New Microscope Combines Various Microscopy Techniques to Produce Clearer Images

Hari Shroff, PhD, and his colleagues from the National Institute of Biomedical Imaging and Bioengineering (NIBIB) have developed an innovative microscope that combines various microscopy techniques to enhance resolution and contrast in thick biological samples.

Two images of edothelial cells from a mouse aorta. The cells are stained and embedded into a collagen gel. The super-resolution images are collected with the microscope described in this paper, and are much sharper than they would be without the method, given the distortion caused by the gel. They are colorcoded according to depth. Source: Robert Fisher, National Heart, Lung, and Blood Institute.

The new microscope is an improvement over an earlier version, where two-photon laser scanning microscopy (2PM) and instant structured illumination microscopy (ISIM) were combined by incorporating adaptive optics (AO) to swiftly correct for distortions.

Shroff and his colleagues addressed a crucial challenge normally experienced by Scientists while trying to image thick tissue samples. Acquiring a clear image of a thick sample might be highly challenging. This is similar to looking through a pool and observing a ball at the bottom. When viewed through the water, the image of the ball is not so clear. A similar problem is encountered by Astronomers when they try to observe distant objects through atmosphere of earth. To overcome the problem of distortion, Shroff included a method known as adaptive optics in his latest super-resolution microscope.

Adaptive optics involves a two-step procedure to produce undistorted images. As each sample is different, initially, Shroff and his colleagues recorded the way in which a specific sample distorts light. Then, they applied this information to produce an undistorted image by calibrating a deformable mirror. A main feature in the technique is two-photon microscopy, which is employed to produce a small light spot deep inside the specimen. The Researchers moved this light through the entire sample and gathered information on the way in which it is was distorted; thus, they could calibrate the shape of the mirror to nullify the distortions and were able to produce an undistorted image of the entire sample.

It is possible to add this new microscope to the arsenal of tools developed in Shroff’s lab over the past years. Much of Shroff’s enhancements to microscopy technology have centered on enabling Researchers to more clearly observe biological specimen in their native 3D environment. According to Shroff, this is highly significant for Scientists.

Life did not evolve on a coverslip. We have been able to view cell biology at high resolution on a microscope slide for a long time, but many times that’s not how those cells exist in nature.

Hari Shroff, PhD, NIBIB

The distinctive features of this microscope persistently enhance the efficiency of super-resolution microscopy. Shroff and his colleagues are already exploring innovative techniques to apply this combination of technologies in order to produce images of high clarity in lesser time.

Efficiency and speed are key. The faster we can image live samples and the less we can interfere with their environment, the better we can understand how biology truly works.

Hari Shroff, PhD, NIBIB

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