Sharp/bright fluorescent images vs pale?


I have troubles to understand why some fluorescent images look sharp and bright (picture on the left) while others look pale (picture on the right). They were imaged with different microscopes, here are parameters that look different in the Zen metadata of both pictures:

On the left:

-Imaged with Axio observer Z1./7

-Objective: Plan-Apochromat 20x/0.8 M27

-Scaling per pixel:0.264*0264

-Bit depth: 14 bit

-Camera: Axiocam 705

On the right:

-Imaged with Axio Imager.M2

-Objective: Plan-Apochromat 20x/0.8 Ph 2 M27

-Scaling per pixel: 0.360*0.360

-Bit depth: 42 Bit

-Camera: Axiocam 503 color

I cannot analyse fluorescence intensity of the picture on the right because the intensity value of pixels are so low (max 30, while on the left they achieve around 140, analysis done in Qupath). Could this be due to a filter or camera settings? I am actually clueless as I don´t know much about the hardware part of the microscope.

Thank you very much for your input!


Welcome @asmae

At first glance at the parameters you supplied - the upright microscope you are using (Imager) does not seem to be outfitted for fluorescence work. First - color cameras are not generally recommended for fluorescence as this signal is weak compared to white light contrasting techniques and the bayer filter on this camera will reduce your collected signal. Your inverted microscope (Observer) has a monochrome camera more suitable to this imaging. Second - the upright uses a phase contrast objective (Ph2 annotation) which contains a phase ring in the back focal plane that will block some percentage of your fluorescence.

The intensity values you indicate also point to your camera settings down-sampling the dynamic range of the camera - likely to 8bit from 14bit. You want to avoid this when working with low levels of signal.

While those options are not ideal - that doesn’t mean you cannot use the upright for fluorescence quantification. You just have to realize that the images you take on this microscope will be sub-optimal compared to a microscope that has fluorescence specific components. You can likely compensate with an increase in exposure time on the color camera to better visualize the signal - but overall the image quality will not be as good as the monochrome camera.

Comparing images taken on two different microscopes such as these is quite difficult - so I would suggest at this stage that you collect all your images on the inverted if you can. Otherwise - optimize the upright as best you can and collect all your data here. Don’t try to mix and match - too many variables here will certainly bias any measurements.

Good luck!



Hello Jason,

Thank you very much for the great infromation!

I normally take out the phase contrast from the objective before imaging, it gets better but not perfect. as for the color camera, I wasnt aware of this! is the down-sampling the dynamic range of the camera something I can modify or it is a default parameter?
Increasing the exposure time does not help either. The signal doesn´t get brighter but just wider and looks like over exposed (see picture)

as for intensity measurements in the upright microscope (the one we have in our lab), I am a bit skeptical because in the Observer microscope, one can clearly see a gradient pattern of fluorescence in different cells (pixel intensity ranges from 0 to 140), the thing that I don´t see in the upright Imager microscope. In the latter, it´s more like: Low intensity cells or high intensity cells (pixel intensity ranges from 0 to 20/30).

If there are more tricks to try to obtain a better image quality I would be grateful to try it. Otherwise maybe I should consider taking my images somewhere else.

Just out of curiosity, is changing the camera to a monochrome one a good option? what are benefits of the color camera?

Thank you very much again for your great answer!

Hey again,

I found we have monochrome camera also and took pictures with it. It looks amazing now!

Thanks alot!