Multiscanning mode laser scanning confocal microscopy system

Authors: MERT AKTÜRK, GÖKHAN GÜMÜŞ, BAYKAL SARIOĞLU, YİĞİT DAĞHAN GÖKDEL

Abstract: In this paper, a table-top, reflective mode, laser scanning confocal microscopy system that is capable of scanning the target specimen alternately through various scanning devices and methods is proposed. We have developed a laser scanning confocal microscopy system to utilize combinations of various scanning devices and methods and to be able to characterize the optical performance of different scanners and micromirrors that are frequently used in scanning microscopy systems such as multiphoton microscopy, optical coherence tomography, or confocal microscopy. By integrating the scanner to be characterized on the same optical path with a galvanometric scan mirror, which is the conventional benchmarking scanning unit in a typical scanning microscope, we obtain two major advantages: (1) microscopy images are automatically acquired from the same location on the target specimen without having any time-consuming alignment problem and accordingly provide a high-quality optical comparison opportunity, and (2) it totally eliminates the utilization of a second scanning microscopy to benchmark the performance of the scanner-based system and considerably reduces the time spent for imaging, which is a crucial factor for a freshly excised tissue, especially under a fluorescence microscope. %First, the prototype of the proposed flexible LSCM system is optically characterized using a USAF resolution target. Subsequently, we provide images of red blood and bacteria cells to demonstrate the system's capability for clinical diagnostics. It is reported that maximum FOV and lateral resolution of the proposed LSCM are 420 $\mu$m $\times$ 360 $\mu$m and 1 $\mu$m with galvanometer and 117 $\mu$m $\times$ 117 $\mu$m and 3.2 $\mu$m with the polymer scanner unit, respectively. % The system is composed of a 658 $nm$ laser source, collimation optics, a 2D galvanometer, a 2D polymer micro-scanner, an objective lens with a numerical aperture of 0.40, a 100 $\mu m$ pinhole, a PMT, a DAQ card and peripheral electronics as well as a Matlab software that simultaneously controls the system through a personal computer. Prototype of the proposed flexible LSCM system is first optically characterized using a USAF resolution target. Subsequently, we provided images of red blood and bacteria cells to demonstrate the systems' capability for clinical diagnostics. It is reported that maximum FOV and lateral resolution of the proposed LSCM are measured to be 420 $\mu$m x 360 $\mu$m and 1 $\mu$m with galvanometer and, and 117 $\mu$m x 117 $\mu$m and 3.2 $\mu$m with the polymer scanner unit, respectively.

Keywords: Laser scanning confocal microscopy, skin cancer, polymer scanner, galvanometric scan mirrors, raster scanning, Lissajous scanning, benchmarking

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