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
A new microscope merges different microscopy methods to increase resolution and contrast in thick biological samples. Developed by Hari Shroff, PhD, and his team at the National Institute of Biomedical Imaging and Bioengineering
(NIBIB), this new microscope improves on a previously developed microscope, combining two-photonlaser scanning microscopy (2PM) and instant structured illumination microscopy (ISIM) by including adaptive optics (AO) to rapidly correct distortions.
Shroff and his team tackled a major problem that researchers encounter when attempting to image thick tissue samples. It can be difficult to get a clear image within a thick sample. This is not unlike looking down into a pool and seeing a ball at the bottom. The image, seen through the water, doesn’t look very crisp. Astronomers have the same problem when attempting to look at distant objects through the earth’s atmosphere. Shroff incorporated a technique called adaptive optics to his latest super-resolution microscope to help solve this problem of distortion.
Adaptive optics uses a two-step process to create clearer images. First, since every sample is different, Shroff’s team measured how a particular sample distorts the light. This information is then used to create a clear image by adjusting a deformable mirror. A key component of the method is two-photon microscopy, used to generate a small point of light deep inside the sample. By moving this light throughout the sample and collecting information on how it is being distorted, Shroff and his team are able to adjust the shape of the mirror to cancel out the distortions, thus creating a clear image of the whole sample.
This new microscope can be added to the arsenal of tools that Shroff’s lab has developed over the years. (Read more about his 2013 advance here and his 2016 advance here.) Most of Shroff’s improvements on microscopy technology have been focused on giving scientists the ability to see biological samples ever more clearly in their native 3D environment. This is important for researchers because, as Shroff said, “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,” said Shroff, “but many times that’s not how those cells exist in nature.”
The unique aspects of this microscope continue to expand the efficacy of super-resolution microscopy. Shroff and his team are already looking at new ways to use this combination of technologies to create clearer images more quickly. “Efficiency and speed are key,” emphasized Shroff. “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.”
Read the paper here: Zheng, W., Wu, Y., Winter, P., Fischer, R., Nogare, D. D., Hong, A., . . . Shroff, H. (2017). Adaptive optics improves multiphoton super-resolution imaging. Nature Methods. doi:10.1038/nmeth.4337