Recently, stimulated emission depletion microscopy has achieved high resolution in fluorescent imaging. In this paper, we present effects of a pupil filter on the performances of stimulated emission depletion microscopy. In stimulated emission depletion microscopy, a saturated zero-centered spot is usually used to achieve a high lateral resolution. Using a half-coated phase plate, a zero-centered spot was made with a narrow and steep gap at the center. Numerical and experimental results show that by simply inserting a central obstacle as a pupil filter, it is possible to reduce the central gap of the zero-centered spot. However in order to compensate inevitable loss of light, which is blocked by the obstacle, increased laser power is required.
In this paper, we describe size-effect of fluorescent microsphere for measuring point-spread function (PSF) in confocal fluorescence microscopy. We present the numerical results for the practically available microsphere size range for measuring PSF, and demonstrate with experiment. Also, the effective PSF is restored with deconvolution technique within an acceptable error. Also the size-effect for measuring phase modulated PSF, which has two vicinal peaks, is described. The numerical and experimental result is also presented.
In this research, the method how to estimate the image quality for different scanning rate is suggested and experimentally shown with the laboratory-built confocal laser scanning microscope. The confocal microscope is designed for in vivo reflectance imaging of a biological tissue, which uses the refractive index mismatch at the boundaries of a tissue to generate an image without any additional staining process. The two-dimensional scanning mechanism is built up with a polygonal mirror and a galvanometric mirror that can be controlled to operate at a specific speed. To examine the effect of scanning rate on the image contrast, confocal scanning images of a biological specimen are acquired with various scanning rate while the other conditions are kept same. The contrast of confocal microscopic image is transformed into the numeric expression to describe the relation between image contrast and scanning rate quantitatively. Results suggest some useful methodology of how to determine the allowable maximum scanning rate for the specific application of confocal microscopy.