Hi Mayank,

EYFP is a very bright fluorescent protein ... in neutral conditions.

In acid condition it is dim.

The pKa of the fluorophore is 6.9. This means that below a pH of 6.9, the tyrosine of the fluorophore deprotonates. In these conditions the quantum yield is much lower. This leads to a dimmer fluorescence of YFP and an enhanced bleaching in acid conditions.

Oliv

Thanks for this precision, I thought peroxisome was an acidic organelle like lytic vacuoles. I was obviously wrong.

I am not sure to correctly understand your question. You want to see weak signal from peroxisome that might be hidden by stronger signals from plastids and you want to know if there are methods to remove stronger signals.

Am I right ?

If you do wide field imaging you can try deconvolution. There are deconvolution plugins in ImageJ. Your images will gain resolution.

If the signal coming from peroxisomes is still too weak, you can modify the gamma curve. Gamma = 0.45 is generally useful to equilibrate the image display between weak signals and strong signals.

If you do confocal microscopy you can pump up the voltage of the PMT until you have a correct signal from the peroxisomes, even if you saturatethe pixels from the plastids.

This phenomenon is due to the fact that the direction of the emitted fluorescence photon is determined by the spatial orientation of the fluorophore. It is enhanced by refraction and diffusion phenomena that occur in cells due to the variations of refractive indexes between organelles. This is vaguely nammed as "blur".

In Bright Field microscopy you can set the incident light ray parallel with Köhler Alignment, but in florescence microscopy, as you cannot control the orientation of the fluorophores, you can do nothing to control the direction of the emitted photons.

But you can either eliminate any phton that originates from other points than focal point with confocal microscopy, or reassignate the right origin to a detected photon by deconvolution.

The word you are looking for is "dynamic range". The dynamic range in your images seems to be too small to see the weak signal (peroxisome) without saturating your strong signal (plastid). If I understood you correctly.

There are several ways you can try to improve on this. First, what bit-depth are you using, usually you can set the readout from the PMT to 8 or 12 bit, 16 bit on some systems. While this not increase the dynamic range as such, it subdivides it into more steps (256 for 8 bit, 4096 steps for 12 bit). Higher bit will make the files larger and might affect acquisition speed, but might give you more details on your weak structures.

Another option is HDRi (high dynamic range imaging), which you might know from your phone. In principle this can also be applied to microscopy images. A series of exposures is taken with different settings and then combined into a single image by software. Some microscope software has this function integrated but it can be done externally (e.g. using imageJ). However, be aware that this is a non-linear operation and the resulting image will not be quantitative anymore.

Thank You Dominik for your answer, this is exactly what I was asking about. I think, this solves my question. I will try the steps you mentioned for better visualisation.

Thanks.

well the dynamic range explanation from Dominik is a very good explanation... but nevertheless there is also another one:

fluorescence resonance energy transfer

google it, but in essence you can measure with it proximity, because you get only two fluorophors in resonance if they are very near together...

now with two organelles? but depending on how the spatial setting is AND the background of course depending on the molecules you could also have fret with floating/diffusing molecules if the concentration is high etc. = and in the very extrem case: one molecule pics up the energy diffuses away and finally sends out the photons, but that would mean you have a very long excitation to emission time - so far for the theory, but the question is of course, which molecules, which setting, i.e. what realistically can happen...