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multi-immersion_objectives [2019/10/14 13:24]
jon [Location of Back Focal Plane]
multi-immersion_objectives [2020/01/07 17:33] (current)
jon [Numerical Aperture and Resolution] note RI
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 __54-12-8__:​ NA = 0.483 * RI __54-12-8__:​ NA = 0.483 * RI
  
-The diffraction-limited resolution is a function of the lens NA which depends on 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:
  
 Res<​sub>​x,​y</​sub>​ = 0.61 * λ / NA Res<​sub>​x,​y</​sub>​ = 0.61 * λ / NA
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 Res<​sub>​z</​sub>​ = 2 * λ * RI / NA<​sup>​2</​sup>​ Res<​sub>​z</​sub>​ = 2 * λ * RI / NA<​sup>​2</​sup>​
  
-From these expressions it is clear that axial resolution is worse than lateral resolution. ​ For 500 nm light with NA 0.4, the lateral resolution is 0.76 μm and the axial resolution (depth of field) is 9.1 μm (at NA 0.7, the numbers are 0.44 μm and 3.0 μm).  The dSPIM/​diSPIM geometry provides the opportunity to overcome poor axial resolution by combining two views of the same object from orthogonal directions so that each feature is seen from at least one high-resolution vantage point, all without needing to move the sample. ​ Some home-built imaging systems use the similar concept but rotate the sample to be imaged from different directions.+From these expressions it is clear that axial resolution is worse than lateral resolution. ​ For 500 nm light at RI 1.45, the lateral resolution is 0.76 μm and the axial resolution (depth of field) is 9.1 μm for the NA 0.4 lens (for the NA 0.7 lens, the numbers are 0.44 μm and 3.0 μm).  The dSPIM/​diSPIM geometry provides the opportunity to overcome poor axial resolution by combining two views of the same object from orthogonal directions so that each feature is seen from at least one high-resolution vantage point, all without needing to move the sample. ​ Some home-built imaging systems use the similar concept but rotate the sample to be imaged from different directions.
  
 In light sheet microscopy, commonly only a small fraction of the objective'​s NA is used for light sheet generation. ​ The baseline axial resolution is the depth of field of the detection objective, but can be improved if the light sheet is thinner than the depth of field. ​ Whether or not this happens depends on the illumination NA and hence imaging FOV.  In many cases light sheet provides no true resolution benefit even though out of focus florescence will be reduced ("​optical sectioning"​) which improves SNR and image quality. ​ There are ways of creating very thin light sheets to increase axial resolution (e.g. Bessel beams) but they generally have other undesirable properties including extra complexity/​cost,​ large amounts of out of focus light, and/or being extremely sensitive to scattering or sample inhomogeneity.  ​ In light sheet microscopy, commonly only a small fraction of the objective'​s NA is used for light sheet generation. ​ The baseline axial resolution is the depth of field of the detection objective, but can be improved if the light sheet is thinner than the depth of field. ​ Whether or not this happens depends on the illumination NA and hence imaging FOV.  In many cases light sheet provides no true resolution benefit even though out of focus florescence will be reduced ("​optical sectioning"​) which improves SNR and image quality. ​ There are ways of creating very thin light sheets to increase axial resolution (e.g. Bessel beams) but they generally have other undesirable properties including extra complexity/​cost,​ large amounts of out of focus light, and/or being extremely sensitive to scattering or sample inhomogeneity.  ​
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multi-immersion_objectives.1571059475.txt.gz · Last modified: 2019/10/14 13:24 by jon