Condensed Mitotic Chromosome Structure at Nanometer Resolution Using PALM and EGFP- Histones

Photoactivated localization microscopy (PALM) and related fluorescent biological imaging methods are capable of providing very high spatial resolutions (up to 20 nm). Two major demands limit its widespread use on biological samples: requirements for photoactivatable/photoconvertible fluorescent molecules, which are sometimes difficult to incorporate, and high background signals from autofluorescence or fluorophores in adjacent focal planes in three-dimensional imaging which reduces PALM resolution significantly. We present here a high-resolution PALM method utilizing conventional EGFP as the photoconvertible fluorophore, improved algorithms to deal with high levels of biological background noise, and apply this to imaging higher order chromatin structure. We found that the emission wavelength of EGFP is efficiently converted from green to red when exposed to blue light in the presence of reduced riboflavin. The photon yield of red-converted EGFP using riboflavin is comparable to other bright photoconvertible fluorescent proteins that allow <20 nm resolution. We further found that image pre-processing using a combination of denoising and deconvolution of the raw PALM images substantially improved the spatial resolution of the reconstruction from noisy images. Performing PALM on Drosophila mitotic chromosomes labeled with H2AvD-EGFP, a histone H2A variant, revealed filamentous components of ∼70 nm. This is the first observation of fine chromatin filaments specific for one histone variant at a resolution approximating that of conventional electron microscope images (10–30 nm). As demonstrated by modeling and experiments on a challenging specimen, the techniques described here facilitate super-resolution fluorescent imaging with common biological samples

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322 tr. ; 21 c

Sensation Seeking and Affective Disorders: Characteristics in the Intensity Dependence of Acoustic Evoked Potentials

Augmenting/reducing of the evoked potential has been shown to be related to sensation seeking (SS) and specific clinical disorders. Buchsbaum demonstrated that patients with bipolar affective disorders (BAD) tend to be augmenters, as is the case with sensation seekers, and patients with unipolar affective disorders (UPD) tend to be reducers. In addition, he reported that prophylactic medication reduced the tendency to augment in bipolar patients. However, evidence for these relations is restricted to a few studies. This study explores whether Buchsbaum’s initial findings can be found in a naturalistic clinical setting. Acoustic evoked potentials were recorded for six levels of intensity (59, 71, 79, 88, 92, 96 dB SPL) from 24 healthy adults, 21 unipolar depressed patients, and 21 patients with BAD. Participants also completed personality questionnaires, especially the Sensation Seeking Scales Form V. Results revealed a positive correlation between SS and augmenting/reducing in healthy controls, thereby replicating earlier findings. Bipolar depressed patients showed larger P1/N1 slopes than healthy controls, when medication was statistically controlled. Unipolar depressed patients showed smaller P2 slopes, but only when medication was not controlled. Implications of these results for further research on augmenting/reducing and affective disorders and their relationship to SS are discussed.Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich

Fluorescence of single molecules in polymer films: Sensitivity of blinking to local environment

Clifford JN, Bell TDM, Tinnefeld P, et al. Fluorescence of single molecules in polymer films: Sensitivity of blinking to local environment. JOURNAL OF PHYSICAL CHEMISTRY B. 2007;111(25):6987-6991.The single-molecule fluorescence blinking behavior of the organic dye Atto647N in various polymer matrixes such as Zeonex, PVK, and PVA as well as aqueous media was investigated. Fluorescence blinking with off-times in the millisecond to second time range is assigned to dye radical ions formed by photoinduced electron transfer reactions from or to the environment. In Zeonex and PVK, the measured off-time distributions show power law dependence, whereas, in PVA, no such dependence is observed. Rather, in this polymer, off-time distributions can be best fitted to monoexponential or stretched exponential functions. Furthermore, treatment of PVA samples to mild heating and low pressure greatly reduces the frequency of blinking events. We tentatively ascribe this to the removal of water pockets within the polymer film itself. Measurements of the dye immobilized in water in the presence of methylviologen, a strongly oxidizing agent, reveal simple exponential on- and off-time distributions. Thus, our data suggest that the blinking behavior of single organic molecules is sensitive to their immediate environment and, moreover, that fluorescence blinking on- and off-time distributions do not inherently and uniquely obey a power law