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Abstract
Here, we describe a cost-effective setup for targeted photoconversion of fluorescent signals into electron dense ones. This approach has offered invaluable insights in the morphology and function of fine neuronal structures. The technique relies on the localized oxidation of diaminobenzidine (DAB) mediated by excited fluorophores. This paper includes a detailed description of how to build a simple photoconversion setup that can increase reliability and throughput of this well-established technique. The system described here, is particularly well-suited for thick neuronal tissue, where light penetration and oxygen diffusion may be limiting DAB oxidation. To demonstrate the system, we use Correlative Light and Electron Microscopy (CLEM) to visualize functionally-labeled individual synaptic vesicles released onto an identified layer 5 neuron in an acute cortical slice. The setup significantly simplifies the photoconversion workflow, increasing the depth of photoillumination, improving the targeting of the region of interest and reducing the time required to process each individual sample. We have tested this setup extensively for the photoconversion of FM 1-43FX and Lucifer Yellow both excited at 473 nm. In principle, the system can be adapted to any dye or nanoparticle able to oxidize DAB when excited by a specific wavelength of light.
Highlights
The study of presynaptic organization requires the analysis of both functional and structural aspects of axons and their synaptic compartments
The functional study of axonal fine structures often requires a combination of fluorescence and electron microscopy
These measurements can be performed in parallel (Clayton et al, 2008; Cheung et al, 2010; Chung et al, 2010; Ratnayaka et al, 2012) or in a correlative way following the same axons and synapses from light to electron microscopy
Summary
The study of presynaptic organization requires the analysis of both functional and structural aspects of axons and their synaptic compartments (for a review see Debanne et al, 2011). Classic EM cannot provide any functional information and, to overcome this limitation, a number of Correlative Light and Electron Microscopy (CLEM) techniques have been developed over the years to study live tissue (Maranto, 1982; Harata et al, 2001; Darcy et al, 2006; Ratnayaka et al, 2011; Shu et al, 2011; Peddie et al, 2017; de Beer et al, 2018). For over 30 years, photoconversion of diaminobenzidine (DAB) has been used to observe, at the ultrastructural level, functionally identified neurons (Maranto, 1982; Smith and Bolam, 1990; Viney et al, 2013). A number of different protocols have been published to describe the technique in a range of different preparations and using different modes of light delivery to drive the photoconversion (Opazo and Rizzoli, 2010; Marra et al, 2014; Sabeva and Bykhovskaia, 2017)
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