Confocal principle

The setup illustrated on the right uses a confocal principle. The laser is directed into the euvette by a diehroie mirror, D, and focused by a lens. The fluorescence light is colleeted by this lens, sent through a set of filters, focused by a second lens, and detected by the PMT module. The setup can be built with a high numeri-eal aperture and a eorrespondingly high collection efficiency. 

Cork T and Kino G S 1996 Confocal Scanning Optical Microscopy and Related Imaging Systems (New York Academic) Gu Min 1996 Principles of Three Dimensional Imaging In Confocal Microscopes (Singapore World Scientific)... 

Figure 1 Principle of confocal Raman microscopy A laser spot in the focal plane passes through the pinhole P. A laser spot at a distance z from the focal plane is projected in the image plane with size P, and is largely blocked by the pinhole P. [L — lens M = beam splitter fi, f2 = focal length of lens L, and L2, respectively b2 — image distance of out-of-focus laser spot). Reproduced from Tabaksblat et al. [14], with permission of the Society for Applied Spectroscopy. 2000.

Principle A confocal microscopy as a modification of luminescent microscope may produce images of high quality from fluorescing cells and permits the study of cells structures (see Chapter 8). 

Principle ROS production can be monitored by imaging the ROS-sensitive fluorescent dye dichlorofluorescein (DCF) in a confocal microscope. 

Fig. 11.3. Principle of confocal microscopy (left) compared with conventional microscopy (right).

As shown in Section 11.2.1.1, more details can be obtained by confocal fluorescence microscopy than by conventional fluorescence microscopy. In principle, the extension of conventional FLIM to confocal FLIM using either time- or frequency-domain methods is possible. However, the time-domain method based on singlephoton timing requires expensive lasers with high repetition rates to acquire an image in a reasonable time, because each pixel requires many photon events to generate a decay curve. In contrast, the frequency-domain method using an inexpensive CW laser coupled with an acoustooptic modulator is well suited to confocal FLIM. 

In fluorescence correlation spectroscopy (FCS), the temporal fluctuations of the fluorescence intensity are recorded and analyzed in order to determine physical or chemical parameters such as translational diffusion coefficients, flow rates, chemical kinetic rate constants, rotational diffusion coefficients, molecular weights and aggregation. The principles of FCS for the determination of translational and rotational diffusion and chemical reactions were first described in the early 1970s. But it is only in the early 1990s that progress in instrumentation (confocal excitation, photon detection and correlation) generated renewed interest in FCS. 

Bacallao, R., Morgane, B., Stelzer, E. H. K., and DeMey, J. (1989) Guiding principles of specimen preservation for confocal fluorescence microscopy, in Handbook of Biological Confocal Microscopy (Pawley, J. B., ed.). Plenum, New York, pp. 197-205. 

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. 

Shooton, D. (ed.) (1993) Electronic Light Microscopy. The Principles and Practice of Video-Enhanced Contrast, Digital Intensified Fluorescence, and Confocal Scanning Light Microscopy. Wiley-Liss, New York. 

Gu, M. 1996. Principles of three-dimensional imaging in confocal microscopes. Singapore World Scientihc, Inc. 

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. 

Raman microspectroscopy results from coupling of an optical microscope to a Raman spectrometer. The high spatial resolution of the confocal Raman microspectrometry allows the characterization of the structure of food sample at a micrometer scale. The principle of this imaging technique is based on specific vibration bands as markers of Raman technique, which permit the reconstruction of spectral images by surface scanning on an area. 

Describe, with a labeled diagram, the principle of confocal fluorescent detection. (4 marks)... 

Figure 3.5-12 Principle of a confocal microscope a LF fiber transporting the laser radiation, D dichroitic mirror, 0 objective, S sample, the Raman radiation produced in the illuminated spot of the sample is focused upon the diaphragm A, only the radiation from the spot is focused at the fiber SF, which transports the Raman radiation to the spectrometer b focal range in the illuminated sample, Ax spatial, Az depth resolution.

FIGURE 66 (A) Principle and confocal luminescence images of the up-conversion... 

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