Extending Spatiotemporal Resolution Of Super-Resolution Microscopy

Optical microscopes have limited spatial resolution～200 nm because of light diffraction.Many super-resolution(SR)fluorescence microscopy techniques that overcome the limit have been developed,achieving widespread biological applications.Nobel Prize(2014 Chemistry)was given to the field as well.Generally,SR techniques include three major families:structured illumination microscopy(SIM),stimulated emission depletion(STED)microscopy,and single-molecule localization microscopy(SMLM).The spatial resolution decreases from SIM to STED to SMLM,with typical lateral resolution of 100 nm,50 nm and 20 nm,respectively.Apart from spatial resolution,continuous imaging duration,three-dimension imaging depth,multicolor capability,photo-toxicity and live-cell capability are important parameters as well-especially temporal resolution(i.e.imaging speed).If imaging is not fast enough,the fast movement of biological structures would induce motion blur,negatively affecting trustiness of images.Currently,spatial and temporal resolution are antagonistic in SR microscopy,limiting the performance of SR microscopy in biological applications.Can the boundaries of spatial resolution be extended further?Can the imaging speed be improved without compromising spatial resolution?These questions concern three major aspects:theory of optics,instruments and fluorophores.In this thesis,theory of optics and instruments are concerned.This thesis conducts the following works:1.Theoretical framework of two-photon MINFLUX.MINFLUX has the highest spatial resolution in current SR techniques,with～1 nm localization precision and～5 nm resolution.However,current MINFLUX doesn’t provide efficient multicolor capability,and contributes to only limited biological findings.This thesis proposes application of two-photon fluorescence to MINFLUX.Two-photon excitation increases gradient of intensity of donut minima through nonlinear effect,doubling the localization precision,as well as resolution,in two-photon MINFLUX.A 0.36 nm precision can be obtained when collected 1000 fluorescence photons per localization.A single two-photon excitation wavelength may efficiently excite multiple fluorescence;registration-free multicolor 2p-MINFLUX is thus anticipated.2p-MINFLUX takes a step closer to the ultimate resolution limit of size of a single molecule.2.Further improvement of imaging speed of SIM.Firstly,this thesis realizes a reduced frame SIM algorithm with construction of an overdetermined equation system in Fourier domain.The reduced frame SIM algorithm successfully reconstructs 2D-SIM images with only 5 raw images out of 9 images of traditional 2D-SIM.The algorithm is verified with imaging of biological samples on commercial SIM microscope and resulted in 107 nm resolution which is comparable to the resolution(89 nm)of traditional 9 frames reconstruction.It is the first reported reduced frame SIM algorithm that perform successfully on biological samples with high NA.Secondly,a parallel acquisition SIM is proposed to decrease the acquisition time of raw frames.Each raw frame has to be read-out completely before exposure of the next frame,which limits the imaging speed.By designing parallel acquisition scheme,multiple raw images are acquired with a single readout.Exploiting the rolling shutter of sCMOS camera,fluorescence exposure and camera readout are synchronized completely,saving the previously wasted time of camera readout.For field of view of 16.5*4.7 μm2,it is anticipated that 296 Hz SIM frame rate and 889 Hz rolling reconstruction rate could be achieved.3.STED engineering prototyping with a single super-continuum laser.A STED microscope with 55 nm spatial resolution is built based on a single super-continuum laser,which is used for both excitation and depletion.It has the advantages of freely choices of excitation and depletion wavelengths,inherent synchronization of excitation and depletion beams and compactness of optics and hardware.In collaboration with Donglilai Optics Inc.,Nanjing,the STED microscope is anticipated as a prototype for turn-key commercial STED microscope.Performances and limitations of the STED microscope are assessed with respect to parameters as wavelengths,power,and repetition rate.Notably,negative influence of resolution caused by saturation of Single-Photon Counting Module(SPCM)is observed and analyzed,and strategies to minimize and correct the saturation are proposed.