Confocal resolution

Several tests have been performed with the biomedical systems of Fuji Film, the BAS 2500 and BAS 5000. The BAS25(K) is similar to the BAS2000 a flat bed scatmer which can read diverse IP-sizes up to 20 x 40 cm. The read out resolution can be set up to 100 pm or 50 pm. The BAS5000, is based on a new confocal read out optic which is... 

Plenary 8. J Grave et al, e-mail address J.Greve tn.utwente.nl (RS). Confocal direct unaging Raman microscope (CDIRM) for probing of the human eye lens. High spatial resolution of the distribution of water and cholesterol in lenses. 

It is interesting to note the analogy of developments in light microscopy during the last few decades. The confocal microscope as a scaiming beam microscope exceeds by far the nomial fluorescence light microscope in resolution and detection level. Very recent advances in evanescent wave and interference microscopy seem to promise to provide even higher resolution (B1.18). 

Figure Bl.18.11. Confocal scanning microscope in reflection the pinliole in front of the detector is in a conjugate position to the illumination pinliole. This arrangement allows the object to be optically sectioned. The lens is used to focus the light beam onto the sample and onto the pinliole. Thus, the resulting point spread fimctioii is sharpened and the resolution increased.

The extension of the voxel in a radial direction gives infomiation on the lateral resolution. Since the lateral resolution has so far not been discussed in temis of the point spread function for the conventional microscope, it will be dealt with here for both conventional and confocal arrangements [13]. The radial intensity distribution in the focal plane (perpendicular to the optical axis) in the case of a conventional microscope is given by... 

B1.18.5.7 THE FUTURE RESOLUTION BEYOND THE DIFFRACTION LIMIT IN CONFOCAL FLUORESCENCE... 

Flell S W and Stelzer E FI K 1992 Fundamental improvement of resolution with a four Pi-confocal... 

Another significant advantage of confocal microscopy is the increase in resolution achieved by the absence of the out-of-focus images. A microscope yielding a resolution of 220 nm, if confocal, yields a resolution of 160 nm. 

A confocal microscope using ultraviolet light and a 1.30-NA objective is expected to produce a resolution of about 0.07 p.m (70 nm), but no such instmment has been developed. There are confocal attachments that fit on almost any compound microscope. If one of the eady twentieth century ultraviolet microscopes or a Burch reflected optics scope can be found, the shorter wavelength and improved contrast would make possible better resolution than any compound light microscope. 

The classical polarizing light microscope as developed 150 years ago is still the most versatile, least expensive analytical instrument in the hands of an experienced microscopist. Its limitations in terms of resolving power, depth of field, and contrast have been reduced in the last decade, in which we have witnessed a revolution in its evolution. Video microscopy has increased contrast electronically, and thereby revealed structures never before seen. With computer enhancement, unheard of resolutions are possible. There are daily developments in the X-ray, holographic, acoustic, confocal laser scanning, and scanning tunneling micro-... 

Takami S., Getchell M. and Getchell T. (1995). Resolution of sensory and mucoid glycoconjugates with terminal O-galactose residues in the mucomicrovillar complex of the vomeronasal sensory epithelium by dual confocal laser-scanning microscopy. Cell Tiss Res 280, 211-216. 

In the z-direction (depth-direction) the resolution is determined by the confocal instrument settings. While it is essential to be aware of the limitations of confocal measurements, as mentioned above, it is possible to create three-dimensional maps. That means probing a sample in the x, y and z-directions. In Figure 2 the data cube of a two-dimensional map is shown. One element (row) of the cube has been picked out and its content enlarged on the right side of the figure. It is evident that each row therefore contains the information of a whole spectrum. 

Noise can be also introduced by biochemical heterogeneity of the specimen. This can be a major cause of uncertainty in biological imaging. The high (three-dimensional) spatial resolution of fluorescence microscopy results in low numbers of fluorophores in the detection volume. In a typical biological sample, the number of fluorophores in the detection volume can be as low as 2-3 fluorophores for a confocal microscope equipped with a high NA objective at a fluorescent dye concentration of 100 nM. This introduces another source of noise for imaging applications, chemical or molecular noise, related to the inherent randomness of diffusion and the interaction of molecules. 

Drummond, D. R., Carter, N. and Cross, R. A. (2002). Multiphoton versus confocal high resolution z-sectioning of enhanced green fluorescent microtubules Increased multiphoton photobleaching within the focal plane can be compensated using a Pockels cell and dual widefield detectors. J. Microsc. 206, 161-9. 

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