Study And Application Of Quantitative Phase Microscopy Through Ultra-Oblique Illumination

The complicated biological process of a cell typically involves many subcellular organelles,proteins and molecules.Fine structures inside a cell can be observed by electron microscope with nanoscale spatial resolution,while the developments of super resolution fluorescence microscope provide a powerful tool for researchers continuously observing the organelles' dynamics inside a live cell for a short time.Using superresolution microscopy,researchers have observed many important biological dynamics inside the live cells.However,fluorescence labeling will affect the normal state of cells to a certain extent,and fluorescence substances will produce phototoxicity and photobleaching to the cell structures when they are excited,which is not conducive to continuous observation of cell dynamics for a long time.Particularly,there are only four or five structures that fluorescence microscopy can observe at the same time,making it difficult to observe multiple organelles,proteins or molecular structures simultaneously.Quantitative phase microscopy as a non-fluorescent labeling imaging method provides a new means for continuous observation of intracellular dynamic processes.Although quantitative phase microscopy has made many advances in terms of spatial resolution,image contrast,and temporal resolution,most of the fine structures and fast dynamics in cells cannot be captured by label-free imaging techniques.In response to this situation,this paper proposes the use of ultra-oblique illumination to achieve label-free observation of submicron-sized organelles.Based on the light scattering model of the weakly scattering object under oblique illumination,the transfer function of the partially coherent oblique illumination imaging system is derived.The relationship between the resolution of the system and the illumination angle is systematically studied.Further,we used an objective with large numerical aperture to realize the super oblique illumination of the sample,and built a label-free quantitative phase microscopy with high spatial resolution,high temporal resolution and high image contrast.In order to improve the imaging speed of the system,we not only upgraded the hardware of the system,including the use of light-emitting diodes with relatively high illumination intensity to achieve multi-angle partial coherent oblique illumination,and a very fast switching spatial light modulator for modulating the phase of incident light.High-sensitivity cameras are used for signal capture.At the same time,we have developed a phase reconstruction algorithm based on three raw images and a cross-reconstruction algorithm based on reusing raw data,which limits the imaging speed of the system to single camera exposure time.In the test of system spatial resolution and image contrast,we respectively imaged 200 nm diameter polystyrene beads and intracellular vesicle structures in live cells,and achieved the lateral spatial resolution of 270 nm and high image contrast.Using ultra-oblique quantitative phase microscopy,we used the label-free imaging method to successfully capture the dynamic structure of the endoplasmic reticulum network in live cells and the transport of vesicles along the endoplasmic reticulum for the first time,indicating that the system has high image contrast and spatial resolution.We also added fluorescent channels to the system to simultaneously perform fluorescence and phase imaging of fluorescently labeled samples such as microspheres,mitochondria and endoplasmic reticulum,verifying the correctness of morphology measurement of micro-submicron cellular structures by label-free microscopy.In addition,in order to verify that the system has high temporal resolution,we observed the dynamic of high-speed vibration of the endoplasmic reticulum.The results show that the high-speed vibration of the endoplasmic reticulum microtubule is detailed at the framerate of 250 fps and recorded.At the same time,we captured the process of mitochondrial fusion and di'vision using label-free imaging methods and observed the special spinning motion of mitochondria for the first time.In summary,the label-free imaging method proposed here provides a new means for further research in life sciences.