Spectroscopy confocal

Single-molecule detection in confocal spectroscopy is characterized by an excellent signal-to-noise ratio, but the detection efficiency is in general very low because the excitation volume is very small with respect to the whole sample volume, and most molecules do not pass through the excitation volume. Moreover, the same molecule may re-enter this volume several times, which complicates data interpretation. Better detection efficiencies can be obtained by using microcapillaries and micro structures to force the molecules to enter the excitation volume. A nice example of the application of single-molecule detection with confocal microscopy is... 

Fluorescence Polarisation coupled with confocal spectroscopy... 

Raman photoluminescence piezospectroscopy of bone, teeth and artificial joint materials has been reviewed by Pezzotti (2005) with emphasis placed on confocal microprobe techniques. Characteristic Raman spectra were presented and quantitative assessments of their phase structure and stress dependence shown. Vibrational spectroscopy was used to study the microscopic stress response of cortical bone to external stress (with or without internal damages), to define microscopic stresses across the dentine - enamel junction of teeth under increasing external compressive masticatory load and to characterise the interactions between prosthetic implants and biological environment. Confocal spectroscopy allows acquisition of spatially resolved spectra and stress imaging with high spatial resolution (Green etal., 2003 Pezzotti, 2005 Munisso etal., 2008). 

No.9, 1997,p.2067-75 STUDY OF THE CONFORMATIONS OF POLY(EPSILON-CAPROLACTAM) AND POLY(EPSILON-CAPROLACTAM)-POLYBUTADIENE BLOCK COPOLYMERS BY FTIR SPECTROSCOPY WITH PHOTOACOUSTIC DETECTION AND BY MICRO-RAMAN CONFOCAL SPECTROSCOPY Schmidt P Fernandez M R Pastor J M Roda J Czech Republic,Academy of Sciences ... 

FTIR spectroscopy with photoacoustic detection and micro-Raman confocal spectroscopy were used to study the conformations of poIy(epsilon caprolactam) and poly(epsilon-caprolactam)-polybutadiene block copolymers. In the block copolymers prepared by anionic polymerisation, the fraction of the planar conformation of poly(epsilon-caprolactam) chains decreased with increasing polybutadiene content. In the surface layers formed by rapid saw cutting and in the islands formed by microtome cutting, the content of the planar conformation was lowered. This was substantially increased by water treatment, especially at elevated temperatures. 15 refs. 

Edel JB, Lahoud P, Cass AEG, deMello AJ (2007) Discrimination between single Escherichia coli cells using time-resolved confocal spectroscopy. J Phys Chem B 111 1129-1134... 

Fries J R, Brand L, Eggeling C, Kdllner M and Seidel CAM 1998 Quantitative identification of different single molecules by selective time-resolved confocal fluorescence spectroscopy J. Phys. Chem. A 102 6602-13... 

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]. 

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. 

Leopold et al. and Nyholm et al. have investigated this oscillatory system by in situ confocal Raman spectroscopy [43], and in situ electrochemical quartz crystal microbalance [44], and in situ pH measurement [45] with the focus being on darification of the osdllation mechanism. Based on the experimental results, a mechanism for the oscillations was proposed, in which variations in local pH close to the electrode surface play an essential role. Cu is deposited at the lower potentials ofthe oscillation followed by a simultaneous increase in pH close to the surface due to the protonation... 

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. 

Diaspro A, Chirico G, Federici F, Cannone F, Beretta S, Robello M (2001) Two-photon microscopy and spectroscopy based on a compact confocal scanning head. J Biomed Opt 6 300-310... 

When investigating opaque or transparent samples, where the laser light can penetrate the surface and be scattered into deeper regions, Raman light from these deeper zones also contributes to the collected signal and is of particular relevance with non-homogeneous samples, e.g., multilayer systems or blends. The above equation is only valid, if the beam is focused on the sample surface. Different considerations apply to confocal Raman spectroscopy, which is a very useful technique to probe (depth profile) samples below their surface. This nondestructive method is appropriate for studies on thin layers, inclusions and impurities buried within a matrix, and will be discussed below. 

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.

The first fluorescence correlation spectroscopy experiments were carried out several decades ago,62 64 but the general use of the technique was made possible with the introduction of lasers with high beam quality and long-term temporal stability, low noise detectors, and high-quality microscope objectives with high numeric apertures.58,63 The most common set-up is using a confocal inverted epi-fluorescence... 

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. 

Under the same optical configuration, FCS (Fluorescence Correlation Spectroscopy) measurements (see Section 11.3) can be carried out on samples at the singlemolecule level under conditions where the average number of fluorescent molecules in the excitation volume is less than 1. It should be noted that at low fluorophore concentrations, the time required to obtain satisfactory statistics for the fluctuations may become problematic in practical applications (e.g. for a concentration of 1 fM, a fluorophore crosses a confocal excitation volume every 15 min). 

Lemasters J. J. (1996) Confocal Microscopy of Single Living Cells, in Wang X. F. and Herman B. (Eds), Fluorescence Imaging Spectroscopy and Microscopy, Chemical Analysis Series, Vol. 137, John Wiley ... 

Finally, in Chapter 11 some advanced techniques are briefly described fluorescence up-conversion, fluorescence microscopy (confocal excitation, two-photon excitation, near-field optics, fluorescence lifetime imaging), fluorescence correlation spectroscopy, and single-molecule fluorescence spectroscopy. 

Gierlinger, N., Hansmann, C., Roder, T., Sixta, H., Gindl, W. and Wimmer, R. (2005). Comparison of UV and confocal Raman spectroscopy to measure the melamine - formaldehyde resin content within ceU walls of impregnated spruce wood. Holzforschung, 59(2), 210-213. 

A number of methods are available for the characterization and examination of SAMs as well as for the observation of the reactions with the immobilized biomolecules. Only some of these methods are mentioned briefly here. These include surface plasmon resonance (SPR) [46], quartz crystal microbalance (QCM) [47,48], ellipsometry [12,49], contact angle measurement [50], infrared spectroscopy (FT-IR) [51,52], Raman spectroscopy [53], scanning tunneling microscopy (STM) [54], atomic force microscopy (AFM) [55,56], sum frequency spectroscopy. X-ray photoelectron spectroscopy (XPS) [57, 58], surface acoustic wave and acoustic plate mode devices, confocal imaging and optical microscopy, low-angle X-ray reflectometry, electrochemical methods [59] and Raster electron microscopy [60]. 

Combining fluorescence spectroscopy with fluorescence microscopy, confocal microscopy could be used to elucidate the pathway of siderophore-mediat iron uptake in the fungus Ustilago maydis, and visualize this pathway by providing unique fluorescent microscopic images. Using these techniques, clear images of two independent iron-uptake mechanisms have become visualized as well as their cellular compartment locahzed. 

Cryo-transmission electron spectroscopy (TEM), scanning electron spectroscopy, and confocal laser scanning microscopy smdies indicated the presence of large. 

Thomas R. (2000) Determination of water contents of granite melt inclusions by confocal laser Raman microprobe spectroscopy. Am. Mineral. 85, 868-872. 

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