In my experiments I used a PDMS biochip seeded with cells and used confocal microscope to observe cells structures like tubulin, Golgi and so on. During the confocal acquisition in time, sometimes I have experimented the lost of the focus. Does anyone had the same problem? How could I solve this?
Hi Brunella, this is weird. The whole point of a confocal microscope is to take advantage of the pinhole in order to obtain highly focused images.
When you say acquisition in time, how long is your experiment? Is it possible that the cells are moving?
Hi karolina,
my experiments takes almost 20 min. I used fixed cells so I don't they can move. Anyway I think this is more due to a mechanical instability of the chip. Indeed, the microfluidic biochip is bonded to a 170 um glass coverslip.
Hi Brunella, Is your confocal from Zeiss? Even if it is not, do you have the Z-stack option? Because you could always run a Z-stack in parallel with your time experiment and you would always get a layer that was focused (use a layer thickness of around 2 um).
TThere are many ways that PDMS or any other elastomer can complicate imagin. The flexible nature of the material can cause movement if there are temperature instabilities during image aquisi Even a fraction of a degree can cause movement on the micron scale. There are schemes to reference one of the surfaces of the glass and get yoreference your stack from that and this works well for cells that are adhered to the glass but not so well for cells that are attached to the elastomer.
Thanks Ron Reiserer. I'm interested only on the cell on the glass surface. Which schemes do you mean?thanks so much in advance.
Each manufacturer has their own version but essentially, they bounce light off of the front surface of the sample (usually the coverglass or slide) and use this as a reference for the z-stack. This way, if the stage has some thermal expansion or the slide bows under some stress, the z-stack can be adjusted to follow the front surface. It is possible to set the software to do this auto focus at the beginning of the experiment or at the beginning of each z-stack. It takes longer to do it every time so most software defaults to once at the beginning. Some software will allow you to specify an interval like every 5 stacks on a time series. It is also possible to to have several x-y positions for imaging and you can select whether or not you get a focus reference at every x-y position or not. The more you reference the longer the imaging will take.
If you notice that the images are popping out of focus in the middle of a z-stack, then you should probably try to let the temperature of the sample stabilize for a couple minutes before imaging. If the images go out of focus between z-stacks, try enabling auto-focus at every acquisition.
More specific information can be given if you post you microscope model and software version.
- Badges
- Science topic
- Similar topics
- Correction