Two photon polymerization, based on two-photon absorption, is a powerful and potential technique to fabricate 3D micro/nanostructures with submicrometric resolution. We use a photopolymerizable resin based on methyl methacrylate monomers as a photosensitive medium, in which the polymerization is triggered by the nonlinear optical effect. Nonlinear effect photoreaction occurs only in a submicrometric volume, voxel, much smaller than the cube of the wavelength, λ<sup>3</sup>. By using a femtosecond laser, 780 nm wavelength, we investigate the effect of different parameters on the resolution of our custom made micro/nanofabrication set up. The fabrication accuracy and the resolution of 3D micro/nanostructures depend on the accuracy of the focal spot position in z-direction, in the glass substrate-resin interface. We control the focal spot position by using ascending scan process meaning the focus spot level. Employing the proposed process, the lateral resolution of individual voxels, is improved almost to 94 nm. The resolution of two photon absorption polymerized voxels is studied as a function of focus spot level, laser power and single-shot irradiation time. Finally, we show 3D microstructures and a micro-device, which present great potential for future applications.
Two-photon excitation microscopy (TPEM) provides spatial resolution beyond the optical diffraction limit using the nonlinear response of fluorescent molecules. One of the strong advantages of TPEM is that it can be performed using a laser-scanning microscope without a complicated excitation method or computational post-processing. However, TPEM has not been recognized as a super-resolution microscopy due to the use of near-infrared light as excitation source, which provides lower resolution than visible light. In our research, we aimed for the realization of nonlinear fluorescence response with visible light excitation to perform super-resolution imaging using a laser-scanning microscope. The nonlinear fluorescence response with visible light excitation is achieved by developing a probe which provides stepwise two-photon excitation through photoinduced charge separation. The probe named nitro-bisBODIPY consists of two fluorescent molecules (electron donor: D) and one electron acceptor (A), resulting to the structure of D-A-D. Excited by an incident photon, nitro-bisBODIPY generates a charge-separated pair between one of the fluorescent molecules and the acceptor. Fluorescence emission is obtained only when one more incident photon is used to excite the other fluorescent molecule of the probe in the charge-separated state. This stepwise two-photon excitation by nitro-bisBODIPY was confirmed by detection of the 2nd order nonlinear fluorescence response using a confocal microscope with 488 nm CW excitation. The physical model of the stepwise two-photon excitation was investigated by building the energy diagram of nitro-bisBODIPY. Finally, we obtained the improvement of spatial resolution in fluorescence imaging of HeLa cells using nitro-bisBODIPY.
The simultaneous observation of multiple fluorescent proteins (FPs) by optical microscopy is revealing mechanisms by which proteins and organelles control a variety of cellular functions. Here we show the use of visible-light based two-photon excitation for simultaneously imaging multiple FPs. We demonstrated that multiple fluorescent targets can be concurrently excited by the absorption of two photons from the visible wavelength range and can be applied in multicolor fluorescence imaging. The technique also allows simultaneous single-photon excitation to offer simultaneous excitation of FPs across the entire range of visible wavelengths from a single excitation source. The calculation of point spread functions shows that the visible-wavelength two-photon excitation provides the fundamental improvement of spatial resolution compared to conventional confocal microscopy.