blue arrow point at the right image, where the sensitivity has been adjusted and only the cells are blue, with red dots, on a black background.
Sensitivity mask covers all cells, although the cells are not confluent (left). After sensitivity adjustments, the mask covers individual cells only (right).
Two images showing blue cells with red dots on a black background. The left image has more blue areas due to a too sensitive mask that covers some of the background as well.
Sensitivity mask covers some of the background (left). After sensitivity adjustmensts, the mask covers individual cells only – their outlines are clear and background noise is minimized (right).

Examples of frames that should be excluded from Analysis

Blue and red dots on black background, showing a holographic image without any cells in it.
No cells or too few cells in the field of view
Blue and red dots on black background, showing a holographic image without any cells in it.
No cells or too few cells in the field of view
BLack image with blue and red dots, and a blue arrow in the upper right corner pointing at a filament.
Fillament/dust disturbing the frame
Black image with a blue arrow pointing at an out-of-focus cell.
Cells are out of focus
Black and white image showing an airbubble.
Small airbubble and no cells in the field of view
Black and white image showing an airbubble.
Airbubble disturbs the mask, thus cells are not covered

In-Depth Assays

HoloMonitor cell morphology assay icon

Cell Morphology

HoloMonitor single-cell analysis and tracking icon

Single Cell Tracking

When the Experiment capture finishes, click the Show Result button to get directly to the Experiment Overview page.

Generate in-depth analysis data from the captured images by selecting one of the applications icons.

In-depth analysis – Identify cells tab

  • First step is to identify the cells.
  • Inspect the image quality and include/exclude frames from the analysis.

Start by selecting a frame of interest, so the image is displayed. You can alter the viewing options.

Adjust the background threshold and object size so the mask best fits the cell segmentation and click Apply (Current/ Checked/ All) to fix the settings to the frames and to display the automatic calculation of cell count (#/frame) and confluence (%).

Manual changes are possible. Add/remove cell markers or grow/ shrink a cell outline, delete a cell area or draw a cell area yourself. These changes will only be saved to the currently selected image.

View the full Cell Morphology and Single Cell Tracking protocols for further in-depth analysis steps.

Screenshot from HoloMonitor App Suite showing the Identify cells tab.
Identify cells tab
Segmented red cells on a black background, encirvled with a yellow border and with a blue dot in the middle.

Threshold: OK
Object size: OK

Segmented red cells on a black background, encirvled with a yellow border and with a blue dot in the middle.

Threshold: OK
Object size: too big,
cells are missed out
or clumped together.

Segmented red cells on a black background, encirvled with a yellow border and with a blue dot in the middle.

Threshold: too high,
missing out on cells
Object size: OK

Segmented red cells on a black background, encirvled with a yellow border and with a blue dot in the middle.

Threshold: OK
Object size: too small,
cells are fragmented

Segmented red cells on a black background, encirvled with a yellow border and with a blue dot in the middle.

Threshold: too low,
too much background
included
Object size: OK

  • It is users responsibility to verify that the software algorithm was correctly applied during image analysis and that results are accurate.
  • Check cell segmentation for images throughout the experiment to make sure the applied threshold fits.
  • The more thorough the user is in this step, the better analysis results are in the following steps.
  • It is important for in-depth analysis that identifying the cells is done correctly.
  • For more information, see Identify cells in the Setup and Operation Manual for HoloMonitor® M4.

In-depth analysis – Wound healing tab

HoloMonitor wound healing assay icon

Wound Healing Assay

Check the image quality before adding frames to the Wound healing analysis.

Adjust the image threshold and object size and click Apply to all frames.

View the full Wound Healing protocol for further in-depth analysis steps.

Purple cells on a dark purple background, showing a wound healing experiment with a threshold set too low.

Threshold: too low, too much background included

Grey cells on a black background. Some cells are purple, showing a wound healing experiment with a threshold set too high.

Threshold: too high, missing out on cells

Purple cells on a black background, showing a correctly set threshold for a wound healing experiment.

Threshold: OK

  • It is users responsibility to verify that the software algorithm was correctly applied during image analysis and that results are accurate.
  • Check the mask coverage for all the images throughout the experiment to make sure the applied threshold fits correctly.
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