| Laser confocal microscope is an advanced microscopic equipment that uses optical imaging system,nano-scanning positioning system,and computer image processing technologyfor three-dimensional fluorescence imaging.It has important applications in the fields of micro-nano optics,material science and biomedicine.In the process of laser confocal microscopy imaging,the manual focusing of confocal microscopes is not only time-consuming and laborious,but also has great subjectivity and human error.Therefore,it is necessary to develop a fast and accurate auto-focus technology for laser confocal microscopy imaging.In addition,the three-dimensional nanomicroscopic imaging of the laser confocal microscope usually needs to collect a large number of imaging pictures to observe and analyze the evolution of cells or tissues and other life evolution process.Since the confocal microscope needs to perform point-by-point scanning imaging,the acquisition process usually takes tens of minutes or even tens of hours.During the entire image scanning acquisition process,various factors such as external environment vibration,system mechanical instability,environmental temperature changes and other factors can cause axial drift of the confocal microscope,resulting in imaging blur due to defocus in the confocal system,thus affecting the results of experimental observations.This dissertation focuses on two key scientific issues: how to realize the automatic focus of the confocal microscope and how to realize the drift compensation in the imaging process of the confocal microscope.The main work and innovations of this thesis are as follows:1.The focus detection principle and algorithm of autofocus are developed,and the automatic focusing function of laser confocal microscope is realized based on the spot image processing mode,which can automatically and quickly find the imaging focal plane,and realize the automatic focusing function with a focusing speed of 0.5 μm/s and a sensitivity of 46.2 nm under fixed steps.2.The method of detecting the position of the center of gravity of the spot has been developed,and the automatic drift compensation function of the microscope has been realized,which can stabilize the experimental sample on the set imaging focal plane for a long time,and the stability accuracy of the system is 98.9 nm,which can meet the needs of confocal microscopy.3.The automatic focus and drift compensation functions are integrated in the same optical system,and the C++ program suitable for the automatic focus and drift compensation system is developed,which can directly perform image algorithm processing and system hardware control,which significantly improves the practicality of the system. |
