Dedicated Setup for the Photoconversion of Fluorescent Dyes for Functional Electron Microscopy

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.

Isolation of Human Small Extracellular Vesicles and Tracking of Their Uptake by Retinal Pigment Epithelial Cells In Vitro

“…Most photoconversion studies describe the importance of the temperature during the cooling stage and the additional oxidation of the DAB solution by bubbling oxygen into the solution [35,36] as key factors for successful photoconversion. Herein, we conclude that, at least for the PKH fluorophore, a strong light source (such as the X-City XYLIS illumination system), at best in combination with the right objectives and adequate cooling of the sample (as a function of the exposure time), are the key elements to obtain a positive DAB precipitate.…”

Nanoscale Remodeling of Functional Synaptic Vesicle Pools in Hebbian Plasticity

“…Samples were then transferred to 100 mM glycine (1 h), then rinsed in 100 mM ammonium chloride (1 min) and washed in PBS. For photoconversion, slices were placed on a dedicated photoconversion setup (Dobson et al, 2019) in an oxygen-bubbled diaminobenzidine solution (DAB, 1 mg/ml) and viewed with a 40x 0.8 N.A. water immersion objective.…”

Correlating cell function and morphology by performing fluorescent immunocytochemical staining on the light-microscope stage

“…In a second variant, non-fluorescent gold nanoparticles are visualized by local surface plasmon resonance of light microscopy and also by electron microscopy (Haruta et al, 2019). In a third variant, an electron-dense product is generated from fluorophores through a process termed photooxidation or photoconversion (Bishop et al, 2011;Darcy et al, 2006a;Dobson et al, 2019;Hoopmann et al, 2012;Opazo and Rizzoli, 2010;Schikorski, 2010Schikorski, , 2014.…”