Immediately after the LM imaging, directly transfer the EM cryo-holder into a Thermo Fisher FEI Tecnai F20 scope. Load the EM grid with frozen-hydrated sample onto the Gatan EM cryo-holder, and subsequently insert the cryo-holder into the pre-cooled cryo-chamber.įor each field of view which covers the whole square of interest and its indexes, collect one fluorescent image in EGFP channel, and one bright-field image in the EGFP channel. Plunge freeze the cultures on EM grids at DIV 16 and store the samples in liquid nitrogen until use. Transfect the cultures with lentivirus encoding PSD95-EGFP constructs for 5–7 days in vitro (DIV). 2018).Ĭulture dissociated embryonic rat hippocampal neurons on poly-lysine-coated gold EM finder grids in 35-mm Petri dishes, following a previously described protocol (Tao et al. Using this correlative system, we have identified and characterized the ultrastructural features of excitatory synapses of cultured hippocampal neurons in their native states (Tao et al. The overall workflow of our cryo-CLEM protocol is summarized in Fig. Based on these carbon holes, accurate correlation between light and electron microscopy was obtained using a custom-developed program. This method uses the patterned carbon holes on Quantifoil EM grids that can be visualized by both bright-field LM and EM as fiducial markers. To overcome this problem, we have developed a new geometry-based method for refined correlation. It is also practically challenging to obtain appropriate distribution of beads on the specific grid areas where sample targets are located. 2015 Schorb and Briggs 2014), which may involve complicated procedures such as correction for chromatic aberration (Schorb and Briggs 2014). This design allows the EM cryo-holder to be shuttled between light and electron microscopes without repeated sample transfer, thus minimizing ice crystal contamination and grid damage.įor correlation between fluorescence and EM images, most existing cryo-CLEM methods rely on the use of fluorescent beads (100–200 nm) as fiducial markers (Liu et al. An EM cryo-holder can directly fit into the cryo-chamber through a side-port to position the EM grid above the objective lens of the light microscope. The hardware design of our system includes a cryo-chamber that was built to fit on an inverted light microscope and to maintain a low temperature (around −192 ☌) inside by continuous liquid nitrogen cooling. In this paper, we describe an efficient cryo-CLEM system and its use for the study of synapses with fluorescent label in cultured hippocampal neurons. Successful implementation of a cryo-CLEM method requires a series of steps, including proper subcellular labeling, frozen-hydrated sample handling, cryo-FLM/cryo-ET imaging, and image correlation. 2018), which utilizes cryo-fluorescence light microscopy (cryo-FLM) and cryo-ET to examine vitrified samples, promises better sample preservation and high resolution compared to conventional CLEM methods. With such correlative methods, specific structural features or cellular components can be localized by FLM and then examined by EM to reveal high-resolution details in the same sample.Ĭryo-correlative light and electron microscopy (cryo-CLEM) (Chang et al. Correlative light and electron microscopy (CLEM) has been developed to combine the advantages of both FLM and EM techniques (de Boer et al. Nevertheless, the lack of efficient ways for cryo-ET to specifically label cellular targets or proteins makes it challenging to identify specific types of cells and subcellular structures. In particular, the recent advent of cryo-electron tomography (cryo-ET) technique achieves nanometer resolution and is ideally suited for the study of three-dimensional (3D) details of native cellular architectures (Baumeister 2002 Frank 2005 Hoenger and McIntosh 2009 Tao et al. For studying finer structures such as vesicles inside neuronal synapses, electron microscopy (EM) has been the primary tool. However, light microscopy (LM) is generally used for studying cellular structures of hundreds nanometers or larger in size due to resolution limitations. The use of fluorescence light microscopy (FLM) in the past decades has yielded a wealth of knowledge of cellular structures and functions (Giepmans et al. Microscopic imaging techniques are essential research tools to delineate structural and molecular information in biological systems.
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