Laser confocal microscope

Figure 12. Schematic diagram of the scanning laser confocal microscope. The out-of-focus information that normally reaches the eyepiece (detector) and leads to difficulties in interpreting optical images is rejected because the optical path does not take it through the pinhole. By scanning the incident laser beam across the sample, a digitized image is constructed from the infocus light rays that pass through the pinhole.

Figure 10.57 Comparison of laser confocal microscope (LCM) PL spectra of an isolated single strand of doped and dedoped PEDOT nanowires without Raman modes. (Reprinted with permission from Synthetic Metals, ight emission of a single strand of poly(3,4-ethylenediox-ythiophene) (PEDOT) nanowire by D. H. Park, H. S. Kim, Y. B. Lee et a ., 158, 3—4. Copyright (2008) Elsevier Ltd)...

Functional Correlation, Fig. 3 Image of a surface turned with a feed of 150 pm and a tool nose 400 pm, rendered from a measurement with scanning laser confocal microscope... 

Cells were plated onto coverslips and allowed to attach for 24 hours. After treated with 100 pM EGCG for 6 hours, ceUs were fixed in 4% paraformaldehyde for 10 min and permeabilized with 0.1% Triton X in PBS for 5 min CeUs were then washed with PBS, blocked with 2% bovine serum albumin for 30 min and incubated with p-FAK antibody for 1 hour at room temperature. After washing three times with 0.05% Triton X in PBS, cells were subsequently incubated with Alexa fluor 488 goat anti-rabbit second antibody for 1 hour at room teirqierature, and nuclei were stained with Topro 3 before mounting. The slides were viewed using a Zeiss LSM 510 laser confocal microscope (Carl Zeiss, Jena, Germai r). 

Evaporative rate analysis (ERA) measures the evaporation rate of a radioactive-tagged material from a surface. Organic contaminants dissolved in the solution reduce the evaporation rate and, by cahbration, the amount of organic present can be determined. The MESERAN (measurement and evaluation of surfaces by ERA), is a commercially available ERA instrument, Ruorescent molecules can be observed at high resolution using a laser confocal microscope (LCM). 

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. 

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

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. 

Spengler and Hubert (2002) describe a confocal laser scanning microscope used in conjunction with a TOF mass spectrometer, and also possessing ion imaging... 

Figure 4. DDC (A), serotonin (B), and tyrosine hydroxylase (C) immunore-activity in the posterior region of a wild-type Drosophila ventral ganglion. Tyrosine hydroxylase (TH) encodes the rate-limiting step in dopamine biosynthesis and is a marker for dopamine cells. B and C are the same CNS assayed for both serotonin and TH. M, medial dopamine neurons VL, ventrolateral serotonin neurons DL, dorsolateral dopamine neurons. Short unmarked arrows in C show vacuolated cells that do not contain DDC immunoreactivity. The immunoreactivity in these cells may represent a nonspecific cross-reactivity of the rat TH antibody. The length bar in A is 50 pM. The images are confocal projections generated on a Molecular Dynamics-2000 confocal laser scanning microscope.

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. 

Historically, this has been the most constrained parameter, particularly for confocal laser scanning microscopes that require spatially coherent sources and so have been typically limited to a few discrete excitation wavelengths, traditionally obtained from gas lasers. Convenient tunable continuous wave (c.w.) excitation for wide-held microscopy was widely available from filtered lamp sources but, for time domain FLIM, the only ultrafast light sources covering the visible spectrum were c.w. mode-locked dye lasers before the advent of ultrafast Ti Sapphire lasers. 

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). 

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