Scientists have developed a technique that improves the spatiotemporal resolution and collection efficiency of light-sheet fluorescence microscopy (LSFM), without modifying the underlying microscope. The technique instead uses reflection to improve speed, resolution and light efficiency. A reflective, mirrored coverslip is used in place of a conventional glass coverslip. Computational imaging algorithms are used to remove unwanted background from the image and process the resulting data. Researchers at the National Institute of Biomedical Imaging and Bioengineering High Resolution Optical Imaging lab (NIBIB HROI) enabled simultaneous collection of four complementary views in 250 ms, doubling speed and improving information content relative to symmetric dual-view LSFM. Further, they enhanced spatial resolution by applying their imaging method to single-view LSFM. This allowed simultaneous acquisition of two high-resolution views that otherwise would be difficult to obtain due to constraints at high numerical aperture. A group at the University of Chicago helped the NBIB HROI team create an algorithm that can identify and remove epifluorescence contamination from both conventional and reflected views and fuse all views for resolution recovery. The images obtained by the combination of the new coverslip and computer algorithms show clearer views of small structures. Courtesy of Yicong Wu, National Institute of Biomedical Imaging and Bioengineering. The researchers demonstrated their technique on a variety of samples, studying mitochondrial, membrane, Golgi and microtubule dynamics in cells and calcium activity in nematode embryos. Using the mirrored coverslips with the computer software, the team was able to improve the speed two-fold and almost double the resolution in comparison with conventional dual-view selective plane microscopy (diSPIM) systems. An additional benefit of the technique is that with mirrored coverslips, the microscope is able to collect more light from the sample without increasing the overall light exposure to the sample. As a result, it increases the efficiency by two to three times compared with diSPIM. The microscope builds on improvements that the HROI lab had previously made with selective plane illumination microscopy (SPIM), a form of microscopy that is less damaging to samples because it irradiates only a portion of the sample being imaged. In 2013, HROI Lab Chief Hari Shroff and his colleague Yicong Wu developed the diSPIM system — a selective plane illumination microscopy system equipped with two lenses that allow it to obtain two views of the sample instead of one. This dual view microscope enables 3D imaging with greater clarity and resolution than traditional single-view imaging. In 2016, Shroff and Wu added a third lens, further improving light efficiency and resolution in 3D imaging. “Once we incorporated three lenses, we found it became increasingly difficult to add more,” said Shroff. “Not because we reached the limit of our computational abilities, but because we ran out of physical space.” The lenses used to image the samples are bulky and the space around the sample becomes more limited with each additional lens. Instead of trying to find ways to add more lenses, Wu and Shroff’s solution was to use mirrored coverslips. In this diagram, you can see how the mirrored coverslip allows for four simultaneous views. Courtesy of Yicong Wu, National Institute of Biomedical Imaging and Bioengineering. “It's a lot like looking into a mirror,” Shroff said. “If you look at a scene in a mirror, you can view perspectives that are otherwise hidden. We used this same principle with the microscope. We can see the sample conventionally using the usual views enabled by the lenses themselves, while at the same time recording the reflected images of the sample provided by the mirror.” The researchers believe that in the future this technique could be adapted to other forms of microscopy. The research was published in Nature Communications (doi: 10.1038/s41467-017-01250-8).