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 may be better so that all sides of the sample can be directly seen. For sub-diffraction resolution on thin samples lattice light sheet is preferred. The LaVision BioTec system is optimized for large fixed samples, though such samples can also be imaged on the diSPIM with suitable objectives.

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.

• diSPIM
  • 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 30-150 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)
• Zeiss Z.1 / OpenSPIM
  • 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
• LaVision BioTec UltraMicroscope
  • 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”)
• Leica TCS SP8 DLS Digital Light Sheet
  • 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
• 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 from above (details) similar to diSPIM
  • 25x 1.1 NA detection objective and custom excitation objective for fixed single-view ⇒ relatively poor axial resolution but better than typical diSPIM because of objective with higher NA
  • beginning to be commercialized by Intelligent Imaging

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