Light-sheet microscopy technique is possible on standard microscopes

An international team of researchers has demonstrated that their single-objective selective-plane illumination microscopy (soSPIM) technique allows researchers to examine the activity of single proteins or entire embryos on existing microscope systems.

Related: Single-objective SPIM simplifies super-resolution 3D cell imaging

The soSPIM technique—developed by Prof. Virgile Viasnoff, principal investigator of the Mechanobiology Institute (MBI) at National University Singapore—uses an array of micromirrored wells, where each mirror is inclined at precisely 45°. Together with a beam steering add-on unit, these micromirrors allow for both the excitation beam and resulting fluorescence signal to pass through a single standard, objective lens. The soSPIM technique exhibited fast response and good sectioning capability for 3D imaging of a whole cell up to 30 µm above the coverslip and could identify single proteins, deep within the cell. When larger fabricated mirrors were introduced, the system was capable of imaging whole Drosophila embryos.

"Bearing in mind that a typical human skin cell is 30 µm in height, most super-resolution microscopy techniques can only capture 2D images near the surface of the glass coverslip or 3D images within the first micrometer," Viasnoff explains. "Although SPIM enables 3D super-resolution imaging of thicker samples at a single-cell level by selectively illuminating a single plane of the specimen with a focused light sheet from one side while capturing the fluorescence signal through a second objective positioned perpendicularly to the light sheet, this approach requires a complicated two-objective system and special sample holder. This is incompatible with standard microscope systems and prohibitively complicated and expensive for most labs."

With the soSPIM technique, cells can be grown in the microfabricated wells, holding them in place for imaging (a). The 45° micromirrors reflect the excitation beam (dotted line) from a single objective, through the sample, and the resultant emitted fluorescence signal is captured by the same objective (b).

By contrast, Viasnoff says, their soSPIM method is versatile enough to identify single proteins anywhere within a cell or assess cellular organization in whole embryos in 3D on a microscope likely to be found in the majority of life science labs. They found that soSPIM's acquisition rate was limited solely by sample brightness and the camera's maximum acquisition rate, so they used a sCMOS camera with a 22-mm-diameter, 5.5 Mpixel sensor that enables a frame rate of 30 frames/s sustained or 100 frames/s burst mode to overcome this limitation. Also, since the microfabricated mirror and sample holder is produced independently of the microscope system, soSPIM is compatible with standard inverted microscopes and high-numerical-aperture immersion objective lenses. This allows more laboratories to study the activity of single proteins within individual cells or tissues, providing new insight into protein function and accelerating understanding of how protein dysregulation leads to disease.

Full details of the work appear in the journal Nature Methods; for more information, please visit http://dx.doi.org/10.1038/nmeth.3402.

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