Proteomic analysis of nuclei dissected from fixed rat brain tissue using expression microdissection.

Expression microdissection (xMD) is a high-throughput, operator-independent technology that enables the procurement of specific cell populations from tissue specimens. In this method, histological sections are first stained for cellular markers via either chemical or immuno-guided methods, placed in close contact with an ethylene vinyl acetate (EVA) film, and exposed to a light source. The focal, transient heating of the stained cells or subcellular structures melts the EVA film selectively to the targets for procurement. In this report, we introduce a custom-designed flashcube system that permits consistent and reproducible microdissection of nuclei across an FFPE rat brain tissue section in milliseconds. In addition, we present a method to efficiently recover and combine captured proteins from multiple xMD films. Both light and scanning electron microscopy demonstrated captured nuclear structures. Shotgun proteomic analysis of the samples showed a significant enrichment in nuclear localized proteins, with an average 25% of recovered proteins localized to the nucleus, versus 15% for whole tissue controls (p < 0.001). Targeted mass spectrometry using multiple reaction monitoring (MRM) showed more impressive data, with a 3-fold enrichment in histones, and a concurrent depletion of proteins localized to the cytoplasm, cytoskeleton, and mitochondria. These data demonstrate that the flashcube-xMD technology is applicable to the proteomic study of a broad range of targets in molecular pathology.

A spin echo sequence with a single-sided bipolar diffusion gradient pulse to obtain snapshot diffusion weighted images in moving media.

In vivo MRI data can be corrupted by motion. Motion artifacts are particularly troublesome in Diffusion Weighted MRI (DWI), since the MR signal attenuation due to Brownian motion can be much less than the signal loss due to dephasing from other types of complex tissue motion, which can significantly degrade the estimation of self-diffusion coefficients, diffusion tensors, etc. This paper describes a snapshot DWI sequence, which utilizes a novel single-sided bipolar diffusion sensitizing gradient pulse within a spin echo sequence. The proposed method shortens the diffusion time by applying a single refocused bipolar diffusion gradient on one side of a refocusing RF pulse, instead of a set of diffusion sensitizing gradients, separated by a refocusing RF pulse, while reducing the impact of magnetic field inhomogeneity by using a spin echo sequence. A novel MRI phantom that can exhibit a range of complex motions was designed to demonstrate the robustness of the proposed DWI sequence.