Because of its optical property of photostability, Nitrogen-Vacancy center (NV center) is desired to be applied for biomedical staining for super-resolution microscopy. In this paper, we report the sub-diffraction imaging of NV centers in nano-diamond and bulk materials. The resolution of ~65nm is achieved in the FND sample with our home built CW STED system.
Fluorescent microscopy has become an essential tool to study biological molecules, pathways and events in living cells, tissues and animals. Meanwhile even the most advanced confocal microscopy can only yield optical resolution approaching Abbe diffraction limit of ~200 nm. This is still larger than many subcellular structures, which are too small to be resolved in detail. These limitations have driven the development of super-resolution optical imaging methodologies over the past decade.<p> </p>In stimulated emission depletion (STED) microscopy, the excitation focus is overlapped by an intense doughnut-shaped spot to instantly de-excite markers from their fluorescent state to the ground state by stimulated emission. This effectively eliminates the periphery of the Point Spread Function (PSF), resulting in a narrower focal region, or super-resolution. Scanning a sharpened spot through the specimen renders images with sub-diffraction resolution. Multi-color STED imaging can present important structural and functional information for protein-protein interaction.<p> </p>In this work, we presented a two-color, synchronization-free STED microscopy with a Ti:Sapphire oscillator. The excitation wavelengths were 532nm and 635nm, respectively. With pump power of 4.6 W and sample irradiance of 310 mW, we achieved super-resolution as high as 71 nm. Human respiratory syncytial virus (hRSV) proteins were imaged with our two-color CW STED for co-localization analysis.
A portable video-rate confocal laser scanning microscope (CLSM) is implemented with polygon mirror and
galvanometric mirror employed as the fast and slow axis scanner, respectively. The system can be applied for
noninvasively imaging skin and other tissue. The dimension of this real-time CLSM is only 33×20×12cm<sup>3</sup> with weigh of
1.780 kg. Here we used a single Complex Programmable Logic Device (CPLD) to generate the control and
synchronization signals for real time confocal microscopy. Utilizing NI image acquisition card, the CLSM system can
acquire and store the real-time images. So that high resolution confocal microscopy is achieved simultaneously.
Based on the electro-optical properties of liquid crystal, we have designed a novel partial gating detector. Liquid crystal
can be taken to change its own transmission according to the light intensity outside. Every single pixel of the image is
real-time modulated by liquid crystal, thus the strong light is weakened and low light goes through the detector
normally .The purpose of partial-gating strong light (>10<sup>5</sup>lx) can be achieved by this detector. The modulation transfer
function (MTF) equations of the main optical sub-systems are calculated in this paper, they are liquid crystal panels,
linear fiber panel and CCD array detector. According to the relevant size, the MTF value of this system is fitted out. The
result is MTF= 0.518 at Nyquist frequency.