BIOPHYSICS:

Since visible light gets as small as approximately 400 nanometers, resolution in conventional optical microscopy is limited to this diameter. In other words, you can't visualize fine details any smaller than this.

This is a problem if you want to investigate intracellular processes on the single molecule level (a typical protein molecule is roughly 5 nanometers in diameter). Furthermore, you can't necessarily extrapolate single-molecule behavior from that of a large population of molecules.

A recent example is the discovery that protein molecules slide down individual DNA strands to find their genetic target. This may have medical consequences; by altering the sliding rate of viral proteins (e.g. by affixing large drug molecules to them), scientists might find a new way to combat viral infections.

The average behavior of many molecules may mask the physiologically relevant behavior of individual molecules. This underlies the need for single-molecule resolution in biological imaging.

A number of research groups have improved the imaging resolution of optical microscopy. Although these developments have been very valuable, they are either slow or cannot image thick samples (e.g. tissue slices).

Addressing this unmet need, Ulrich Kubitscheck (Rheinische Friedrich-Wilhelms Universität, Germany) and coworkers have expanded the utility of light sheet fluorescence microscopy. They have used this optical mocroscopy technique to probe the mobility of individual RNA molecules up to 200 micrometers within fly salivary glands.

What is light sheet fluorescence microscopy?

Fluorescence imaging is common in biological laboratories, because it enables one to highlight specific features of a cell or some other structure of interest. What's special about light sheet fluorescence spectroscopy?

A unique feature of light sheet fluorescence microscopy is that, instead of shining a laser above or below the sample to be imaged, a laser beam is instead shined from the side. This greatly reduces interfering background fluorescence from the rest of the sample, and also enables three-dimensional imaging and long imaging times (the rest of the sample is not damaged by the laser).

This microscopy technique also enabled the scientists to switch back and forth between other useful imaging modes (e.g. differential interference contrast) that conventionally do not operate from the side. Differential interference contrast is a common imaging technique that amplifies differences in refractive index by artificially displaying them as differences in height, thereby highlighting fine intracellular detail.

The scientists found that the illumination diameter of their light sheet was between 2.9 and 3.0 (± 0.1) micrometers when using either a 488 (blue), 532 (green), or 638 (orange) nanometer laser. For reference, conventional illumination diameters in optical microscopy may be around a few centimeters in the x-y plane, illuminating much more than a cell-sized field of view (a typical human cell may be several tens of micrometers in diameter), hindering resolution.

The optimum field of view in light sheet fluorescence microscopy is 84x20 micrometers. As mentioned, this is a useful field of view for imaging a typical human cell.

Single-molecule tracking in solution.

The scientists started out with something easy: tracking single fluorescent dextran molecules (a polymer of glucose) in solution. There should be no background fluorescence in these experiments, enabling fast single-molecule tracking.

They found that the signal-to-noise ratio was improved by a factor of 4, relative to conventional imaging, enabling more precise localization of individual molecules. Furthermore, their imaging rate of 100 frames per second is much faster than the seconds to hours per frame required by some other specialized single-molecule imaging techniques.

The scientists furthermore tracked several molecules in solution: a large dextran molecule, a protein molecule, and a DNA molecule (their theoretical diffusion coefficients span a factor of 100). Each of these molecules was easily tracked at a frame rate of 483 frames per second.

Single-molecule tracking deep within living cells.

After their successful early-stage tests, the scientists proceeded to something more challenging and physiologically-useful: tracking individual mRNA molecules (messenger RNA, important in protein synthesis) within intact fly salivary glands. Why is this so difficult?

mRNA molecules can be up to around two hundred micrometers deep within the cells under study. Therefore, fluorescence from these molecules would be overwhelmed with fluorescence from everything else in the cell using conventional optical imaging.

At an imaging rate of 50 frames per second, the scientists tracked individual mRNA molecules within their cells. Their certainty in localization was 40 ± 12 nanometers, much smaller than the diameter of the cells under study.

They determined three different states of the mRNA molecules. Forty-five percent of the time, its diffusion coefficient was fast at 2.0 ± 0.2 square micrometers per second.

Forty-five percent of the time, its diffusion coefficient was four times slower. Ten percent of the time, it was 25 times slower.

Based on similar experiments with dextran polymers, the scientists concluded that the slowest speed was due to interactions with intracellular structures within the cell nucleus, and the other two speeds were a consequence of nonspecific interactions with everything else in the cell. Why is any of this interesting?

Such results are of interest for understanding the possible physiological consequences of nonspecific crowding within cells. Intracellular communication, enzymatic reaction cascades, and spontaneous intracellular organization may all be regulated (at least in part) by crowding; single-molecule microscopy can easily have a role to play in investigating these questions.

Implications.

This research focused on demonstrating the power of light sheet fluorescence microscopy for tracking individual molecules deep within living tissue. It could easily be extended to studying intricate physiological processes (e.g. enzyme kinetics) on a single-molecule level, without damaging the cells or their delicate multicellular spatial arrangements that may be critical for proper function.

for more information:
Ritter, J. G., Veith, R., Veenendaal, A., Siebrasse, J., & Kubitscheck, U. P. (2010). Light Sheet Microscopy for Single Molecule Tracking in Living Tissue PLoS ONE, 5 (7) DOI: 10.1371/journal.pone.0011639