Spectroscopic Approach to Correction and Visualisation of Bright-Field Light Transmission Microscopy Biological Data

The most realistic information about the transparent sample such as a live cell can be obtained only using bright-field light microscopy. At high-intensity pulsing LED illumination, we captured a primary 12-bit-per-channel (bpc) response from an observed sample using a bright-field microscope equipped with a high-resolution (4872x3248) image sensor. In order to suppress data distortions originating from the light interactions with elements in the optical path, poor sensor reproduction (geometrical defects of the camera sensor and some peculiarities of sensor sensitivity), we propose a spectroscopic approach for the correction of this uncompressed 12-bpc data by simultaneous calibration of all parts of the experimental arrangement. Moreover, the final intensities of the corrected images are proportional to the photon fluxes detected by a camera sensor. It can be visualized in 8-bpc intensity depth after the Least Information Loss compression [Lect. Notes Bioinform. 9656, 527 (2016)].