Hazards of fumes associated with sample preparation for scanning electronic microscopes (SEM)

Electron microscope images of insects.Thanks to SEMs, we’ve seen wonders. From water bears to bed bugs, our eyes have been opened – and we’re fascinated!

Those compelling views do not happen nonchalantly.

Appropriate sample preparation for SEM determines how much we see.

And because sample prep involves chemicals, our interests lie in understanding any respiratory hazards that may be involved.

Chemicals, more chemicals, then air in, air out

There is no one method of preparation that guarantees successful imaging. Users often must experiment with different substances and techniques to get the results they desire.

Sentry Air graphic listing SME specimen prep respiratory hazards.Many documents stress the development of patience in order to obtain desired results. To really see the details of an insect close up, for example, that insect has to be prepared to suit the properties of the insect AND operational peculiarities of scanning electron microscopes.

In general:

_Insect specimen must be ‘fixed’
_Buffer fixing chemical
_Dry the specimen
_Mount the specimen
_Coat the specimen

Sentry Air's Model 200 Floor Sentry air purifier.

A formaldehyde discussion at leicabiosystems.com includes:
“The OSHA permissible exposure limit (PEL) is 0.75 ppm (8 hour TWA) and a short-term exposure limit (STEL) of 2 ppm (15 min exposure) and these recommendations are supported by a regular monitoring program.”
“In a well-equipped laboratory with modern fume-extraction equipment these target levels should not be exceeded.”
The Model 200 Floor Sentry – AKA modern fume-extraction and local exhaust ventilation (LEV) equipment — is used in many labs because it can be placed where specific specimen preparation tasks are being performed.
Unlike a hood, it goes where the work is, not where hood location dictates.

Fixing

This process dispatches live insect specimens without deforming them or otherwise distorting their inherent characteristics.

Preservation of cellular structure is typically accomplished with chemicals, including solutions containing glutaraldehyde and formaldehyde. Aldehydes interact with proteins in the specimen to stabilize cell substances and structures.

Stabilization of lipids may be accomplished with osmium tetroxide (OsO4). OSHA’s permissible exposure limits (PEL) for this chemical are a time-weighted average (TWA) of 0.002 mg/m3 over an 8-hour day.

The CDC’s NIOSH has a lower recommended exposure limit (REL) of 0.0002 ppm TWA and a 15-minute short-term exposure limit (STEL) of 0.0006 ppm.

Descriptions of formaldehyde as a fixative include cautions on its hazardous and potentially carcinogenic nature.

While OSHA has not issued a PEL for glutaraldehyde, it has published a best practices guide for safe use of glutaraldehyde:

“Local exhaust ventilation located at the level of vapor discharge is the preferred method of reducing glutaraldehyde vapor concentrations because it captures and removes vapor at the source before it can escape into the general work environment.” (Page 11)

 

Buffer the fixing chemical

Fixative chemicals may need to be buffered to prevent the specimen from becoming too acidic.

Buffering chemicals, such as phosphate salts and sodium cacodylate, can be included with commercially prepared fixatives.

In some labs, the addition of buffering agents is part of sample preparation.

Use PPE and caution

Make sure you read the Safety Data Sheet (SDS) for the chemicals you handle.

Sodium cacodylate, for example, is a suspected human carcinogen.

The term phosphate salts refers to different combinations of the chemical phosphate with salts and minerals.

The many online SDSs of phosphate salts say inhalation can irritate mucus membranes and may cause chest pains and difficulty breathing. Some recommend local exhaust ventilation (LEV).

If you are using a phosphate salt to prepare SEM samples, be sure you follow any cautions in the SDS.

Dry the specimen

google search of dyers used in SEM sample preparation.Complete drying of the specimen eliminates any off-gassing of the chemicals used to fix the specimen.

Skilled drying also eliminates deformations caused by surface tension on the specimen.

While some specimens may be air dried, many SEM specimen prep processes use specialized dryers whose manufacturers often recommend dehydration start with acetone or ethanol.

In the dryer, specimens may be exposed to liquid carbon dioxide CO2 plus optimal temperature and air pressure to minimize shrinkage associated with water and chemicals.

When dried in this manner, often referred to as critical point drying, specimens are so dry they could begin accumulating moisture from the air. Quickly mounting and coating them is recommended.

Ambient fumes from acetone or ethanol can be collected by ambient air cleaners as well as a properly positioned Model 200 Floor Sentry.

Mount, then coat the specimen

Double-sided adhesive tab on aluminum SEM stub (A) and removal of protective layer (B).

Double-sided adhesive tab on aluminum SEM stub (A) and removal of protective layer (B).
Image source: Scanning Electron Microscopy. Authors: Elizabeth R. Fischer, Bryan T. Hansen, Vinod Nair, Forrest H. Hoyt, David W. Dorward.

Enhancing the conductivity of a specimen is a technique used to overcome charging, a negative build-up at the point where the SEM’s beam hits the sample that can cause unexpected artifacts in the image.

Secure mounting on sample stub may involve conductive cement on tape to provide some electrical grounding.

Researchers also sputter-coat a conductive layer of metal onto the sample to inhibit charging.

Finally, the SEM

A potential respiratory hazard awaits.

Turns out, a SEM’s beam can be affected by dirty air in the specimen chamber. Therefore, after the specimen in the chamber, air in the chamber is evacuated, i.e., pumped out to leave a vacuum.

The Australian Microscopy & Microanalysis Research Facility has a neat simulation that shows venting and evacuating the chamber.

And that’s why a researcher called us

He was concerned about nanoscale material becoming airborne in the evacuation process.

Image of a SEM online simulation.

From the sim site:
Principles of SEM operation
A scanning electron microscope is a machine comprised of an electron generating component called the gun, a column through which the electron beam travels, a series of lenses to shape the electron beam, the sample chamber at the base, and a series of pumps to keep the system under vacuum.

Nano concerns

Concern about exposure to nanoparticles is reasonable.

SEM users do not limit their investigations to insects. They may examine specimens of powder, bone, minerals and modern manufactured materials, to name a few.

A study published in 2004 discussed the movement of inhaled ultrafine particles (UFP) to the brain.

Articles published in the online journal Particle and Fibre Toxicology have listed studies that investigate the relationships between various types of nano-scale particles and human health conditions such as diabetes, vasodilation, and glucose metabolism.

Seeking control of exposure in your lab?

If nano-scale substances are becoming an issue in your workplace, please give us a call at 800.799.4609, email sales@sentryair.com, or use the comment form below.