VitroJet: new features and case studies.

Single-particle cryo-electron microscopy has become a widely adopted method in structural biology due to many recent technological advances in microscopes, detectors and image processing. Before being able to inspect a biological sample in an electron microscope, it needs to be deposited in a thin layer on a grid and rapidly frozen. The VitroJet was designed with this aim, as well as avoiding the delicate manual handling and transfer steps that occur during the conventional grid-preparation process. Since its creation, numerous technical developments have resulted in a device that is now widely utilized in multiple laboratories worldwide. It features plasma treatment, low-volume sample deposition through pin printing, optical ice-thickness measurement and cryofixation of pre-clipped Autogrids through jet vitrification. This paper presents recent technical improvements to the VitroJet and the benefits that it brings to the cryo-EM workflow. A wide variety of applications are shown: membrane proteins, nucleosomes, fatty-acid synthase, Tobacco mosaic virus, lipid nanoparticles, tick-borne encephalitis viruses and bacteriophages. These case studies illustrate the advancement of the VitroJet into an instrument that enables accurate control and reproducibility, demonstrating its suitability for time-efficient cryo-EM structure determination.

Cryo-EM structures from sub-nl volumes using pin-printing and jet vitrification.

The increasing demand for cryo-electron microscopy (cryo-EM) reveals drawbacks in current sample preparation protocols, such as sample waste and lack of reproducibility. Here, we present several technical developments that provide efficient sample preparation for cryo-EM studies. Pin printing substantially reduces sample waste by depositing only a sub-nanoliter volume of sample on the carrier surface. Sample evaporation is mitigated by dewpoint control feedback loops. The deposited sample is vitrified by jets of cryogen followed by submersion into a cryogen bath. Because the cryogen jets cool the sample from the center, premounted autogrids can be used and loaded directly into automated cryo-EMs. We integrated these steps into a single device, named VitroJet. The device's performance was validated by resolving four standard proteins (apoferritin, GroEL, worm hemoglobin, beta-galactosidase) to ~3 Å resolution using a 200-kV electron microscope. The VitroJet offers a promising solution for improved automated sample preparation in cryo-EM studies.

Development and evaluation of matrix application techniques for high throughput mass spectrometry imaging of tissues in the clinic.

Matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI) is a sensitive label-free technique that can be used to study a wide variety of clinical phenotypes. In this context, MSI offers huge diagnostic potential by supporting decision making in the determination of personalized treatment strategies. However, improvements in throughput and robustness are still needed before it finds a place in routine application. While the field has seen tremendous improvements in the throughput of data acquisition, robust and high-throughput sample preparation methods compatible with these acquisition methods need to be developed. To address this challenge, we have developed several methods to reduce the matrix application time to less than 5 min, while maintaining sensitivity and reproducibility. Workflows incorporating these methods provide a pipeline analysis time for MSI sample preparation and acquisition of less than 30 min. The reduced time for these analyses will contribute towards the integration of MSI into routine molecular pathology for clinical diagnostics.

Toward noninvasive blood pressure assessment in arteries by using ultrasound.

A new method has been developed to measure local pressure waveforms in large arteries by using ultrasound. The method is based on a simultaneous estimation of distension waveforms and velocity profiles from a single noninvasive perpendicular ultrasound B-mode measurement. Velocity vectors were measured by applying a cross-correlation based technique to ultrasound radio-frequency (RF) data. From the ratio between changes in flow and changes in cross-sectional area of the vessel, the local pulse wave velocity (PWV) was estimated. This PWV value was used to convert the distension waveforms into pressure waveforms. The method was validated in a phantom set-up. Physiologically relevant pulsating flows were considered, employing a fluid which mimics both the acoustic and rheologic properties of blood. A linear array probe attached to a commercially available ultrasound scanner was positioned parallel to the vessel wall. Since no steering was used, the beam was perpendicular to the flow. The noninvasively estimated pressure waveforms showed a good agreement with the reference pressure waveforms. Pressure values were predicted with a precision of 0.2 kPa (1.5 mm Hg). An accurate beat to beat pressure estimation could be obtained, indicating that a noninvasive pressure assessment in large arteries by means of ultrasound is feasible.