The Application of Near-Field Scanning Optical Microscope
With the improvement of resolution and image quality of the near-fleld scanning optical microscope, the scope of its application is becoming more and more widely, from the single molecule fluorescence spectrum detection to the film and materials research, especially attracted the attention of people in the field of biological medicine and bio-chemistry.
The connections of near field scanning optical microscope with monochromator or optical multichannel spectrum analysis instrument produce a multi-functional system with spatial resolution and time resolution, provides possibilities for studying inhomogeneity of nanoscale samples through realizing the detection of spectrum from micro region of the sample. This trend of continuous improvement of the near-field scanning optical microscopy receives a strong attention in biology research. Combined Near field scanning optical microscopy together with fluorescence resonance energy transfer (FRET) not only provides high spatial resolution of sample topography and fluorescence image, but also increases longitudinal sensitivity for dynamic measurement at a single point by utilizing distance dependence FRET techniques; The use of ultrafast lasers to make the near-fleld scanning optical microscope to study ultrafast two-photon induced single molecule fluorescence; The combination of near field scanning optical microscopy and CLSM (confocal laser scanning microscope) can study the nucleus, DNA, the cell membrane and membrane protein fluorescence images, accurately determine the cell and the location of the organelles within the structure of proteins. This method also enables in-situ detection of samples in the liquid environment, imaging the myofibril with the fluorescent tag in the physiological saline. The structure of z-line protein of myofibril with antibody markers is easy to recognize in the near field fluorescence images. Successful observations include tobacco Mosaic virus, the salmonella flagella filaments in the water, bacteria and LB film [14, 17, 28, 29, 53, 54].
Despite that the near-field scanning optical microscope (NSOM) has achieved outstanding results in many field, various technical indicators are still under constant improvement, making it gradually become a powerful tool for life science research. A new form of aperture-type NSOM was proposed whose resolution was not determined by the resolution of the recovered image but the sharpness of the corners of the rectangular aperture and the step size of the scan, this feature of DNSOM (differential near-field scanning optical microscope) makes it potentially advantageous for nanometer-level imaging, especially when resolution and light throughput are at a premium .