Dear friend Luigi Marongiu

An excellent video to watch:

https://youtu.be/_91FqqGEi4Y?si=paxVJpBHSEqsiLgM

I wanted to reach out after hearing about your recent work with SEM on viral samples. It’s clear you’re putting in a lot of effort—SEM can be especially challenging when working with something as small and delicate as viruses. I thought I’d share a few protocol pointers and troubleshooting tips that might help refine your process.

First, regarding sample attachment: poly-L-lysine is a solid choice, but it’s worth double-checking both the concentration and the coating time to ensure a uniform layer. This can make a noticeable difference in how well the viruses adhere to your slides. For fixation, your use of glutaraldehyde is spot-on; sometimes, extending the fixation time or gently warming the sample can further improve structural preservation.

When it comes to rinsing, be cautious—vigorous washing can easily dislodge viruses. Gentle aspiration rather than dipping is often safer for these fragile samples. During dehydration, handle the slides carefully, as ethanol series steps can also lead to loss of material. Some researchers find that critical point drying, rather than hexamethyldisilazane, helps minimize damage from surface tension.

A couple of additional suggestions: applying a thin carbon or gold coating can enhance virus visibility under SEM, and a quick check under a light microscope before imaging can confirm that your viruses are adhering as expected. Don’t get discouraged—optimizing SEM prep for phages often takes a bit of trial and error.

If you have any questions or want to discuss your protocol further, feel free to reach out. Wishing you the best as you continue refining your technique!

A few articles to read/cite:

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Article Morphology of single inhalable particle inside public transi...

Article Qualitative Evaluation of Particulate Matter Inside Public T...

Article Hierarchical Ni-Mn Double Layered/Graphene Oxide with Excell...

Best,

Kaushik

"Glutaraldehyde"! So the "viruses" are in fact deactivated, with some bad luck even partially destroyed. I would expect that such an aldehyde-covered particle sticks rather well to most surfaces.

I did a lot of virus SEM (with TMV); I usually employ very low concentrations and let a droplet dry on a hydrophilic surface (e.g., oxidized Si wafer), rather than washing. Depending on what you are interested in, coating with a conductive layer might be the best option. I would call that a standard protocol. I would guess that positive staining would be another option; I have no experience with it, though.

Without coating, you have to fight very hard. Check out this paper for E-SEM in water vapour (no coating, but....):

Article The Condensation of Water on Adsorbed Viruses

Better go for AFM!

Btw, E-SEM would also work with your (completely nonconductive) glass cover slips. Standard SEM would result in extreme charging, resolution can be as poor as 0.1mm (sic!).

Without water, "normal" SEM in vacuum, I would use a conductive support, e.g. a Si wafer. Try low voltage (

You're asking an excellent and surprisingly common question for anyone doing SEM of viruses, especially phages. Let's address both your protocol and some best-practice recommendations:

1. General Principles for SEM of Viruses/Phages

• Viruses (phages) are extremely small (~50–300 nm), making them easy to lose during sample processing.
• SEM sample prep generally involves adsorbing the particles on a substrate, fixing them, dehydration, and coating.
• Poly-L-lysine coated coverslips are commonly used to promote attachment of negatively charged particles like viruses.
• Critical steps for good retention: gentle handling, avoid harsh rinsing, and minimal manipulation after attachment.

2. Assessment of Your Protocol

Potential Issues:

• Drying 48h in fridge: Prolonged drying can result in aggregation, deformation, or loss (due to incomplete binding).
• Fixation: You used a high glutaraldehyde concentration (10% of 25% = 2.5%), and fixation after drying, not before. For viruses, immediate fixation after adsorption (without drying) is usually preferred.
• Washing/Rinsing: Dipping steps can dislodge loosely bound virions.
• Adsorption: Virus adsorption may be insufficient if the suspension is not allowed proper contact time, or if the poly-L-lysine layer is not fresh/active.

3. Best-Practice SEM Protocol for Virus/Phage Imaging

Here’s a standard protocol used in many EM labs (example; also protocol):

1. Substrate Preparation

• Clean cover slips (glass or silicon chips).
• Coat with fresh filtered 0.01% poly-L-lysine for 10 min, air dry.

2. Virus Adsorption

• Drop 10–20 µL of virus suspension onto coverslip. Incubate 10–30 min at room temp in a humid chamber (prevents drying).
• (Optional) Lightly rinse ONCE in buffer (e.g., PBS or Tris) to remove unbound particles.

3. Fixation

• Add 2–2.5% glutaraldehyde in buffer (PBS or 0.1 M cacodylate), 10–20 min at room temp. Do NOT let sample dry before fixation.
• Carefully remove fixative and gently rinse once with buffer.

4. Dehydration

• Stepwise ethanol dehydration (ideally with minimal disturbance): 30%, 50%, 70%, 90%, 100% (5–10 min each).
• Remove ethanol and critical point dry (alternatively, air dry in a desiccator with HMDS as you did; air drying alone can lead to collapse).

5. Coating

• Sputter-coat with a thin layer (3–5 nm) of gold or platinum.

4. Critical Points to Maximize Virus Retention:

• Immediate fixation after adsorption. Never let your sample dry before fixation, as dehydration prior to fixation can wash away or distort virions.
• Short handling times. Prolonged processing, especially with repeated dips/rinses, increases loss.
• Minimal rinsing. Use gentle buffer overlays or very light rinsing.
• Drop-wise addition of reagents, not dipping, is often best for fragile viral preps.
• Humidity control: During adsorption, prevent evaporation so viruses settle and bind, not dry out.

5. Possible Cause of Your Issue

You let the virus drop dry for 48 hours before fixation (“in the fridge”). Unfixed viral particles often do not adhere strongly, and during drying, they may be lost, aggregated, or distorted. Subsequent dipping then likely washed away what little was left.

6. Summary Table: Comparison

StepYour ProtocolRecommendedAdhesion48h drying, poly-L-lysine10–30 min adsorption, humid, poly-L-lysineFixationAfter drying, 2.5% glutaraldehydeImmediately after adsorption, 2–2.5% glutaraldehydeWashingDipping in waterMinimal buffer rinse, no dippingDehydrationEthanol series, HMDSEthanol series, HMDS or CPDObservationNo virus visibleGood retention with above steps

7. Key References & Protocols

• “Sample preparation for electron microscopy of viruses and virus-infected cells”: doi:10.1002/jemt.10263
• “A Simple Method for High-Resolution SEM Imaging of Virions”: PMC3495024

Fix as soon as the virus is adsorbed—don't let the sample dry out before fixation. Minimize rinsing, and handle gently throughout. This should dramatically improve your virus yields on SEM. If you're having trouble visualizing the phages still, considering labeling them with gold nanoparticles or immunolabeling can help confirm their presence.