A microscope that may help the study of neurological diseases has been developed at The Chinese University of Hong Kong (CUHK). A team led by Prof. Shih-Chi Chen, Associate Professor in the Department of Mechanical and Automation Engineering at CUHK, has recently developed the first digital holography-based (DH) two-photon excitation (TPE) microscope to generate simultaneous video-rate fluorescent imaging and multi-point optical stimulation. This allows the tracking of nerve cells activities and thus may help the study of neurological diseases, such as glaucoma, a very common eye disease. By scanning the retinal ganglion cells using the DH-TPE microscope, scientist can understand the molecular mechanisms of optic nerve degeneration in glaucoma and find ways to tackle it. Prof. Chen’s team is now working on this project with Prof. Christopher Kai Shun Leung, Professor, Department of Ophthalmology and Visual Sciences at the Faculty of Medicine at CUHK, and the Hong Kong Eye Hospital. 3-D imaging tracks inter-cells activities which helps the study of glaucoma The DH-TPE microscope is powered by a digital micromirror device (DMD), a chip used in our everyday projector that contains millions of micromirrors switching at tens of kilohertz speed. By controlling the amplitude and phase of the input laser via binary holograms and the fast-switching micromirrors, the laser beam can be split into up to 20 focal points for simultaneous optical stimulation and real-time fluorescent imaging. Each focus can be independently controlled to scan along arbitrarily defined paths or surfaces at 22.7 kHz. More importantly, the DMD-scanner is also an ultrafast beam shaper, and by superposing the scanning and wavefront-correction holograms, the point spread function can be engineered or even shaped into other novel beam modes to achieve efficient 3-D imaging. Compared to state-of-the-art commercial TPE microscopes, the DH-TPE microscope presents a suite of distinctive imaging functionalities that have never been realised in the past, including (1) random-access imaging, (2) multi-plane imaging, (3) 3-D programmable imaging plane, (4) point-specific wavefront correction, and (5) simultaneous video-rate fluorescent imaging and multi-point optical stimulation. Through funding support from the Innovation and Technology Commission (ITC), Prof. Chen’s team is setting up the DH-TPE microscope at the Hong Kong Eye Hospital, collaborating with a team led by Prof. Christopher Kai Shun Leung. The two teams are working together to exploit the unique capability of the DH-TPE microscope to study and understand the basic mechanisms of a few important diseases. For example, one objective is to study metabolic dysfunction in the retinal ganglion cells via in vivo two-photon imaging to study the molecular mechanisms of optic nerve degeneration in glaucoma. Glaucoma, characterised by progressive loss of retinal ganglion cells, is the leading cause of irreversible blindness worldwide with a significant social and economic burden. The reduced form of nicotinamide adenine dinucleotide (NADH) is an intrinsic fluorophore and a co-factor in major metabolic pathways for energy production. The levels of NADH decrease in the retinal ganglion cell following optic nerve injury and the imaging of NADH fluorescence intensity can serve as a non-invasive indicator of retinal ganglion cell death in glaucoma. The unique design of the DH-TPE microscope allows point-specific wave-front correction of ocular aberration that will facilitate non-invasive, label-free imaging of the retina. The investigation of metabolic dysfunction of retinal ganglion cells will provide mechanistic insights into the development of neuroprotective and neuroregenerative therapies for patients with glaucoma and non-glaucomatous optic neuropathies.
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