Confocal setups

An alternative setup is the classical one with two detectors and analyzers which have been used by Kask et al. 1987 and Tsai et al. 2008. Relating to the work of Aragon and Pecora (1975 [14]) the analysis with this setup leads to a complex solution with higher order eigenvalues. Our setup can easily be verified in the confocal setup [15], which started the new era of FCS. 

For the analysis in which the correlation function has to be recorded in the ns regime a typical Flanbury Brown and Twiss [18] set up. Figure 4.4 shows a confocal setup (discussed later) in combination with a beam splitter and a time to amplitude converter (TAG). Since both pathways for the emitted photons acting as start or stop for the time amplitude converter (TAG) are equivalent the correlation curves registered in a multichannel analyzer (MGA) are symmetric relative to 0 time. 

Fig. 4.4. Confocal setup for excited states correlation analysis using a TAG. (a) Time response (FWHM 0.5 ns) of the system using an Avalanche Photodiode (APD). (b) Rotational diffusion of Texas red labeled pancreatic porcine lipase and antibunching of Texas red [19] (c)...

Furthermore, in the electrochemical system, most of the electrolytes are corrosive, and to protect the objective, a cover glass or quartz window has to be employed between the electrolyte and the objective, this results in 50% loss of the signal. An alternative way to protect the objective is to wrap it with a very thin and highly transparent polyvinyl chloride (PVC) film. A benefit of the confocal setup is that there is no signal from PVC in this configuration. By this approach, the Raman signal only suffers 20% loss. The disadvantage of this approach is that without the quartz window, the solution has to be exposed to the air, and impurities in the air wQl contaminate the solution. For systems that are very sensitive to any contamination, a thin quartz window, with thickness of 0.2 to 0.5 mm, has to be employed at the cost of the intensity. 

Figure 9. Fluorescence spectra of two single pentacene molecules in />-terphenyl recorded with a confocal setup (see text). Spectrum A was taken in 33 minutes with a sample contacted to a silica plate. The excitation frequency of this molecule coincided with the unperturbed 0 site maximum. The spectrum presents all features of an Oi molecule when compared to a bulk spectrum with only some small deviations [39]. Spectrum B was recorded over 40 minutes with a sample glued to a pinhole. The excitation frequency of tlie molecule was in the middle of the 0 -O2 interval. According to its spectrum which shows some small differences to spectrum A it clearly belonged to site O2 (from Ref. 39).

Fig. 10 Fluorescence images of single fluorescent molecules in the same area observed by conventional confocal (a) and STED configurations (b), and the rectangiflar areas in both images are enlarged (c). Panel d indicates the cross-section profiles for panel (c). The STED microscopy clearly resolves the three molecules separated by 160 nm, which were observed as one ellipsoidal spot by the confocal setup. The histograms of the full width at half maximum (FWHM) of the observed fluorescence spots for the - (e) andy-directions (f) indicate the sharpening of the focal spot in the STED configuration. Reprinted with permission of [40], copyright (2003) American Institute of Physics...

In the microspectrometer schematic of Figure 7.1, the two objectives share a common focus at the specimen, i.e. they are confocal. However, we add an additional constraint that the two apertures (labelled upper and lower in the figure) are both present and matched to define the same sample region when using the term confocal. Whether used with a single aperture, or in a confocal setup, the instrument spectroscopically samples just one location at a time. 

Figure 7.3 Confocal setup for Raman microspectroscopy. Holes D1 and D2 are optically conjugated.

A slight enhancement of the lateral resolution. Theoretically, the confocal setup may decrease the spreading function, but it should be mentioned that this requires very small diaphragms to be used, which in turn may decrease the amount of detected intensity. 

The name already implies that in this method, as in a traditional optical miaoscope, the laser beam or an arc lamp illuminates a wide area that is several tens of miaometers in diameter in a uniform fashion. This is in contrast with scanning confocal miaoscopy where the laser beam is focused to a near-difffaction-limited spot on the sample. To get a uniform collimated circular polarized beam, the laser beam is introduced into the miaoscope with some slight modifications as compared to the confocal setup (vide infra). To get sufficient signal, the power density on the sample is similar as in a confocal setup, adjusted between 1 and lOkWcm". However, in the case of wide-field illumination, the laser power is spread over the entire illumination area. Therefore, high power continuous lasers are generally used. Typical output powers required are 50-100 mW. 

Around the glass transition temperature, the polymer is more mobile than far below Tg and the cooperative motion of polymer chains can induce rotational motion of the dye molecules embedded in the polymer matrix. The rotational motion can be studied using polarization measurements (confocal setup) or defocused wide-field imaging. 

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). 

These differences add up to one major distinction on wide-field imaging setups, it suffices to calibrate the setup just once for a given set of filters and fluorophores, and then use it for weeks or months without bothering about it. In contrast, for confocal filterFRET imaging, calibrations must be made every time a gain setting or laser line is adjusted, and preferably, for every image. 

Figure 14.3. Sketch of experimental setup. CSBLM (confocal scanning-beam laser microscope) sends laser beam to anode. (From Ref. 6. By permission of the Electrochemical Society.)...

With the help of a micro-Raman setup the laser spot can be focused down to about 1 pm in diameter. This allows for the differentiation of single bacterial cells or a biochemical analysis of subcellular components within bacterial (diameter approx. 1 pm) or yeast cells (diameter approx. 5-10pm). A confocal Raman setup achieves an even better spatial resolution [6, 7]. This possibility enables Raman mapping or imaging experiments with spatially resolved information of the whole sample in axial and lateral directions. 

FIG. 16.2 A typical optical setup of reflection confocal microscope. 

Here, C f,t) is the concentration of fluorescent molecules at position f and time t. CEF f) is the collection efficiency function of the confocal microscope setup and Iexc r) denotes the excitation intensity. Q = f, is a bright-... 

Although STED and confocal microscopy are easily combined to each others advantage in the same setup, STED is not an extension of confocal microscopy, because it does not require the imaging of the fluorescence onto a pinhole. In principle, one could detect the fluorescence signal right at the sample. Therefore, parallelized camera-based STED microscopy will also be possible with arrays of doughnuts or lines (Fig. 19.Id) [88]. 

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