Li-Chung Cheng

Measurements of multiphoton action cross sections for multiphoton microscopy

We report quantitative measurements of two-, three-, and four-photon excitation action cross sections of several commonly used fluorophores and fluorescent proteins at three different excitation wavelengths of 800 nm, 1300 nm, and 1680 nm. The measured cross section values are consistent with simple quantum mechanic estimations. These values indicate that the optimum repetition rate for deep tissue 3-photon microscopy is approximately 1 to 2 MHz. We further demonstrate that it is feasible to perform 4-photon fluorescence microscopy of GFP labeled microglia in mouse brain in vivo at 1700 nm. 4-photon excitation increases the accessibility of fluorophores at the long wavelength spectral window of 1700 nm.

Fast multiphoton microfabrication of freeform polymer microstructures by spatiotemporal focusing and patterned excitation

One of the limits of conventional scanning multiphoton microfabrication is its low throughput due to point-by-point processing. In order to surpass this limit, a multiphoton microfabrication system based on spatiotemporal focusing and patterned excitation has been developed to quickly provide three-dimensional (3D) freeform polymer microstructures. 3D freeform polymer microstructures using Rose Bengal as the photoinitiator are created by sequentially stacking two-dimensional fabricating patterns. The size of each fabrication area can be larger than 300 × 170 μm2 (full width at half maximum). Compared to conventional scanning multiphoton excitation and fixed mask pattern generation, this approach offers freeform microstructures and a greater than three-order increase in fabrication speed. Furthermore, the system is capable of optically sectioning the fabricated microstructures for providing 3D inspection.

Nonlinear structured-illumination enhanced temporal focusing multiphoton excitation microscopy with a digital micromirror device

In this study, the light diffraction of temporal focusing multiphoton excitation microscopy (TFMPEM) and the excitation patterning of nonlinear structured-illumination microscopy (NSIM) can be simultaneously and accurately implemented via a single high-resolution digital micromirror device. The lateral and axial spatial resolutions of the TFMPEM are remarkably improved through the second-order NSIM and projected structured light, respectively. The experimental results demonstrate that the lateral and axial resolutions are enhanced from 397 nm to 168 nm (2.4-fold) and from 2.33 μm to 1.22 μm (1.9-fold), respectively, in full width at the half maximum. Furthermore, a three-dimensionally rendered image of a cytoskeleton cell featuring ~25 nm microtubules is improved, with other microtubules at a distance near the lateral resolution of 168 nm also able to be distinguished.

Wavefront sensorless adaptive optics temporal focusing-based multiphoton microscopy.

Temporal profile distortions reduce excitation efficiency and image quality in temporal focusing-based multiphoton microscopy. In order to compensate the distortions, a wavefront sensorless adaptive optics system (AOS) was integrated into the microscope. The feedback control signal of the AOS was acquired from local image intensity maximization via a hill-climbing algorithm. The control signal was then utilized to drive a deformable mirror in such a way as to eliminate the distortions. With the AOS correction, not only is the axial excitation symmetrically refocused, but the axial resolution with full two-photon excited fluorescence (TPEF) intensity is also maintained. Hence, the contrast of the TPEF image of a R6G-doped PMMA thin film is enhanced along with a 3.7-fold increase in intensity. Furthermore, the TPEF image quality of 1μm fluorescent beads sealed in agarose gel at different depths is improved.

High-throughput multiphoton-induced three-dimensional ablation and imaging for biotissues.

In this study, a temporal focusing-based high-throughput multiphoton-induced ablation system with axially-resolved widefield multiphoton excitation has been successfully applied to rapidly disrupt biotissues. Experimental results demonstrate that this technique features high efficiency for achieving large-area laser ablation without causing serious photothermal damage in non-ablated regions. Furthermore, the rate of tissue processing can reach around 1.6 × 10(6) μm(3)/s in chicken tendon. Moreover, the temporal focusing-based multiphoton system can be efficiently utilized in optical imaging through iterating high-throughput multiphoton-induced ablation machining followed by widefield optical sectioning; hence, it has the potential to obtain molecular images for a whole bio-specimen.

Widefield multiphoton microscopy with image-based adaptive optics

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.

Fast multiphoton microfabrication of freeform polymer microstructures by spatiotemporal focusing and patterned generation

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.

SPATIOTEMPORAL FOCUSING-BASED WIDEFIELD MULTIPHOTON MICROSCOPY FOR FAST OPTICAL SECTIONING OF THICK TISSUES

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.

Widefield multiphoton excited fluorescence microscopy for animal study in vivo

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.