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Light-sheet microscopy
What is light-sheet microscopy?
A light-sheet microscope is an observation technique that illuminates a sample with sheet-shaped light to acquire 2D images. By changing the position where the sheet light is applied, it also enables 3D imaging.
While light-sheet microscopy is often compared to confocal microscopy because it can capture 3D images, it has two significant features:
- Separate optical paths for excitation light illumination and fluorescence detection.
- No excitation light hitting the upper and lower focal planes.
In confocal microscopy, due to the epifluorescence illumination, excitation light hits the upper and lower sides of the focal plane, causing sample fading and phototoxicity. In contrast, light-sheet microscopy illuminates the sample’s focal plane parallelly, avoiding excitation light exposure to the upper and lower sides of the focal plane, which reduces fading and phototoxicity (see Figure 1).
Additionally, while confocal microscopy requires scanning in the XY direction during imaging, light-sheet microscopy illuminates the XY plane all at once, eliminating the need for scanning and enabling faster imaging (see Figures 2 and 3).
Figure 1: Differences in illumination methods between confocal microscopy and light-sheet microscopy
Figure 2: Excitation light illumination image in confocal microscopy
Figure 3: Schematic diagram of light-sheet microscopy (SPIM)
The disadvantage of light-sheet microscopy compared to confocal microscopy lies in its lower resolution. In light-sheet microscopy, you need two objective lenses—one for excitation light illumination and another for fluorescence detection—both of which must be brought close to the sample. However, when attempting to use high-NA (numerical aperture) objective lenses, interference occurs between the objective lenses due to their working distances. As a result, you have no choice but to use low-magnification, low-NA objective lenses with a certain working distance.
Additionally, while sheet light allows clean image acquisition immediately after exiting the excitation objective lens, at positions farther from the emission side of the objective lens, the image quality decreases due to scattering of excitation light by the sample and increased background light. To overcome this, recent advancements include light-sheet microscopes with objective lenses positioned in two directions, enabling excitation light to be irradiated from both directions (see Figure 3).
| Observation method | Confocal microscopy | Light-sheet microscopy |
|---|---|---|
| Capture speed | Slow | Fast |
| Resolution | High | Low |
| Sample phototoxicity | High | Low |
Types of light-sheet microscopy
SPIM（selective plane illumination microscopy）
SPIM (selective plane illumination microscopy) is a light-sheet microscopy technique that creates a sheet of light by expanding a laser using a cylindrical lens. Unlike DSLM (digital scanned light-sheet microscopy), SPIM eliminates the need for laser scanning, resulting in a simpler optical system and easier synchronization with cameras.
DSLM（digital scanned light-sheet microscopy）
DSLM (digital scanned light-sheet microscopy) creates a pseudo-light-sheet by scanning lasers. By combining laser scanning timing with the light-sheet readout mode (mentioned later), DSLM can acquire lower-noise images compared to SPIM.
Example images
Free-swimming Volvox carteri scaned by ezDSLM
Data courtesy of Laboratory for Spatiotemporal Regulations Exploratory Research Center on Life and Living Systems (ExCELLS) / National Institute for Basic Biology (NIBB), Dr. Atsushi Taniguchi, Dr. Shigenori Nonaka
Free-swimming Paramecium bursaria scaned by ezDSLM
Data courtesy of Laboratory for Spatiotemporal Regulations Exploratory Research Center on Life and Living Systems (ExCELLS) / National Institute for Basic Biology (NIBB), Dr. Atsushi Taniguchi, Dr. Shigenori Nonaka
GFP-expressing blood cells of Oryzias latipes scanned by ezDSLM
Data courtesy of Laboratory for Spatiotemporal Regulations Exploratory Research Center on Life and Living Systems (ExCELLS) / National Institute for Basic Biology (NIBB), Dr. Atsushi Taniguchi, Dr. Shigenori Nonaka
Sample provided by Spectrography and Bioimaging Facility Core Research Facilities / National Institute for Basic Biology (NIBB), Dr. Joe Sakamoto
3D multicolor observation of Amoeboid movement and the cytoplasm flow
Data courtesy of Laboratory for Spatiotemporal Regulations Exploratory Research Center on Life and Living Systems (ExCELLS) / National Institute for Basic Biology (NIBB), Dr. Atsushi Taniguchi, Dr. Shigenori Nonaka
Quantitative high-speed imaging of developmental processes
Data courtesy of Dr. Philipp L. Keller, Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, Va 20147, USA
Simultaneous two-color imaging of the beating zebrafish heart
Data courtesy of the group of Dr. Jan Huisken from the Max Planck Institute of Molecular Cell Biology and Genetics
Technical introduction: Light-sheet Readout Mode
The Light-sheet Readout Mode is a method for improving the signal-to-noise ratio (S/N) of sCMOS cameras used in light-sheet microscopy. In this mode, it is possible to adjust the timing of camera readout synchronously with the movement of excitation light, allowing for high-quality image acquisition with an unaffected S/N due to scattering.
Hamamatsu Photonics has obtained a patent for this.
Patent numbers:
JP06475307, JP05770958, JP06240056, JP05639670
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Highlights:
- Ideal for ultra low light and replacing EM-CCDS
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The LCOS-SLM optical phase modulator significantly enhances light-sheet microscopy by enabling precise control over the phase and intensity of the light sheet. This leads to sharper, high-contrast images and minimizes phototoxicity by optimizing light distribution to only target the region of interest.
With its rapid wavefront modulation capabilities, the LCOS-SLM improves image acquisition speed, essential for capturing dynamic biological processes in live samples. Overall, the LCOS-SLM supports research applications with enhanced image quality and reduced sample stress, pushing the state-of-the-art of clarity and efficiency in light-sheet microscopy applications.
The MEMS mirrors elevate light-sheet microscopy by providing precise, rapid beam steering, ideal for capturing live-cell events with minimal photodamage. The Hamamatsu MEMS mirrors, with their compact form and reliability, add value by enabling real-time adjustments critical for advanced biological research.
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