Comparison of Light Sheet Microscopy Methods

Commercial Light Sheet Microscopes

Type # Views Mounting Software Comments
diSPIM 2 fixed (isotropic) Coverslip or dish with media Free/open + various proprietary Modular/flexible configuration, allows simultaneous photo-manipulation
Zeiss Z.1 (similar to OpenSPIM) Unlimited (isotropic) Capillary with agarose Single proprietary Rotating sample allows imaging scattering samples from both sides
Leica TCS SP8 DLS 1 fixed Dish with media Single proprietary Add-on to existing Leica confocal
3i Lattice Light Sheet 1 fixed Small coverslip in dish Single proprietary Lattice illumination allows for improved axial resolution for thin samples
LaVision BioTec Ultramicroscope 1 fixed Dish with media Single proprietary Optimized for large fixed samples (low mag, low res)

In general the diSPIM approach is ideal for cells or small groups of cells (e.g. c. elegans embryos). For thicker samples (e.g. Drosophila embryos) where the light sheet cannot penetrate across the sample the Zeiss/OpenSPIM approach has the advantage that all sides of the sample can be directly seen via rotating the sample. For sub-diffraction resolution on thin samples lattice light sheet gives better resolution (though much of the advantage can be gained simply by using the lattice light sheet objectives on the diSPIM). The LaVision BioTec system is optimized for large fixed samples, though such samples can also be imaged on the diSPIM with an appropriate objective for cleared tissue.

Instruments which rely on a single fixed view lead to relatively poor axial resolution and poor imaging of scattering samples.

The diSPIM sample mounting is easy and extremely flexible compared with Zeiss/OpenSPIM. Compared with other commercial light-sheet solutions a diSPIM system is quite inexpensive and extremely flexible/customizable.

Detailed Comparison

    • sample mounting like inverted microscope; light sheet objectives lowered into open chamber with dipping media
    • two fixed orthogonal views without moving the sample (ideal for fast-moving samples)
    • two views can be combined computationally for isotropic resolution
    • scattering slightly mitigated slightly by having two views; typically 30-300 um imaging depth depending on sample
    • inverted microscope objective allows simultaneous photomanipulation or other techniques
    • 40x 0.8 NA water-dipping objectives most common (others possible)
      • yields >400 um diagonal field of view with standard sensor/tube lens
      • yields 380 nm lateral resolution at 500 nm wavelength (improved slightly by combining views computationally)
    • OpenSPIM can be thought of as simplified “build-your-own Zeiss Z.1”
    • sample is suspended from glass capillary into special chamber which holds dipping media
    • sample can be rotated to see all different sides (unlimited views around Z axis) but requires time to rotate
    • different views can be combined computationally for isotropic resolution
    • scattering mitigated by multiple views; typically ~2x deeper than diSPIM
    • 20x water-dipping objective most common for imaging (others possible); usually imaging objective has large NA and illumination objective has low NA
    • usually used for larger samples, including cleared tissue
    • available magnifications 1.26x - 12.6x (“cellular resolution”)
    • one fixed view ⇒ relatively poor axial resolution, e.g. >4 um (“cellular resolution”)
    • add-on module for Leica confocal microscope
    • uses axially-arranged objectives with special mirrors to create orthogonal light sheet
    • one fixed view ⇒ relatively poor axial resolution, scattering not mitigated
    • light sheet parallel to dish bottom, so flat samples must be tilted with respect to dish
  • lattice light sheet
    • uses structured light sheet “lattice” from interfering Bessel beams
    • scattering strongly affects lattice formation as well as imaging; typically <20 um imaging depth
    • can obtain sub-diffraction images like SIM by combining multiple exposures with shifted lattice; cost is extra time/dose like SIM
    • objectives dipped in media at angle from above (details) similar to diSPIM
    • 25x 1.1 NA detection objective and custom excitation objective for fixed single-view; axial resolution is improved beyond the objective's capability (but not quite isotropic) because of the optical sectioning of the lattice illumination
    • commercialized by Intelligent Imaging under license from Zeiss

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