If you want to keep something alive while you look at it under a microscope, one good way is light sheet microscopy. For a variety of reasons, using a horizontal light sheet to image specimens—as opposed to piercing cones of light—is becoming a life-science-friendly means of imaging that both speeds up the process and decreases the deleterious effects other microscopy techniques have on living specimens.
Until the end of the 20th century, the technology remained essentially dormant. Then, in 1993, Arne Voie and colleagues at the University of Washington built the first light-sheet-based fluorescence microscope (LSBM) and created a 3-D image of a biological sample. As described in the Journal of Microscopy, their prototype used orthogonal-plane fluorescence optical sectioning to produce fluorescent optical sections of an excised guinea-pig cochlea (2).
In 2002, Jules Jaffe and colleagues at the Scripps Institution of Oceanography wanted to develop a superior way to observe aquatic microbes or larger organisms in their natural habitat, but found themselves limited by existing technology. So, they built a new version of the microscope: the thin light sheet microscope, which used a laser light sheet of 23 micron thickness to illuminate only particles within a specific depth of field.
“The idea was that if you’re working in a medium with lots of stuff that scatters information, you’re going be confusing that in one image,” says Jaffe. “With standard microscopes you get a lot of scatter and out-of-focus blurry things that only tell you something’s there that’s interfering with what you really want to see at that depth plane.” In a paper published in Optics Express (3), Jaffe and colleagues showed the difference in the end results of imaging with their new microscope: easily discernable discrete bacteria in sea water.
Light Sheet Renaissance
A major milestone in light sheet microscopy, one that brought a renaissance in the field, came after Jaffe’s paper. A group led by Ernst Seltzer from the European Molecular Biology Laboratory in Heidelberg, Germany published a paper in Science in 2004 that not only described another iteration of light sheet microscopy—selective plane illumination microscopy (SPIM)—but also demonstrated that heartbeats and other developmental processes in living embryos could be imaged with good spatial resolution and physical coverage of large specimens by combining light sheet illumination with sequential stacking of multiple views (4).
Then, in 2008, a student from Seltzer’s lab, Philipp Keller, along with colleagues, introduced another version of light sheet florescence microcopy named digital scanned laser light sheet florescence microscopy (DSLM) that now serves as the standard for the technology and the platform for new improvements (5). In that paper, Keller and colleagues tracked the in vivo development of zebrafish embryos over a 24-hour time frame and also labeled and tracked individual nuclei.
“Philipp’s innovation and his landmark demonstration of the technology on zebrafish embryogenesis really resonated with biologists and inspired imagination—everyone was talking about it,” says Zhirong Bao, a computational biologist and developmental biologist at Memorial Sloan Kettering Cancer Center in New York. He has co-authored several papers with Keller and uses LSBM to trace neuronal development in embryos—specifically for systematic single-cell studies of Caenorhabditis elegans embryogenesis.
“The idea is to capture the dynamic process in sufficient detail to understand the entire process with a gentle enough touch that you’re not causing any toxic effects as a result of the observation process,” says Keller, who now has his own lab at Howard Hughes Medical Institute, Janelia Farm Research Campus.
Speed and Reduced Toxicity
With light sheet microscopy, however, light is beamed in a flat sheet coming in at a 90° angle so that only the targeted slice or plane is illuminated rather than the entire sample. This has two main advantages: speed and reduced phototoxicity.
With regard to speed, in contrast to point-by-point scanning of non-light sheet microscopy, LSBM collects the signal from the entire plane at the same time. To study the neural development of C. elegans, speed is necessary because as the embryo starts to mature, its neuronal wiring begins to make connections with muscle and then the organism starts to twitch. “Once an embryo starts to twitch and move, the image becomes blurred, and that is why you need super fast imaging,” says Bao.
Phototoxicity is reduced because of the specificity of the illumination process and the lack of cumulative light exposure effects caused by 3-D imaging. The advantage factor you gain in LSBM is almost exactly the number of images taken—that is, if 500 images are needed to get sufficient physical coverage, then you get approximately 500 times less photo damage to the live specimen. Other advantages of LSBM include the use of highly sensitive cameras and lower laser power. And because florescence is only excited in the image plane, less photobleaching effects occur.
In 2010, Keller, Bao, and colleagues combined DSLM with structured illumination which yielded improvements in image quality and allowed the team to record brain development in live zebrafish embryos over the course of three days (6). This technique enhanced visualization of structures less transparent than the early zebrafish embryo. In addition, the team combined light sheet-based structured illumination with multi-view imaging to record Drosophila embryogenesis and created a digital fly embryo.
“What [Shroff] has come up with is ingenious,” says Bao. “He placed the sample between two objectives that sit perpendicular to one another and with the sample sitting between the two. That obviates the need to rotate the sample. It allows the conventional mounting that is widely used in biology labs.”
This iteration of LSBM—termed inverted selective plane illumination microscopy (iSPIM)—was described in a 2011 paper published in PNAS (7). Bao feels this will help to push the commercialization of the light sheet microscopy.
To date, no commercial product is on the market, forcing each lab who wants to use the technique to build a microscope from scratch. Commercialization is difficult because it not simply a matter of building one piece of equipment. There are also endless varieties of computational software required to drive the microscope for the different tasks of image acquisition and analysis.
But some manufacturers are interested in building the instruments. For example, Carl Zeiss is working to develop a commercial version, and Shroff is developing a commercial product with Applied Scientific Instrumentation in Oregon.
In the end, the implications are huge: the ability to track a live embryo over time could advance drug development, disease modeling, and neurobiology. “Just like there are basic laws of physics, there must be basic laws of biology that we don’t yet understand,” says Keller.
- Keller, P. J., and H.-U. U. Dodt. 2012. Light sheet microscopy of living or cleared specimens. Current opinion in neurobiology 22(1):138-143.
- Voie, A. H., D. H. Burns, and F. A. Spelman. 1993. Orthogonal-plane fluorescence optical sectioning: three-dimensional imaging of macroscopic biological specimens. Journal of microscopy 170(Pt 3):229-236.
- Fuchs, E., J. Jaffe, R. Long, and F. Azam. 2002. Thin laser light sheet microscope for microbial oceanography. Opt. Express 10(2):145-154.
- Huisken, J., J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. Stelzer. 2004. Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science 305(5686):1007-1009.
- Keller, P. J., A. D. Schmidt, J. Wittbrodt, and E. H. K. Stelzer. 2008. Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy. Science 322(5904):1065-1069.
- Keller, P. J., A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt, and E. H. K. Stelzer. 2010. Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy. Nat Meth 7(8):637-642.
- Wu, Y., A. Ghitani, R. Christensen, A. Santella, Z. Du, G. Rondeau, Z. Bao, D. Colón-Ramos, and H. Shroff. 2011. Inverted selective plane illumination microscopy (iSPIM) enables coupled cell identity lineaging and neurodevelopmental imaging in caenorhabditis elegans. Proceedings of the National Academy of Sciences 108(43):17708-17713.