• 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 >100 um imaging depth
  • inverted microscope objective allows simultaneous photomanipulation or other techniques
  • 40x 0.8 NA water-dipping objectives most common (others possible)
    • yields 330 um 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 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

In general the diSPIM approach is ideal for cells or small groups of cells (e.g. c. elegans embryos). For thicker samples where the light sheet cannot penetrate across the sample the Zeiss/OpenSPIM approach is probably better so that all sides of the sample can be directly seen. For sub-diffraction resolution on thin samples lattice light sheet is preferred. Compared with Zeiss/OpenSPIM the diSPIM sample mounting is easy and extremely flexible. Instruments which rely on a single fixed view lead to relatively poor axial resolution and problems with scattering samples. Compared with other commercial light-sheet solutions a diSPIM is quite inexpensive.

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