A modern microscope is a joy to behold. Resolutions between 0.5-1 micrometer are common, but that sort of focus comes at a cost. When creating images at high resolution, it becomes difficult to track what is going on just a micron away. This means that a biologist must really know where the action is going to occur before focusing in and taking a look.

This problem is particularly severe for certain forms of microscopy where the depth of field (the portion of the sample that's in focus) is very small. However, the development of a new type of lens has now been shown to improve the situation substantially.

A current-generation microscope obtains its pictures by raster scanning a laser rapidly over a sample, while simultaneously collecting light emitted by the sample. In the case of two-photon microscopy, a single dye molecule must simultaneously absorb two photons in order to emit a single, higher-energy photon. This requires quite high intensity, so light is only emitted from the tiny region where the laser is focused. As a result, the images are both high resolution and high contrast.

Unfortunately, the laser is only focused to a single plane, meaning that a single raster scan only produces an image of a slice of the sample. Often, it is useful to have an extended depth of focus—the tradeoff is that the resolution of the image is much poorer—in order to ensure that you really are looking where you want to look.

In the case of two-photon microscopy, an extended depth of field requires changing optics in the microscope, which is not really ideal. To overcome this problem, researchers have developed an acoustic lens. This lens consists of a piezoelectric ring, the center of which is filled with an oil. When a voltage is applied to the piezoelectric ring, it expands or contracts, depending on the voltage's polarity.

Like dropping a stone in a pond, this sends a ripple through the oil. If the voltage varies with a high enough frequency, the outward and inward moving ripples interfere with each other, creating a set of permanent ripples in the oil. These ripples refract the light, creating what is called a Bessel beam.

Bessel beams are very special in that, ideally, they never expand. When this beam is sent through the microscope objective, it remains focused over a much greater distance. Used in a two-photon microscope, it ensures that light is emitted from a relatively thick slice from a sample. Furthermore, changing from an extended depth of field to a high resolution image is as simple as switching the sound wave on and off.

The researchers showed that they can do this by scanning every line twice, once with the lens switched on and once with the lens switched off. You only get half the absolute frame rate, but two different sequences of images, one narrowly focused, the other with an extended depth.

The very cool thing is that this technology can be retrofitted to every two-photon microscope in the world with relatively little trouble. Expect to see this as an add-on in the next couple of years.

Optics Letters, 2009, DOI: 10.1364/OL.34.001684