KEYWORDS: 3D microstructuring, Pulsed laser operation, Digital micromirror devices, Optical amplifiers, Microfabrication, Beam controllers, Control systems, 3D image processing, Ultrafast phenomena, Imaging systems
A developed temporal focusing-based multiphoton excitation system with additional patterned excitation and local laser control can provide high-throughput fabrication of three-dimensional gray-level biomicrostructures via two-photon cross-linking with rose bengal (RB) as the photoactivator. Multiple bovine serum albumin (BSA) structures of different concentrations were simultaneously achieved by selecting different pulse numbers in the designated regions with an appropriate femtosecond laser power within a few seconds. Intensity of the RB two-photon excited fluorescence (TPEF) is correlated to the concentration of the fabricated cross-linked BSA microstructure. Hence, the fabricated BSA microstructure can be monitored online by utilizing the RB TPEF as contrast agent. It suggests that this approach with its unique capability of high-speed, gray-level, and online-inspection fabrication meets the requirements of the biomedical researches involved in extracellular matrix.
One of the limits of a conventional multiphoton microfabrication is its low throughput due to the sequential nature of
scanning process. In this study, a multiphoton microfabrication system based on spatiotemporal focusing and patterned
excitation has been developed to provide freeform polymer microstructures fast. The system integrates a 10 kHz
repetition rate ultrafast amplifier featuring strong instantanrror device generating designed patterns at the focal plane. As the result, three-dimensional freeform polymer microstructures using Rose Bengal as the photoinitiator are created by sequentially stacking up two-dimensional (2D) structures layer-by-layer. The size of each 2D fabrication area can be larger than 100 × 100 μm2. Compared with scanning process or fixed mask pattern generation, this approach provides two- or three-fold fabrication speed and freeform microstructures. Furthermore, the system is capable of optical sectioning the fabricated microstructures for providing 3D inspection.
Conventional multiphoton microscopy employs beam scanning; however, in this study a microscope based on
spatiotemporal focusing offering widefield multiphoton excitation has been developed to provide fast optical sectioning images. The microscope integrates a 10 kHz repetition rate ultrafast amplifier featuring strong instantaneous peak power (maximum 400 μJ/pulse at 90 fs pulse width) with a TE-cooled, ultra-sensitive photon detecting, electron multiplying charge-coupled device camera. This configuration can produce multiphoton excited images with an excitation area larger than 200 × 100 μm2 at a frame rate greater than 100 Hz. Brownian motions of fluorescent microbeads as small as 0.5 μm have been instantaneously observed with a lateral spatial resolution of less than 0.5 μm and an axial resolution of approximately 3.5 μm. Moreover, we combine the widefield multiphoton microscopy with structure illuminated technique named HiLo to reject the background scattering noise to get better quality for bioimaging.
Unlike conventional multiphoton microscopy according to pixel by pixel point scanning, a widefield multiphoton
microscope based on spatiotemporal focusing has been developed to provide fast optical sectioning images at a frame
rate over 100 Hz. In order to overcome the aberrations of the widefield multiphoton microscope and the wavefront
distortion from turbid biospecimens, an image-based adaptive optics system (AOS) was integrated. The feedback
control signal of the AOS was acquired according to locally maximize image intensity, which were provided by the
widefield multiphoton excited microscope, by using a hill climbing algorithm. Then, the control signal was utilized to
drive a deformable mirror in such a way as to eliminate the aberration and distortion. A R6G-doped PMMA thin film is
also increased by 3.7-fold. Furthermore, the TPEF image quality of 1 μm fluorescent beads sealed in agarose gel at
different depths is improved.
KEYWORDS: Luminescence, Multiphoton microscopy, Multiphoton fluorescence microscopy, Microscopy, 3D image processing, Electron multiplying charge coupled devices, Objectives, Signal detection, In vivo imaging, Pulsed laser operation
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.
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