Total Sample Conditioning and Preparation of Nanoliter Volumes for Electron Microscopy

Abstract

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. A microcapillary is used to aspirate 3-5 nL of sample. The microcapillary tip is immersed into a reservoir of negative stain or trehalose, where the sample becomes conditioned by diffusive exchange of salt and heavy metal ions or sugar molecules, respectively, before it is deposited as a small spot onto an EM grid. We demonstrate the use of the method to prepare protein particles for imaging by transmission EM and nanocrystals for analysis by electron diffraction. Furthermore, the minute sample volume required for this method enables alternative strategies for biological experiments, such as the analysis of the content of a single cell by visual proteomics, fully exploiting the single molecule detection limit of EM.

Miniaturizing EM Sample Preparation: Opportunities, Challenges, and “Visual Proteomics”

“…This problem was recently diminished by using an even smaller stationary sample plug (Figure B). The sample plug has a volume of 5 nL and is loaded in the very tip of a microcapillary . The microcapillary is then immersed in a tube containing the conditioning solution, such as a replacement buffer or negative stain.…”

“…The microcapillary is then immersed in a tube containing the conditioning solution, such as a replacement buffer or negative stain. The diffusion coefficients of buffer components and large proteins, allows the tiny sample plug to be conditioned within a few minutes by diffusive‐exchange without significant sample loss . The conditioned sample is then applied to an EM grid, omitting any further washing or blotting steps.…”

“…The basic procedure for quantitative EM would be as follows: A defined nanoliter volume of sample is aspirated into a microcapillary using a high‐precision pump and conditioned with heavy metal salts inside the capillary tip via diffusive exchange (Figure B). Afterward (Figure A), the sample is deposited on an EM grid as a small spot with an area of roughly 0.5 mm 2 . If the sample is deposited on a substrate without grid bars, e.g., a slot grid or a large silicon nitride window, the complete negatively stained sample will be available for imaging by automated data collection software .…”

“…Since ET is limited to very thin (1 μm or below) cells or sections of cells obtained, e.g., by lamella milling, and the process of data acquisition and analysis is very time consuming and cumbersome, a complementary lyse‐and‐spread visual proteomics approach was developed . This method uses a single‐cell lysis device combined with microcapillary sample deposition to collect the lysate of a single cell, condition it, and spread it over a grid for imaging by negative stain EM . Alternatively, cryo‐EM grids can be prepared from the cell lysate of individual cells .…”

Shake-it-off: a simple ultrasonic cryo-EM specimen-preparation device

“…Time-resolved EM experiments have been performed with pressure-driven microfluidic (Feng et al, 2017;Kaledhonkar et al, 2018) and voltage-assisted (Kontziampasis et al, 2019) spraying devices. Sample loss has been addressed by using microcapillaries to apply small volumes of specimen solutions, with the cryoWriter device capable of applying less than 5 nl of sample to a conventional EM grid (Kemmerling et al, 2013;Arnold et al, 2016Arnold et al, , 2017Schmidli et al, 2019). This method has allowed the impressive determination of a structure of the human 20S proteasome to $3.5 Å resolution after isolation from 1 ml of cultured human cells (Schmidli et al, 2019).…”