Study On Fast Axial Scanning Method

The two photon fluorescence microscopy has been widely used in observation and study of living tissue's life phenomena,with advantages of high detection depth,high resolution,low optical damage,etc.The neural network is distributed in three dimensional space,and Ca²⁺concentration on both sides of the nerve fiber can be changed in nerve signal transduction.The changes of fluorescence signals can be detected through fluorescence labeling of Ca²⁺,so that the various life activities of nerve cell can be studied.Nerve signal conduction is very fast,signal change time is in milliseconds.Accordingly,the fluorescence signals of Ca²⁺also change rapidly in three-dimensional space,and three-dimensional random scanning imaging technology is required to detect such fast signals.To do this,scanning speed of fluorescence microscopy system should be as fast as possible?kHz?.Currently,the two-dimensional random scanning technique is rapid enough.Therefore,axial scanning speed is the bottleneck that limits scanning speed of the whole system.In order to realize three-dimensional random scanning of fluorescent signal,a new fast axial scanning method must be developed,which makes optical focal point move fast in direction of optical axis.The existing axial scanning methods have problems of slow scanning speed,low signal to noise ratio of fluorescent signal and small scanning range,etc.It is necessary to develop new methods for fast axial scanning,which can achieve 3D imaging of fast fluorescence signals in neural cells.Based on the theory of two-photon fluorescence microscopy,new fast axial scanning methods are studied in this paper.The main innovative results are:?1?The fast axial scanning method based on moving a few lenses of objective lens is proposed.The objective is divided into a focusing lens group and a main lens group.The light path between focus lens group and main lens group is near parallel.If length of the parallel light path is changed,the change of image quality is small,and the optical properties are stable.The focus lens group is composed of one or two lenses,which weighs only 0.1g.The focus lens group is driven by the lens driver,so that the focus of objective lens can be rapidly scanned in axial direction,and system enables to adapt to a large scanning range.The system positioning accuracy is high,the axial scanning frequency is about 8.76 kHz,and the scanning range is 0-505.3?m.?2?A lens driver based on ring displacement amplification mechanism is designed.The ring displacement amplification mechanism is used to carry out two stages amplification of displacement,which makes the structure simple and compact,and the displacement amplification rate is high.The motion transmission is continuous without friction,which is beneficial to modular installation.Three displacement drive devices can be controlled separately,which provide larger driving force and higher axial scanning speed.The displacement output platform can realize axial scanning along z axis or rotation along y and x axis.Moreover,lens,mirrors and other optical elements can be mounted on it,so that the lens driver has a wide range of uses.?3?A fast axial scanning method based on digital micro-mirror device is proposed.The scanning system is composed of modules in series.By controlling each module individually and changing divergence angle of the incident light,different focal depths can be obtained.Therefore,the system can realize fast axial scanning of biological samples.The scanning frequency can reach 10 kHz,the optical aperture is large,the maximum scanning range can reach 771.1?m when module number is 5,and the axial distribution of scanning points is uniform.By increasing number of modules,the system's axial scanning range and imaging resolution can be further improved.The system has advantages of simple structure,modular design,convenient user upgrading and transformation.The axial scanning system is independent of the lateral scanning system,has flexible operation and strong practicability,can satisfy the confocal and two-photon microscopy system for axial scanning speed and scanning range.