Holomonitor
! this is a short guide for operating the Holomonitor . a more detailed guide can be downloaded from
this link: Holomonitor M4 user manual
The holomonitor is designed to acquire live cell imaging without any need for labeling.
The instrument is placed inside an incubator and can track cell movement and changes in its
morphology through time.
The optimal plate for image acquisition is a 35-40mm diameter, 5mm height Plate.
4 plates can be positioned on the holomonitor stage.
! A special plate cover designed to prevent condensation is available.
This “Anti condensation plate cover” can be sterilized and used multiple times as long as no scratches
are formed on the view window.
For more info on how to use it check the Holomonitor PHi lid instruction page
A six wells plate can also be viewed.
Before startup:
There is an anti-condensation cap on the holomonitor lens. One should remove it before starting the
experiment and replace it back once the experiment is finished.
Start the laser 30 minutes before the experiment
Start the computer
Startup:
1. Place your plate on the holomonitor stage
2. Start the software Hstudio
The instrument will go through a homing routine. When it ends the Live capture option is
activated
The “live capture” tab:
3. In the “live capture” tab, the calibration wizard will start up
Follow the wizard instructions to perform a calibration
If all values are in the green everything is fine
4. On preset choose the Holographic X20
5. At the right side window write down the Project (lab name) and Group (experiment name)
6. Adjust the Focus in the “Microscope setting” window
The flat line is the distance between the stage and the lens.
Start from the top and go down. The wheel to the left is the
for fine tuning.
Time Lapse:
There are two parameters to define:
1. Total time
Once you define these parameters the software will calculate how many
pictures it should take.
2. Interval
A single field needs a minimum interval of 1sec
Capture pattern:
1. Define the time it waits after the stage movement before it takes a picture
2. Select the wells (the position of the plates on the stage)
3. Select fields within the plate
(one can also choose a random pattern option)
Once one scans the plate and find cells one can choose to “remember” this position
4. Define Setup storage: one can choose to define each image taken on the plate (at each
position) as a separate experiment.
If using the Anti condensation cover plate, make sure you choose acquisition points that
coincide with the view window.
Color adjustment of cell height:
On the coloring side window one can define any number of colors. The position of the color on the x axis
assigns the colors to heights on the y axis. One can define a set of colors and save it for future analysis
The picture is composed of the fraction index of the cells and the medium. One can change the fraction
index and it will affect the picture:
Cell ref index
1.38
Medium ref index
1.34
These values are the average default values (the DDW ref index=1)
One can change the focus after acquisition with the Software focus
Once one adds a color one can apply it to other or all the pictures
One can also choose all the pictures from a specific point in the field (by choosing one point and then
choosing every n pictures….)
Analysis:
1. Cell segmentation (identify cells)
Choose a picture
Show threshold (marks all that it defines as cells)
Show cell marker (places a point in each cell it identifies)
Show outline (marks the cell outline)
“Adjustment “ enables choosing from different algorithms
Once one chooses an algorithm one can also adjust its parameters (size and background)
Then apply all
2. Track cells
Choose the pictures you want (not enough to check the box)
Each cell is then marked with a small + sign
Once one clicks one cell the software tracks it
To view it one can move the timeline bar or plot the tracking movement on a graph.
It is possible to track few cells in the same picture
Once one labels the cell a list of warnings appears representing instances where there is a
potential problem like change in volume due to cell division….
One needs to go over each warning and check it to make sure one tracks the right cell
If the cell runs out of the picture on can unset it and the tracking will stop
One can get the following parameters:
Morphology changes
Migration (the distance the cell moved between two points)
Motility (the distance between the two points)
Export data – mark the cell and export to get all the possible parameters as plot features
Then one can save the plot
Data analysis tab:
For analyzing a large number of cells where statistics matters
For example, analysis of 10 pictures at one time point and another 10 pictures at another time point and
compare morphology parameters….
One can also gate a population so it
will be marked differently in the
picture
One can also do a histogram
3. Cell count ;
For counting cells one needs at least 5 pictures and 3 fields
Define plate size and medium volume to get cell count parameters
Export results\images
Results- Export options are to excel or to ICEF (a format that can be read by FACS softwares)
Images – choose pictures and export
Movie – to choose a single field: “select every x image”. Export and define the number of frames per
second, choose ones focus point on the picture and apply to all.
Shutdown:
Once the experiment is over one can shut down the laser and cover the lens with the cap
The Phi lid should be sterilized and kept according to instructions until the next experiment