Quasi-spectral characterization of intracellular regions in bright-field light microscopy images

Abstract

Investigation of cell structure is hardly imaginable without bright-field microscopy. Numerous modifications such as depth-wise scanning or videoenhancement make this method being state-of-the-art. This raises a question what maximal information can be extracted from ordinary (but well acquired) bright-field images in a model-free way. Here we introduce a method of a physically correct extraction of features for each pixel when these features resemble a transparency spectrum. The method is compatible with existent ordinary bright-field microscopes and requires mathematically sophisticated data processing. Unsupervised clustering of the spectra yields reasonable semantic segmentation of unstained living cells without any a priori information about their structures. Despite the lack of reference data (to prove strictly that the proposed feature vectors coincide with transparency), we believe that this method is the right approach to an intracellular (semi)quantitative and qualitative chemical analysis.

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

“…Since this calibration method [23] is wavelength-wise, it allows the segmentation of objects of a particular wavelength in a microscopy image like our spectral method (following our calibration approach, ref. [15]). Unlike our method, the calibration filters are not placed in the focal plane of the imaging system.…”

“…In label-free bright-field microscopy, the signal is generated mainly by diffraction of a wide range of light wavelengths in the sample. Light diffraction is a spectrally dependent phenomenon that can be utilized for determination of the transmission spectra of observed objects [15]. Therefore, in order to describe the spectral properties of the observed sample, in the proposed microscope arrangement [13,14], the object response is captured by a color digital camera.…”

Estimation of rheological parameters for unstained living cells

“…To suppress the image distortions, the microscope optical path and camera chip was calibrated and the obtained time-lapse micrographs were corrected by a radiometric approach described in detail in [7].…”