Confocal microscopes

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). 

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)... 

Ojcius D M, Niedergang F, Subtil A, Hellio R and Dautry-Varsat A 1996 Immunology and the confocal microscope Res. Immunol. 147 175-88... 

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. 

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 sample is measured with a confocal microscope, by using a chromatic coding of the height, and with a contact-less optical needle focused on the surface. We determine by this way the surface topography and its roughness. 

Confocal Microscope with a Chromium Forsterite Ultrafast Laser as an Excitation Source... 

Fluorescence intensity detected with a confocal microscope for the small area of diluted solution temporally fluctuates in sync with (i) motions of solute molecules going in/out of the confocal volume, (ii) intersystem crossing in the solute, and (hi) quenching by molecular interactions. The degree of fluctuation is also dependent on the number of dye molecules in the confocal area (concentration) with an increase in the concentration of the dye, the degree of fluctuation decreases. The autocorrelation function (ACF) of the time profile of the fluorescence fluctuation provides quantitative information on the dynamics of molecules. This method of measurement is well known as fluorescence correlation spectroscopy (FCS) [8, 9]. 

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. 

Hammerle, S. P., Rothen-Rutishauser, B., Kramer, S. D., Gunthert, M., Wunderli-Allenspach, H., P-glycoprotein in cell cultures a combined approach to study expression, localisation, and functionality in the confocal microscope, Eur. J. Pharm. Sci. 2000, 32, 69-77. 

FRAP (fluorescence recovery after photo bleaching) analysis was originally developed in the mid-1970s to study the diffusion of biomolecules in living cells (Edidin et al, 1976). Flowever, due to the increased availability of GFP tags and advances in the bleaching capabilities of confocal microscopes, this... 

Take images using a confocal microscope. We use a Leica DM RBE TCS SP1 microscope (Leica, Wetzlar, Germany) and process images in Adobe Photoshop... 

Chapter 8 written by Steve Vogel et al. also deals with sensitized emission based FRET methodology, but now using a spectral imaging detector device. Because a spectral detector and spectral unmixing software nowadays are standard options on the major commercial confocal microscopes, here a complete description is given how to quantify FRET from unmixed spectral components. 

Lifetime imaging can be implemented both in wide field and in scanning microscopes such as confocal microscopes and two-photon excitation microscopes. The most common implementations in time-domain fluorescence lifetime imaging microscopy (FLIM) are based on TCSPC [8, 9] and time-gating (TG) [2, 10],... 

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. 

Ghiggino, K. P., Harris, M. R. and Spizzirri, P. G. (1992). Fluorescence lifetime measurements using a novel fiberoptic laser scanning confocal microscope. Rev. Sci. Instrum. 63, 2999-3002. 

Several other approaches to solve the quantitation problem have been proposed. Hoppe et al. [2] determined y/ by calibrating it against constructs with known FRET efficiency. We and others [3, 6] have used data from a cell before and after acceptor photobleaching to relate the FRET-induced sensitized emission in the S channel to the loss of donor emission in the D channel by factors termed or G, respectively. For the CFP/YFP pair this works very well on confocal microscopes with a 514-nm Argon ion laser line, but on wide-held systems, selective acceptor photobleaching reportedly causes problems [ 14]. F inally, G can also be determined by comparison of several constructs that differ in FRET efficiency, a bit analogous to the Yellow Cameleon calibration described above [10,14],... 

Because on CCD setups excitation for D, S, and A images is usually filter-selected from a single white light source the relative intensity of excitation is approximately fixed. Confocal microscopes use separate laser lines, often from distinct lasers, that can (and for optimal imaging should) be independently adjusted. Thus, on CCD setups y (Eq. (7.6)) is constant for a given set of filters whereas on the confocal, it varies from image to image (also, see Sect. 7.4.2). 

A final distinction is that on confocal microscopes S and A images are commonly acquired with the exact same emission filter settings whereas for CCD microscopes they typically involve physically separate- and therefore slightly different—filter cubes.6 This simplifies the calculation of leak-through terms [3], In Appendix of this chapter, we rather generalized the treatment of filterFRET by not making assumptions on the filter settings for S and A. 

On a variety of confocal microscopes, we and others [3, 18] observed considerable drift as well as oscillations (on a time scale of... 

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