Multiphoton excited (MPE) fluorescence microscopy has been extensively used for biological imaging. With superior features such as minimum invasiveness, low photobleaching, and deep penetration depth, multiphoton microscopy has proven to be particularly suitable for three-dimensional (3D) imaging of thick tissues and living animals . Moreover, photopolymerization or photocrosslinking is a process which uses a combination of light with low molecular weight photoinitiators or photoactivators to trigger the respective reactions of polymerization or crosslinking . MPE photochemistry is the preferred technique to fabricate 3D tissue scaffolds and multiphoton microsurgery is also implemented in living tissues and cells.

                 (a)                                     (b)

(a)Cultured COS7 cells transfected with eGFP. (b) SHG image of Collagen gel.

• Our instrument mainly including a femtosecond laser (Tsunami, Spectra-Physics, USA),which has a pulse width of less than 100 fs and a repetition rate of 80 MHz.To overcome the group velocity dispersion of the femtosecond laser through the AOM and the objective, an prism pair was used for optimized pulse width in the wavelength region between 700 to 840 nm. An acousto-optic modulator (AOM) (23080-x-1.06-LTD, Neos, USA) for rapid on/off switching of the laser and pulse selection; and processing of the single photon counting (SPC) signals.Selected experimental parameters such as laser power, scanning rate, imaging, and sample positioning can be adjusted by the use of custom LabVIEW program. In this manner, imaging with nonlinear optical signals (two-photon fluorescence (TPF)/second harmonic generation (SHG)) and 3D microfabrication can be achieved.

• Another illustrates a schematic of the developed high-throughput multiphoton-induced reduction and ablation system . Briefly, the system include a titanium-sapphire (ti-sa) ultrafast amplifier, an ultrafast oscillator as the seed beam of the amplifier, an upright optical microscope, a triple-axis sample positioning stage, an Andor EMCCD camera, a DMD, and a data acquisition (DAQ) card with a field-programmable gate array (FPGA) module. The ultrafast amplifier has a maximum peak power of 400 μJ/pulse and a pulse width of 90 fs at an average power of 4.0 W and a repetition rate of 10 kHz. First, the beam pass through a Michelson-interferometry-based autocorrelator in order to measure the pulse width of the amplifier on the sample surface later, and then oblique incidents on the DMD chip, which generates the designed processing pattern. Following this, the pulsing beam is spatially dispersed via a grating. A set of relay lenses was inserted to lengthen the beam path and also adjust the pattern size incidents on the sample surface. Finally, the dispersed frequencies propagate through the 4f setup to realize temporal focusing excitation.

Graphene-based materials recently have become more interesting due to their unique high conductivity, chemical stability, optical property, and intrinsic flexibility. Graphene oxide (GO) is an oxidization of graphene which has properties of inexpensive, scalable, and good water-soluble compared to those of graphene; hence it has another advantages for further applications.

• A developed temporal focusing-based femtosecond laser system provides high-throughput multiphoton reduction and ablation of graphene oxide (GO) films. Integrated with a digital micromirror device for locally controlling the laser pulse numbers, GO-based micropatterns can be achieved instantly. Furthermore, the degree of reduction and ablation can be implemented via controlling the laser wavelength, power, and pulse number. Compared to other laser direct writing methods, this approach offers a high-throughput and multiple-function way to accomplish large-area and micro-scale patterns on GO films. The high-throughput micropatterning of GO via the temporal focusing-based femtosecond laser system matches the requirement of mass production for GO-based applications in electronic microdevices.

• Three-dimensional (3D) polymer microstructures containing graphene oxide (GO) nanosheets were fabricated by two-photon polymerization (TPP) using Rose Bengal (RB) as the photoinitiator. In order to prevent the photothermal effect and products of reduced grapheme oxide (rGO) from GO nanaosheets in TPP processing.Hence, an optimal fabrication parameter was found to fabricate the polymer microstructure containing GO nanosheets without causing a serious thermal effect and restraining more products of rGO during the TPP processing. Moreover, increasing the laser power to reduce GO in the localized area of the fabricated polymer microstructure. As a result, the existence of GO and rGO nanosheets in designated positions of the fabricated microstructures can be achieved. Fig. 2 shows the SEM and OM images of GO  polymer micro-woodpile.

Fig. 2

• We develop a method to directly selfcrosslinking the graphene quantum dot  ( GO QD) for three-dimensional (3D) graphene oxide-based microstructures, and then apply them in 3D microelectronic device, photonics crystal, and tissue scaffold. With the established theoretical study, we using Rose Bengal (RB, Avocado Research Chemicals Ltd，UK) as the photo activator., then by photochemistry to directly crosslinking GO 3D microstructures without polymer monomer via femtosecond laser 3D lithography technology.As a result, and the GO QD microstructures can be achieved. Fig. 3 shows the SEM image of  directly crosslinking GOQD microstructures.

Fig. 3

And the patent is pending for this research. 

• Two photon induced fluorescence from biocompatible NGO in ovarian cancer cells.