Timelapses
Time-lapses are images taken at regular intervals over time. They are a very powerful method to illustrate biological processes such as cell division, bacterial growth... They are almost exclusively done on live samples kept in appropriate conditions.
The key variables in microscope timelapses are:
Sample preparation – How the sample is mounted, labeled, or stained
Temperature & environment – For live samples, conditions like temperature, humidity, and CO₂ levels affect biological processes
Time interval and duration – How often images are captured and the total length of imaging to capture the full process of interest
Imaging settings (magnification, illumination, detection) – Determines the level of detail visible (resolution vs field of view, illumination intensity, exposure time, photo-toxicity...)
Focus & stability – The sample must be and stay in focus for the full duration.
- Processing data - Processing timelapse data can be challenging
We will review here this information below
Focus & Stability
After sample care (environment and phototoxicity), focus stability is probably the biggest problem with timelapse imaging. Most microscope loose focus over time. This phenomenon called drift occurs naturally because of gravity. This phenomenon can be quite strong especially when the instrument is turned on. It is strongly recommended to turn on the microscope for about 3 hours before processing with an important timelapse experiment.
Some microscope includes a laser based technology that ensure a reliable measurement of the distance between the objective and the sample. This measurement is used to compensate any drift occurring with time. Interestingly the companies have different names for the same technology:
- Perfect Focus for Nikon
- TrueFocus for Evident/Olympus
- Definite Focus for Zeiss
- Adaptive Focus Control for Leica
- PureFocus for Prior Scientific
- fCRISP Autofocus System from Applied Scientific Instrument
regardless this technology rely on periodic measurement the distance between the objective and the sample to compensate for naturally occurring drift.
Processing timelapse data
We strongly recommend to save raw acquisition files during acquisition using the proprietary format from the acquisition software. You can then use FIJI to open and process it. While scientific analysis must be performed on raw data, timelapses are also used to illustrate a process. For that purposes, timelapse can be processed to improve portability, compress size, better interoperability etc... We will provide some clues to that purposes.
Not sure what is FIJI? Feel free to check our Microscopy software list to learn about it. |
Raw data file
1024 x 1022 16-bit 9601 frames -> 19.6 GB !
Reducing bit-depth
In an image each pixel is storing an information relative to brightness (for greyscale images). This information is usually an integer between 0 and X. The higher X, the higher number of possible values can be address to that pixel and thus the higher storage space is required to store this number. This is called bit-depth. A 8-bit image can store 255 value for each pixel. a 16-bit image can store 65000 values for each pixel etc... unfortunately human eyes are not able to appreciate more than few dozen of grey level. So reducing bit-depth is a easiest way to reduce the size of your data with actually changing how the images look.
In FIJI:
Drop the raw image file into the FIJI bar to import it
Select Image>Adjust>Brightness and contrast to open the Brightness and Contrast windows (Crtl+Shift+C)
Click Auto or adjust the brightness and contrast how you like it
Select Image>Type>8-bit to convert your image into 8-bit format (for multicolor images you should use RGB instead)
How many shades of grey can you identify? Please test yourself here by selecting ALL the squares that do not match the background. You need to submit your answers 8 times to get your result. My best was 42... |
1024 x 1022 8-bit 9601 frames -> 9.8 GB
Cropping
Cropping your image to your region of interest will effectively reduce the file size without affecting your resolution.
In FIJI:
Click on the rectangle selection tool
Click and drag while maintaining the shift key (this key will lock the aspect ratio 1:1 : square) around your region of interest
Select Image>Crop to crop your image
Cropped to 900 x 900 8-bit 9601 frames -> 7.6 GB
Downsizing
Another option consist in resizing (downsizing) your image. This means converting a number of pixel to a smaller number which effectively reduce the file storage size but also the resolution.
In FIJI:
Image>Adjust>Size
Check the following settings:
Constrain aspect ratio
Average when downsizing
Interpolation: Bicubic
Enter the number of pixel for the height
500 for web usage
720 for presentation
1080 for full HD
Click OK
900 x 900 downsized to 500 x 500 8-bit 9601 frames -> 2.3 GB
Timestamps
Timestamps are critical information because they indicate what is the real speed of the event that are being displayed. Without it, the observer has no idea if it is a fast process that is slowed down or vice-versa. Timestamps are usually stored within the raw file metadata, to read it:
In FIJI:
Image>Properties...
Look at the frame interval
(if it is not present you should complain to the company that has provided the acquisition software)
In the example the frame interval is 3 seconds.
In FIJI:
Image>Stacks>Series Labeler
- Select the following settings
- Stack Type: Time series
- label Format: Digital
- Label unit: s
- Start: 0
- Interval: 3
- Every n-th: 1
- Localization preset: Lower right
- Select Background
- Select Previw
- Click OK
An alternative in FIJI:
Image>Stacks>Time Stamper
- This will add the timestamps as an overlay (floating layer that is independant of your image). You may need to Flatten the overlay to actually write the timestampes on the image itself. If so Image>Overlay>Flatten
900 x 900 downsized to 500 x 500 8-bit 9601 frames with labelled timestamps