I am working with biological samples(mammalian cell lines) to acquire their Raman spectra. I have Raman substrates (gold coated with glass) on top of which I have a polyacrylamide hydrogel. My cells adhere on top of this hydrogel. I am having trouble using the HORIBA confocal microscope (60x water immersion objective, 785 nm laser) to find and focus on my cells. I try to focus first in the microscope mode and switch to laser mode only when I have found the cell and focused on it. I typically first focus on the glass and then move the objective up to find my cells. I am able to focus well on the glass surface. However, when I move the objective up to find the surface of my hydrogel, it gets darker and the image pixelates. I am not able to see anything and focus on it. As I keep going higher, it gets darker and the objective by then is out of the liquid media. Is there a better approach to focus the microscope and find my cells on the hydrogels? Is there a way to tell if I am too close to or too far away from the focal plane of the microscope?
Your concern is genuine. Microscopy techniques are widely used in life sciences to study cells and tissues. You please refer to these articles for details
Article Label-free live cell imaging by Confocal Raman Microscopy id...
https://pubs.rsc.org/en/content/articlehtml/2016/an/c6an00152a
https://www.nature.com/articles/s41598-018-30407-8
Article Confocal Raman microscopy in life sciences
The problem is that hydrogels have almost the same refractive index as water- this is a video which shows it- https://www.youtube.com/watch?v=_1plSYKR7Oo
You should be able to focus on the cells with the laser on, looking for the point at which the spot is focused...
Piyush Kumar
Hi Piyush -Even just with the camera on, you should be able to see when the laser is focused. I should say - this is easier to see if you reduce the power to ~1% with the filter.
why bother to get focus on the cell first? btw, in most cases, the best signals are not obtained when focus on the surface of the sample!
My suggestion is first to get focus on the glass substrate as you said you can get it done easily. Move your sample stage up gradually (coaresly) and see the changes of your signals/noise untill you can see some Raman peaks turning up. Then tuning the sample stage (finely) to optimise the signals.
You have to control your laser power to avoid damaging your sample! A good practice is to check gels without cells first to get feeling, e.g. sample stage position, power control, signals from the gels if applicable, etc. if your gel does have Raman response, then my suggestion above should be different peaks turning up from those of the gel itself.
worthe mentioning there are at least 6 ineterefaces, inc two from glass slide, two from gel, two from your cells if no overlapping or stacking.
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