Characterization On The Beam Propagation Properties For In Situ RNA Imaging In Thick Samples

Researchers have been trying to explore the mysteries of brain for a long time.In brain science,neuronal cell-type classification is a hot topic.RNA imaging in situ is a key technique to obtain 3D spatial distribution of multiple RNAs without destroying the structural integrity of brain slices,and thus improves the accuracy of neuronal cell-type classification.Currently,there are many ways of imaging RNA in situ;however,due to the illumination limitation,the imaging thickness of RNA imaging in situ is still insufficient.This paper investigated the beam propagation characteristics during the imaging process of in situ RNA imaging in thick samples,which was based on optical simulation and experiment and aimed to improve the efficiency of illumination and optical system optimization.The main contents are as follows:(1)To meet the illumination requirement of in situ RNA imaging in thick samples(a uniform light sheet with an illumination volume of 200 ?m × 10 ?m × 50 ?m),this paper designed and verified a Gaussian-beam based illumination scheme.This paper studied the waist diameter of light sheet by the physical optics propagation(POP)analysis in ZEMAX simulation,and then used the MZDDE toolbox to establish a connection between ZEMAX and Matlab,which helps realization of a rapid reconstruction of 3D light sheet.This work is helpful for fully understanding the characteristics of illumination beams,and for the design of the illumination scheme based on cylindrical lens.Additionally,this paper proved that a cylindrical lens with a focal length of 100 mm can realize the illumination requirement mentioned above when the incident Gaussian beam spot diameter is 8 mm.(2)To meet the goals of fast system optimization and improved compactness in multicolor light sheet illumination system,this paper used both simulation and experiments to study the beam propagation characteristics of light sheet from a multi-mode fiber.Results from both simulation and experiments showed that the thickness of light sheet from multimode fiber coupling is much higher than that from Gaussian light sheet,and the influence factors include focusing objective,fiber core diameter and the conjugate ratio of light path.Specifically,with a smaller fiber core diameter and/or a larger NA in the focusing objective,the beam waist thickness becomes smaller.The waist diameter of light sheet is proportional to the optical fiber core diameter,and the ratio equals to the light path conjugate magnification.Based on these findings,this paper designed a light sheet illumination scheme using a square multi-mode fiber with a core diameter of 100 ?m × 100 ?m,and achieved a single illumination volume of 200 ?m × 10 ?m × 50 ?m.(3)This paper investigated the characteristics of light sheet beam propagation inside biological tissues using fractal propagation method(FPM),and then extended the beam propagation simulation for a whole optical system by combining ZEMAX optical path tracing with FPM analysis.Results showed that,with the increase of propagation distance in scattering tissue,the waist diameter of light sheet grew,but the diameter can be maintained at about 10 ?m in over 50 ?m propagation distance for brain slices.In addition,this paper verified that FPM is applicable to the simulation of the beam propagation in a whole RNA imaging system.Further,after studying the relationship between the PSF and image quality,this paper found out that the image background comes from the stack of defocus signals.When the objective NA meets the requirements,researchers should select an objective with low magnification and high depth of focus,which is helpful for reducing the fluorescence background in raw images.This paper used both simulation and experiments to study the beam propagation characteristics during the imaging process of in situ RNA imaging in thick samples.The study on the characteristics of light sheet beam propagation greatly improves the efficiency of the optical system design.The study on the beam propagation characteristics of a whole RNA imaging system is helpful for understanding the source of high background and improving the efficiency in system optimization,and has good potential to promote light sheet illumination in more application scenarios where tissue scattering is unavoidable.