Optical microscopic techniques, resolution limits

Optical and Confocal Microscopy A Brief Overview 1525 Table 16.1 Optical microscopic techniques and their resolution limits. 

Laser microprobe mass analyzers permit mass spectrometric analysis of very small volumes (0.01-1 pm3) of thin Sections. The method is based on laser induced ion production from a microvolume and analysis of the evaporated ions in a time-of-flight mass-spectrometer. The technique allows detection of all elements and isotopes with a sensitivity approaching the ppm range and an extremely low limit of detection 10 15 to 10-20 g. Transmission type instruments such as the LAMMA 500 are designed for the analysis of particles of 3 pm in diam. The lateral resolution is about 0.5-1 pm. Because the area to be analyzed is selected by an optical microscope, distribution of chemical constituents can be precisely correlated with morphologic structures (Hillenkamp et al., 1982 39), Simons, 198440), Kaufmann, 1984)41 >. 

At high concentration, when molecules are no longer isolated in space, a conventional optical microscope is unable to resolve them within the diffraction limit. Efforts have been made to circumvent the diffraction limit by engineering the point spread function using nonlinear optical techniques. Spatial resolution of 20 nm in a cell has been demonstrated without using a proximal probe.67... 

Like STM, AFM produces 3-D images. Unlike STM, AFM does not require the sample to be conducting and so has wider applicability. Unlike optical microscopes, AFM does not use a lens, so the resolution of the technique is limited by probe size (and strength of interaction between surface and probe tip) rather than by diffraction effects. The key to the sensitivity of AFM is in carefully monitoring the movement of the probe tip. 

The inventions of the scanning tunnelling microscope (STM) [16] and the atomic force microscope (AFM) [17] have allowed sub-micrometre and, at times, atomic-scale spatially-resolved imaging of surfaces. Spatially-resolved temperature measurements using optical systems are diffraction limited by the wavelength of the radiation involved, which is about 5-10 pm for infrared thermography and about 0.5 pm for visible light [18]. The spatial resolution of near-field techniques (such as AFM) is only limited by the active area of the sensor (which in the case of STM may be only a few atoms at the end of a metal wire). 

During the last few years optical visualization techniques have also been introduced. Among them the total internal reflection fluorescence excitation (TIFR) microscopy [4] and optical interference-enhanced reflection microscopy [5] appear to be the most promising nonintrusive techniques. Their resolution, however, does not even approach the resolution of atomic force microscope and optical techniques may thus serve as an image survey of nanobubbles at 300 nm level (diameter) which is so far their resolution limit. 

While some breakthrough technologies of scanning near-field optical microscope have been developed to overcome the diffractimi limit to increase resolution, these near-field techniques require the distance between the tip and the specimen surface to be in the order of mie to several percents of a wavelength, normally less than 1 pm. However, in nanofluidics and lab-on-a-chip, the channels are mostly enclosed ones and the solid walls are more than 100 pm thick, and hence, the near-field scanning microscopes cannot be appUed to nanofluidics. For lab-on-a-chip appUcatimi, far-field nanoscopy, where the working distance is similar to that of conventional cmifocal microscope, is required for nanofluidics apphcatimis. 

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