Correlative Cryo-Fluorescence Light Microscopy and Cryo-Electron Tomography of Streptomyces

Abstract

Light microscopy and electron microscopy are complementary techniques that in a correlative approach enable identification and targeting of fluorescently labeled structures in situ for three-dimensional imaging at nanometer resolution. Correlative imaging allows electron microscopic images to be positioned in a broader temporal and spatial context. We employed cryo-correlative light and electron microscopy (cryo-CLEM), combining cryo-fluorescence light microscopy and cryo-electron tomography, on vitrified Streptomyces bacteria to study cell division. Streptomycetes are mycelial bacteria that grow as long hyphae and reproduce via sporulation. On solid media, Streptomyces subsequently form distinct aerial mycelia where cell division leads to the formation of unigenomic spores which separate and disperse to form new colonies. In liquid media, only vegetative hyphae are present divided by noncell separating crosswalls. Their multicellular life style makes them exciting model systems for the study of bacterial development and cell division. Complex intracellular structures have been visualized with transmission electron microscopy. Here, we describe the methods for cryo-CLEM that we applied for studying Streptomyces. These methods include cell growth, fluorescent labeling, cryo-fixation by vitrification, cryo-light microscopy using a Linkam cryo-stage, image overlay and relocation, cryo-electron tomography using a Titan Krios, and tomographic reconstruction. Additionally, methods for segmentation, volume rendering, and visualization of the correlative data are described.

Correlated fluorescence microscopy and cryo-electron tomography of virus-infected or transfected mammalian cells

“…It should be noted that within the field of cryo-EM the procedures and instrumentation used for CLEM and cryo-CLEM may vary and are dependent on the biological questions to be addressed 22,23 . In this protocol, we describe the CLEM and cryo-CLEM approaches that we have adapted for use on specific biological targets of interest to us, and with the hardware and software available to us 14,15 .…”

“…In addition, (cryo-)CLEM procedures were used to determine the arrangement of HIV-1 particles anchored to cell plasma membranes via tetherin, a host cellular restriction factor that inhibits enveloped virus release 14 . Cryo-CLEM of prokaryotes has also been described 22,27–29 and sections of this protocol that describe imaging and analysis parameters are also relevant to studies of these systems. With further development of cryo-CLEM technologies 13,24,26 and macromolecular-labeling strategies 30–32 , we anticipate that CLEM and cryo-CLEM methods will be routinely used for investigations of virus replication and broader topics in structural cell biology.…”

CorRelator: An interactive and flexible toolkit for high-precision cryo-correlative light and electron microscopy

“…Cryo-correlative light and electron microscopy (cryo-CLEM) is the coupling of (cryo-) fluorescence light microscopy (FLM) and cryo-electron microscopy (cryo-EM). Cryo-CLEM has proven to be a powerful method for in situ structural studies, including time-dependent events, especially those associated with viral entry, replication, and egress (Briegel et al, 2010;Fu et al, 2019;Hampton et al, 2016;Jun et al, 2019;Koning et al, 2014;Koster & Grünewald, 2014;Schorb et al, 2017;Wolff et al, 2016;Zhang, 2013). Fluorescent labeling and light-level imaging of viral and cellular factors enables one to capture dynamic events during virus-infection of cultured cells for real time rapid identification of targets, and for orienting on their spatial positions.…”

The application of CorrSight™ in correlative light and electron microscopy of vitrified biological specimens

“…Light microscopy (LM), more specifically, fluorescence microscopy (FM) is well developed to label specific molecules and localize them in a large viewing area with a resolution of several hundreds of nanometers. The advantages of specific labeling and localization in LM and high spatial resolution in EM can be combined, leading to the development of a powerful technique as the correlative light and electron microscopy (CLEM) (Anderson et al 2017;Compera et al 2015;Faas et al 2013;Koning et al 2014;Sjollema et al 2012).…”