Stronger together: Analytical techniques for recombinant adeno associated virus.

With recent FDA approval of two recombinant adeno-associated virus (rAAV)-based gene therapies, these vectors have proven that they are suitable to address monogenic diseases. However, rAAVs are relatively new modalities, and their production and therapy costs significantly exceed those of conventional biologics. Thus, significant efforts are made to improve the processes, methods, and techniques used in manufacturing and quality control (QC).

"Differential Visual Proteomics": Enabling the Proteome-Wide Comparison of Protein Structures of Single-Cells.

Proteins are involved in all tasks of life, and their characterization is essential to understand the underlying mechanisms of biological processes. We present a method called "differential visual proteomics" geared to study proteome-wide structural changes of proteins and protein-complexes between a disturbed and an undisturbed cell or between two cell populations. To implement this method, the cells are lysed and the lysate is prepared in a lossless manner for single-particle electron microscopy (EM).

Microfluidic protein isolation and sample preparation for high-resolution cryo-EM.

High-resolution structural information is essential to understand protein function. Protein-structure determination needs a considerable amount of protein, which can be challenging to produce, often involving harsh and lengthy procedures. In contrast, the several thousand to a few million protein particles required for structure determination by cryogenic electron microscopy (cryo-EM) can be provided by miniaturized systems.

Miniaturized Sample Preparation for Transmission Electron Microscopy.

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein complexes to atomic resolution. However, protein isolation techniques and sample preparation methods for EM remain a bottleneck. A relatively small number (100,000 to a few million) of individual protein particles need to be imaged for the high-resolution analysis of proteins by the single particle EM approach, making miniaturized sample handling techniques and microfluidic principles feasible.

Miniaturizing EM Sample Preparation: Opportunities, Challenges, and "Visual Proteomics".

This review compares and discusses conventional versus miniaturized specimen preparation methods for transmission electron microscopy (TEM). The progress brought by direct electron detector cameras, software developments and automation have transformed transmission cryo-electron microscopy (cryo-EM) and made it an invaluable high-resolution structural analysis tool. In contrast, EM specimen preparation has seen very little progress in the last decades and is now one of the main bottlenecks in cryo-EM.

Total Sample Conditioning and Preparation of Nanoliter Volumes for Electron Microscopy.

Electron microscopy (EM) entered a new era with the emergence of direct electron detectors and new nanocrystal electron diffraction methods. However, sample preparation techniques have not progressed and still suffer from extensive blotting steps leading to a massive loss of sample. Here, we present a simple but versatile method for the almost lossless sample conditioning and preparation of nanoliter volumes of biological samples for EM, keeping the sample under close to physiological condition.