Fluorescence Microscopy With High Spatiotemporal Resolution

Fluorescence microscopy imaging is one of the most important imaging modalities in the realm of biomedical science.The advances of fluorescence microscopy promote biomedical research.Acquiring fluorescence images with high spatiotemporal resolution remains a great challenge,yet it is a long demand for scientists.To address this problem,we develop a novel multispectral confocal microscope derived from the principle of high spatiotemporal resolution fluorescence imaging.The imaging system has high spatial resolution and spectral imaging capacity.The multispectral confocal system consists of a confocal microscope and a tunable filter system.The thesis will first introduce the basic theory related to the imaging system.Then,the system setup will be presented.Finally,we will present the experiments using our system for validations.Firstly,we introduce the principle of tunable filter and confocal microscope and describes the dispersion of light and digital micromirror devices in detail.In particular,we study the prism and trace the ray in prism dispersion and obtain the basic principles of prism together with key parameters.Secondly,in order to improve the system's spectral resolution,we designed a tunable filter system with optical structure comprised of two lenses and a prism.Our design thought not only makes the optical path owns low magnification but also increases the length of the dispersion path.Based on the design,we used Zemax to simulate the optical path.After that,we built the tunable filter and confocal system and integrate them into a multispectral confocal microscope.Lastly,multiple performance tests and biological experiments were conducted.Performance tests include energy efficiency test,spectral resolution test,fluorescence emission spectra acquisition.Biological experiments cover confocal microscope imaging,multi-color imaging,and multispectral imaging.In the spectral resolution test,we designed a collection optical path for the tunable filter,which reduces the diffraction effect of the digital micromirror device and enables the commercial spectrograph to measure the resolution correctly.In addition,we redefined the theoretical spectral resolution for our system and calculate those values using Zemax.Besides,the method of constructing fluorescence emission spectra was proposed and utilized to measure the spectra of fluorescence dye.Finally,multi-color imaging showed that the system can distinguish different objects through spectra,and multispectral imaging proved that it can acquire spatial and temporal information simultaneously.