cryo-em core @ vai
optimizing cryo-preparation to improve ultrastructural imaging of C. elegans.
Abstract: Proper sample fixation is crucial to ensure that cellular structures are imaged in their most native state. Traditional chemical fixation introduces toxic chemicals that disrupt structural integrity, presenting a need for improved preservation methods. By optimizing the cryoprotectant, FS medium, and incubation time in HPF/FS, we enhanced cytoplasmic clarity, membrane integrity, and organelle visibility in TEM imaging of C. elegans oocytes.
background & rationale
previously, HPF/FS with C. elegans led to some fixation artifacts (osmium contamination, poor preservation). we wanted to first troubleshoot our protocol with c. elegans, with the intention of applying it to 2-cell embryos if successful.
method
we experimented with a variety of parameters:
cryoprotectant: including dextran into the m9 buffer helped immobilize the worms during the high pressure freezing process.
freeze substitution medium & protocol: initially, our use of aqueous OsO₄ and tannic acid with an incubation time of ~5.5 days resulted in osmium precipitates and poor imaging resolution. this was due to excessive staining; to eliminate these fixation artifacts, we just OsO₄ as the medium (switching to crystalline instead of aqueous), and decreased the incubation time to ~4 days.
we then tried our protocol on 2-cell embryos but… that didn't work out so well ☹️
results
our optimized protocols worked the best in preservation and resolution quality, compared to our previous HPFFS procedure and certainly to chemical fixation.
reflection
i'm truly grateful for the cryo-em core @ vai for this wonderful summer internship opportunity! and none of this would've been possible without my amazing mentor, Ishara, who has been so patient and supportive in his guidance.
in my hands-on experience with sample prep, TEM imaging, and a fair share of cryo-EM methods, ive learned just how enriching the structural biology field can be. i hope to further my knowledge by using cryo-em to investigate the molecular constituents of neurodegenerative diseases!
psst.. there's more!
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jamming to flipturn ⟣ reading the analog sea review ⟣ watching the apothecary diaries
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