Hi Meen,

You can try using Fiji for getting some basic information out of your stacks:

https://imagej.net/Fiji

It seems your stacks are already very well aligned, so you probably don't need to worry about registering your recordings. As you mention, you should definitely start by normalizing your different sessions. You can try at first to do a z-scoring in Fiji by:

1) using the Analyze > Summarize tool to find the mean and standard deviation of pixels within each of your sessions,

2) Use Process > Math > Subtract tool to subtract the mean from all pixels

3) Use Process > Math > Divide tool to divide all pixels by the stdev

Doing so should make your pixel statistics to be centered around the mean (i.e. mean = 0) and have a standard deviation of 1. Notice that it is likely that the images will not be properly displayed since ImageJ expects values in the range of 0-65k for 16-bit images.

From here, there are many ways of extracting your signals, which will depend on exactly what you want to do. For example, you can select particular regions of the image using the drawing tools and then using

-- Analyze > Measure to get the mean intensity value in that region, or

-- Image > Stacks > Plot Z-axis Profile to make a graph of the average pixel intensity in that region

About the noise, that is likely an acquisition issue. When you do calcium imaging, microscopes are set up to acquire images quickly (usually around 10 images per second), which results in a decrease in resolution. You can often get a "pretty image for publication" by taking a subset of your recording (usually in the beginning, when your signal has nor suffered from bleaching) and averaging those images together, or by running a quick recording at the beginning or the end of your sessions with increased laser power and/or reduced frame rate.

Hope this helps!

Ronan

Hi Meen,

The attached TIF has two frames. Each frame contains the same features (dendrites), but the second frame appears dimmer. Since the alignment seems extremely similar, I assume they are from the same imaging session? What is the difference between the two frames?

I noticed that the pixels values all fall into the range 0-255 (8-bit), but most fall into a much smaller subrange. Indeed, simply adjusting the lookup table (ImageJ->Image->Adjust->Brightness/Contrast) improves the appearance. Most in vivo scopes can digitize with 16-bit resolution, and you will get much better results if you adjust the imaging parameters to make full use of that range.

As it stands, I think you will have too much difficulty reliably identifying features in these stacks, due to the low signal of the dendrites. I would recommend modifying your acquisition, rather than focusing on analysis of these or similar frames. But, that all depends on your question - maybe you don't need to identify or trace features.

What kind of fluorescence imaging system are you using? Two-photon? What fluorophore are you using? What is the NA and magnification of your objective lens? Does the objective lens have good transmission in the excitation and detection bands? Is the excitation wavelength optimal? Is the detection path collecting most of the emitted light? Normally I would say increase the excitation laser power until you have good signal, but in vivo you can't do that freely, as there is a limit to how much power can be used without causing damage/injury. That limit depends on the wavelength and other laser parameters. You may also be able to increase the gain of your detectors, but generally I would avoid that, as it will also increase noise.

As Ronan already said, one way to increase S/N is to increase the time you spend collecting photons. This can be done by repeatedly acquiring the same frame over and over, and then averaging. On some systems (non-resonant), you can instead or in addition directly adjust the dwell time per pixel. If you are not imaging activity (it seems you are looking at a structural dye), there may not be a need to image at a rapid frame rate - unless there is substantial brain movement, in which case higher frame rates will generally make motion correction easier.

In summary: please tell us more about your setup, prep and main questions, and we can give more specific feedback. Going just on what you've shown, it looks like you have acquired only a very narrow bit depth, and that you could benefit from modifying your acquisition settings to increase the signal. On the plus side, it looks to me like your resolution is quite good.

Use ImageJ frame arithmetics, subtract background etc, ImageJ is free . kmake line or retangular profiles you will have a pixel number related to the brightness, not the amout of free calcium directly