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multi-immersion_objectives [2025/02/06 17:14] – [Chemical Compatibility] jonmulti-immersion_objectives [2026/10/07 14:18] (current) – [Known Safe Media] jon
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 ====== Multi-Immersion Objectives ====== ====== Multi-Immersion Objectives ======
  
-Applied Scientific Instrumentation (ASI) worked with Special Optics to develop various immersion/dipping objective lenses for light sheet microscopy.  They are intended for use with cleared tissue samples, but are useful for other applications where very long working distance or RI matching is important.  These objectives work in any refractive index media without a correction collar, and so can be considered not just multi-immersion but "all-immersion" objectives.((they are actually dipping lenses, as they are not correcting for imaging through a coverslip))  The first objective (54-10-12) has nominal NA 0.4 and was released October 2017.  The second design (54-12-8) has nominal NA 0.7 and higher magnification and was released June 2019.  The third objective (57-12-19) has nominal NA 0.3 and lower magnification and was released January 2023.  Another design has been proposed but not yet manufactured.  ASI is the sole distributor of these objectives but will sell them freely to anyone interested, including home builders and companies.+Summary specifications (click link to jump to details) 
 + 
 +^  ^  [[multi-immersion_objectives#specifications2|57-12-19]]  ^  [[multi-immersion_objectives#specifications|54-10-12]]  ^  [[multi-immersion_objectives#specifications1|54-12-8]]  ^  [[multi-immersion_objectives#specifications3|57-12-5]]  ^ 
 +|  [[multi-immersion_objectives#numerical_aperture_and_resolution|Nominal Numerical Aperture]]|  0.3  |  0.4  |  0.7  |  1.2  | 
 +|  [[multi-immersion_objectives#magnification|Approximate Magnification]]|  10x  |  17x  |  24x  |  40x  | 
 +|  Working distance [mm]|  18.1  |  12.1  |  10.0  |  3.3  | 
 +|  Nominal Refractive Index|  1.53  |  1.45  |  1.45  |  1.52  | 
 +|  Refractive Index Range|  1.33 – 1.56  |||| 
 +|  Field of view [mm]|  3.0  |  1.2  |  1.0  |  0.6  | 
 +|  Parfocal length [mm]|  75  |  62  |  83  |  83  | 
 + 
 + 
 +===== History ===== 
 + 
 +Applied Scientific Instrumentation (ASI) worked with Special Optics to develop several dipping objective lenses for light sheet microscopy.  They are intended for use with cleared tissue samples, but are useful for other applications where very long working distance or RI matching is important.  These objectives work in any refractive index media without a correction collar, and so can be considered not just multi-immersion but "all-immersion" objectives.((they are actually dipping lenses, as they are not correcting for imaging through a coverslip))  The first objective (54-10-12) has nominal NA 0.4 and was released October 2017.  The second design (54-12-8) has nominal NA 0.7 and higher magnification and was released June 2019.  The third objective (57-12-19) has nominal NA 0.3 and lower magnification and was released January 2023.  A fourth design (57-12-5) with very high NA is available starting July 2025. 
 + 
 +ASI is the sole distributor of these objectives but will sell them freely to anyone interested, including home builders and companies.
  
 The original goal of the first objective was isotropic ~1 micron resolution at least 5 mm deep into slabs of cleared tissue.  Doing this with a dSPIM/diSPIM geometry (two orthogonal identical objectives) requires a modest NA objective with extremely long working distance and tapered shape.  We designed the objective lens accommodate a wide variety of imaging media, which is an important feature given the variety and rapid development of clearing protocols. The original goal of the first objective was isotropic ~1 micron resolution at least 5 mm deep into slabs of cleared tissue.  Doing this with a dSPIM/diSPIM geometry (two orthogonal identical objectives) requires a modest NA objective with extremely long working distance and tapered shape.  We designed the objective lens accommodate a wide variety of imaging media, which is an important feature given the variety and rapid development of clearing protocols.
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 The {{:documentation:57-12-19-RevA.pdf|mechanical drawing}} and corresponding {{:documentation:57-12-19-RevA.STEP.zip|3D CAD file}} are available.  Zemax black box files are available too ([[https://www.asiimaging.com/technical-drawings/models/57-12-19_forward_BB%20(CATALOG%20MODEL).zip|forward]], [[https://www.asiimaging.com/technical-drawings/models/57-12-19_reversed_BB%20(CATALOG%20MODEL).zip|reverse]]). The {{:documentation:57-12-19-RevA.pdf|mechanical drawing}} and corresponding {{:documentation:57-12-19-RevA.STEP.zip|3D CAD file}} are available.  Zemax black box files are available too ([[https://www.asiimaging.com/technical-drawings/models/57-12-19_forward_BB%20(CATALOG%20MODEL).zip|forward]], [[https://www.asiimaging.com/technical-drawings/models/57-12-19_reversed_BB%20(CATALOG%20MODEL).zip|reverse]]).
-===== 57-12-5 Specifications (tentative) =====+===== 57-12-5 Specifications =====
  
-In spring 2020 we designed an objective lens and in early 2021 we tried unsuccessfully unto get enough researcher commitment to justify a production run   ([[https://www.asiimaging.com/frontpage-article/blog/crowd-funding-a-high-na-multi-immersion-objective-lens/|details]]).  In late 2024 we are revising the design based on feedback and going to make another push for pre-orders.  Here are the revised specifications.+The first lenses are made!  Here are the specifications.
  
 ^  Specification            ^  Value                ^  Comments                                                     ^ ^  Specification            ^  Value                ^  Comments                                                     ^
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 |  Chemical Resistance     |  very high           | Tentatively same as others but needs to be tested (see [[multi-immersion_objectives#known_safe_media|list]])  | |  Chemical Resistance     |  very high           | Tentatively same as others but needs to be tested (see [[multi-immersion_objectives#known_safe_media|list]])  |
 |  Effective Focal Length  |  4.93 mm @ RI 1.52   | 36x in water, 42x in ECi w/ 200 mm TL                    | |  Effective Focal Length  |  4.93 mm @ RI 1.52   | 36x in water, 42x in ECi w/ 200 mm TL                    |
-|  Working Distance        |  3.4 mm (for all RI) | exact value TBD, originally 3.4 mm | +|  Working Distance        |  3.3 mm (for all RI) | able to co-focus with 54-10-12 | 
-|  Field of View           |  0.6 mm Ø            | FOV reduced in water   | +|  Field of View           |  0.3 - 0.6 mm Ø            | FOV 0.6 mm in RC1.52 and similar, ~0.3mm in water, simulate if this is critical   | 
-|  Spherical Correction    |  480 – 1300 nm       | Diffraction-limited for most media to 910 nm full NA, 910 – 1300 nm NA 0.8 |+|  Spherical Correction    |  480 – 1300 nm       | Diffraction-limited for most media to 910 nm full NA, NA 0.8 from 910 – 1300 nm |
 |  Chromatic Correction    |  480 – 720 nm        | Performance varies by media, optimized for RC1.52 and organic solvents in general| |  Chromatic Correction    |  480 – 720 nm        | Performance varies by media, optimized for RC1.52 and organic solvents in general|
 |  Transmission    |  Expected ~75%        | Internal coatings 400 – 1300 nm      | |  Transmission    |  Expected ~75%        | Internal coatings 400 – 1300 nm      |
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 |  Mechanical Angle        |  59°          | Measured from center of FOV  | |  Mechanical Angle        |  59°          | Measured from center of FOV  |
  
-2D drawing, 3D model, and Zemax black box files are available from ASI upon request.+The {{https://www.asiimaging.com/technical-drawings/pdf/57-12-5%20REV%20A.pdf|mechanical drawing}} and corresponding {{https://www.asiimaging.com/technical-drawings/models/57-12-5%20REV%20A%20(CATALOG%20MODEL).zip|3D CAD file}} are available.  Zemax black box files are available too ([[https://www.asiimaging.com/technical-drawings/models/57-12-5%20BB-forward%20(CATALOG%20MODEL).zar|forward]], [[https://www.asiimaging.com/technical-drawings/models/57-12-5%20BB-reverse%20(CATALOG%20MODEL).zar|reverse]]). 
 + 
  
 ===== Magnification ===== ===== Magnification =====
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 __57-12-19__: EFL<sub>obj</sub> = 30.0 mm / RI __57-12-19__: EFL<sub>obj</sub> = 30.0 mm / RI
  
-__54-12-5__: EFL<sub>obj</sub> = 7.50 mm / RI (not yet manufactured)+__57-12-5__: EFL<sub>obj</sub> = 7.50 mm / RI
  
  
 For the 54-10-12 and 54-12-8 the nominal RI is 1.45 (FocusClear/CLARITY), so for example the focal length is 12.0 mm for the 54-10-12 in FocusClear.  Taking a 200 mm focal length tube lens, which ASI uses by default on SPIM systems, this results in a magnification of 16.7x.  However, if a different magnification is needed then a different tube lens can be used.  ASI offers a variety of suitable [[mim_ramm_vts#mim_tube_lenses_and_assemblies|tube lenses]] as do others. For the 54-10-12 and 54-12-8 the nominal RI is 1.45 (FocusClear/CLARITY), so for example the focal length is 12.0 mm for the 54-10-12 in FocusClear.  Taking a 200 mm focal length tube lens, which ASI uses by default on SPIM systems, this results in a magnification of 16.7x.  However, if a different magnification is needed then a different tube lens can be used.  ASI offers a variety of suitable [[mim_ramm_vts#mim_tube_lenses_and_assemblies|tube lenses]] as do others.
  
-For the 54-12-8 at RI 1.45, the focal length is 8.4 mm and nominal magnification is 24x, and for the 54-9-4 at RI 1.45, the focal length is 3.83 mm and nominal magnification is ~52.2x.+For the 54-12-8 at RI 1.45, the focal length is 8.4 mm and thus nominal magnification is 24x (assuming 200mm tube lens).
  
 The 57-12-19 has a nominal RI of 1.53 and is specifically intended for ECi/BABB/EasyIndex media. The 57-12-19 has a nominal RI of 1.53 and is specifically intended for ECi/BABB/EasyIndex media.
  
-The 57-12-5 has a nominal RI of 1.52 and is specifically intended for organic media such as RapidClear 1.52 and EasyIndex.+The 57-12-5 has a nominal RI of 1.52 and is specifically intended for organic media such as RapidClear 1.52 and EasyIndex.  The diffraction-limited FOV is reduced in water.
  
 Because the magnification depends on the imaging medium, magnification ideally would be measured empirically.  However, for most situations estimating the magnification based on the presumed RI of the imaging medium is probably sufficient. Because the magnification depends on the imaging medium, magnification ideally would be measured empirically.  However, for most situations estimating the magnification based on the presumed RI of the imaging medium is probably sufficient.
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 __54-12-8__: NA = 0.483 * RI __54-12-8__: NA = 0.483 * RI
  
-__54-12-19__: NA = 0.196 * RI+__57-12-19__: NA = 0.196 * RI
  
-__54-9-4__: NA = 0.793 * RI (not yet manufactured)+__57-12-5__: NA = 0.790 * RI
  
 The diffraction-limited resolution is a function of the lens NA which depends on the RI of the immersion medium as described above.  The pre-factors can differ depending on the criteria used to define resolution, but common expressions are as follows for the resolution in the lateral (x,y) and axial (z) directions where λ is the wavelength of light and RI is the refractive index of the mounting medium: The diffraction-limited resolution is a function of the lens NA which depends on the RI of the immersion medium as described above.  The pre-factors can differ depending on the criteria used to define resolution, but common expressions are as follows for the resolution in the lateral (x,y) and axial (z) directions where λ is the wavelength of light and RI is the refractive index of the mounting medium:
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 All of these objective lenses have been designed with large working distance.  A unique feature is that the working distance is exactly the same for all media. All of these objective lenses have been designed with large working distance.  A unique feature is that the working distance is exactly the same for all media.
  
-For most objective lenses, the WD depends on the medium's RI due to refraction at the objective/media surface.  Realizing this was going to be a practical issue we specifically designed both objectives to have uniform WD for all RI.  The optical implementation of this feature is straightforward: the first element of the objective is concave with radius of curvature centered on the focal plane.  Thus to first order there is no refraction at that surface and hence the WD is constant for all RI.  For the same reason, there is no intrinsic spherical aberration as the medium RI changes, unlike objective lenses with a flat first surface.+For most objective lenses, the WD depends on the medium's RI due to refraction at the objective/media surface.  Realizing this was going to be a practical issue we specifically designed all of these objectives to have uniform WD for all RI.  The optical implementation of this feature is straightforward: the first element of the objective is concave with radius of curvature centered on the focal plane.  Thus to first order there is no refraction at that surface and hence the WD is constant for all RI.  For the same reason, there is no intrinsic spherical aberration as the medium RI changes, unlike objective lenses with a flat first surface.
  
  
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 For some applications (e.g. light sheet illumination) it is important to maintain "4f" or telecentric spacing between optical elements, i.e. space adjacent lenses so that their focal planes line up.  If an external lens is positioned with focal position at this plane, parallel rays into the lens pair will emerge from the objective parallel.  Although the working distance of these objectives does not depend on the refractive index of the medium, the location of the back focal plane does depend on it slightly. For some applications (e.g. light sheet illumination) it is important to maintain "4f" or telecentric spacing between optical elements, i.e. space adjacent lenses so that their focal planes line up.  If an external lens is positioned with focal position at this plane, parallel rays into the lens pair will emerge from the objective parallel.  Although the working distance of these objectives does not depend on the refractive index of the medium, the location of the back focal plane does depend on it slightly.
 +
 +Per optical simulations for the 57-12-19 objective lens, the location of the back focal plane referenced to flange is approximately 40.07 mm - 78.77 mm * RI + 19.77 mm * RI^2 (negative inside flange). Specifically, this is ~29.8 mm inside the flange for water, ~32.7 mm inside the flange for FocusClear, and ~35.0 mm inside the flange for ethyl cinnamate (RI 1.56). To a rough approximation the back focal plane location is about 14mm closer to the sample compared to the 54-10-12 objective and about 9mm closer to the sample compared to the 54-12-8.
  
 Per optical simulations for the 54-10-12 objective lens, the location of the back focal plane referenced to flange is approximately 31.07 mm - 38.80 mm * RI + 9.42 mm * RI^2 (negative inside flange).  Specifically, this is ~4  mm inside the flange for water, ~5.4 mm inside the flange for FocusClear, and ~6.5 mm inside the flange for ethyl cinnamate (RI 1.56). Per optical simulations for the 54-10-12 objective lens, the location of the back focal plane referenced to flange is approximately 31.07 mm - 38.80 mm * RI + 9.42 mm * RI^2 (negative inside flange).  Specifically, this is ~4  mm inside the flange for water, ~5.4 mm inside the flange for FocusClear, and ~6.5 mm inside the flange for ethyl cinnamate (RI 1.56).
  
-Per optical simulations for the 54-12-8 objective lens, the location of the back focal plane referenced to flange is approximately -11.75 mm - 22.90 mm * RI + 5.87 mm * RI^2 (negative inside flange).  Specifically, this is ~31.8  mm inside the flange for water, ~32.5 mm inside the flange for FocusClear, and ~33.2 mm inside the flange for ethyl cinnamate (RI 1.56). The difference in back focal plane position between the two objectives depends on RI, but is to a good approximation the location is 27 mm deeper inside the objective for the 54-12-8 as measured from the flange, or 5 mm closer to the sample.+Per optical simulations for the 54-12-8 objective lens, the location of the back focal plane referenced to flange is approximately -11.75 mm - 22.90 mm * RI + 5.87 mm * RI^2 (negative inside flange).  Specifically, this is ~31.8  mm inside the flange for water, ~32.5 mm inside the flange for FocusClear, and ~33.2 mm inside the flange for ethyl cinnamate (RI 1.56). The difference in back focal plane position between the 54-10-12 and 54-12-8 depends on RI, but is to a good approximation the location is 5 mm closer to the sample for the 54-12-8.
  
-Per optical simulations for the 57-12-19 objective lens, the location of the back focal plane referenced to flange is approximately 40.07 mm - 78.77 mm * RI + 19.77 mm * RI^2 (negative inside flange). Specifically, this is ~29.8 mm inside the flange for water, ~32.7 mm inside the flange for FocusClear, and ~35.0 mm inside the flange for ethyl cinnamate (RI 1.56). To a rough approximation the back focal plane location is about 14mm closer to the sample compared to the 54-10-12 objective and about 9mm closer to the sample compared to the 54-12-8.+Per optical simulations for the 57-12-5 objective lens, the location of the back focal plane referenced to flange is ~44.0 mm inside the flange for water and ~44.9 mm inside the flange for the design medium which is RC1.52.
  
 ===== Spherical and Chromatic Aberrations ===== ===== Spherical and Chromatic Aberrations =====
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 The objectives have minimal spherical aberration in the NIR spectrum to allow for multi-photon excitation, though they are corrected chromatically only in the visible.  Internal anti-reflective coatings are optimized for visible, but it is possible to make a batch with coatings optimized for NIR (e.g. 800-1600 nm); contact ASI if you are interested in this possibility. The objectives have minimal spherical aberration in the NIR spectrum to allow for multi-photon excitation, though they are corrected chromatically only in the visible.  Internal anti-reflective coatings are optimized for visible, but it is possible to make a batch with coatings optimized for NIR (e.g. 800-1600 nm); contact ASI if you are interested in this possibility.
  
-Chromatic aberrations are rooted in dispersion, which describes how the exact RI changes with wavelength (commonly reported as the Abbe number).  For a specific medium the dispersion can be corrected, but since these objectives are designed to work in many media it cannot be perfectly corrected for all of them.  Chromatic correction could have been improved with a correction collar; this was deemed to add too much complexity for the corresponding benefit.  Hence, during the design we could only optimize the chromatic correction for one media, but it turns out that both TDE and CLARITY/Focus Clear are very well corrected for.  The middle dotted line on the plot below shows the approximate "perfect" correction line.  Chromatic aberrations can be categorized as lateral color, meaning different wavelengths have slightly different magnification, and axial color, meaning that the focus point is shifted slightly. These chromatic effects scale with the distance from the "perfect" correction line in the plot.  The lateral color for water, a rather extreme case as seen from the plot, amounts to ~0.3% change in magnification between 480 nm light and 640nm light for the 54-10-12.  We expect that the lateral color can be corrected in post-processing if needed. The axial color remains within the diffraction limit for all media simulated.+Chromatic aberrations are rooted in dispersion, which describes how the exact RI changes with wavelength (commonly reported as the Abbe number).  For a specific medium the dispersion can be corrected, but since these objectives are designed to work in many media it cannot be perfectly corrected for all of them.  Chromatic correction could have been improved with a correction collar; this was deemed to add too much complexity for the corresponding benefit.  Hence, during the design we can only optimize the chromatic correction for one media, but TDE and CLARITY/Focus Clear are very well corrected for simultaneously. The middle dotted line on the plot below shows the approximate correction line used for 54-10-12 and 54-12-8, whereas the bottom line going through methyl salicylate was chosen for the 57-12-19 (i.e. emphasizing organic solvents). 
 + 
 +Chromatic aberrations can be categorized as lateral color, meaning different wavelengths have slightly different magnification, and axial color, meaning that the focus point is shifted slightly. These chromatic effects scale with the distance from the "perfect" correction line in the plot.  In the 54-10-12, the lateral color for water, (a rather extreme case as seen from the plot). amounts to ~0.3% change in magnification between 480 nm light and 640nm light.  We expect that the lateral color can be corrected in post-processing if needed. The axial color generally remains within the diffraction limit for all media for all the objectives but should be simulated if this is important.
  
 {{:documentation:abbenumber_vs_ri.png?300|}} {{:documentation:abbenumber_vs_ri.png?300|}}
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   * water with salt, sugars, and/or other non-aggressive solutes including routine-use biological buffers   * water with salt, sugars, and/or other non-aggressive solutes including routine-use biological buffers
-  * FocusClear (CLARITY)+  * FocusClear (CLARITY protocol)
   * glycerol   * glycerol
   * CUBIC-1, CUBIC-2, CUBIC-R1, CUBIC-R2   * CUBIC-1, CUBIC-2, CUBIC-R1, CUBIC-R2
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   * TDE (2,2-thiodiethanol)   * TDE (2,2-thiodiethanol)
   * ECi (ethyl cinnamate)   * ECi (ethyl cinnamate)
-  * benzyl benzoate and BB-PEG (PEGASOS)  +  * PEGASOS (benzyl benzoate and BB-PEG) 
-  * BABB+  * BABB (benzyl alcohol and benzyl benzoate)
   * DBE (dibenzyl ether)   * DBE (dibenzyl ether)
   * 100% ethanol   * 100% ethanol
 +  * DMSO (dimethyl sulfoxide)
 +  * formamide
   * other proprietary organic media   * other proprietary organic media
Address: 29391 W Enid Rd. Eugene, OR 97402, USA | Phone: +1 (541) 461-8181
multi-immersion_objectives.1738880057.txt.gz · Last modified: by jon