Fluorescence imaging with Micro-Manager

Hi everybody,

I am trying to take picture of immunofluorescence reaction with Micro-Manager, but I am having a lot of problems with it. We have a Retiga 2000R from QImaging. It is a mono camera, but we have a cube for fluorescence attached to our Leica microscope to see fluorescent staining. I already installed the QCam driver on the pc and MM recognize it and let me see my slides. However, I can’t find a way to set channels or pseudocolors in order to see the fluorescent staining correctely. When I tried using the Multi-D acquisition, I obtained very “dirty” and blurred images. Is there a way to fix it and finally take good pictures?

Thanks a lot in advance for the help!

Hi Dario,

It sounds like this could either be a problem with configuring your Leica scope to be controlled from Micro-Manager, or a problem with setting up the images to display the way you need to. Or it may be something else.

Were you able to configure the Leica microscope in the Hardware Configuration Wizard, the way you added the QCam camera?

Could you maybe post a screenshot of the ‘“dirty” and blurred’ images, and the settings you used to acquire them?

Welcome to the forum!

Hi Mark,

Thanks for your reply and I am very happy to be part of the forum!

I configured the QCam camera, but I wasn’t able to do the same with the microscope.

I am trying again to solve the prblem today and I thought that maybe it’s only a problem due to gain/exposure/offset. Modifying them, indeed, I can reach a little bit better results (attached). What do you think? Could it be only this kind of issue?

Thanks a lot,
Dario

Hi Dario,

From an imaging standpoint, those images look great to me! Strong signal, no background, no noise, high resolution. If you’re not happy with the quality I’d blame the specimen (of course I would, I’m a microscopist lol). The images are saturated, though, so reduce the exposure times. I believe Micro-Manager has a pixel under/over-exposure indicator for the Live image.

In the red image, the fact that the nuclei are focused in the middle and blurred on the sides makes me think the slide is not flat but slanted. Do the nuclei go in/out of focus across the field of view from side to side when you focus up and down? If so then maybe the slide is not mounted flat in the holder or the slide holder itself isn’t mounted flat into the stage (I see the latter all the time on microscopes around campus).

-Esteban

Thanks for your reply Esteban!

You are right: the nuclei go in/out of focus across the field of view since this tissue was not mounted correctely on the slide (I had some problems with the mounting medium we had in the lab). Now, I should have fixed it and I will try again.

The signal is good and strong (maybe too much), but I would like to se the tissue behind, so that I can localize the brain region. Is in your opinion only a problem of setting? In your opinion, should I only reduce the exposure times or also other parameters?

Thanks a lot in advance!
Dario

Hi Dario,

Sorry I didn’t respond sooner, somehow I stopped getting image.sc emails. Reducing the exposure is the only parameter that I see is sub-optimal in those images. If you’re not seeing what you need then it’s a specimen prep issue; maybe you need to clear the specimen?

-Esteban