One of the limiting factors in cell biology and microbiology has been the resolution of microscopes. The simplest way of thinking about it is that every cellular interaction requires contact between two or more chemicals, but where do they meet? How do those chemicals find each other? Do the chemicals go through intermediate structures during the interaction? All these questions require imaging a living (or recently living) cell with a spatial resolution on the order of a few nanometers (10-9m). Unfortunately, optics tells us that there is a fundamental limit to the smallest object that can be resolved, and, depending on the color of the light used to make the image, this clocks in at around 300nm.
In 2006 (where did 2007 go?), we reported on a new imaging technique, called stochastic optical reconstruction microscopy (STORM), that used statistics to give the diffraction limit the finger. To recap, the chemicals of interest are stained with a pair of dyes that will glow when illuminated by a laser. However, the excitation of one dye can switch the other dye off. By carefully controlling the illuminating lasers, one can be assured that only a few dye molecules will emit light in a given time period. By analyzing the statistics of where the photons come from, one can see inside the blobs of light to resolve structures on the scale of 20-30nm, which is smaller than some proteins. Unfortunately, the resulting image is still 2D, and scanning the focal volume of the microscope gives great lateral resolution but very poor depth resolution.
Enter a new piece of research, published in ScienceExpress, where the scientists take advantage of astigmatism. Astigmatism is generated by using a lens that isn't cylindrically symmetric and focuses at a slightly shorter distance in one axis than in the other. To illustrate this, imagine a perfectly circular laser beam passing through an astigmatic lens. The circle is immediately deformed into an ellipse, with the shorter focal length axis squeezing the circle more than the longer focal length axis. At the first focal position, the squeezing along one axis reaches its maximum and begins to expand, while the other axis is still being squeezed. At the average position between the two focal lengths, the circle is recovered. After this, the ellipse flips as one axis of the ellipse expands faster than the other.
Now, astigmatism is normally the enemy of a good imaging system, because it distorts the details of the picture. However, if you already know that you are looking at a bunch of circular dots, the astigmatism can be used to calculate how far away the dot is from the focus. This is exactly the situation for STORM, where the image is constructed from a bunch of circular dots. To take advantage of this, a controlled amount of astigmatism was introduced to the optical system, and the ellipticity of each dot was used to calculate the depth from which the emitting dye was located. In doing so, the researchers obtained a depth resolution of 60-70nm, which is pretty good.
This research has come along way since we first covered it. It took six hours to create an image back in 2006, but this microscope has a 20Hz frame rate and resolution to drool over. I wouldn't be surprised to see this starting to turn up in commercial instruments in the next few years, making some happy scientist happier and rich.
ScienceExpress, 2008, DOI: 10.1126/science.1153529