Development of High-speed Laser Scanning Microscope for In Vivo Deep Brain Imaging

Sponsored by the Shun Hing Institute of Advanced Engineering

Principal Investigator: Professor Shih-Chi Chen, Department of Mechanical and Automation Engineering


Prof. Wing Ho Yung, School of Biomedical Sciences

Project Team:


This work aims to develop new imaging techniques, including tunable frame rate (30 - 17,280 fps) and omnidirectional imaging, for a custom-designed laser scanning confocal and two-photon excitation (TPE) microscope. The new functions will be used for in vivo deep brain imaging on mice. Current microscopes typically run at a fixed frame rate with a flat imaging plane. However, all biological subjects are “3-dimensional (3-D)” in nature and various biological events, e.g. blood flow or neuron signaling, occur at different time scales. Accordingly, a versatile microscope with capabilities of frame-rate tuning and a 3-D programmable imaging plane is highly desirable. The frame-rate tuning function can be achieved by a new synchronization circuit and related software development. It is worth to note that ultra-high frame rates, i.e. 1000 - 10,000 fps, are achieved by trading off the imaging area, and thus at any frame rate, the “pixel dwell time” of the system remains constant, keeping a low signal-to-noise ratio. 3-D programmable imaging plane is achieved by the introduction of a high-speed piezoelectric objective scanner. During the in-plane raster scan procedure, the objective lens can be moved to any arbitrary position in the Z axis, thus enabling the “omnidirectional scan”.

These new functions will be used to investigate deep regions in brain in vivo and enable many new studies that cannot be realized in the past. Specifically, we will follow neuron axons (not in the same plane) in a mouse brain and identify their related neural circuits and simultaneously observe their signaling processes at 1000 fps. We will perform deep brain calcium imaging of visual and motor cortical columns (~800µm deep) and record from multiple hypercolumns in a single scan. Lastly, we will study and image dendritic spines and track the formation and disappearance of individual spines. These results will generate significant impact by elucidating the learning processes involved in visuomotor tasks.




[1] D. Zhang, J. Cheng, and S. Chen, “Multi-depth Real-time Confocal Imaging,” Proceedings of the 2013 International Symposium on Optomechatronic Technologies (ISOT), Jeju, Korea, Oct. 28-30, 2013 (Best Paper Award).

[2] J. B. Pawley, ed., Handbook of Biological Confocal Microscopy, 3rd Ed. Springer, 2006.