Resolution subcellular

Vibrational spectroscopy provides for the analysis of the chemical composition, molecular structures, conformations, and interactions in a sample. Two methods that are commonly used for molecular vibrational analysis are infrared (IR) and Raman spectroscopy. One of the limitations of the former, which relies on measuring the absorption or reflection of mid-IR radiation by molecular bonds, is the low spatial resolution afforded by the technique, allowing only chemical information from a large group of cells to be obtained. Raman spectroscopy, on the other hand, offers high spatial resolution subcellular chemical analysis of an individual cell. It relies on the scattering of photons by molecular bonds, which yields chemical information about the molecular stmcture and conformations in the sample. It has seen an increase in its use for biological studies because it offers many attractive features. The method is able to... 

Looger, L. L., Lalonde, S. and Frommer, W. B. (2005). Genetically encoded FRET sensors for visualizing metabolites with subcellular resolution in living cells. Plant Physiol. 138, 555-7. 

Taken together, the field is now well placed to design new biosensors, examine protein-protein and protein-lipid interactions, and sensitively determine protein conformation in living tissues at submicron resolution. These interactions are either impossible or extraordinarily difficult to examine in other ways, and the subcel-lular resolution of FRET-FLIM that allows detection of interactions in specific subcellular compartments may provide insight that... 

Gordienko D V, Bolton TB, Cannell MB 1998 Variability in spontaneous subcellular Ca2+ release in guinea-pig ileum smooth muscle cells. J Physiol 507 707-720 Ho R, Shao Z 1991 Axial resolution of confocal microscopes revisited. Optiik 88 147—154 Holz GG, Leech CA, Heller RS, Castonguay N, Habener JF 1999 cAMP-dependent mobilization of intracellular Ca2+ stores by activation of ryanodine receptors in pancreatic [j cells. A Ca2+ signaling system stimulated by the insulinotropic hormone glucagon-like peptide-1-(7-37). J Biol Chem 274 14147-14156 Lipsius SL, Hiiser J, Blatter LA 2001 Intracellular Ca2+ release sparks atrial pacemaker activity. News Physiol Sci 16 101-106... 

In contrast to many chemotherapeutic agents in cancer therapy, boron compounds for BNCT do not require a tumoricidal action in their own right. For their successful application in the therapy of patients, it is important to deliver, to the tumor, a radiation dose which is higher than the radiation dose to the surrounding tissue. The demonstration that this is actually achieved lies ultimately in the treatment of the tumor in question. Because of the short range of the particles produced in the 10B(n,a)7Li reaction, it is very important where, on a cellular and subcellular dimension, the neutron capture reaction takes place. Different methods for boron detection and quantification give different resolution of the boron distribution. It is instructive to compare these methods, both for their precision and lower detection limits, as well as for their ability to yield an image of the boron distribution in tissue (Table 2.2-1). 

Bader AN, Hofrnan EG, Voortman J, en Flenegouwen PM, Gerritsen HC (2009) Homo-ERET imaging enables quantification of protein cluster sizes with subcellular resolution. Biophys J 97 2613-22... 

Conventional, high-resolution immunoelectron microscopy has been extensively used for the subcellular distribution of proteins to obtain information on their functions. However, this approach is time consuming. Processing time can be substantially reduced by applying microwave heating the total time is reduced to 4-5 hr while the conventional... 

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. 

Altelaar, A. F., van Minnen, J, Jimenez, C. R., Heeren, R. M., and Piersma, S. R. (2005). Direct molecular imaging of Lymnaea stagnalis nervous tissue at subcellular spatial resolution by mass spectrometry. Anal. Chem. 77 735-741. 

In EMP the cross section of the electron probe is often of the order of 2 nm, and with a section thickness approximately 100 nm a resolution at the subcellular level can be obtained. Considering the fact that physiologically interesting elements generally are freely dispersed in the cytosol, it is clear that local variations in concentration in biological tissues are to be expected. Therefore, analysis data are retrieved from sets of spots in regions of the tissue deemed to be representative of the structure under investigation. 

It should be obvious from the above that membrane proteomics strictly follows Murphy s law. This is due to the fact that there is a mutual exclusion, for physicochemical reasons, between on the one hand the conditions that must be used to solubilize in water all membrane proteins, including the most hydrophobic ones, and thus give a fair representation of the protein population in the sample, and on the other hand the conditions prevailing in the high resolution peptide separation methods. On top of this problem, there is a second problem linked to the membrane versus aqueous phase volume in many subcellular preparations, which makes transmembrane proteins rare compared to water-soluble proteins. 

Chen, X., Velliste, M., Weinstein, S., Jarvik, J.W. and Murphy, R.F. (2003) Location proteomics-building subcellular location trees from high resolution 3d fluorescence microscope images of randomly-tagged proteins. Proc. SPIE 4962, 298—306. 

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