A Near-Infrared HyperSpectral Reflective Confocal Microscopy (NIHS-RCM) is proposed in order to get high resolution images of deep biological tissues such as skin. The microscopy system uses a super-continuum laser for illumination, an acousto-optic tunable filter (AOTF) for rapid selection of near-infrared spectrum, a resonant galvanometer scanner for high speed imaging (15f/s) and near-infrared avalanche diode as detector. Porcine skin and other experiments show that the microscopy system could get deep tissue images (180 μm), and show the different ingredients of tissue with different wavelength of illumination. The system has the ability of selectively imaging of multiple ingredients at deep tissue which can be used in skin diseases diagnosis and other fields.
On the base of the vectorial diffraction theory, the diffraction integral represents are obtained to generate a hollow spot for STED. In the paper, the incident light is modulated by phase and polarization to focus a hollow spot. The 2-dimension hollow spot is obtained by modulating the circularly polarized beam with 0-2π vortex phase or 0/π circular phase. Addition In addition, we get the 2-dimension hollow spot by focusing azimuthally polarized beam or a radially polarized beam modulated with 0/π circular phase. The intensity distributions and size of these 2-dimension hollow spots are discussed. Then the 3-dimension hollow spot is got by taking advantage of two kinds of 2-dimension hollow spots. There are two methods to generate a hollow spot by the diffraction integral represents. One is modulating two circularly polarized beams with 0-2π vortex phase and 0/π circular phase respectively. The other is choosing an azimuthally polarized beam and a radially polarized beam modulated with 0/π circular phase as depletion beams. So we obtain a 2-dimension hollow spot by modulating a beam, and obtain a 3-dimension hollow spot by modulating two beams. These hollow spots can be useful for STED to realize 2-dimension and 3-dimension super-resolution.
Laser scanning confocal microscope has been widely used in biology, medicine and material science owing to its advantages of high resolution and tomographic imaging. Based on a set of confirmatory experiments and system design, a novel confocal microscopic imaging system is developed. The system is composed of a conventional fluorescence microscope and a confocal scanning unit. In the scanning unit a laser beam coupling module provides four different wavelengths（405nm，488nm，561nm and 638nm）which can excite a variety of dyes. The system works in spot-to-spot scanning mode with a two-dimensional galvanometer. A 50 microns pinhole is used to guarantee that stray light is blocked and only the fluorescence signal from the focal point can be received . The three-channel spectral splitter is used to perform fluorescence imaging at three different working wavelengths simultaneously. The rat kidney tissue slice is imaged using the developed confocal microscopic imaging system. Nucleues labeled by DAPI and kidney spherule curved pipe labeled by Alexa Fluor 488 can be imaged clearly and respectively, realizing the distinction between the different components of mouse kidney tissue. The three-dimensional tomographic imaging of mouse kidney tissue is reconstructed by several two-dimensional images obtained in different depths. At last the resolution of the confocal microscopic imaging system is tested quantitatively. The experimental result shows that the system can achieve lateral resolution priority to 230nm.
According to Wolf and Richards vectorial diffraction theory, an electric field intensity model of focal spot for high numerical aperture radially polarized beam is established to analyze the intensity distributions of the focal spot and the polarization components of the electric field along the x, y and z axis, separately. In the reflection-mode confocal of imaging system, the intensity distributions of focal spot is obtained utilizing the gold nanoparticles, and the intensity distributions of the polarization components of the electric field along the x, y and z axis are obtained utilizing the gold nanorods. In the incident light, the polarization component along the z axis is nonexistent in front of the objective. But there is the polarization component along the z axis, which is relative to the numerical aperture, in the focal spot behind the objective. When the numerical aperture increases from 0.8 to 1.4, the ratio of the polarization component maximum along the z axis to that along the x axis or y axis increases from 0.57 to 3.16. The results show that the focal spot of radially polarized beam through high numerical aperture objective have the polarization component along the x, y and z axis, separately, and polarization component along z axis is much more than the other.