Microscopy, confocal

Confocal microscopy is a related new technique that provides three-dimensional (3D) optical resolution. Image formation in a confocal microscope is significantly different from a conventional light microscope. Compared with a conventional compound microscope, a modern confocal microscope has two distinctive features in its structure a laser light source and a 

A variant of the confocal microscope is the laser scanning microscope (LSM, Fig. 8.9). Here, the effective focus acts as a three-dimensional probe, which is scanned in subsequent height steps two-dimensionally through a transpar- 

1) To first order one would expect multiphoton excitation of fluorescence markers to decrease the resolution, since the excitation wavelength increases (Sheppard 1996). 

The limiting spatial resolution of confocal microscopy is demonstrated in Fig. 8.12a with the help of objects that have diameters far below the wavelength of the imaging light here, fluorescing spheres of 110 nm diameter. The lateral resolution is of the order of a few hundred nanometers, while the vertical resolution of the order of a micrometer. This latter distortion of the spheres towards elongated objects results from the fact that the axial resolution is a factor of three to four smaller than the focal resolution. 

beam-splitter 5, scanning mirrors 6, focal plane, movable. 

A way of improving the axial resolution is to illuminate the sample with coherent light from the back side. Coherent in this context means that a fixed phase relation exists between front- and back-side illumination. This can be achieved by illuminating from front- and back-side with light from the same laser source. In this 47r confocal microscopy the axial higher order maxima 

The source in the confocal microscope is a laser, which uses the objective lens in the discriminator to focus it onto different planes on the sample. 

The sample is prepared as required, placed on a slide and mounted on a stage. Discriminator 

The objective lens is the discriminator. It forms a real intermediate image that is then detected by the photomultiplier tube (PMT). 

The detector is the PMT, which picks up the image and transfers the information to the computer for magnification. 

One of the developments in the area of confocal microscopy is the use of fluorescence to highlight or visualise certain features that either fluoresce naturally or are derivatised with probes prior to microscopic examination. Another new development is two-photon microscopes, which allow operation in the UV spectrum and, hence, deeper penetration of structures such as living cells, skin and other biological samples. 

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This overview will first deal with the optical aspects of conventional microscopes and the various means to improve contrast. Confocal microscopy, which in the last decade has become an important tool, especially for biology, is discussed in the final section. 

The progress tiiat has been achieved by confocal microscopy [13,14,15 and 16] is due to the rejection of... 

Confocal microscopy requires serial data acquisition and processing and hence comprises a complete system whose cost exceeds that of a conventional microscope. 

B1.18.5.6 CONFOCAL MICROSCOPY WITH MULTIPHOTON-EXCITATION FLUORESCENCE... 

Tdrdk P, Sheppard C J R and LaczikZ 1996 Dark field and differential phase contrast imaging modes in confocal microscopy using a half aperture stop Optik 103 101-6... 

V Kriete (ed) 1992 Visualization In Biomedical Microscopy, 3-d Imaging and Computer Visualization (Weinheim VCH) Wilson T (ed) 1996 Confocal Microscopy (New York Academic)... 

Schroth D 1997 The confocal laser scanning microscopy. A new tool in materials testing Matehalpruefung 39 264 Chestnut M H 1997 Confocal microscopy of colloids Curr. Opin. Colloid Interface Sc/. 2 158-61... 

Ribbe A E 1997 Laser scanning confocal microscopy in polymer science Trends Polym. Sc/. 5 333-7 Oliveira M J and Hemsiey D A 1996 Optical microscopy of polymers Sonderb. Prakt. Metallogr. 27 13-22 Nie Sh and Zare R N 1997 Optical detection of single molecules Ann. Rev. Biophys. Biomol. Struct. 26 567-96 Masters B R 1994 Confocal redox imaging of cells Adv. Mol. Cell Biol. 8 1-19... 

Lemasters J J 1996 Confocal microscopy of single living cells Chem. Anal., NY 137 157-77... 

Bhawalkar J D, Swiatkiewicz J, Pan S J, Samarabandu J K, Liou W S, He G S, Berezney R, Cheng P C and Prasad P N 1996 Three-dimensional laser scanning two-photon fluorescence confocal microscopy of polymer materials using a new, efficient upconverting fluorophore Scanning 18 562-6... 

Fleury L, Gruber A, Draebenstedt A, Wrachtrup J and von Borczyskowski C 1997 Low-temperature confocal microscopy on individual molecules near a surface J. Phys. Chem. B 101 7933-8... 

Two-photon excited fluorescence detection at the single-molecule level has been demonstrated for cliromophores in cryogenic solids [60], room-temperature surfaces [61], membranes [62] and liquids [63, 64 and 65]. Altliough multiphoton excited fluorescence has been embraced witli great entluisiasm as a teclmique for botli ordinary confocal microscopy and single-molecule detection, it is not a panacea in particular, photochemical degradation in multiphoton excitation may be more severe tlian witli ordinary linear excitation, probably due to absorjDtion of more tlian tire desired number of photons from tire intense laser pulse (e.g. triplet excited state absorjDtion) [61],... 

Hell S W and Nagorni M 1998 4Pi confocal microscopy with alternate interference Opt. Lett. 23 1567-9... 

Brakenhoff G J, Squier J, Norris T, Bliton A C, Wade M FI and Athey B 1996 Real-time two-photon confocal microscopy using a femtosecond, amplified Ti sapphire system J. Microscopy 181 253-9... 

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 also laser-scanning confocal microscopes rapidly overtaking the TSM as a means of confocal microscopy. There is also a computer software program that produces a "confocal" image by recogni2ing the shapes of out-of-focus detail, ie, halos, and subtracting these from the in-focus image. 

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. 

Korlach, J., Schwille, P., Webb, W. W. and Feigenson, G. W. (1999) Characterization of lipid bilayer phases by confocal microscopy and fluorescence correlation spectroscopy. Proc. Natl. Acad. Sci. USA, 96, 8461-8466. 

There are several other techniques Uke the fluorescent dye displacement assays, footprinting, Fourier transform infrared spectroscopy. X-ray crystallography, electron microscopy, confocal microscopy, atomic force microscopy, surface plasmon resonance etc used for hgand-DNA interactions that are not discussed here. 

Y. Nicolas, M. Paques, A. Knaebel, A. Steyer, J.-P. Munch, T. B. J. Blijdenstein, G. A. van Aken 2003, (Microrheology structural evolution under static and dynamic conditions by simultaneous analysis of confocal microscopy and diffusing wave spectroscopy), Rev. Sci. Instrum. 74, 3838. 

To provide a complete assessment of all these variables, the final evaluation of safety must be made in the in vivo model using the preparation under the proposed conditions for use, following tissue compatibility with many of the techniques already discussed. Confocal microscopy is a relatively new noninvasive technique that allows a detailed examination of the endothelium in the live animal, and thus may prove useful in following changes in this delicate tissue over time. As in ex vivo models, the... 

Fricker G, Miller D, Bauer B, Nob-mann S, Gutmann H, Torok M et al. Transport of xenobiotics across isolated brain microvessels studied by confocal microscopy. Presented at the 3rd FEBS Advanced Fecture Course - ABC2001 Gosau, Austria. 

Volume 307. Confocal Microscopy Edited by P. Michael Conn... 

This work and SGC were supported by a Wellcome Trust project grant 080349/Z/06/Z to MPA. We thank G. Pereira and G. Pavitt for reagents and D. Jackson and C. Tang for their assistance with the confocal microscopy. 

Pawley, J. (2006). Handbook of Biological Confocal Microscopy, 3rd edition. Springer Verlag, New York. 

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