I am currently using a 20W LED light source, but even with an exposure time of 99998, I still cannot capture the fluorescent dye. I have also used a 3W LED with equal intensity across various wavelengths, but I still cannot capture clear fluorescent images. Do you have any suggestions for a better fluorescent light source?
I don’t remember the recommended LED, but the star type LED is not commonly more than 3W. 3W is a lot of light! (Edit: I checked and the specified LEDs linked from the Deltastage reflection instructions are 1 to 5W for different colours, 3W should be plenty)
If you are not seeing anything, that sounds like alignment rather than power. What does it look like if you put a piece of paper on the stage? Have you got a brighr spot centred on the lens? Can you make it brighter by adjusting the LED position in the illuminator? Microscope slides made from fluorescent plastic are available that can also help with getting good alignment and maximum signal before looking at real samples.
Star type LEDs come with different built-in lenses, that will make a difference. (Edit: I checked this on the specified LEDs, there is a dome lens, but the beam angle is not stated)
Finally, as in the recent thread on a reflective microscope, you can check the imaging part separately from the illumination. Try a transmissive sample with the standard condenser illumination, or put a mobile phone screen face down on the stage set to a white background and try to focus on the pixels in the screen. Getting the basic focus might make a huge dufference to the apparent brightbess of a fluorescent sample. Finally of course the sample must be on the underside of the micrope slide, facing the objective lens.
Have you got pictures of your setup that you could share?
I used the high-resolution microscope with an infinity objective and beamsplitter parts.
This is the image I used cy5 filter sets. I can’t see the fluorescence image through this 20W light.
Also, I want to know whether I can get the parameter after auto gain & shutter speed.
I also tried the one you said to focus on the pixels on the phone screen. But after I focus on the pixels I still cannot see the fluorescence. 
I’ve used an Olympus light source to get the ROX image without the pinhole. Then I’ll get the image below. Is the field like that because I use a 4x objective lens? If I put the pinhole back I can’t get the fluorescence image again. So I think the light source is quite important. Can anyone give me a suggestion to fix the image below?
ROX image
HEX image
4x lens is very low magnification. Most of the testing of the fluorescence system has been at 40x or more I think. Filling the large field of view of 4x may be challenging.
Which pinhole are you referring to?
I use low magnification for ddPCR fluorescence imaging. Is there any other suggestion for getting images of bigger sizes? Maybe without an objective lens is okay.
The pinhole I refer to is the one on reflection illumination.
When you say that you are using an Olympus light source, is that directed from above for transmission, or directed through the reflection port? Then ‘with pinhole’ you mean a star-LED in the parts from reflection_illuminator.stl. A photograph of the setups might help to understand the issues.
I am not familiar with the ROX and HEX terminology.
In the reflection illumination there are two places where the aperture could be clipping your illumination, the small aperture by the LED, and the larger aperture close to the beamsplitter. If you are using an infinity corrected objective that larger aperture needs to be as large as the aperture at the back of the lens and the light needs to fill it, I think.
Through the reflection port. I used from above for transmission before but it doesn’t have dark field like below.
Great. So the imaging part is working as expected, it is a question of the illumination.
When you took the two images with the Olympus light source, did the light illuminate around the big hole in the bracket for the reflection illumination, or was it all going through the hole? Also was the light collimated at that point or was it focussed somewhere?
I put the light guide into the hole till it reaches the excitation filter.
The light does not focus on the sample and the picture doesn’t light at the middle of the view but in the corner like below.
Also, it shows part of the cube shape on it .How could I fix it?