Demonstration of correlative atomic force and transmission electron microscopy using actin cytoskeleton

Abstract

In this study, we present a new technique called correlative atomic force and transmission electron microscopy (correlative AFM/TEM) in which a targeted region of a sample can be observed under AFM and TEM. The ultimate goal of developing this new technique is to provide a technical platform to expand the fields of AFM application to complex biological systems such as cell extracts. Recent advances in the time resolution of AFM have enabled detailed observation of the dynamic nature of biomolecules. However, specifying molecular species, by AFM alone, remains a challenge. Here, we demonstrate correlative AFM/TEM, using actin filaments as a test sample, and further show that immuno-electron microscopy (immuno-EM), to specify molecules, can be integrated into this technique. Therefore, it is now possible to specify molecules, captured under AFM, by subsequent observation using immuno-EM. In conclusion, correlative AFM/TEM can be a versatile method to investigate complex biological systems at the molecular level.

Atomic force microscopy of food assembly: Structural and mechanical insights at the nanoscale and potential opportunities from other fields

“…7) or to correlate oil-gelatin emulsion network structure with nanomechanical AFM indentation experiments (Filip et al, 2006). Protein fibers or fibrillation have been observed, e.g., in cytoskeleton or other structures using coupled AFM and super-resolution optical microscopies (Chacko, Zanacchi, & Diaspro, 2013;Cosentino, Canale, Bianchini, & Diaspro, 2019;Janel, Werkmeister, Bongiovanni, Lafont, & Barois, 2017), coupled AFM and total internal reflection fluorescence microscopy (TIRFM -Fukuda et al, 2013) or using coupled AFM and transmission electron microscopy (TEM - Yamada, Konno, & Shimabukuro, 2017). Protein identification or structures were investigated using coupled AFM and infrared spectroscopy (Ji et al, 2019;Paluszkiewicz et al, 2017) or using coupled AFM and mass spectroscopy using heated cantilevers (Andrade, Silva, Azevedo, Cunha, & Sousa, 2006;de Vries, 2015;Somnath, Jesse, Van Berkel, Kalinin, & Ovchinnikova, 2016).…”

AFM‐TEM correlation microscopy and its application to lipid nanoparticles

“…Atomic‐force microscopy (AFM) and TEM are among the most popular nanoscale imaging methods. However, to our knowledge, only five works describe correlation microscopy using these two methods (Hermelink et al, 2017; Lin & Goh, 2002a; Lin & Goh, 2002b; Mulvaney & Giersig, 1996; Yamada et al, 2017). TEM implies application of highly specialized substrates (grids and windows), which must be electron‐transparent.…”

“…TEM implies application of highly specialized substrates (grids and windows), which must be electron‐transparent. Thus, the most authors use AFM to image the objects deposited onto them, and only one article describes AFM imaging of a sample deposited onto pre‐treated glass and the subsequent preparation of a replica suitable for TEM (Yamada et al, 2017). With this approach, AFM can be operated in liquid, and the sample will still fit the TEM requirements.…”

Extreme stiffness of neuronal synapses and implications for synaptic adhesion and plasticity

“…We combined this AFM method with fluorescence microscopy to identify mature synapses and monitor their activity, and with TEM to reliably identity synapses by observing the synaptic cleft, presynaptic vesicles, and postsynaptic density, and also to correlate AFM stiffness images to synaptic ultrastructure. Although AFM is commonly used in combination with fluorescence microscopy to study biological cells, combined use of AFM and TEM has been limited to imaging of filamentous biomolecules deposited on substrates 11,12 . To achieve combined AFM and TEM imaging on the same individual synapse, AFM stiffness mapping of the delicate synapses has to be performed without compromising their structural integrity prior to TEM analysis.…”