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.
Micro- and nano-scale molds were fabricated using nanocrystalline diamond (nano-diamond)
and diamond-like carbon (DLC) films for imprinting lithography. Patterning was first transferred
to the resist on nano-diamond and DLC thin films by photolithography and imprint lithography
methods, and then deep etching with inductively-coupled plasma reactive ion etching (ICP-RIE) was
applied to transfer patterns to nano-diamond and DLC films for the fabrication of diamond molds.
Nano-diamond films of about 1.5μm in thickness were deposited on silicon substrates by hot
filament chemical vapor deposition (HFCVD) by controlling CH<sub>4</sub>/H<sub>2</sub> ratios and substrate
temperatures. Thick diamond-like carbon films containing silicon oxide nanoparticles were
deposited on silicon substrates by PECVD using gaseous HMDSO (Hexamethyldisiloxane) reactants
to release the film stress. E-beam writer was used to pattern the resist on the Cr film-covered thick
DLC film. By using ICP-RIE, Cr film was first patterned with the patterned e-beam resist as the
etching mask, and then DLC thick film was etched to form nanoimprint mold using the patterned Cr
as the etching mask. High fidelity nano-patterns were transferred with nano-imprinting lithography
using the nano-diamond and DLC molds. Good mold releasing behavior and high mold strength
were observed for the nanocrystalline diamond and DLC molds due to their highly hydrophobic
surface and high toughness, respectively.