All common methods of fluorescence imaging detect fluorescence through the same objective that is used to pass on the excitation light, which means that the light doesn't need to be able to pass through the sample.

If the opaque substrate is on the bottom of your culture dish, you have two options: 1) Use an upright microscope, so that the light comes from the top and doesn't need to pass through the substrate. 2) Find a way to mount your culture upside-down on the inverted microscope.

Of course no matter what you do, you won't be able to acquire transmitted light for contrast imaging.

Thanks for your answer.

If I use the second method, can I observe on an inverted type microscope in the same way as usual? (In the same way I put the sample and observe it)

Yes, you would need to mount the culture dish such that the cells face the objective, and the substrate is not between the objective and the cells. If that's possible, you should be able to just image it like any other sample.

Thank you again for the answer, but I will add one more question.

I reconsidered answer # 1, and I tried to take a bright field time-lapse image. Based on your answer, if you don't have fluorescent dye on an inverted type microscope,  Is it hard to observe anyway? Even if the magnification is 40x or less (probably about 10x magnification)?

When I read another paper that did a similar experiment, the researchers used Method 2) that you recommended, but it didn't tell me how to shoot in the inverted type.

The question is whether I need an inverted type with other special options or just use an upright type to take bright field images.

I don't know if I've passed the question because I'm not good at English, but the key is whether I can use a bright field image from the method you recommended 2) to see it in a normal inverted type microscope, or if you don't need any additional options, or upright type is better.

Bright field imaging was always based on transmitted light on the inverted microscopes that I've seen, so that of course wouldn't work for your sample.

If you want bright field, just use an upright microscope like the ones commonly used for dissection or cell counting.

I have tried to take brightfield images of cells on opaque surfaces with both inverted confocal and with upright microscope, however the contrast is very bad and I could not get a lot of information.

I observe the cells using inverted confocal fluorescent microscope.

For imaging live cells, I first transfect them with fluorescent proteins (GFP for example) and when it is time to look at them, i have to put a coverslip on top of the opaque sample, so the cells are in between the substrate and the coverslip, and then turn it around. In the microscope, the coverslip should be on the bottom (close to the objective) and the cells will be suspended on top, attached to the substrate. It could help to put paraffilm or something else at the sides, so the cells can survive for longer. I also read about using vaseline at the sides of the substrate to prevent fluid loss (sometimes the fluid between the substrate and the coverslip will leak out, killing the cells).

If you want to image for long times, i suggest you use an imaging chamber (with CO2 and 37 degree incubation) and maybe more appropriate media with no phenol red. Hope that helps, good luck!