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Comparison of Light Sheet Microscopy Methods
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- sample mounting like inverted microscope with water/buffer immersion
- two fixed orthogonal views
- two views can be combined computationally for isotropic resolution
- scattering slightly mitigated slightly by having two views
- inverted microscope objective allows simultaneous photomanipulation or other techniques
- 40x NA 0.8 water-dipping objectives most common (others possible)
- yields 330 um field of view with standard sCMOS sensor and standard 200 mm tube lenses
- yields 380 nm lateral resolution at 500 nm wavelength (improved slightly by combining views computationally)
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- 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)
- different views can be combined computationally for isotropic resolution
- scattering greatly mitigated by multiple views
- 20x water-dipping objective most common for imaging (others possible); usually imaging objective has large NA and illumination objective has low NA
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- usually used for larger samples, including cleared tissue
- available magnifications 1.26x - 12.6x (“cellular resolution”)
- one fixed view ⇒ relatively poor axial resolution (“cellular resolution”)
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- 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
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. 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.
