Unmixing fluorescent species from spectral data using Fiji

In this video, we will cover how to process fluorescence spectral imaging data using Fiji/ImageJ. These types of data sets could typically come from Zeiss LSM 780 microscopes.

[@daliborpanek1002]

Thanks a lot for this tutorial! Exactly what I need.

[@fathiamir5417]

Thanks for this tutorial. I do pump-probe microscopy and now I have an image that certain regions have different temporal decay profiles. As the focus laser beam is much larger than the features on the film-in this case, crystals with different sizes- most of the pixels have a linear combination of (what I am speculating based on the histogram) 2 different exponential decay. I played around with spectral unmixing a while ago, so I thought maybe I can use the same here. I want to simulate 2 decay profiles and use them as my reference to unmix the whole image. If I may, what are your thoughts on this?
Finally, thanks for your very helpful tool and tutorial. Certainly, it helped me a lot.

[@jezzmus55]

Hi, thank you for the valuable information. My situation is that I am taking calcium movies and hemodynamics measurements using dsRED for labeling pericytes and FICT for labeling blood in a two-photon microscope. dsRED is in channel 1 and FITC in channel 2, but FITC bleeds into channel one complicating my analysis, could I use this unmixing to make a better distinction if I already have the two channels established in my final movie?

[@meetings5145]

Hi,
After linear unmixing using the spectral image and plot, i get a 'Concentrations' plot but i do not get the unmixed image ? My image has 32 frames and is 512 by 512 in resolution. ON using the v1 linear unmixing plugin, i get a multichannel image but it is all black!

[@StowersInstitute]

It seems that you’ve run the linear unmixing plugin on the spectral plot window itself and not the image. Try again, but be sure to click on the image window before clicking ‘Run’ on the plugin. Clicking on the image window first, will ensure that the plugin runs on the image and not the plot window.”

[@camillaromagnoli9060]

Hi, thanks for the video, where can I find the plugins?

[@StowersInstitute]

Be sure to check out the first video in this series which shows how to set up Fiji and get the plugins we are using.

[@ebineshs]

What difference it makes doing Linear unmixing instead a mere LUT for each channel by splitting the channel ? Please Help me in this

[@StowersInstitute]

Hello, thank you for your question! Remember the data we’re talking about here is fundamentally different from a standard multichannel fluorescence image. Here each pixel is not just a fluorescence intensity, but a fluorescence spectrum. Spectral unmixing is the method by which you can take those fluorescence spectra, and turn them into an unmixed multichannel image.

[@ebineshs]

@@StowersInstitute Thanks for the Reply! It's clear. But whenever I do Unmixing spectral image with ACE, the overlapping region is being removed. I am not sure what happens actually here in this method. Should I be aware of what need to be removed ! Looking for good reading suggestions to fully understand and apply Unmixing perfectly. Thanks a lot for your time in helping us