Proc. SPIE. 7765, Nanobiosystems: Processing, Characterization, and Applications III
KEYWORDS: Multiphoton microscopy, Microscopy, Luminescence, Objectives, Electron multiplying charge coupled devices, In vivo imaging, Signal detection, Pulsed laser operation, Multiphoton fluorescence microscopy, 3D image processing
Unlike conventional multiphoton excited microscopy according to pixel-by-pixel point scanning, a widefield
multiphoton excited microscopy based on spatiotemporal focusing has been developed to construct three-dimensional
(3D) multiphoton fluorescence images only with the need of an axial scanning. By implementing a 4.0 W 10 kHz
femtosecond laser amplifier with an instant strong peak power and a fast TE-cooled EMCCD camera with an
ultra-sensitive fluorescence detection, the multiphoton excited fluorescence images with the excitation area over 100 μm
x 100 μm can be achieved at a frame rate up to 80 Hz. A mechanical shutter is utilized to control the exposure time of 1
ms, i.e. average ten laser pulses reach the fluorescent specimen, and hence an uniform enough multiphoton excited
fluorescence image can be attained with less photobleaching. The Brownian motion of microbeads and 3D neuron cells
of a rat cerebellum have been observed with a lateral spatial resolution of 0.24 μm and an axial resolution of 2.5 μm.
Therefore, the developed widefield multiphoton microscopy can provide fast and high-resolution multiphoton excited
fluorescence images for animal study in vivo.