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Water is weird. Compared to other common liquids of similar molecular weight, water has high surface tension, high density, unusually high boiling and melting points, and significant heat capacity — all thanks to hydrogen bonding.

Hydrogen bonding is the force of attraction between the partially positive hydrogen atom of one water molecule and the partially negative oxygen atom of another. This force may be weak compared to covalent bonds, but it wields powerful effects in biological systems and can make the nanoscale investigation of such systems quite challenging.

/Wikimedia Commons

The blue dotted lines represent the hydrogen bonds in liquid water.

Challenges of imaging in water

My personal encounter with the peculiarities of hydrogen bonding occurred during graduate research 15 years ago where I focused on studying collagen — a key structural protein — using atomic force microscopy or AFM. AFM is an advanced imaging technique that uses a sharp, nanometer-scale tip attached to a flexible cantilever with a laser beam pointed on top. Much like an old-fashioned gramophone needle traces the grooves of a record to produce sound, the tip scans the surface and the degree of deflection of the tip is translated into detailed topographical images of the sample.

In biological studies, AFM imaging is often performed in water to preserve the sample’s native, hydrated state. I had used AFM for imaging dry samples, but I found the transition to imaging in fluids imaging to be quite challenging. After all, you can’t expect to be able to swim in water just because you practiced swimming on land.

During my very first practice session, I pipetted a small droplet of water — about 50 microliters — onto a plastic dish, carefully positioning the AFM cantilever above it and setting it to automatically approach the droplet. To my bewilderment, as soon as the cantilever touched the water surface, I heard a squeal from the machine, a sign that the laser signal was no longer pointing on the cantilever. I looked through the optical microscope and found the delicate cantilever had snapped. I chalked it up to a fluke occurrence and started over. After several additional attempts sent more cantilevers to the graveyard, I sought assistance from a senior researcher, who helped me figure it out.

If you’ve ever tried to pull apart two wet glass slides, you will be familiar with the resistance offered by the water layer and the force required to break them apart. This is due to the cohesive hydrogen bonding between water molecules and the adhesive hydrogen bonding between the glass slides. The effect is especially pronounced in humid environments or when using small volumes of water.

In my case of the curiously cleaved cantilevers, the culprit was also hydrogen bonding. When the AFM tip approaches water, hydrogen bonds between water molecules form a meniscus around the tip. The water meniscus pulls the tip downward. This capillary force acts much like a small suction cup. While the force is minuscule in everyday terms (of the order of nanonewtons), it was enough to break the soft AFM cantilever was using. I used a little bit more water and switched to a stiffer cantilever. These changes minimized the destructive capillary forces, allowing me to finally capture images without breakage.

Hydrogen bonding in AFM imaging

/Wikimedia Commons

Indu Sridharan used an atomic force microscope like the one on the left for the experiments described in this essay.

Hydrogen bonding between water and the sample can also impair the resolution and quality of the AFM images. On one of my first attempts at imaging collagen fibers in water, I had gone through the usual process of setting up the cantilevers and careful approach to the sample. I began imaging and waited patiently for the topographical images of collagen fibers to show up on my screen in real time as the tip scanned the collagen surface.

Instead of the trademark elongated, banded cylindrical structures of collagen fibers I had expected, the image was a mess of blobs and amorphous streaks; it looked like bits of scrambled noodles on a plate. I watched through the optical microscope and saw that the tip was getting stuck every so often to the surface like a pen hitting a sticky spot on paper.

This was due to the thin layer of water molecules adhering to both the AFM tip and the collagen surface. These molecules, held together by hydrogen bonds, created an adhesive layer that caused the AFM tip to drag across the sample. In addition, the hydrated collagen fibers had swollen slightly, making them easier for the tip to penetrate.

The hydrogen bonding network in water, although essential for maintaining the physiological state of the sample, made it tough to obtain accurate topographical images.

A few adjustments reduced the impact of hydrogen bonding and improved image resolution. These included using a buffer solution such as phosphate buffered saline, instead of water, and imaging with a specially designed AFM with built-in fluid cells for controlling microfluidic behavior.

Hydrogen bonding in biological processes

The very hydrogen bonds that complicated my imaging experiments are indispensable for life.

Within cells, water’s dynamic hydrogen-bonding network facilitates critical biochemical reactions. For instance, in cellular respiration and adenosine triphosphate production, protons hop from one water molecule to the next, driven by hydrogen bonds. This rapid proton transport is essential for enzymatic reactions and helps maintain pH balance within cells. Without hydrogen bonding, many of the chemical processes necessary for life would be too slow to sustain cellular function.

Hydrogen bonding is also key to enzyme function. During enzyme catalysis, water molecules often stabilize the enzyme substrate complex by forming hydrogen bonds, lowering the energy required for the reaction to proceed. In some reactions, water molecules directly participate by donating or accepting protons, facilitating the conversion of substrates into products.

A double-edged sword

As a budding scientist, the challenges I faced with fluid imaging showed me that imaging biological samples is more of an art than science, requiring a blend of technical skill and creative problem solving. My challenges with AFM imaging also provided an up-close and personal experience with the wonderful weirdness of water.

The unique properties of water, driven by hydrogen bonding, are both essential for sustaining life and a source of complexity in the study of biological samples. However, understanding and embracing these properties can lead to technological innovations that overcome the challenges and reveal deeper insights into the nanoscale world of biology.