Research On Deep-depth, Near-infrared Two-zone Confocal Imaging System Based On Wavefront Shapin

Laser scanning confocal microscopy has the advantage of optical sectioning and high imaging resolution,and is widely used in nanomaterials,basic medicine and biology.However,the scattering of incident light by biological tissues makes it impossible for confocal microscopy to focus in deeper areas,which seriously affects the image quality in deep imaging and also puts forward new requirements for laser scanning confocal microscopy.Imaging in the near-infrared band,where biological tissues have less influence on light,is an effective way to improve the depth of imaging.Nevertheless,as the imaging depth increases,the incident light inevitably scatters,affecting the image quality.Traditional adaptive optics improves image quality in deep imaging by detecting aberrations generated by samples and compensating for incoming light wavefronts using spatial light modulators(SLMs).This method requires direct measurement of wavefront distortion using wavefront sensors and guide stars,which may affect the life activities of living samples due to the implantation of guide stars.In view of the above problems,this paper carried out a research on the nearinfrared-Ⅱ confocal imaging system based on wavefront shaping,and introduced wavefront shaping into the confocal imaging system to realize the light focusing after penetrating biological samples,so as to improve the imaging quality.On the one hand,the near-infrared-Ⅱ dye was used to obtain the fluorescence of the near-infrared-II with an emission wavelength of 1118 nm,which effectively reduced the scattering effect of biological tissues on light.On the other hand,the method of indirect wavefront shaping was developed to avoid the use of "guide stars" and wavefront sensors.Fast non-intrusive wavefront shaping is achieved while reducing system complexity.The main results of this paper are as follows:1.The near-infrared laser scanning confocal system based on spatial light modulator was designed,the selection of components was completed according to the system requirements,and the parameters and functions of each module were studied.In the designed hardware optical path system,an 808 nm continuous laser is used as the excitation light source,and the spatial light modulator is used as the wavefront shaping carrier to realize the modulation of the incident light.The scanning galvanometer realizes the bidirectional scanning of the beam,while the photomultiplier tube completes the reception and amplification of the weak fluorescence signal.Finally,a MATLAB program is written to realize the reconstruction of the electrical signal amplified by the photomultiplier tube to obtain the scanned image.2.Complete the construction of the imaging system,write the galvanometer control program to realize the control of the voltage applied to the scanning galvanometer;At the same time,the spatial light modulator control program is also written,and the wavefront shaping algorithm based on the "genetic algorithm" is implanted in it to realize the correction of the incident light.Finally,the galvanometer control program,spatial light control program,and image reconstruction program are integrated on labview into an easy-to-operate program panel.3.Prosthesis and in vivo experiments were designed to test the application of wavefront shaping in biological imaging.First,an prosthesis experiment is performed on the electrospinning sample.The experimental results show that our system can correct for aberrations caused by the system,defocus,and scattering media,increasing the signal intensity to 2.02x,2.18x,and 1.63x,respectively,before correction.In live mouse experiments,intracranial vascular imaging of mice penetrating intact skulls was achieved,and the blood vessel signal intensity at 320 nm below the skull was increased to 2.85 times that before correction.These experimental results show that the developed system can overcome the scattering of turbid media and even biological tissues to obtain high-resolution images,which is expected to be widely used in mouse brain vascular marker imaging,brain neuronal marker tracking and even optogenetic analysis.