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Table of Contents
Objectives
The choice of light sheet objectives is limited because they must be co-focused without bumping into each other. A detailed overview is given in Supplementary Note 6 in the Power/Huisken review paper (link to supplemental).
The most commonly-used objectives for (symmetric) diSPIM are 40x water-dipping objectives with a NA of 0.8 (Nikon CFI Apo 40XW NIR). The Olympus 20x/0.5 water objective is another possibility) as is the Nikon 10x/0.3 water. ASI and Special Optics have co-developed an objective for cleared tissue that is suitable for the diSPIM geometry and can image cleared tissue up to 5 mm deep in slab form or within a 12 mm spherical envelope.
Single-sided systems (iSPIM) have much more flexibility because the illumination objective can be a low-NA long-WD objective. A popular pair for high-resolution imaging is the same objective pair as used on the Lattice light sheet, specifically the Nikon 25x/1.1 objective paired with Special Optics 54-10-7 which is 28.6x/0.66.
Close-up Drawings
Resolution
Attained resolution can never be better than the diffraction-limited resolution determined by the objective NA and wavelength. Typical equations are 0.61*lambda/NA lateral and 2*lambda/NA^2 axial but there are other expressions depending on the definition chosen. Remember that with diSPIM you get 2 orthogonal views which can be computationally combined to give “lateral” resolution in 3 axes 1).
Aberrations in the optics or in the sample will degrade attained resolution.
Undersampling on the camera or in your Z step size will also decrease attained resolution. The Nyquist criterion applies: the sampling (e.g. pixel size) must be at least twice the smallest feature you want to resolve.
Resolution does not depend on whether stage scanning or piezo/slice scanning is used as long as the sampling is done correctly. What does is the spatial relationship between planes.
Magnification
Like all infinity microscopes, the magnification is given by the ratio of the effective focal lengths of tube lens and objective. By default ASI uses 200 mm focal length tube lenses (Nikon glass) but a offers a variety of tube lenses so the magnification can easily be chosen otherwise. Typical reasons to adjust the magnification include to adequately sample on the camera (especially for sensors with larger pixels or using low-mag high-NA objectives) or to increase the field of view by intentionally spatially undersampling 2).
Note that using Olympus objectives with Nikon tube lens will result in 1.11x increase in magnification. The effective focal length of the cleared tissue objective depends on the media refractive index.
