Confocal illumination

The proof that single molecules could be measured by confocal illumination was given in a study where fluorescence bursts of Rhodamine molecules passing through a sharply focused laser beam were analyzed. From the burst... 

The principal concept was already described in the early 70 s by the first paper on this topic by Magde et al. (2J. A real renaissance arrived in 1993 with the introduction of the confocal illumination scheme for FCS by Rigler et al. fSJ, Since then FCS has been developed towards probably the most important technique for single molecule detection (SMD). FCS allows measuring rates of binding/unbinding reactions SJ, coefficients of translational and rotational diffusion (6, 7, 8, 9J, conformational states and a manifold of photophysical parameters (JO, II, 12) of the induced fluorescent process. 

For FFS the confocal illumination scheme led to a highly confined excitation and detection volume. Alternative optical schemes for confining the excitation volume, for instance evanescent field excitation, were demonstrated already in the 80 s (13, 14), However, they did not show a performance comparable to confocal FFS for single molecule detection. 

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 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. 

There are two types of scanning acoustic microscopes. If the illumination and the reception of the acoustic waves are performed by two identical lenses arranged confocally, the SAM is called a transmission SAM. The lens geometry used for transmission imaging is shown schematically in Fig. 41 [93]. 

Cogswell, C. J. and Larkin, K. G. (1995). The specimen illumination path and its effect on image quality. In Handbook of Biological Confocal Microscopy (Pawley, J. B., ed.). Plenum press, New York, pp. 127-37. 

Two-photon excitation provides intrinsic 3-D resolution in laser scanning fluorescence microscopy. The 3-D sectioning effect is comparable to that of confocal microscopy, but it offers two advantages with respect to the latter because the illumination is concentrated in both time and space, there is no out-of-focus photo-bleaching, and the excitation beam is not attenuated by out-of-focus absorption, which results in increased penetration depth of the excitation light. 

When fluorescently labeled biological specimens are viewed with a conventional wide-field microscope, a haze of out-of-focus fluorescence is usually created hy the overlapping structures within the sample. As we focus through the specimen, our hrains have a remarkable ability to discern substantial structural detail. However, the resolution of the images we record on film is degraded hy the out-of-focus fluorescence. The confocal microscope can reject out-of-focus information and enhance the contrast of an image because the illumination and the detection are confined to an identical (small) region of the specimen. An overview of the basic principles of a confocal microscope is presented in Fig. 1 and outlined helow. 

Fig. 2. Depth discrimination (z-axis resolution) properties of a confocal microscope. The illumination and detection images in a confocal microscope are diffraction-limited and confined to a small region of the specimen (1). Only light emitted in the plane of focus and on the optical axis will pass the detector pinhole and form an image. Light emitted from other areas of the specimen does not enter the detector pinhole.

Lin et al. 1985 Wade and Meyyappan 1987 Wey and Kessler 1989) the insonification is broadcast throughout the specimen, and the detection is by a focused optical probe that measures local surface tilt on the surface of the specimen. But in the scanning acoustic microscope both the illumination and the detection are performed by focusing elements and, since these are focused at the same point, the configuration may be described as confocal. The first con-focal acoustic microscopes worked in transmission and, although this is now of mainly historical interest, the transmission arrangement will be described first because in some respects it is simpler and will serve to introduce some principles. 

The structure (e.g., number, size, distribution) of fat crystals is difficult to analyze by common microscopy techniques (i.e., electron, polarized light), due to their dense and interconnected microstructure. Images of the internal structures of lipid-based foods can only be obtained by special manipulation of the sample. However, formation of thin sections (polarized light microscopy) or fractured planes (electron microscopy) still typically does not provide adequate resolution of the crystalline phase. Confocal laserscanning microscopy (CLSM), which is based on the detection of fluorescence produced by a dye system when a sample is illuminated with a krypton/argon mixed-gas laser, overcomes these problems. Bulk specimens can be used with CLSM to obtain high-resolution images of lipid crystalline structure in intricate detail. 

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