Research On Light Sheet Based Two-Dimensional Light Scattering Cytometry And Its Application For Cell Senescence Detection

The aging of population has becoming an issue of common concern in the world.With the exacerbation of aging problem,the hazard caused by the aging-associated diseases is also increasing.Thus it is of great significance to investigate the aging mechanism as well as the prevention and therapy of the aging-associated diseases.Cell senescence is the basics of individual aging and bound up with the occurrence of various diseases.It has the promising potential to find applications in the cancer treatment.However,the study of cell senescence is still in the early stage,thus it is extremely important to explore new methods for detection of cell senescence,which may help to investigate the application of cell senescence in disease diagnosis and treatments.Conventionally,the most extensively used method for senescent cell detection is to measure the SA-β-gal(Senescence associated β-galactosidase)activity through histochemical staining or fluorescence staining.The staining-based methods are sophisticated,time-consuming and costly,especially difficult to be used for detection of living cell.Light scattering is sensitive to the cell structure and cellular components,enabling the real-time,noncontact and label-free detection of living cells.It may provide a new possibility for the fast and noninvasive detection of senescent cells.To address the problems in the senescent cell detection,the 2D light scattering technology and the light-sheet illumination are combined with microfluidics to develop the low-cost and label-free devices for single cell analysis.The main works and contributions of this dissertation are summarized as follows.(1)The light-sheet illumination is adopted to address the noise issue in the light scattering measurements.The light sheet has the ability of selective illumination,thus only the scatterers of interest can be excited while other areas are out of excitation.Therefore,the background noise can be reduced and the signal to noise ratio can be enhanced.Then a light-sheet-based 2D light scattering static cytometer is developed.The operation of the cytometer is validated by performing measurements on the light sheet and polystyrene microspheres.The device is applied for particle differentiation and size determination of yeast cells,with the resolution achieving submicron level.The innovations of this work lie in three aspects.Firstly,the light sheet technology is firstly combined with 2D light scattering to enhance the signal-to-noise ratio.Secondly,the developed cytometer needs no flow-control and thus is low-cost,easy-to-use and less-sample consuming.Additionally,the device enables the measurements of single particles with submicron resolution.(2)The 2D light scattering static cytometer is applied for label-free characterization of senescent cells combined with machine learning method,with the accuracy up to 90%.Thus,the 2D light scattering is proposed as a new method for fast label-free detection of senescent cells,without the need of any histochemical or fluorescence staining(3)A compact hydrodynamic focusing unit based on glass microcapillaries is developed to generate steady 3D focusing fluid for cell control.Different from conventional microfluidic cytometry,the unit is free of microfabrication.Therefore,it does not require microfabrication instruments or ultraclean room.This inexpensive hydrodynamic focusing unit is disposable,and thus it is able to avoid cross-contamination.Moreover,the designed unit is independent and compatible with other techniques and devices.(4)Light-sheet-based 2D light scattering microfluidic cytometer is developed.The disposable hydrodynamic focusing unit coupling well with light-sheet illumination is developed to improve the efficiency of label-free detection for single cells.The cytometer is successfully applied for classification of the mixed particles,with the resolution reaching up to 100 nm by introducing the Euclidean distance-based similarity method.With the advancement of machine learning method,the nicrofluidic cytometer achieves an automatic,label-free classification of senescent cells from normal cells.It provides a very high accuracy for automatic,label-free identification of senescent cells This device can also be applied for label-free detection of other types of cells.