Research On Methods Of Improving The Image Quality Of The Digital Scanned Laser Light Sheet Microscopy

Digital scanned laser light sheet microscopy(DSLM)sweeps out a virtual lightsheet by scanning a laser beam through the sample,which is beneficial to reduce the striping artifacts and provide a better illumination efficiency and form a homogenous light-sheet.It’s an ideal tool to provide the three-dimensional and live imaging of the biological specimen for the long-term tracking of biological dynamics.This thesis focuses on the research that how to improve the imaging quality of the DSLM.The main innovative progresses are shown as follows:(1)For Gaussian beams,usually the light sheet has to be thicker to cover the field of view(FOV),which compromise the sectioning ability.To address this issue,we propose to utilize the spherical aberration to extend the depth-of-field(DOF)of the Gaussian beam.As a result,the FOV is extended and the light-sheet thickness is kept small.In the applications of microscopes,spherical aberration is introduced when focusing beams through an interface between materials of mismatched refractive indices.The DOF extension of the Gaussian beam is achieved when using air objectives to focus light into the samples dipped in the immersion medium with a higher refractive index.The results show that the FOV of the light sheet is increased by 5 times.Also,the thickness is reduced by almost 1.3-fold when compared with that of the light-sheet with the equal FOV produced by the objective with low numerical aperture in the unaberrated case.(2)As a non-diffracting beam,the Bessel beam can produce a thin light-sheet over a large field,and it enables anti-scattering and deeper penetration into thick cells or tissues.However,the high-intensity sidelobes of the Bessel beam will reduce the image contrast and sacrifice the optical sectioning capability.To address this issue,a leaky filter is designed to be inserted in the Gaussian beam to create a similar Bessel beam with lower side lobes.By optimizing the parameters of the leaky filter,the first sidelobe energy decreased from 41.18% to 33.25% when compared with the Bessel light-sheet created with annular aperture.(3)Using the hollow Gaussian beam as the depletion beam of the stimulated emission depletion microscopy,we propose applying the stimulated emission depletion microscopy technology to the digitally scanned light sheet fluorescence microscopy with Bessel beams.As a result,the axial resolution of the Bessel beam scanned light sheet fluorescence microscopy can be enhanced and the sidelobes of the light sheet with Bessel beam can be effectively suppressed.Compared with the traditional digitally scanned light sheet fluorescence microscopy with Bessel beam,the thickness of light sheet is reduced by almost 2.2 times and the first sidelobe is decreased from the original value of 42% to 6.5%.