Improving Sample Preparation In Electron Microscopy And Imaging Technologies

The most beautiful, complicated and mystifying events in nature exist on a scale that we cannot directly witness. This is evident in the biological sciences, where living organisms can be only microns in size, and where standard microscopes are not sufficient to observe the details present at this scale. Harmonious to the need for advanced imaging, microscope technology has progressed immensely in the past few decades and has provided fuel for curiosity by allowing scientists to observe the microscopic world in more detail.

Techniques like electron microscopy allow the magnification of samples several thousand times, providing accurate visual accounts that are typically acceptable as supporting evidence of the items/processes under observation. It would be correct to assume that the impressive equipment associated with these technologies are usually only sustainable at high-level institutions that are capable of financing the maintenance and expertise required to accommodate these technologies.


An added downside to electron microscopy and similar imaging technologies are the costs and procedures involved with preparing samples to be viewed with these microscopes. They are characteristically tedious, time-consuming, and involve reagents and secondary equipment that are expensive and often dangerous.

To reduce the complexity and expense associated with conventional biological sample preparation for electron microscopy, we developed a protocol that eliminates the fixative Osmium tetraoxide (OsO4) and critical point drying, both expensive components of conventional sample preparation procedures.

To show that omitting these aspects of sample preparation is feasible, we visualized the interactions of an E. coli strain engineered to delivery DNA to mammals, with human cancer cells. Our results showed that basic morphology and interactions of the bacteria and human cells were preserved enough to produce satisfactory images even when using attenuated sample preparation procedures (Figure 1). Utilizing this affordable approach, we captured changes to the patterns of invasive bacteria-human cell adherence induced using Lipofectamine and PULSin, commercial cationic lipids that are routinely used for DNA and protein transfection respectively.

The behavior of this E. coli vector when interacting with human cells, especially its efficacy of attachment to the cell membrane translates directly to the success of eventual gene transfer from the vector to the host. We were able to manipulate these interactions using the aforementioned cationic lipids and observe significant changes in how the vector binds to the cell membrane of the host cells (Figure 2).


Omitting sample fixation with OsO4 and critical point /freeze-drying during preparation for electron microscopy reduced the preparation time and experimental cost of the procedure several-fold, and nonetheless yielded accurate representations of the biological interactions in question, with decent quality. We found mainly that thorough sample fixation using paraformaldehyde or glutaraldehyde, and gentle but precise dehydration of samples in ethanol gradients were the critical steps to produce samples of comparable integrity to those prepared using conventional high-end reagents and equipment.

The protocol we have highlighted represents a quick and affordable way to prepare biological samples for electron microscopy of basic structures in biological specimen without sacrificing pertinent morphological details. While this procedure may not be universally applicable to all biological specimen, it may be applied to most cultured mammalian and bacterial cells and may be useful routinely where advanced microscopic analysis of specimen is otherwise abandoned due to the intricacy and expense traditional of sample preparation.

Figure 1: Electron microscope micrograph obtained using simplified sample preparation, showing an HT1080 fibroblast (brown) being invaded by an engineered E. coli vector (Blue). Credit: Kumaran Narayanan
Figure 2: SEM of HeLa with attached E. coli DH10B fixed on plastic coverslips and dehydrated using EtOH gradients without the use of osmium tetroxide or critical point/freeze drying. (abc) HeLa cells allowed to attach overnight, (def) HeLa cells treated with 1 mg PULSin Рinvasive E. coli DH10B complex at an MOI of 1000 showing diffused adherence patterns and (ghi) HeLa cells treated with 1 mg lipofectamine Рinvasive E. coli DH10B at an MOI of 1000 showing polarized adherence. Republished with permission from Wiley from:

This work was conducted by A. Osahor, K. Deekonda, and K. Narayanan from Monash University Malaysia, C. W. Lee from the University of Malaya, E. U. Sim from Universiti Malaysia Sarawak, and A. Radu from the Icahn School of Medicine at Mount Sinai-New York. This work was funded by a MOSTI eScience grant 02-02-10- SF0252 from the Ministry of Science, Technology and Innovation, Malaysia to K.N.



African Antelope Types

Antelopes are many different species of ungulate that can be found in parts of Eurasia and Africa.¬†African antelopes can be […]

Transparent Polymer Solar Cells: Harvesting Light Everywhere

In the past two decades, polymer solar cells have gradually attracted considerable attention as a promising alternative energy candidate. Polymer […]

Understanding How The Brain Controls Movement In Humans

How the human brain controls limb movements is still not fully understood. Many existing models relating brain activity to human […]

New Mineral Discovered From Earth’s Mantle – Locked Away In Rare Diamonds

Scientists have recently found evidence of a new mineral inside a diamond. This mineral had never been seen before this […]

Bonobos Are Eager To Help Strangers Without Being Asked

While altruism is often practiced between close friends, people will also sometimes help random strangers they meet. New research shows […]

A Novel Theory For Aging Including Cancer

During the last century in medicine, much progress was made in controlling infectious diseases. The associated human mortality has considerably […]

A Bacterial Cell Imaging Method Using CRISPR And Microfluidics

With the rise in bacterial strains that are resistant to available antibiotics, there is a growing need to better understand […]

Science Trends is a popular source of science news and education around the world. We cover everything from solar power cell technology to climate change to cancer research. We help hundreds of thousands of people every month learn about the world we live in and the latest scientific breakthroughs. Want to know more?