Photobleaching property of confocal laser scanning microscopy with masked illumination

Confocal laser scanning microscopy (CLSM) has become the tool of choice for high-contrast fluorescence imaging in the study of the three-dimensional and dynamic properties of biological system. However, the high cost and complexity of commercial CLSMs urges many researchers to individually develop low cost and flexible confocal microscopy systems. The high speed scanner is an influential factor in terms of cost and system complexity. Resonant galvo scanners at several kHz have been commonly used in custom-built CLSMs. However, during the repeated illumination for live cell imaging or 3D image formation, photobleaching and image distortion occurred at the edges of the scan field may be more serious than the center due to an inherent property (e.g. sinusoidal angular velocity) of the scan mirror. Usually, no data is acquired at the edges due to large image distortion but the excitation beam is still illuminated. Here, we present the photobleaching property of CLSM with masked illumination, a simple and low cost method, to exclude the unintended excitation illumination at the edges. The mask with a square hole in its center is disposed at the image plane between the scan lens and the tube lens in order to decrease photobleaching and image distortion at the edges. The excluded illumination section is used as the black level of the detected signals for a signal quantizing step. Finally, we demonstrated the reduced photobleaching at the edges on a single layer of fluorescent beads and real-time image acquisition without a standard composite video signal by using a frame grabber.