The Research And Functional Verification Of Calibration Method On Fluorescence Microscope

Fluorescence microscope is a kind of optical microscope and it is a basic tool of immunofluorescence cytochemistry.Its main function is to study the absorption,transportation,distribution and location of chemical substances in cells.It has been widely used in some fields,such as,molecular genetics,cell biology,medical diagnostics and drug screening.However,during the use of the fluorescence microscope,there will be some problems,for example,aging of components and unstable optical path,which will influence the performance of the instrument,and reduce the accuracy of test data.So,it is necessary to carry out daily calibration of the instrument.At the same time,because the fluorescence measurement method belongs to relative measurement,the fluorescence intensity of the same product detected on different instruments will be different,resulting in no comparable data between instruments.Therefore,it is necessary to calibrate the instrument.At present,many foreign scientific research institutions have used the calibration method of reference materials to trace the measurement value of such instruments.The calibration method of reference materials is mainly divided into the calibration method of fluorescent reference materials and the calibration method of fluorescent calibration slide.But now our domestic scientific research institutions are rarely engaged in the relevant work.Although there are some researches on the calibration method of fluorescent reference materials,there is a lack of research on the calibration method of fluorescent calibration slide.In this environment,based on the calibration method of fluorescence calibration slide,this paper studies the calibration of fluorescence microscope,develops the calibration method and makes the fluorescence calibration slide.The main content of this paper is divided into three parts:(1)Firstly,it uses the calibration method of fluorescence calibration slide,to calibrate the uniformity of fluorescence light path of fluorescence microscope.There are two methods to obtain the fluorescence intensity,one of them is using detection device,another one is using Image J software.Then it compares and analyzes the results of the two methods and determines the calibration method.(2)According to the calibration method,the calibration slide is designed and made.By the way,the minimum resolution of the simultaneous calibration slide is 4μm.(3)The calibration method is verified and analyzed by using the calibration slide,and the function of the calibration slide is verified at the same time.Through the research and experimental results,it is proved that the calibration method proposed in this paper can meet the calibration requirements of the fluorescence light path of the fluorescence microscope.And,the correlation coefficient between fluorescence intensity and concentration is 0.9989.This calibration method has higher detection accuracy.The designed fluorescence calibration glass has good uniformity in some areas,and can select uniform areas to calibrate the fluorescence microscope.At the same time,it has good short-term stability,and can display the calibration with the microscope Image J data at the interval of minutes.The stability of the process meets the calibration function.