### Table of Contents

# Objectives

The choice of light sheet objectives is limited because they must be co-focused without bumping into each other. See more details below on this page in the section Mechanical angle.

The most commonly-used objectives for (symmetric) diSPIM are 40x water-dipping objectives with a NA of 0.8 (Nikon CFI Apo 40XW NIR). Other possibilities include the Olympus 20x/0.5 water (UMPLFLN20XW) ^{1)} and the Nikon 10x/0.3 water (CFI Plan Fluor 10XW).

ASI and Special Optics have co-developed two multi-immersion objectives designed originally for cleared tissue (but useful in any media) suitable for the diSPIM geometry. The 54-10-12 with nominal NA 0.4 can image cleared tissue up to 5 mm deep in slab form or within a 12 mm spherical envelope, and the 54-12-8 with nominal NA 0.7 can go 2 mm deep or 10 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

Resolution is usually defined as the smallest feature that can be distinguished from a neighboring feature. The diffraction-limited resolution is determined by the objective NA and wavelength; equations are k1*lambda/NA for lateral resolution and k2*lambda*RI/NA^2 for axial resolution (pre-factors vary depending on the criteria, but commonly are 0.61 and 2). With dual-view systems you can collect datasets from 2 orthogonal directions which can be computationally combined to give “lateral” resolution in 3 axes ^{2)}. Deconvolution can also improve resolution to a certain extent.

Aberrations in the optics or in the sample will degrade the attained resolution. As you image deeper into samples the resolution usually degrades due to scattering, inhomogeneous refractive index within the sample (e.g. cell membrane vs. cytoplasm), and/or mismatch between sample and media refractive index (typically cells are RI ~1.4 vs. media ~1.33).

Undersampling on the camera or in Z step size will decrease attained resolution. The Nyquist criterion applies: the sampling (e.g. pixel size) must be at least half the size of the smallest feature you want to resolve. On the other hand, oversampling in large excess to the Nyquist criterion generates “empty data” where the same amount of information could be contained in a Nyquist-sampled dataset as well as increasing the effect of shot noise. Thus using improper magnification can degrade resolution. With a 2k x 2k sensor, the Nyquist criterion tells us that the resolution can be no better than the 0.1% of the field of view.

Resolution does not depend on whether stage scanning or piezo/slice scanning is used as long as the sampling is done correctly. Rather the acquisition method changes 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. Field of view is just camera sensor size divided by magnification. The sensor size is readily available. The most typical sCMOS cameras have 6.5um square dexels and the sensor comprises an array of 2048×2048 dexels (“dexel” is the more precise term for detection pixel). Other sensor sizes are being used; watch out for 11um square dexels which usually require increasing magnification in order to sample sufficiently for diffraction-limited resolution.

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 (for sensors with larger pixels or using low-mag high-NA objectives) or to increase the field of view by intentionally spatially undersampling ^{3)}.

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.

### Mechanical Angle

For traditional light sheet microscopy with two orthogonal objective lenses, the objectives have to be able to co-focus before they mechanically bump ^{4)}. Regardless of working distance, the most important/fundamental factor in whether or not two objectives can be co-focused orthogonally is simply whether the sum of their mechanical half-angles is less than 90° ^{5)}. For any objective lens, the mechanical angle must be at least as big as the optical angle, i.e. it must be at least big enough to capture the cone of rays corresponding to its numerical aperture (NA) across the entire field of view. The mechanical angle is computed as arctan(dia/2/WD) where dia is the diameter of the first surface (assuming the rest of the objective lens fall inside the line from the focal plane to this first surface as is usually the case). The optical (half) angle is computed as arcsin(NA/RI) where RI is the medium refractive index. Some objective lenses have mechanical angles only barely larger than the lower bound optical angle, but others are much less efficient in a mechanical/bulkiness sense.

A detailed overview and helpful table of many (more) objective lenses can be found in Supplementary Note 6 in the Power/Huisken review paper (link to supplemental).

Here are a few objectives that have been used with iSPIM/diSPIM-type systems. Notice that all are used as dipping lenses, even the few that have correction collars.

Objective | Optical angle (NA) | Mechanical angle | Comments |
---|---|---|---|

Nikon 40x/0.8 W | 37° | 42.5° | common high-resolution diSPIM |

Nikon 10x/0.3 W | 13° | 36° | common low-resolution diSPIM |

Nikon 16x/0.8 W | 37° | 45° | does it actually fit with pair? (if you try it please speak up) |

Olympus 20x/0.5 W | 22° | 45° | fits better than expected (WD is probably a bit more than spec) |

Olympus 20x/1.0 W | 49° | 52° | |

Nikon 25x/1.1 W | 56° | 58° | usual lattice detection |

Olympus 60x/1.0 W | 49° | 53° | sometimes oSPIM detection |

Olympus 60x/1.1 W | 56° | 57° | usual oSPIM detection, potential lattice detection |

SO 54-10-7 29x/0.66 W | 30° | 30° | traditional lattice illumination |

TL20X-MPL 20x/0.6 W | 27° | ~30° | new (2020) cost-effective lattice illumination |

Nikon 20x/1.0 glyc | 43° | ~54° | cleared tissue confocal used in light sheet (RI 1.44 - 1.50) |

Olympus 25x/1.0 glyc | 43° | 56° | cleared tissue confocal used in light sheet (RI 1.41 - 1.52) |

SO 54-10-12 17x/0.4 MI | 16° | 22° | ASI multi-immersion #1 (RI 1.33 - 1.56, nominal 1.45) |

SO 54-12-8 24x/0.7 MI | 29° | 36° | ASI multi-immersion #2 (RI 1.33 - 1.56, nominal 1.45) |

SO 54-9-4 52x/1.15 MI | 52° | 57° | (preliminary) ASI multi-immersion #3 (RI 1.33 - 1.56, nominal 1.45) |

### 4f spacing

On the diSPIM as well as other light sheet implementations it is beneficial to have the illumination objective back focal plane at “4f” or telecentric spacing from the scanner tube lens. This ensures that tilt at the MEMS (or galvo) results in pure translation of the beam at the sample. If 4f spacing is not followed then the beam will not scan parallel to itself, resulting in a wedge-shaped sheet.

Different objectives have different positions of their back focal plane (BFP). Olympus lists the BFP position of their objectives and so does ASI. Nikon considers the BFP position confidential but it can be measured empirically.

Most often ASI installs spacers between the scanner tube lens body and the Cube III containing the dichroic to adjust the total space, which is the easiest approach if the same objectives are always used. ASI makes a 0-30mm continuously adjustable spacer which is useful if you are switching between objectives with different BFP positions or if you need to exactly tune the spacing (e.g. for using the virtual slit approach where the camera's rolling shutter is exactly synchronized with the motion of the beam).

The approximate spacers are listed here:

Illumination Objective | Config | Spacer (approx) |
---|---|---|

Nikon 40x/0.8 W | standard diSPIM | 10 mm |

Nikon 10x/0.3 W | standard diSPIM | 10 mm |

Olympus 20x/0.5 W | standard diSPIM | 0 mm |

SO 54-10-12 17x/0.4 MI | standard diSPIM | 25 mm |

SO 54-12-8 24x/0.7 MI | standard diSPIM | 20 mm |

SO 54-10-12 17x/0.4 MI | ct-dSPIM, no spacer | 50 mm |

SO 54-12-8 24x/0.7 MI | ct-dSPIM, RAO-ADJ-10 spacer | 25 mm |

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