Researchers at NIH have been tackling problems in biological microscopy with the development of two new microscopes. The new technologies will allow researchers to see fast moving organisms at higher resolutions and to capture three-dimensional images with minimal light damage to the cells.
The first microscope addresses an issue that is familiar to even the casual photographer: how to take photos of moving objects that do not turn out blurry. Biologists study structures such as proteins and viruses that move very quickly within and between cells. Photographing them in a live setting is extremely difficult, but it is crucial to understanding how the different parts of cells function in real time.
Issues arise because there is always a give-and-take between how fast your camera is — how sensitive it is to motion - and how clear the picture is going to come out. With higher resolutions, the motion blur becomes more noticeable.
“Time resolution, spatial resolution and axial resolution have been parameters that have imposed constraints on light microscopy for decades,” J. Michael McCaffery, director of the Integrated Imaging Center at Hopkins, said.
“Different tools which have been developed to overcome these constraints have pertained to improving camera technology or detector technology, making cameras that are faster and more sensitive,” McCaffery said.
The team from NIH’s National Institute of Biomedical Imaging and Bioengineering lab, headed by Hari Shroff and Andrew York, developed instant linear structured illumination microscopy (iSIM) to tackle this conundrum of detector sensitivity. The iSIM technology takes real-time images of small, rapidly moving structures at very high resolutions, allowing them to record, for example, individual blood cells flowing in the vessels of a live zebrafish embryo.
“Size is one of the main things limiting microscopy,” Robert Horner, a biology professor here at Homewood campus, said. You can’t see objects that are smaller in size than the wavelength of light used to observe them, so there will always be some threshold point where two structures are indistinguishable — either because they are too small in comparison to the light wavelength, or the camera’s resolution is insufficient.
Shroff and his lab were able to produce such high resolution images because rather than improving on the imaging software that compiles hundreds of images, they improved on the microscope itself. They built a microscope with better lenses and mirrors so that every image would be clear from the moment of capture. When computer software would be used after the fact to put the images together into video, there would be no worry about blurred motion because each individual image was taken at a high resolution.
The second new microscope also takes images with improved clarity — but in 3D. One method used by light microscopes to image objects in 3D is to rotate the sample while taking photos at multiple angles. However, when a cell or biological compound is continuously exposed to a light source, as in the typical method, it can be damaged.
An effect of this is seen when scientists “mark” a certain compound with a specific dye. “You can use a laser to excite the sample, but the laser will bleach the dye that you’re using to track a compound,” Horner said.
Another effect of light damage can be seen in the way ultraviolet light from the sun causes mutations in DNA molecules.
Dual-view selective plane illumination microscopy (diSPIM), developed by Shroff and Yicong Wu, reduces damage to cells by installing two cameras at perpendicular angles to each other to simultaneously capture images of the cell. The sample is not rotated so light exposure is significantly minimized, but the two views at 90° angles are sufficient to create a 3D image of the sample.
A major advantage of diSPIM is that a traditional single-camera microscope can be converted into the new dual-camera form with relatively minor modifications.
“One of the issues with camera technology is that you have to have the microscope that will allow you to benefit from the increased detection capabilities of the camera,” McCaffery said. “The camera is nearly as expensive as the microscope, which didn’t use to be the case at all.”
Microscope technologies can be prohibitively expensive, with the microscope and camera having separate costs each in the range of hundreds of thousands or even millions of dollars.
“It will be worth the investment for the kinds of things that biologists and biophysicists are looking to discover and to visualize,” McCaffery said.
Despite the advent of new microscopy technologies, students and researchers alike will always benefit from traditional microscopes, Horner said. “If students can learn the limitations of the microscopes and learn what they’re seeing, then they’ll accomplish what we want them to do here as undergraduates.”
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