How to focus on the glass surface?


I have to glass slides with a fluorescent protein adsorbed on the surface. The protein density is different between glass slides. I want to quantify the fluorescence intensity for each slide.
Is there any trick on how to focus a confocal microscope (laser scanning) on the surface of the glass slide?

Thanks in advance.

Hi @Oleg,
Try using reflectance imaging to find the surface. You’ll need to set one of your detectors to detect laser light reflecting off the interface between the glass surface and the sample. Your confocal system may not have an option to set this up automatically, so you may need to do it manually. Just be very careful because the laser light is very intense and you don’t want to fry your detector (use lower laser power, low gain, low pixel dwell time, etc.). As you adjust your z, you should see an intensity maximum at the air-water interface. Note that you’ll also see an intensity maximum at other refractive index interfaces if you move too far out of focus (e.g. glass/oil, glass/air). Good luck!


Hi @Oleg I second @Kris_Kubow suggestion. If you let us know what kind of scope you have we might be able to point you in the right direction.

Another option is to take an extended Z stack with the channel of interest. Minimize xy frame size (large pixel size) to go faster, and look at the orthogonal projection. You can usually see the autofluorescence of the glass and transition to another phase. Your signal should be a thin layer (make fine Z steps not to miss it). You might need to open the pinhole a bit to help see more light. Once you found the layer, move to a fresh area - chances are you will bleach your sample a bit.


Better to use a gene chip imager if you can find one that is still in service. These have much greater dynamic range and are tolerant of focus. Just stuff the slide in and get 16 bit quantitation right across the whole area of the slide. Otherwise you will have bleach effects and just very local information.

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Similar to this suggestion - Nikon A1R confocals with peizo focus and in resonant scan mode have a xz scan mode which lets you see in real time your planes of focus and you can derive your chosen focal plane from the image (or a z-stack range also)

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I want to try to adhere purified FRET sensor on the glass. Then measure FRET change under some treatment.
I have access to the TIRF setup, so I’m thinking whether it would be better to use TIRF instead of a confocal microscope.

P.S. Solution is not an option.

Super Critical Angle Fluorescence (SAF) Microscopy would be very suitable for this - it’s when you use TIRF but the beam is definitely bouncing off the glass - only the first 100nm above the coverglass will be illuminated / detected. Perfect for adsorbed proteins.
You only need to find a microscope with the correct filters and lasers if you are doing Cyan/Yellow fp based FRET. Also calibrating your TIRF angle - again - newer systems can automate this alignment process now

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