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Laser scanning confocal microscopy (LSCM) has been the dominating imaging technique for examining fluorescently labelled biological samples in the past few decades. The major advantage of LSCM is attributed to the use of a pinhole in the signal detection light path can largely reduce out-of-focus background signals, thus creating a thin optical section with not only good lateral but also axial resolution. However, one inherent drawback of the technique is that the use of excitation laser in the visible range cannot efficiently penetrate biological tissues due to light scattering, limiting the usable imaging depth to only ~100 µm.

Multiphoton microscopy (MPM; also termed two-photon microscopy) is an advanced fluorescence imaging technique that comes around such problem. In MPM, the pulsed excitation laser source is in the far-red or infrared range, enabling much better penetration power (up to ~500 to 1000 µm) compared to LSCM. Such improvement is significant, as it allows researchers to observe and examine biological structures at depths that are otherwise unreachable using LSCM. On the other hand, although a pinhole is not used in MPM, optical sectioning can still be achieved by focusing the laser to a thin volume where photons are dense enough to excite the fluorophores.

The Nikon AX R MP Microscope is of an upright configuration, allowing researchers to image relatively large samples (e.g., 3-dimensional cell cultures, organoids, isolated whole organs), and more importantly, live animals (e.g., mice, fish and C. elegans). On the other hand, the resonant scanner, in combination with the ultra-sensitive non-descanned detectors (NDDs), will allow for the detection of highly dynamic signals, making the system ideal for such applications as in vivo fluorescent calcium imaging. Furthermore, the microscope is equipped with a pulsed laser with a wide emission spectrum (i.e., from 690 to 1300 nm) and a second output at 1045 nm, allowing for such applications as multi-channel simultaneous multiphoton imaging and simultaneous photo-activation and imaging. Potential applications include in vivo optogenetic experiments, photo-stimulation and uncaging using live animals.


Dr. Michael Yuen

Senior Scientific Officer

Dr. Alan Leung

Scientific Officer


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