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For decades, optical microscopy was restricted to analyzing very thin specimens or physically compressed thicker specimens. Although this is a tried-and-true approach, researchers have increasingly needed microscopes capable of resolving fine detail of intact, often living, samples in three dimensions.
Researchers have been able to use non-optical techniques, such as ultrasound tomography and micro-computed tomography (CT), for in vivo 3D imaging, but these are relatively low-resolution technologies compared to 2D microscopy. Researchers using 2D microscopy could find out more about a specimen by just focusing through the layers of a thin sample, but unfortunately, a 3D perception of a sample created by this approach would only exist in the researcher’s mind.
Thanks to motorized focusing technology and automated image analysis, 3D microscopy has become a viable technology.
Challenges of 3D Microscopy
Imaging cells and their various components in 3D has many inherent challenges, and ones that are significantly magnified compared to the 2D viewing of cells using traditional glass slides.
Perhaps the most prominent challenge of 3D imaging is that the resolution along the Z-axis in a typical microscope is considerably worse than in the X- or Y-axis. Caused by the three-dimensional diffraction pattern associated with an objective lens, a distorting effect called “Z-stretch” is another big challenge for 3D microscopy. Poor axial resolution and Z-stretch can cause major problems when tracking the movement of items under the microscope.
Contributing to the issue of axial resolution is the fact that locations of interest can be hundreds of microns away from an objective lens. Focused viewing at these lengths calls for longer working ranges for objective lenses, but these ranges would mean a reduction of the numerical aperture; and thus, light collection and resolving power. Moreover, for fluorescence tactics, excitation light penetration in biological specimens can be troublesome as a result of photonic interaction with the sample, which causes scattering.
These difficulties have been addressed by a technique called structured illumination microscopy (SIM) in addition to point source localization methods.
There have been many recent developments in 3D microscopy that have addressed the technology’s various challenges and made it a more attractive investigative tool.
Watching Cell Division in Detail
In September 2019, researchers from GE Healthcare announced a super-solution 3D microscope that uses confocal technology and SIM. The company said the microscope will allow researchers to view cells in very fine detail without limiting speed, depth or quality. For example, GE Healthcare researchers were able to see the fine details of cell division with high quality and contrast.
The microscope’s confocal approach uses a forward-thinking way of evaluating and expunging out-of-focus light that can affect conventional line-scanning confocal pictures. The microscope considerably cuts down issues related to photobleaching or surplus light exposure, which is an issue in many current imaging solutions. The superior quality afforded by this microscope is particularly valuable for cells grown in culture, where out-of-focus light drastically and adversely affects contrast.
GE Healthcare said this technology could be used in molecular biology, cellular biology, virology, and oncology research.
High-Speed 3D Microscopy
In September 2019, Columbia University researchers revealed the second iteration of their 3D SCAPE microscope and described how they used it to capture previously unseen visual imagery, such as neurons firing inside a moving worm.
Importantly, the SCAPE 2.0 microscope is as much as 30 times faster than the original model. Having rapid, 3D imaging is critical because the operations that drive life are dynamic and constantly in a state of flux, from how cells communicate to the ways that animals move. The more rapidly we can capture images, the more of these operations we can see, especially since fast 3D imaging allows us to see entire biological systems, as opposed to just a single layer.
SCAPE sends an angled sheet of light through a sample and sweeps this sheet of light through the sample to create a 3D image. The system’s top feature is the way it can quickly move the light sheet and focus the image using just one moveable mirror. Due to it only using a small amount of light, SCAPE has a very low impact on living specimens.
Sources and Further Reading
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