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Abstract
A compact and position-addressable blue ray scanning microscope (BSM) based on a commercially available Blu-ray disk pickup head (PUH) is developed for cell imaging with high resolution and low cost. The BSM comprises two objective lenses with numerical apertures (NAs) of 0.85 and 0.6 for focusing blue and red laser beams, respectively, on the sample slide. The blue and red laser beams are co-located adjacent to each other and move synchronously. A specially designed sample slide is used with a sample area and an address-patterned area for sample holding and address recognition, respectively. The blue laser beam is focused on the sample area and is used for fluorescent excitation and image capturing, whereas the red laser beam is focused on the address-patterned area and is used for address recognition and dynamic focusing. The address-patterned area is divided into 310 sectors. The cell image of each sector of the sampling area has a corresponding address pattern. Fluorescence images of monkey-derived kidney epithelial cells and fibroblast cells in which the F-actin is stained with fluorophore phalloidin CF 405 are measured by the BSM, with results comparable to those measured by a Leica TCS CP2 confocal microscope. The cell image of an area of interest can be easily tracked based on the coded address, and a large-area sample image can be accurately reconstructed from the sector images.
Highlights
Fluorescence microscopy is commonly used in life sciences for microstructural observations of entire species at the molecular level [1, 2]
A specially designed sample slide is used with a sample area and an address-patterned area for sample holding and address recognition, respectively
The blue laser beam is focused on the sample area and is used for fluorescent excitation and image capturing, whereas the red laser beam is focused on the address-patterned area and is used for address recognition and dynamic focusing
Summary
Fluorescence microscopy is commonly used in life sciences for microstructural observations of entire species at the molecular level [1, 2]. The high resolution achieved for images in the focal plane can be blurred by information from the out-of-focus light, which makes it difficult to detect cellular and sub-cellular structures. To overcome this problem, laser scanning point confocal microscopy (LSCM) is used to collect only information from the in-focus light and to reduce the out-of-focus noise, thereby increasing the signal-to-noise (S/N) ratio of the obtained fluorescence images [3]. Imaging of living cells is generally performed using a microscopy system after removal of the samples of interest from chambers after the desired period of culture. A compact and high-resolution microscope that can be embedded into a cell culture chamber and utilized for fluorescence image capturing with an accurate positionaddressable function is urgently needed
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