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hardware:objectives [2020/01/24 18:41]
Jon Daniels [Mechanical Angle] |
hardware:objectives [2024/11/01 22:02] (current)
Jon Daniels [Mechanical Angle] |
| 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 [[hardware:objectives#mechanical_angle|Mechanical angle]]. | 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 [[hardware:objectives#mechanical_angle|Mechanical angle]]. |
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| 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) ((By the mechanical drawings the Olympus 20x/0.5 water objectives will exactly touch when co-focused, but in practice it seems the working distance is slightly longer than specified so the spacing of the objectives at the tip is similar to the Nikon 40x/0.8 pair.)) and the Nikon 10x/0.3 water (CFI Plan Fluor 10XW). ASI and Special Optics have co-developed two different [[http://asiimaging.com/docs/cleared_tissue_objective|multi-immersion objectives]] designed originally for cleared tissue (but useful in any media) that is suitable for the diSPIM geometry. The first 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 second with nominal NA 0.7 can go 2 mm deep or 10 mm spherical envelope. | 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) ((By the mechanical drawings the Olympus 20x/0.5 water objectives will exactly touch when co-focused, but in practice it seems the working distance is slightly longer than specified so the spacing of the objectives at the tip is similar to the Nikon 40x/0.8 pair.)) and the Nikon 10x/0.3 water (CFI Plan Fluor 10XW). |
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| 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. | ASI and Special Optics have co-developed two [[http://asiimaging.com/docs/cleared_tissue_objective|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. |
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| | 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 either the Special Optics 54-10-7 which is 28.6x/0.66, or else paired with the Thorlabs 20x/0.6. |
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| ==== Close-up Drawings ==== | ==== Close-up Drawings ==== |
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| * {{ :hardware:close_up_of_nikon_40x_dispim_objectives.pdf | two Nikon 40x/0.8}} | * {{ :hardware:close_up_of_nikon_40x_dispim_objectives.pdf | two Nikon 40x/0.8 W}} |
| * {{ :hardware:close_up_of_nikon_10x_dispim_objectives.pdf | two Nikon 10x/0.3}} | * {{ :hardware:close_up_of_nikon_10x_dispim_objectives.pdf | two Nikon 10x/0.3 W}} |
| * {{ :hardware:objective_assembly_lattice_light_sheet_objective_pair.pdf | Nikon 25x/1.1 with Special Optics 54-10-7}} | * {{ :hardware:objective_assembly_lattice_light_sheet_objective_pair.pdf | Nikon 25x/1.1 Wwith Special Optics 54-10-7}} |
| * {{ :hardware:config1_54-10-12_qty_2.pdf| two Special Optics 54-10-12}} | * {{ :hardware:config1_54-10-12_qty_2.pdf| two Special Optics 54-10-12}} |
| * {{ :hardware:config8_54-10-12_and_54-12-8.pdf| Special Optics 54-10-12 with Special Optics 54-12-8}} | * {{ :hardware:config8_54-10-12_and_54-12-8.pdf| Special Optics 54-10-12 with Special Optics 54-12-8}} |
| * {{ :hardware:config9_54-12-8_qty_2.pdf| two Special Optics 54-12-8}} | * {{ :hardware:config9_54-12-8_qty_2.pdf| two Special Optics 54-12-8}} |
| | * {{ :hardware:config11_57-12-19_qty_2.pdf| two Special Optics 57-12-19}} |
| | * {{ :hardware:Oly20x_Thor20x.pdf| Olympus 20x/1.0 W with Thorlabs 20x/0.6}} |
| ==== Resolution ==== | ==== Resolution ==== |
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| ==== Magnification ==== | ==== Magnification ==== |
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| 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 2048x2048 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. | Like all infinity microscopes, the magnification is given by the ratio of the effective focal lengths of tube lens and objective. The captured field of view is just camera sensor size divided by magnification. The sensor size is readily available. The most common sCMOS cameras have 6.5um square pixels and 2048x2048 pixels, but there are a wide variety of cameras. Some cameras have 11um pixels which usually require increasing magnification in order to sample sufficiently for diffraction-limited resolution. |
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| By default ASI uses 200 mm focal length tube lenses (Nikon glass) but a offers a [[http://asiimaging.com/docs/mim_ramm_vts#mim_tube_lenses_and_assemblies|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 ((the main concern in this situation becomes vignetting, see http://asiimaging.com/docs/mim_ramm_vts#infinity_space_limitations)). | By default ASI uses 200 mm focal length tube lenses (Nikon glass) but a offers a [[http://asiimaging.com/docs/mim_ramm_vts#mim_tube_lenses_and_assemblies|variety of tube lenses]] so the magnification can easily be chosen. 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. When decreasing magnification, beware of the objective's intrinsic field of view (usually specified by field number, of vignetting (see http://asiimaging.com/docs/mim_ramm_vts#infinity_space_limitations), and ensure that the spatial sampling on the camera is sufficient (see https://asiimaging.com/docs/infinity_microscope_basics#spatial_sampling). |
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| Note that using Olympus objectives with Nikon tube lens will result in 1.11x increase in magnification. The [[http://asiimaging.com/docs/cleared_tissue_objective#magnification|effective focal length of the cleared tissue objective]] depends on the media refractive index. | Note that using Olympus objectives with Nikon tube lens will result in 1.11x increase in magnification. The [[http://asiimaging.com/docs/cleared_tissue_objective#magnification|effective focal length of the cleared tissue objective]] depends on the media refractive index. |
| ==== Mechanical Angle ==== | ==== Mechanical Angle ==== |
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| For traditional light sheet microscopy with two orthogonal objective lenses, the objectives have to be able to co-focus before they mechanically bump ((For low-NA illumination you can sometimes extend the working distance a bit of the illumination objective by introducing diverging rays into its back aperture, but this is usually only a small win.)). 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° ((However if the working distance of one is very long then perhaps they can co-focus with only use of the optical angle.)). 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). 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. | For traditional light sheet microscopy with two orthogonal objective lenses, the objectives have to be able to co-focus before they mechanically bump ((For low-NA illumination you can sometimes extend the working distance a bit of the illumination objective by introducing diverging rays into its back aperture, but this is usually only a small win.)). The question of whether or not two objectives can be co-focused orthogonally can usually be simplified to whether the sum of their mechanical half-angles is less than 90°((if the working distance of one is very long then perhaps they can co-focus with only use of the optical angle.)). For any objective lens, the mechanical angle must be at least as large as the optical angle, i.e. it must be 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. |
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| A detailed overview and helpful table of many (more) objective lenses can be found in Supplementary Note 6 in the Power/Huisken review paper ([[https://media.nature.com/original/nature-assets/nmeth/journal/v14/n4/extref/nmeth.4224-S1.pdf|link to supplemental]]). | A detailed overview and helpful table of many (more) objective lenses can be found in Supplementary Note 6 in the Power/Huisken review paper ([[https://media.nature.com/original/nature-assets/nmeth/journal/v14/n4/extref/nmeth.4224-S1.pdf|link to supplemental]]). |
| | Olympus 20x/0.5 W | 22° | 45° | fits better than expected (WD is probably a bit more than spec) | | | 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° | | | | Olympus 20x/1.0 W | 49° | 52° | | |
| | | Nikon 20x/1.0 W | 49° | 53° | | |
| | | Leica 20x/1.0 W | 49° | 52° | | |
| | | Zeiss 20x/1.0 W | 49° | 51° | 421452-9800-00 | |
| | Nikon 25x/1.1 W | 56° | 58° | usual lattice detection | | | Nikon 25x/1.1 W | 56° | 58° | usual lattice detection | |
| | Olympus 60x/1.0 W | 49° | 53° | sometimes oSPIM detection | | | Olympus 60x/1.0 W | 49° | 53° | sometimes oSPIM detection | |
| | Olympus 60x/1.1 W | 56° | 57° | usual oSPIM detection, potential lattice detection | | | Olympus 60x/1.1 W | 56° | 57° | usual oSPIM detection, potential lattice detection | |
| | | Nikon 60x/1.0 W | 49° | 57° | | |
| | SO 54-10-7 29x/0.66 W | 30° | 30° | traditional lattice illumination | | | 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 | | | TL20X-MPL 20x/0.6 W | 27° | ~30° | new (2020) cost-effective lattice illumination, note only 7mm FN at full NA | |
| | Nikon 20x/1.0 glyc | 43° | ~54° | cleared tissue confocal used in light sheet (RI 1.44 - 1.50) | | | 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) | | | 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° | 20° | ASI multi-immersion #1 (RI 1.33 - 1.56, nominal 1.45) | | | SO 54-10-12 17x/0.4 MI | 16° | 32° | ASI multi-immersion #1 (RI 1.33 - 1.56, nominal 1.45), mechanical limitation in body | |
| | SO 54-12-8 24x/0.7 MI | 29° | 36° | ASI multi-immersion #2 (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 57-12-19 10x/0.3 MI | 11° | 28° | ASI multi-immersion #3 (RI 1.33 - 1.56, nominal 1.45), mechanical limitation in body | |
| | | SO 54-9-4 52x/1.15 MI | 52° | 57° | (preliminary) ASI multi-immersion #3 (RI 1.33 - 1.56, nominal 1.45) | |
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| | ==== Working Distance and Imaging Depth ==== |
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| | The imaging depth of a pair of co-focused objectives into a flat pancake-like sample without hitting the top of the sample is proportional to the residual mechanical angle that the two objectives leave and also proportional to their working distance, with minor factor related to the relative mechanical angle/overall rotation. There may also be second-order mechanical effects and such combinations should be modeled. ASI has drawings of many different objective pairs and can prepare others. Please inquire. |
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| 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. | 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. |
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| Most often ASI installes spacers between the scanner tube lens body and the Cube III containing the dichroic to adjust the total space, which is the easiest approach ifthe 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). | 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). |
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| The approximate spacers are listed here: | The approximate spacers are listed here: |
| | SO 54-12-8 24x/0.7 MI | standard diSPIM | 20 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 54-10-12 17x/0.4 MI | ct-dSPIM, no spacer | 50 mm | |
| | SO 54-12-8 24x/0.7 MI | ct-dSPIM, RAO-ADJ-10 spacer | 50 mm | | | SO 54-12-8 24x/0.7 MI | ct-dSPIM, RAO-ADJ-10 spacer | 25 mm | |
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