Abstract
Contrast in fluorescence microscopy images allows for the differentiation between different structures by their difference in intensities. However, factors such as point-spread function and noise may reduce it, affecting its interpretability. We identified that fluctuation of emitters in a stack of images can be exploited to achieve increased contrast when compared to the average and Richardson-Lucy deconvolution. We tested our methods on four increasingly challenging samples including tissue, in which case results were comparable to the ones obtained by structured illumination microscopy in terms of contrast.
Highlights
Microscopy allows biologists to study life by observing at a magnified scale the objects of interest such as a cell and its inner components
We have included MUSICAL result with subpixelaiton of 1 for the different samples in order to analyze the difference between the proposed indicator functions (CE1, Contrast enhancement 2 (CE2), Contrast enhancement 1 (CE1) and CE2s) and the original one
MUSICAL results are included as the starting point as any implementation available could be used to obtain these results by setting the subpixelation to 1
Summary
Microscopy allows biologists to study life by observing at a magnified scale the objects of interest such as a cell and its inner components. Fluorescent optical microscopy allows the visualization of structures of interest such as microtubules or mitochondria through special molecules called fluorophores that are attached to the sample during its preparation. The ability to resolve between different intensities in the image, the so-called contrast, may be affected by several factors such as blurriness due to point-spread-function of the system (PSF), Poisson noise due to photon counting, electronic noise produced by spurious charges and dark noise. While it is possible to reduce noise during acquisition by increasing the excitation laser power and/or increasing the dwell time, this can produce saturation in the sensor and photobleaching on the sample and it is not always possible nor desirable. The alternative is stacking which consist on taking several images of the sample at low power and low dwell time, and average them. Small structures can be affected as their signal becomes closer to the noise level
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