Intensity loss by refractive index mismatch

[Rusty Nicovich]

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In my lab we're working on quantifying the relative success of several
tissue clearing methods we are trialing.  The quick-and-dirty way of doing
the imaging is with a low NA air objective, which does allow us to image
through the few-hundred-micron depth of the cleared tissue.  Then taking
the intensity of the included probe (in this case a fairly uniform genetic
label) gives us a nice readout of the drop off as a function of distance.

For optimization purposes I'd like to be able to separate intensity
drop-off from scattering from that caused by the refractive index mismatch
between air and the cleared tissue.  I know that this mis-match has to be a
contributing factor, but what I don't know is a straightforward expression
for this relationship.  These papers are a good start:

http://www.eng.ox.ac.uk/som/publications/som_2001_2.PDF
http://www3.mpibpc.mpg.de/groups/hell/publications/pdf/J._Micr._169_391-405.pdf

but I'm not seeing a convenient relationship between the contributing
factors (ie refractive indices, objective NA, and depth) and intensity.  Is
there such a relationship that can be derived and concisely (even
approximately) given in a single equation?

Thanks,
Rusty

[mmodel]

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Hi Rusty -
You can find the formula for reflected intensity at the boundary in optics textbooks. But the other reason why refractive mismatch causes intensity loss is spherical aberration, which gets worse with depth and is hard to quantify.

Mike

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Rusty Nicovich
Sent: Tuesday, August 8, 2017 8:10 PM
To: [hidden email]
Subject: Intensity loss by refractive index mismatch

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In my lab we're working on quantifying the relative success of several tissue clearing methods we are trialing.  The quick-and-dirty way of doing the imaging is with a low NA air objective, which does allow us to image through the few-hundred-micron depth of the cleared tissue.  Then taking the intensity of the included probe (in this case a fairly uniform genetic
label) gives us a nice readout of the drop off as a function of distance.

For optimization purposes I'd like to be able to separate intensity drop-off from scattering from that caused by the refractive index mismatch between air and the cleared tissue.  I know that this mis-match has to be a contributing factor, but what I don't know is a straightforward expression for this relationship.  These papers are a good start:

https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.eng.ox.ac.uk%2Fsom%2Fpublications%2Fsom_2001_2.PDF&data=01%7C01%7Cmmodel%40KENT.EDU%7C027831c780684bbd820f08d4debb13bd%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1&sdata=3yGsjMhfrwFl3eLr46v8ObzFOpaJ7pNlPBz4han6gDo%3D&reserved=0
https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww3.mpibpc.mpg.de%2Fgroups%2Fhell%2Fpublications%2Fpdf%2FJ._Micr._169_391-405.pdf&data=01%7C01%7Cmmodel%40KENT.EDU%7C027831c780684bbd820f08d4debb13bd%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1&sdata=X7P0SJ7C3xxTD8C04%2Bgz1oBAaD8fdiGpLI3L8DQIL1o%3D&reserved=0

but I'm not seeing a convenient relationship between the contributing factors (ie refractive indices, objective NA, and depth) and intensity.  Is there such a relationship that can be derived and concisely (even
approximately) given in a single equation?

Thanks,
Rusty

[Benjamin Smith]

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One way to quickly separate the two would be to cut the tissue into a cube
(a vibratome would be great for this) and place the tissue in a dish filled
with the clearing solution, and measure the transmission with, and without,
the tissue.  Since the sides facing the transmitted light are planar and
orthogonal to the light, the Fresnel reflections due to the light
transitioning from the clearing solution to the tissue, and vice versa,
should be reduced to a negligible amount, such that almost all of the loss
will now be due to scattering.  To really hedge your bets, collimate the
light as much as possible (use a low NA objective and close the aperture
stop), so that all the light passes through the tissue orthogonal to the
tissue surface.

To directly quantify scattering in the above setup, put a second light
meter/camera orthogonal to the tissue and the collimated beam (basically
darkfield imaging).  This detector will pickup only the scattered light, so
as the transmission intensity goes down, the scattered intensity should go
up in a proportional manner.  In an ideal situation, the two should be
perfectly inversely proportional (as long as the tissue is optically
isotropic).  This will allow you to infer how much of the total loss was
due to scattering.  Without any tissue, the side detector should see
nothing, so this will give you the baseline loss without scattering.  Then,
put in the tissue, and this will give you how much of the total loss was
due to scattering.  As long as you keep the tissue cubes are similar
dimensions (and again assuming the tissues are optically isotropic), you
can then directly quantify the relative scattering loss between tissues.

Hope this helps,
  Ben Smith

On Tue, Aug 8, 2017 at 5:47 PM, MODEL, MICHAEL <[hidden email]> wrote:

> *****
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> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi Rusty -
> You can find the formula for reflected intensity at the boundary in optics
> textbooks. But the other reason why refractive mismatch causes intensity
> loss is spherical aberration, which gets worse with depth and is hard to
> quantify.
>
> Mike
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Rusty Nicovich
> Sent: Tuesday, August 8, 2017 8:10 PM
> To: [hidden email]
> Subject: Intensity loss by refractive index mismatch
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
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> Post images on https://na01.safelinks.protection.outlook.com/?url=
> http%3A%2F%2Fwww.imgur.com&data=01%7C01%7Cmmodel%40KENT.EDU%
> 7C027831c780684bbd820f08d4debb13bd%7Ce5a06f4a1ec44d018f73e7dd15f2
> 6134%7C1&sdata=tMYpwlL69VmX2HyC4hj80CachT1hVsJPJnB7d9fNVfs%3D&reserved=0
> and include the link in your posting.
> *****
>
> In my lab we're working on quantifying the relative success of several
> tissue clearing methods we are trialing.  The quick-and-dirty way of doing
> the imaging is with a low NA air objective, which does allow us to image
> through the few-hundred-micron depth of the cleared tissue.  Then taking
> the intensity of the included probe (in this case a fairly uniform genetic
> label) gives us a nice readout of the drop off as a function of distance.
>
> For optimization purposes I'd like to be able to separate intensity
> drop-off from scattering from that caused by the refractive index mismatch
> between air and the cleared tissue.  I know that this mis-match has to be a
> contributing factor, but what I don't know is a straightforward expression
> for this relationship.  These papers are a good start:
>
> https://na01.safelinks.protection.outlook.com/?url=
> http%3A%2F%2Fwww.eng.ox.ac.uk%2Fsom%2Fpublications%2Fsom_
> 2001_2.PDF&data=01%7C01%7Cmmodel%40KENT.EDU%7C027831c780684bbd820f08d4debb
> 13bd%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1&sdata=
> 3yGsjMhfrwFl3eLr46v8ObzFOpaJ7pNlPBz4han6gDo%3D&reserved=0
> https://na01.safelinks.protection.outlook.com/?url=
> http%3A%2F%2Fwww3.mpibpc.mpg.de%2Fgroups%2Fhell%2Fpublications%2Fpdf%2FJ._
> Micr._169_391-405.pdf&data=01%7C01%7Cmmodel%40KENT.EDU%
> 7C027831c780684bbd820f08d4debb13bd%7Ce5a06f4a1ec44d018f73e7dd15f2
> 6134%7C1&sdata=X7P0SJ7C3xxTD8C04%2Bgz1oBAaD8fdiGpLI3L8DQIL1o%3D&reserved=0
>
> but I'm not seeing a convenient relationship between the contributing
> factors (ie refractive indices, objective NA, and depth) and intensity.  Is
> there such a relationship that can be derived and concisely (even
> approximately) given in a single equation?
>
> Thanks,
> Rusty
>

--
Benjamin E. Smith, Ph. D.
Imaging Specialist, Vision Science
University of California, Berkeley
195 Life Sciences Addition
Berkeley, CA  94720-3200
Tel  [phone]
Fax [phone]
e-mail: [hidden email]
http://vision.berkeley.edu/?page_id=5635 <http://vision.berkeley.edu/>

[George McNamara]

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Hi Rusty,

I recommend going the other way and finding a refractive index matched
immersion lens for "the best you can do". See also:

http://onlinelibrary.wiley.com/doi/10.1002/jemt.20396/abstract


  2,2′-Thiodiethanol: A new water soluble mounting medium for high
  resolution optical microscopy

one click at
https://biochimie.umontreal.ca/wp-content/uploads/sites/37/2012/08/Thiodiethanol-new-water-mounting-media-2007.pdf 
(probably also available through S. Hell's web site).

I note that Fig 6 is a 1.46 NA objective lens, not the low NA
configuration you were specifically asking about. Fig 6 makes clear that
R.I. matching the immersion medium and mounting medium is ideal. That
paper did not do deconvolution (ultimate paragraph mentions it), now in
2017 can get 'instant gratification' with GPU deconvolution from
microvolution, SVI Huygens or AutoQuant. Perfect R.I. matching and
deconvolution is an ideal team, work with widefield, light sheet(s),
confocal, multiphoton.

//

If you are using an Olympus microscope, check out the 30x Silicone oil
objective lens [30x, 1.05 NA, 0.8mm working distance], using R.I.
matched mounting media (the match to estimate a different mag should be
easy). See:

https://microscopy-news.com/archive/olympus-30x-and-60x-silicone-oil-objectives-offer-brighter-higher-resolution-performance-for-live-cell-and-time-lapse-imaging/

http://confocal-microscopy-list.588098.n2.nabble.com/Silicone-oil-immersion-lenses-for-live-cell-imaging-td7578566.html

also

http://www.microscopy-analysis.com/editorials/editorial-listings/olympus-launches-40x-silicone-oil-objective-high-resolution-live-cell

"Silicone oil is very stable and does not have any of these issues; in
addition, it more closely matches the refractive index (N=1.404) of
intracellular components, making the new objective useful for imaging
into cells during for long-term, time-lapse experiments.

The silicon objective has a correction collar, so users can correct for
the spherical aberration that is present when imaging into a specimen
beyond a cover slip. Image resolution and contrast, along with
fluorescence performance, are optimized and maintained by adjusting this
collar."


Disclosure: 'my' image core at Johns Hopkins University hosts an Olympus
Imaging Center.

enjoy,

George



On 8/8/2017 8:10 PM, Rusty Nicovich wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> In my lab we're working on quantifying the relative success of several
> tissue clearing methods we are trialing.  The quick-and-dirty way of doing
> the imaging is with a low NA air objective, which does allow us to image
> through the few-hundred-micron depth of the cleared tissue.  Then taking
> the intensity of the included probe (in this case a fairly uniform genetic
> label) gives us a nice readout of the drop off as a function of distance.
>
> For optimization purposes I'd like to be able to separate intensity
> drop-off from scattering from that caused by the refractive index mismatch
> between air and the cleared tissue.  I know that this mis-match has to be a
> contributing factor, but what I don't know is a straightforward expression
> for this relationship.  These papers are a good start:
>
> http://www.eng.ox.ac.uk/som/publications/som_2001_2.PDF

Refractive-index-mismatch induced aberrations in single-photon and
two-photon microscopy and the use of aberration correction
> http://www3.mpibpc.mpg.de/groups/hell/publications/pdf/J._Micr._169_391-405.pdf
>
> but I'm not seeing a convenient relationship between the contributing
> factors (ie refractive indices, objective NA, and depth) and intensity.  Is
> there such a relationship that can be derived and concisely (even
> approximately) given in a single equation?
>
> Thanks,
> Rusty

--


George McNamara, PhD
Baltimore, MD 21231
[hidden email]
https://www.linkedin.com/in/georgemcnamara
https://works.bepress.com/gmcnamara/75   (may need to use Microsoft Edge or Firefox, rather than Google Chrome)
http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650
http://confocal.jhu.edu

July 2017 Current Protocols article, open access:
UNIT 4.4 Microscopy and Image Analysis
http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/abstract
supporting materials direct link is
http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/full#hg0404-sec-0023
figures at
http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/figures