Experimental Study On Interferometric Quantitative Phase Microscopy For Three-dimensional Surface Reconstruction Of Biological Cells

Optical microscopy has become the most common and important analytical method and testing tool in the biomedical and life sciences due to its non-contact,non-radiative,real-time observation.Phase microscopy,especially quantitative phase microscopy,has shown great advantages in microscopic imaging of transparent biological cells,and has become one of the hotspots and development directions of current biological cell imaging research.This technology overcomes the problem that the use of labeling dyes in fluorescent microscopes may cause damage to cells,furthermore,it not only enables the quantitative measurement of refractive index,thickness and other information of transparent biological cells or tissues,but also provides us with information on the morphology,structure or dynamic behavior of biological cells.This technology provides a very effective means for our research in the microscopic field and is of great significance.Most quantitative phase microscopy techniques are based on light interference phenomena.This kind of technology mainly includes several processes such as acquisition of interference pattern,reconstruction of phase information and subsequent phase unwrapping.The acquisition of interference pattern is the basis of quantitative phase imaging technology.Obtaining a high quality interference pattern is the key to the success of subsequent phase reconstruction.Based on this,our work focuses on the design and experimental implementation of the interferometric phase measurement system in order to obtain high-quality interference patterns to achieve quantitative phase microscopic imaging of biological cells.The research work of this dissertation mainly has the following aspects:1.Phase imaging of biological cells(paramecium cells)is successfully obtained by performing interferometric quantitative phase microscopy based on single optical element.We respectively use a single wedged glass plate and a single cube beamsplitter to achieve two quantitative phase microscopy methods.For the method of using only a single wedged glass plate,the basic idea is to adjust the sample to touch only one half of the illumination beam,and then use a wedged glass plate to deflect this part of the beam carrying sample information toward another part of the beam,so that the two parts of the beam meet and overlap together to form interference.For the method of using only a single cube beamsplitter,we place the cube beamsplitter obliquely so that the incident beam is incident only on one side of the central semi-reflective layer of the cube beamsplitter.Carefully adjust and control the cube beamsplitter to form a suitable angle between the central semi-reflective layer of the cube beamsplitter and the optical axis of the incident beam.Thus,the two beams generated by the cube beamsplitter will meet and overlap to form interference.Since the images of the beams produced by the cube beamsplitter are opposite each other,we create two channels interference when we adjust the sample to a suitable position.The method based on a single optical component is very simple and easy,and can effectively control the cost,which is very practical.2.Three interferometric quantitative phase microscopy methods with two interference channels were implemented,and phase imaging of biological cells(paramecium cells)is successfully obtained.In this type of method,a conventional cube beamsplitter placed obliquely is used to generate two parallel plane waves parallel to each other.Based on this,a wedged glass plate,a Fresnel's biprism,and a Fresnel bimirror are respectively used to form interference.For the first method,a very common wedged glass plate is used to deflect only one of the beams to the other beam so that they overlap to form interference;for the second method,a Fresnel's biprism is used instead of the wedged glass plate,and the ridge of the Fresnel's biprism is adjusted to coincide with the central semi-reflective layer of the cube beamsplitter,so that the two beams generated by the cube beamsplitter are respectively incident on the different sides of the Fresnel's biprism and are deflected toward each other,and then they are brought together to form interference;for the third method,a conventional Fresnel bimirror is used instead of the above two devices.The Fresnel bimirror is placed at the output port of the oblique cube beamsplitter,so that the two beams generated by the cube beamsplitter are respectively reflected by the two mirrors of the Fresnel bimirror,and then the two beams meet and overlap to form interference.3.An improved Michelson interferometer is proposed by replacing two plane mirrors of an ordinary Michelson interferometer with two right-angle prisms.Based on this improved Michelson interferometer,two Michelson-type lateral shearing interferometric quantitative phase microscopy methods are realized by rotating or translating right-angle prisms,respectively,and phase imaging of biological cells(paramecium cells)is successfully obtained.For the method of rotating the right-angle prism,we realize the lateral shearing interference by rotating the right-angle prism in the horizontal plane with the ridge of the right-angle prism as the rotation axis;for the method of translating the right-angle prism,along the direction perpendicular to the optical axis of the incident beam,we translate the right-angle prism in the horizontal plane to achieve lateral shearing interference.4.All our methods are based on a traditional inverted microscope configuration,which allows our method to be easily integrated into off-the-shelf optical microscopy equipment,greatly expanding the range of applications of our proposed methods.In addition,all our proposed interference devices are easy to operate,inexpensive,compact,and portable,and have very good practicality.