Hyperspectral Microscopy Imaging And Its Application In Cell Testing

Spectral imaging is an optical technique that intergates traditional imaging and spectral measurements.Hyperspectral imaging,as a branch of spectral imaging technology,has been developing rapidly in the fields of agriculture,aerology,and geology since the 1980 s.Recenly,it combinines with life sciences showing an important role especailly in cytology.However,it is difficult for the existing cell detection techniques to obtain the spatial information and constituent information of the sample at the same time.This thesis focuses on the application of hyperspectral imaging in cytology,investigates the forefront of hyperspectral microscopy imaging(HMI),and discusses the advantages and disadvantages of related works.Then,a HMI system based on liquild crystal tunnable filter(LCTF)was set up in the lab,which has reliably good performance for bee pollen classification and pigment studies of Haematococcus pluvialis.The specific contents and achievements of this paper are as follows:1.Bee pollen sample of six spicies were selected for bee pollen classification.The pollen wall composition of each spicies is distinct from one another,so their spontaneous fluorescence characteristics could be one of the classification basises.In the experiment,a 409 nm LD was used as laser source,and a total of 37 hyperspectral single band images ranging from 465 nm to 645 nm(?? = 5 nm)were captured by using the HMI system.One hundred pixels in the region of interest(ROI)were randomly selected from each single bee pollen species,the fluorescence spectral information in all the selected pixels was stored in an 600×37 matrix.The hyperspectral data set was classified using a Fisher linear classifier.The performance of the Fisher linear classifier was measured by the leave-one-out cross-validation method,which yielded an overall accuracy of 89.2%.Finally,additional blinded samples were used to evaluate the established classification model,which demonstrated that bee pollen mixtures could be classified efficiently with the HMI system.2.To study the spatial distribution of the pigment concentration of H.pluvialis,the absorption spectra of the three main pigments chlorophyll a,?-carotene and astaxanthin were measured and their absorption coefficients were obtained.The ultrathin sections of H.pluvialis cells were prepared and observed by transmission electron microscopy,which is used to assist in proof.Then,the hyperspectral images of 34 bands from 510 nm to 675 nm(?? = 5 nm)were collected based on HMI system with a white LED as the light source.After hyperspectral image preprocessing,the spatial distribution of the cells in different cell cycles and stress conditions was determined by the steepest descent method according to the established absorption model.Finally,the consistency of the spatial distribution of the pigment with the transmission electron microscopy and the literature results is compared and analyzed,and the feasibility of this method is shown.