objective inverters, and CLARITY vs. scale

[Feinstein, Timothy]

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Hello all,

A user is eager to try both optogenetics and one of the new brain-clarifying techniques, CLARITY, but we only have inverted microscopes at the moment.  If anyone has experience with objective inverters like this one, could you let me know whether you have found it useful for traditional and multiphoton confocal imaging?

http://www.lsmtech.com/product_objective_inverter.html

Also, has anyone compared CLARITY vs. scale or any other clarifying technique?  Any relative strengths and weaknesses to help in choosing between the two would be much appreciated.

Thanks and all the best,


TF

Timothy Feinstein, Ph.D. | Confocal Director
333 Bostwick Ave., N.E., Grand Rapids, Michigan 49503
Phone: [phone] | Email: [hidden email]<mailto:[hidden email]>

[Periasamy, Ammasi (ap3t)]

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The LSM Tech objective inverter works ok for intravital imaging.
You have to choose the right objective lens to transmit the IR and collect the visible spectrum signal.

Dr. Ammasi Periasamy
Professor & Center Director
W.M. Keck Center for Cellular Imaging (KCCI)
(University of Virginia Imaging Center)
Mail or FedEx or UPS:
Keck Center for Cellular Imaging
University of Virginia
Biology, Gilmer Hall, 485 McCormick Rd.
Charlottesville, VA 22904, USA
Office Location:
Physical and Life Sciences Building (PLSB-B005), White Head Rd.,
Voice: 434-243-7602 (Office); 982-4869 (lab)
Fax:[phone]; Email:[hidden email]
http://www.kcci.virginia.edu/contact/peri.php
************************
13th Annual Workshop on FRET & FLIM Microscopy, March 10-14, 2014
http://www.kcci.virginia.edu/workshop/workshop2014/index.php
*************************

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Feinstein, Timothy
Sent: Tuesday, October 22, 2013 9:47 AM
To: [hidden email]
Subject: objective inverters, and CLARITY vs. scale

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Hello all,

A user is eager to try both optogenetics and one of the new brain-clarifying techniques, CLARITY, but we only have inverted microscopes at the moment.  If anyone has experience with objective inverters like this one, could you let me know whether you have found it useful for traditional and multiphoton confocal imaging?

http://www.lsmtech.com/product_objective_inverter.html

Also, has anyone compared CLARITY vs. scale or any other clarifying technique?  Any relative strengths and weaknesses to help in choosing between the two would be much appreciated.

Thanks and all the best,


TF

Timothy Feinstein, Ph.D. | Confocal Director
333 Bostwick Ave., N.E., Grand Rapids, Michigan 49503
Phone: [phone] | Email: [hidden email]<mailto:[hidden email]>

[Craig Brideau]

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We are trying Clarity, Scale, and SeeDB in our lab to compare the three
methods.  The three techniques are a trade-off between how involved the
process is and how clear the tissue ends up.  SeeDB is a Fructose solution
method that gets the tissue more translucent.  It helps you get a bit
further (a few extra 10's of um approx) than untreated and it is very
simple to set up and takes very little time (a few days).  Clarity is
fairly complex to implement since you need to set up a pair of electrodes
to pull ions out of the tissue.  The process also generates hydrogen
bubbles which can be tricky to deal with.  It took us several iterations of
chamber design to get something that would work.  It does seem to give the
best results when it works right, but the process is a bit twitchy and
takes some practice to achieve the best result.  It also takes a while, so
if you screw up you just wasted 2 or 3 weeks. Scale seems to be in between;
we haven't finished our tests with it yet so I will have more information
on results shortly, but basically it is like Clarity but you don't have to
worry about electrodes so it is simpler to set up.  It is still time
consuming but may be more repeatable.  I will let you know how it works out.

Craig


On Tue, Oct 22, 2013 at 9:11 AM, Periasamy, Ammasi (ap3t) <
[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> The LSM Tech objective inverter works ok for intravital imaging.
> You have to choose the right objective lens to transmit the IR and collect
> the visible spectrum signal.
>
> Dr. Ammasi Periasamy
> Professor & Center Director
> W.M. Keck Center for Cellular Imaging (KCCI)
> (University of Virginia Imaging Center)
> Mail or FedEx or UPS:
> Keck Center for Cellular Imaging
> University of Virginia
> Biology, Gilmer Hall, 485 McCormick Rd.
> Charlottesville, VA 22904, USA
> Office Location:
> Physical and Life Sciences Building (PLSB-B005), White Head Rd.,
> Voice: 434-243-7602 (Office); 982-4869 (lab)
> Fax:[phone]; Email:[hidden email]
> http://www.kcci.virginia.edu/contact/peri.php
> ************************
> 13th Annual Workshop on FRET & FLIM Microscopy, March 10-14, 2014
> http://www.kcci.virginia.edu/workshop/workshop2014/index.php
> *************************
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Feinstein, Timothy
> Sent: Tuesday, October 22, 2013 9:47 AM
> To: [hidden email]
> Subject: objective inverters, and CLARITY vs. scale
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello all,
>
> A user is eager to try both optogenetics and one of the new
> brain-clarifying techniques, CLARITY, but we only have inverted microscopes
> at the moment.  If anyone has experience with objective inverters like this
> one, could you let me know whether you have found it useful for traditional
> and multiphoton confocal imaging?
>
> http://www.lsmtech.com/product_objective_inverter.html
>
> Also, has anyone compared CLARITY vs. scale or any other clarifying
> technique?  Any relative strengths and weaknesses to help in choosing
> between the two would be much appreciated.
>
> Thanks and all the best,
>
>
> TF
>
> Timothy Feinstein, Ph.D. | Confocal Director
> 333 Bostwick Ave., N.E., Grand Rapids, Michigan 49503
> Phone: [phone] | Email: [hidden email]<mailto:
> [hidden email]>
>

[Sean Speese-2]

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For those interested in imaging deeper into tissue with oil immersion lenses, we
have obtained nice results using the CFM media series from Citifluor.  I think
this media has been mentioned on the listserve in the past. In particular, we
have looked at SCALE versus CFM3 in brain slice tissue expressing a nuclear
GFP .  We donated our image of these results to Citifluor for its website
(http://www.citifluor.com/non_hardening_antifadents.php).  I have also had
great success with clearing Drosophila tissue with CFM mounting medias.  In my
hands, CFM clears tissue much faster than Scale buffer.  It seems to work on
the order of minutes for clearing, and due to the medias RI of 1.52, there is
little to no spherical aberration as you move away from the back of the
coverslip.  In quantitative experiments I have conducted, I saw almost no loss
of light from molecular probes beads at the back of the coverslip versus ones
140 um away from the coverslip, when mounted in CFM. I am uncertain of why
CFM clears so well, but there is a "proprietary" surfactant in the media that may
have something to do with its clearing properties.  

The only things to watch out for are 1) some shrinkage that occurs in CFM
medias (~25-30% in our quick and dirty estimate) and 2) It seems that the
media can disrupt weaker antibody-epitope interactions.  To get around this we
post-fix our samples after washing out the secondary.  Generally 20-30 minutes
in 4% para or 4% para/.1% Glut.  3) Lastly, we have found that GFP signals will
degrade after some time in CFM, so best to image right away.  

There is no commercial interest here, just a satisfied customer.  

Sean

Sean Speese, Ph.D | OHSU

[Feinstein, Timothy]

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Many thanks to everyone who responded both on and off the list.  Your
feedback has been a great help.

All the best,


TF

Timothy Feinstein, Ph.D. | Confocal Director, Van Andel Institute
333 Bostwick Ave., N.E., Grand Rapids, Michigan 49503
Phone: [phone] | Email: [hidden email]







On 10/22/13 11:03 PM, "Sean Speese" <[hidden email]> wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>For those interested in imaging deeper into tissue with oil immersion
>lenses, we
>have obtained nice results using the CFM media series from Citifluor.  I
>think
>this media has been mentioned on the listserve in the past. In
>particular, we
>have looked at SCALE versus CFM3 in brain slice tissue expressing a
>nuclear
>GFP .  We donated our image of these results to Citifluor for its website
>(http://www.citifluor.com/non_hardening_antifadents.php).  I have also
>had
>great success with clearing Drosophila tissue with CFM mounting medias.
>In my
>hands, CFM clears tissue much faster than Scale buffer.  It seems to work
>on
>the order of minutes for clearing, and due to the medias RI of 1.52,
>there is
>little to no spherical aberration as you move away from the back of the
>coverslip.  In quantitative experiments I have conducted, I saw almost no
>loss
>of light from molecular probes beads at the back of the coverslip versus
>ones
>140 um away from the coverslip, when mounted in CFM. I am uncertain of
>why
>CFM clears so well, but there is a "proprietary" surfactant in the media
>that may
>have something to do with its clearing properties.
>
>The only things to watch out for are 1) some shrinkage that occurs in CFM
>medias (~25-30% in our quick and dirty estimate) and 2) It seems that the
>media can disrupt weaker antibody-epitope interactions.  To get around
>this we
>post-fix our samples after washing out the secondary.  Generally 20-30
>minutes
>in 4% para or 4% para/.1% Glut.  3) Lastly, we have found that GFP
>signals will
>degrade after some time in CFM, so best to image right away.
>
>There is no commercial interest here, just a satisfied customer.
>
>Sean
>
>Sean Speese, Ph.D | OHSU

[Trevor Wardill]

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Dear Craig and Confocal ListServe,

You may also consider trying thiodiethanol (TDE) as a clearing agent.  We
have had much success with this for both Dragonfly ganglia and cephalopod
clearing tissue up to 500 um thick and imaging with confocal.  We have
published this protocol at Cold Spring Harbor Protocols. You can optimize
all the imaging at the refractive index of oil and avoid many aberrations.
The only issue is that you need a long working distance oil objective to get
the best images.  Zero thickness cover glass also help increase the working
distance (but alter optics slightly).

Gonzalez-Bellido PT, Wardill TJ. 2012. Labeling and confocal imaging of
neurons in thick invertebrate tissue samples. Cold Spring Harb Protoc.
http://dx.doi.org/10.1101/pdb.prot069625

You can do this clearing in about 5 hours or considerably faster using a
specialized fixation microwave.  It does not seem to alter any labeling in
our hands for many months.

Regards,

Trevor Wardill and Paloma Gonzalez-Bellido
Marine Biological Laboratory
Woods Hole MA.