Light confocal

The TSM can be modified for reflected light confocal microscopy (TSRLM). In that case, one Nipkow disk above the objective serves both for the illuminating beams and the reflected image-forming rays. 

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.

Kim Ki H, So P T C, Kochevar I E, Masters B R and Gratton E 1998 Two-photon fluorescence and confocal reflected light imaging of thick tissue structures Proc. SPIE 3260 46-57... 

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-... 

The experimental set-up for the FCS measurement is illustrated schematically in Figure 8.6. A CW Ar laser (LGK7872M, LASOS lasertechnik GmbH) at 488 nm was coupled to a single mode optical fiber to isolate the laser device from an experimental table on which the confocal microscope system was constructed. This excitation laser light transmitted through the optical fiber was collimated with a pair of lenses, and then was guided into a microscope objective (lOOX, NA 1.35, Olympus). 

With the aim of elucidating molecular dynamics in a small domain, we have constmcted several microspectroscopic systems, that is, (i) the confocal microscope with the excitation light source being a femtosecond NIR laser emitting a 35 fs pulse, and (ii) the fluorescence correlation spectroscopic system with optical tweezers. 

When investigating opaque or transparent samples, where the laser light can penetrate the surface and be scattered into deeper regions, Raman light from these deeper zones also contributes to the collected signal and is of particular relevance with non-homogeneous samples, e.g., multilayer systems or blends. The above equation is only valid, if the beam is focused on the sample surface. Different considerations apply to confocal Raman spectroscopy, which is a very useful technique to probe (depth profile) samples below their surface. This nondestructive method is appropriate for studies on thin layers, inclusions and impurities buried within a matrix, and will be discussed below. 

---