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multi-immersion_objectives [2019/04/19 18:29]
jon ↷ Page name changed from cleared_tissue_objective to multi-immersion_objectives
multi-immersion_objectives [2019/05/21 11:01] (current)
jon
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 ====== Multi-Immersion Objectives ====== ====== Multi-Immersion Objectives ======
  
-Applied Scientific Instrumentation (ASI) worked with Special Optics to develop two immersion/​dipping objective lenses for light sheet microscopy. ​ They were intended for use with cleared tissue samples, but are useful for live cell imaging when a very long working distance or RI matching is important. ​ These objectives work in any refractive index media without a correction collar. ​ The first objective (54-10-12) has nominal NA 0.4 and has been offered since October 2017 at the price of \$15k. ​ The second design (54-12-8) has nominal NA 0.7 and is expected to be available May 2019 for \$24k. ​ ASI is the sole distributor of these objectives but will sell them freely to anyone interested, including home builders and companies.+Applied Scientific Instrumentation (ASI) worked with Special Optics to develop two immersion/​dipping objective lenses for light sheet microscopy. ​ They were intended for use with cleared tissue samples, but are useful for live cell imaging when a very long working distance or RI matching is important. ​ These objectives work in any refractive index media without a correction collar.((but not through a coverslip; this would require a correction collar)) ​ The first objective (54-10-12) has nominal NA 0.4 and has been offered since October 2017 at the price of \$15k. ​ The second design (54-12-8) has nominal NA 0.7 and is expected to be available May 2019 for \$24k. ​ ASI is the sole distributor of these objectives but will sell them freely to anyone interested, including home builders and companies.
  
-The original goal 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 each 45 degrees above the sample) 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 first objective lens has been very well received but some users have expressed interest in higher resolution, which led to the design of the second objective lens with higher NA at the expense of some working distance and field of view.  These are unique objectives because of the combination of multi-immersion capability, very long working distance, and mechanical profile amenable to light sheet imaging. ((Other objectives being used for light sheet imaging of cleared tissue ​at present ​were designed for confocal imaging, with relatively large NA and "​fat"​ form factor.))+The original goal 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 each 45 degrees above the sample) 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 first objective lens has been very well received but some users have expressed interest in higher resolution, which led to the design of the second objective lens with higher NA at the expense of some working distance and field of view.  These are unique objectives because of the combination of multi-immersion capability, very long working distance, and mechanical profile amenable to light sheet imaging. ((Other objectives being used for light sheet imaging of cleared tissue were designed for confocal imaging, with relatively large NA and "​fat"​ form factor.))
  
 {{ :​documentation:​cleared_tissue_objective.png?​200|}} {{ :​documentation:​cleared_tissue_objective.png?​200|}}
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 |  Numerical Aperture ​     |  0.4 @ RI 1.45       ​| ​ 0.37 – 0.43 over RI range                                   | |  Numerical Aperture ​     |  0.4 @ RI 1.45       ​| ​ 0.37 – 0.43 over RI range                                   |
 |  Immersion Media RI      |  1.33 – 1.56         ​| ​ will also work in air or any media RI                       | |  Immersion Media RI      |  1.33 – 1.56         ​| ​ will also work in air or any media RI                       |
-|  Chemical Resistance ​    ​| ​ very high           ​| ​ Aqueous and organic solvents including DBE and more (see [[cleared_tissue_objective#​known_safe_media|list]]) ​ |+|  Chemical Resistance ​    ​| ​ very high           ​| ​ Aqueous and organic solvents including DBE and more (see [[multi-immersion_objectives#​known_safe_media|list]]) ​ |
 |  Effective Focal Length ​ |  12 mm @ RI 1.45     ​| ​ 15.3x – 17.9x over RI range w/ 200 mm TL                    | |  Effective Focal Length ​ |  12 mm @ RI 1.45     ​| ​ 15.3x – 17.9x over RI range w/ 200 mm TL                    |
 |  Working Distance ​       |  12 mm (for all RI)  |  > 5 mm imaging depth with flat sample, 12 mm Ø sphere ​      | |  Working Distance ​       |  12 mm (for all RI)  |  > 5 mm imaging depth with flat sample, 12 mm Ø sphere ​      |
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 |  Numerical Aperture ​     |  0.7 @ RI 1.45       ​| ​ 0.64 – 0.75 over RI range                                   | |  Numerical Aperture ​     |  0.7 @ RI 1.45       ​| ​ 0.64 – 0.75 over RI range                                   |
 |  Immersion Media RI      |  1.33 – 1.56         ​| ​ will also work in air or any media RI                       | |  Immersion Media RI      |  1.33 – 1.56         ​| ​ will also work in air or any media RI                       |
-|  Chemical Resistance ​    ​| ​ very high           ​| ​ Aqueous and organic solvents including DBE and more (see [[cleared_tissue_objective#​known_safe_media|list]]) ​ |+|  Chemical Resistance ​    ​| ​ very high           ​| ​ Aqueous and organic solvents including DBE and more (see [[multi-immersion_objectives#​known_safe_media|list]]) ​ |
 |  Effective Focal Length ​ |  8.4 mm @ RI 1.45    |  22x – 26x over RI range w/ 200 mm TL                    | |  Effective Focal Length ​ |  8.4 mm @ RI 1.45    |  22x – 26x over RI range w/ 200 mm TL                    |
 |  Working Distance ​       |  10 mm (for all RI)  |  2 mm imaging depth with flat sample, 10 mm Ø sphere ​      | |  Working Distance ​       |  10 mm (for all RI)  |  2 mm imaging depth with flat sample, 10 mm Ø sphere ​      |
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 ===== Spherical and Chromatic Aberrations ===== ===== Spherical and Chromatic Aberrations =====
  
-Correction collars are commonly used to correct high-NA objectives for spherical aberrations when imaging through a variable-thickness coverslip and/or at different temperatures. ​ Other multi-immersion objectives have a correction collar for different media RI.  Even though our objectives have no correction collar, spherical aberrations are still within the diffraction limit for all media and wavelengths simulated due to design features [[cleared_tissue_objective#​ri_range|discussed below]], most notably the curved first surface. ​ The objectives have minimal chromatic aberration in the NIR spectrum to allow for multi-photon excitation.+Correction collars are commonly used to correct high-NA objectives for spherical aberrations when imaging through a variable-thickness coverslip and/or at different temperatures. ​ Other multi-immersion objectives have a correction collar for different media RI.  Even though our objectives have no correction collar, spherical aberrations are still within the diffraction limit for all media and wavelengths simulated due to design features [[multi-immersion_objectives#​ri_range|discussed below]], most notably the curved first surface. ​ The objectives have minimal chromatic aberration in the NIR spectrum to allow for multi-photon excitation.
  
 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 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 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 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.
multi-immersion_objectives.1555698587.txt.gz · Last modified: 2019/04/19 18:29 by jon