Development of Optical Sectioning Microscope using Binary Di raction Hologram

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An optical microscope is an imaging instrument that is used to view the features of an object, which can not be seen with an unaided eye, by illuminating the object with visible light. Based on the illumination type, optical microscopes are divided into two broad categories, namely, the wide eld microscope and the point scanning microscope. In a wide eld microscope the entire target area is illuminated and imaged simultaneously, while, in a point scanning microscope only one object point is illuminated and imaged, and the image of the target area is formed by imaging each object point in a sequential manner. An important member of the point scanning microscopes is the laser scanning confocal microscope (LSCM). Here a laser is focused onto the sample plane using an objective lens to illuminate the object point and the same lens is normally used to collect the light from the sample plane. The light from the sample point is eventually focused on a point photodetector. The point photodetector is kept optically conjugate to the illumination spot on the sample plane. Consequently the photodetector receives light primarily from a volume very close to the centre of the illumination spot. Owing to the unique position of the point detector, the LSCM provides very large axial resolution and moderately large lateral resolution in images, compared to the wide eld microscopes. The large axial resolution is also indicated by the capability of the microscope, to separate signals coming from two close by planes in the sample volume which are perpendicular to the optic axis, known as the optical sectioning capability. The LSCM, due to its ability to form optically sectioned images in a non-invasive way, has become an extremely important and essential imaging tool in a large number of application areas. Realising the importance of the microscope, there have been constant e orts to come up with newer and improved versions of the confocal microscope. However there still exists a number of issues with the conventional LSCM. One such issue is the use of galvo scanner mirror in the LSCM to scan the illumination beam. Such scanners su er from limited accuracy and frame to frame beam repeatability. Further there is a need to use an additional 4f relay system between the two scanner mirrors. Not only that aligning a 4f relay system is a tedious task, it also adds extra sources of aberrations into the illumination beam. The other issue with a conventional LSCM is the illumination beam whose intensity is kept constant for an entire image frame.
Supervisor: Bosanta R. Boruah