Masked illumination scheme for a galvanometer scanning high-speed confocal fluorescence microscope
School of Information and Mechatronics, Gwangju Institute of Science and Technology, Gwangju, Republic of Korea.Scanning (Impact Factor: 1.89). 11/2011; 33(6):455-62. DOI: 10.1002/sca.20264
High-speed beam scanning and data acquisition in a laser scanning confocal microscope system are normally implemented with a resonant galvanometer scanner and a frame grabber. However, the nonlinear scanning speed of a resonant galvanometer can generate nonuniform photobleaching in a fluorescence sample as well as image distortion near the edges of a galvanometer scanned fluorescence image. Besides, incompatibility of signal format between a frame grabber and a point detector can lead to digitization error during data acquisition. In this article, we introduce a masked illumination scheme which can effectively decrease drawbacks in fluorescence images taken by a laser scanning confocal microscope with a resonant galvanometer and a frame grabber. We have demonstrated that the difference of photobleaching between the center and the edge of a fluorescence image can be reduced from 26 to 5% in our confocal laser scanning microscope with a square illumination mask. Another advantage of our masked illumination scheme is that the zero level or the lowest input level of an analog signal in a frame grabber can be accurately set by the dark area of a mask in our masked illumination scheme. We have experimentally demonstrated the advantages of our masked illumination method in detail.
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ABSTRACT: Two-photon microscopy is a very attractive tool for the study of the three-dimensional (3D) and dynamic processes in cells and tissues. One of the feasible constructions of two-photon microscopy is the combination a confocal laser scanning microscope and a mode-locked Ti:sapphire laser. Even though this approach is the simplest and fastest implementation, this system is highly cost-intensive and considerably difficult in modification. Many researcher therefore decide to build a more cost-effective and flexible system with a self-developed software for operation and data acquisition. We present a custom-built two-photon microscope based on a mode-locked Yb3+ doped fiber laser and demonstrate two-photon fluorescence imaging of biological specimens. The mode-locked fiber laser at 1060 nm delivers 320 fs laser pulses at a frequency of 36 MHz up to average power of 80 mW. The excitation at 1060 nm can be more suitable in thick, turbid samples for 3D image construction as well as cell viability. The system can simply accomplish confocal and two-photon mode by an additional optical coupler that allows conventional laser source to transfer to the scanning head. The normal frame rate is 1 frames/s for 400 x 400 pixel images. The measured full width at half maximum resolutions were about 0.44 μm laterally and 1.34 μm axially. A multi-color stained convallaria, rat basophilic leukemia cells and a rat brain tissue were observed by two-photon fluorescence imaging in our system.
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ABSTRACT: Most of the two-photon fluorescence microscopes are based on femtosecond Ti:Sapphire laser sources near the 800 nm wavelength. Here, we introduce a new confocal two-photon microscope system using a mode-locked Yb(3+)-doped fiber laser. The mode-locked fiber laser produces 13 ps pulses with large positive chirping at a repetition rate of 36 MHz with an average power of 80 mW. By using an external grating pair pulse compressor, the pulse width and the frequency chirping of the laser output are controlled for optimum two-photon excitation. For a given objective lens, the optimum condition was obtained by monitoring the two-photon-induced-photocurrent in a GaAsP photodiode at the sample position. The performance of this pulse width optimized two-photon microscope system was demonstrated by imaging Vybrant DiI-stained dorsal root ganglion cells in 2 and 3 dimensions.
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