Research On Super-resolution Microscopy And Technology Based On The Modulation Of Detection Field In Time And Space

In the field of microscopic imaging,electronic microscope is widely used for its popularity of superior imaging resolution,which can be achieved to sub-nanometer.However,strict imaging condition like vacuum imaging environment limits the application of electronic microscope in long-term observation of biological live-cell samples.Far-field fluorescence optical microscope would be a better choice for biological live-cell imaging for its non-invasive,deep penetration depth properties.Meanwhile,fluorescence optical microscopy owns the superiorities of high specificity and multiple-parameters due to the use of fluorescence dyes,leading to the fact that far-field fluorescence optical microscope is a significant tool for modern biomedical research.However,due to the existence of the diffraction limit that the resolution of the far-field optical microscope is theoretically limited to around half-excitation-wavelength scale,traditional far-field fluorescence optical microscope cannot meet the demands of smaller molecules.To improve the imaging resolution of the fluorescence optical microscopy to realize the super--resolution imaging of the molecule architecture,more and more super-resolution microscopy has been proposed over decades,the most popular of which is the point-scanning based super-resolution microscopy.Point-scanning technique used the focused spot to scan through the sample to realize the scanning imaging.Moreover,the conjugation between the point-excited sample plane and the pinhole(detection plane)can enhance the imaging resolution.However,there still exists many problems in current super-resolution fluorescence microscopy,such as limited resolution,compromise between the resolution and signal-to-noise ratio(SNR),university of samples,monotonous parameter,slow imaging speed,etc.This thesis focuses on time and spatial optical modulation on detection field.By taking the parallel detection technology and stimulated emission depletion(STED)microscopy as the starting points,we aim to achieve the improvement of imaging resolution and simplify the super-resolution optical setup.In the meantime,by utilizing the property of the fluorescence multiple-parameters,this thesis also includes ameliorative methods to obtain the fluorescence lifetime information,leading to the upgrade super-resolution imaging system from another dimension.The main work and innovations of this thesis are as follows:1.Image scanning microscopy based on parallel detection and spatial modulation has been proposed.In-depth theoretical research of image scanning microscopy with pixel reassignment has been studied.In the meantime,fluorescence emission difference(FED)microscopy based on parallel detection has been proposed to improve the imaging resolution.The nonlinearity induced from saturated fluorescence signal has been applied in FED microscopy with parallel detection to impro ve the resolution by a factor of 2 while FED imaging setup could be upgraded with only one excitation light beam path.2.A novel rapid fluorescence lifetime imaging microscopy(FLIM)based on parallel detection has been proposed.The detector array and time-correlated single photon counting(TCSPC)array are used to gain the time information,suppressing the pile-up effect resulting from the detector and TCSPC.The lifetime imaging speed could be increased by at least 3 times.Besides,pixel reassignment has also been applied to enhance the fluorescence lifetime imaging resolution.Meanwhile,combined with spectroscopic technology,the lifetime imaging speed can be further improved significantly by an order of magnitude.3.A STED imaging system based on adaptive optics has been built.Spatial optical modulation on detection field can be achieved by using the spatial light modulator(SLM)to correct the aberration on the depletion beam,and taking advantage of deformable mirror that can correct the aberration in the fluorescence path.The imaging quality and resolution can be improved by custom STED,which may provide the prospect of the application on super-resolution deep imaging of live cell.4.Time modulation is realized with two time-gate and time-delay circuit boards to obtain fluorescence lifetime information in custom STED system,therefore time-gated STED can be achieved to improve the imaging resolution.Meanwhile,a novel STED microscopy with ratiometric photon reassignment based on time-gated detection has been proposed to lower the requirement of depletion power,so that the photobleaching effect can be inhibited.