TIRF depth calibration

[Graham Wright]

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Hello,

Does anyone have a routine way of calibrating the effective penetration
depth of TIRF imaging?

I've found this paper:

Gell, et al. 2009 TIRF microscopy evanescent field calibration using tilted
fluorescent microtubules. Journal of Microscopy 234: 38-46
http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2818.2009.03147.x/references

but wondered if other people have different ways?

Thanks,
Graham

---
Graham Wright

Institute of Medical Biology
8A Biomedical Grove, #06-06 Immunos, Singapore 138648

E:   [hidden email]
W:  http://www.imb.a-star.edu.sg/imu/

[Sebastian Rhode]

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Hi Graham,

one method which definitly works (I tried it out by myself) is the use of an
combined TIRF-AFM setup. You just have to couple fluorescent beads to the
tip of your AFM an record pictures while approaching/or moving away the
coverslip surface. Unfortunately an AFM is really expensive.

So I found some other methods, which might work as well --> see
TIRF_Introduction.pdf, which I send to you directly (LIST server does not
accepted this pdf-file).

One methodes uses an objective piezo-drive and a pencil and the second one
stained beads or a stained solution with intransparent beads.

I case of questions, feel free to contact me directly.

Cheers,
Sebastian


Dr. Sebastian Rhode
Project Manager
Research & Development

TILL Photonics GmbH
Lochhamer Schlag 21
D- 82166 Gräfelfing, Germany
Phone   [phone]
Fax     [phone]
www.till-photonics.com

[John Oreopoulos]

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Sebastian, I remember reading a publication from about ten years ago that talked about mounting a pencil in a piezoelectric micro-manipulator and sticking a fluorescent bead on the end of the pencil tip. This was very similar to the AFM method. Is it the same thing?

Graham, there is a fairly detailed discussion on this topic from a few years ago on the archive that talks about a few other ways to measure the actual TIRF penetration depth (as opposed to calculating it based on an assumed refractive index and crudely measured incident angle):

http://lists.umn.edu/cgi-bin/wa?A3=ind0702&L=CONFOCALMICROSCOPY&E=quoted-printable&P=827770&B=--Apple-Mail-181-588547976&T=text%2Fhtml;%20charset=ISO-8859-1

I've tried the AFM method as well - it works, but my main complaint with this and some of the other protocols is that they require complicated and expensive equipment, and can be difficult to get right. The method involving fluorescent microtubles by Jorg Enderlein's group is a fairly new one that is elegantly simple, but again requires you to have access to some fairly special reagents that might not be found in every lab. A few months ago I came across yet another older method that had evaded my previous searches on the topic. This one is similar to the others that involve imaging fluorescent microbeads, but I like this because all it requires is a microscope with a motorized drive on the z-axis:

Steyer, J.A. and W. Almers, Tracking single secretory granules in live chromaffin cells by evanescent-field fluorescence microscopy. Biophysical Journal, 1999. 76(4): p. 2262-2271.

John Oreopoulos
Research Assistant
Spectral Applied Research
9078 Leslie Street, Unit 11
Richmond Hill
Ontario, Canada


On 2010-11-24, at 6:54 AM, Sebastian Rhode wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Graham,
>
> one method which definitly works (I tried it out by myself) is the use of an
> combined TIRF-AFM setup. You just have to couple fluorescent beads to the
> tip of your AFM an record pictures while approaching/or moving away the
> coverslip surface. Unfortunately an AFM is really expensive.
>
> So I found some other methods, which might work as well --> see
> TIRF_Introduction.pdf, which I send to you directly (LIST server does not
> accepted this pdf-file).
>
> One methodes uses an objective piezo-drive and a pencil and the second one
> stained beads or a stained solution with intransparent beads.
>
> I case of questions, feel free to contact me directly.
>
> Cheers,
> Sebastian
>
>
> Dr. Sebastian Rhode
> Project Manager
> Research & Development
>
> TILL Photonics GmbH
> Lochhamer Schlag 21
> D- 82166 Gräfelfing, Germany
> Phone   [phone]
> Fax     [phone]
> www.till-photonics.com

[lechristophe]

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To join, leave or search the confocal microscopy listserv, go to:
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There is also a simple method in Fiolka et al. Miocroscoc. res Tech 2007 :
http://www.neuro.nano-optics.ethz.ch/publications/fiolka.pdf

<http://www.neuro.nano-optics.ethz.ch/publications/fiolka.pdf>involving an
inclined coverslip with attached fluorescent beads. I didn't try it myself
so I can't tell if it works, but seems simpler than AFM or microtubules
method.

Christophe

On Wed, Nov 24, 2010 at 16:32, John Oreopoulos
<[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Sebastian, I remember reading a publication from about ten years ago that
> talked about mounting a pencil in a piezoelectric micro-manipulator and
> sticking a fluorescent bead on the end of the pencil tip. This was very
> similar to the AFM method. Is it the same thing?
>
> Graham, there is a fairly detailed discussion on this topic from a few
> years ago on the archive that talks about a few other ways to measure the
> actual TIRF penetration depth (as opposed to calculating it based on an
> assumed refractive index and crudely measured incident angle):
>
>
> http://lists.umn.edu/cgi-bin/wa?A3=ind0702&L=CONFOCALMICROSCOPY&E=quoted-printable&P=827770&B=--Apple-Mail-181-588547976&T=text%2Fhtml;%20charset=ISO-8859-1
>
> I've tried the AFM method as well - it works, but my main complaint with
> this and some of the other protocols is that they require complicated and
> expensive equipment, and can be difficult to get right. The method involving
> fluorescent microtubles by Jorg Enderlein's group is a fairly new one that
> is elegantly simple, but again requires you to have access to some fairly
> special reagents that might not be found in every lab. A few months ago I
> came across yet another older method that had evaded my previous searches on
> the topic. This one is similar to the others that involve imaging
> fluorescent microbeads, but I like this because all it requires is a
> microscope with a motorized drive on the z-axis:
>
> Steyer, J.A. and W. Almers, Tracking single secretory granules in live
> chromaffin cells by evanescent-field fluorescence microscopy. Biophysical
> Journal, 1999. 76(4): p. 2262-2271.
>
> John Oreopoulos
> Research Assistant
> Spectral Applied Research
> 9078 Leslie Street, Unit 11
> Richmond Hill
> Ontario, Canada
>
>
> On 2010-11-24, at 6:54 AM, Sebastian Rhode wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Hi Graham,
> >
> > one method which definitly works (I tried it out by myself) is the use of
> an
> > combined TIRF-AFM setup. You just have to couple fluorescent beads to the
> > tip of your AFM an record pictures while approaching/or moving away the
> > coverslip surface. Unfortunately an AFM is really expensive.
> >
> > So I found some other methods, which might work as well --> see
> > TIRF_Introduction.pdf, which I send to you directly (LIST server does not
> > accepted this pdf-file).
> >
> > One methodes uses an objective piezo-drive and a pencil and the second
> one
> > stained beads or a stained solution with intransparent beads.
> >
> > I case of questions, feel free to contact me directly.
> >
> > Cheers,
> > Sebastian
> >
> >
> > Dr. Sebastian Rhode
> > Project Manager
> > Research & Development
> >
> > TILL Photonics GmbH
> > Lochhamer Schlag 21
> > D- 82166 Gräfelfing, Germany
> > Phone   [phone]
> > Fax     [phone]
> > www.till-photonics.com
>

[John Oreopoulos]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

And for the more adventurous types out there, you can measure your penetration depth using fluorescence correlation spectroscopy (FCS) if you have the right hardware:

Harlepp, S., et al., Subnanometric measurements of evanescent wave penetration depth using total internal reflection microscopy combined with fluorescent correlation spectroscopy. Applied Physics Letters, 2004. 85(17): p. 3917-3919.

John Oreopoulos
Research Assistant
Spectral Applied Research
9078 Leslie Street, Unit 11
Richmond Hill
Ontario, Canada


On 2010-11-24, at 10:37 AM, Christophe Leterrier wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> There is also a simple method in Fiolka et al. Miocroscoc. res Tech 2007 :
> http://www.neuro.nano-optics.ethz.ch/publications/fiolka.pdf
>
> <http://www.neuro.nano-optics.ethz.ch/publications/fiolka.pdf>involving an
> inclined coverslip with attached fluorescent beads. I didn't try it myself
> so I can't tell if it works, but seems simpler than AFM or microtubules
> method.
>
> Christophe
>
> On Wed, Nov 24, 2010 at 16:32, John Oreopoulos
> <[hidden email]>wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Sebastian, I remember reading a publication from about ten years ago that
>> talked about mounting a pencil in a piezoelectric micro-manipulator and
>> sticking a fluorescent bead on the end of the pencil tip. This was very
>> similar to the AFM method. Is it the same thing?
>>
>> Graham, there is a fairly detailed discussion on this topic from a few
>> years ago on the archive that talks about a few other ways to measure the
>> actual TIRF penetration depth (as opposed to calculating it based on an
>> assumed refractive index and crudely measured incident angle):
>>
>>
>> http://lists.umn.edu/cgi-bin/wa?A3=ind0702&L=CONFOCALMICROSCOPY&E=quoted-printable&P=827770&B=--Apple-Mail-181-588547976&T=text%2Fhtml;%20charset=ISO-8859-1
>>
>> I've tried the AFM method as well - it works, but my main complaint with
>> this and some of the other protocols is that they require complicated and
>> expensive equipment, and can be difficult to get right. The method involving
>> fluorescent microtubles by Jorg Enderlein's group is a fairly new one that
>> is elegantly simple, but again requires you to have access to some fairly
>> special reagents that might not be found in every lab. A few months ago I
>> came across yet another older method that had evaded my previous searches on
>> the topic. This one is similar to the others that involve imaging
>> fluorescent microbeads, but I like this because all it requires is a
>> microscope with a motorized drive on the z-axis:
>>
>> Steyer, J.A. and W. Almers, Tracking single secretory granules in live
>> chromaffin cells by evanescent-field fluorescence microscopy. Biophysical
>> Journal, 1999. 76(4): p. 2262-2271.
>>
>> John Oreopoulos
>> Research Assistant
>> Spectral Applied Research
>> 9078 Leslie Street, Unit 11
>> Richmond Hill
>> Ontario, Canada
>>
>>
>> On 2010-11-24, at 6:54 AM, Sebastian Rhode wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Hi Graham,
>>>
>>> one method which definitly works (I tried it out by myself) is the use of
>> an
>>> combined TIRF-AFM setup. You just have to couple fluorescent beads to the
>>> tip of your AFM an record pictures while approaching/or moving away the
>>> coverslip surface. Unfortunately an AFM is really expensive.
>>>
>>> So I found some other methods, which might work as well --> see
>>> TIRF_Introduction.pdf, which I send to you directly (LIST server does not
>>> accepted this pdf-file).
>>>
>>> One methodes uses an objective piezo-drive and a pencil and the second
>> one
>>> stained beads or a stained solution with intransparent beads.
>>>
>>> I case of questions, feel free to contact me directly.
>>>
>>> Cheers,
>>> Sebastian
>>>
>>>
>>> Dr. Sebastian Rhode
>>> Project Manager
>>> Research & Development
>>>
>>> TILL Photonics GmbH
>>> Lochhamer Schlag 21
>>> D- 82166 Gräfelfing, Germany
>>> Phone   [phone]
>>> Fax     [phone]
>>> www.till-photonics.com
>>

[mcammer]

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To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Related to the axial resolution issue (which I'll briefly address at the end), I have a question regarding the uniformity of excitation over the TIRF field.

At the end of http://lists.umn.edu/cgi-bin/wa?A3=ind0702&L=CONFOCALMICROSCOPY&E=quoted-printable&P=827770&B=--Apple-Mail-181-588547976&T=text%2Fhtml;%20charset=ISO-8859-1
there is a reference to a solution to non-uniformity of excitation across the TIRF field due to the coherent light source.  The solution is to spin a wedge in the light source and try to have multiple spins per exposure time.  

I recall back in the early 1990s reading a paper on Nomarski optics to image microtubule dynamics in vitro (maybe M Sheetz or RD Vale?) which used a green laser for illumination and spinning diffusion filter in the light path to remove coherence and, thus, speckle.

Does anybody have such a modification to the light path and would you please share your results?


On the axial resolution issue, about six years ago to estimate depth penetration of the TIRF field in the simple case of a glass, thin protein coating, and PBS interface with the Olympus TIRF system of the time, we stuck 4 um Tetraspeck beads to a 1.5 coverslip with polylysine.  As we adjusted the micrometer the beads appeared smaller and larger.  Using simple trig, we estimated the depth of the field.  Of course, this was far from perfect since the beads were big and tend to squish down where they stick, but we got a rough idea.  

-Michael




-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of John Oreopoulos
Sent: Wednesday, November 24, 2010 10:33 AM
To: [hidden email]
Subject: Re: TIRF depth calibration



------------------------------------------------------------
This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email.
=================================

[lechristophe]

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Michael, no direct experience with what you suggest but these two
papers may help :

The article I linked in my previous post (Fiolka Microsc Res Tech
2007) is primarily about uniformizing the field of view in TIRF by
azimuthal rotation of the laser beam (that circles around the edge of
the objective) :
http://www.neuro.nano-optics.ethz.ch/publications/fiolka.pdf

The rotating prism method is used and details provided in this paper
by the Axelrod lab : Mattheyses J Microsc Res 2006
http://deepblue.lib.umich.edu/bitstream/2027.42/55799/1/20334_ftp.pdf

Christophe


On Wed, Nov 24, 2010 at 17:24, Cammer, Michael
<[hidden email]> wrote:

>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Related to the axial resolution issue (which I'll briefly address at the end), I have a question regarding the uniformity of excitation over the TIRF field.
>
> At the end of http://lists.umn.edu/cgi-bin/wa?A3=ind0702&L=CONFOCALMICROSCOPY&E=quoted-printable&P=827770&B=--Apple-Mail-181-588547976&T=text%2Fhtml;%20charset=ISO-8859-1
> there is a reference to a solution to non-uniformity of excitation across the TIRF field due to the coherent light source.  The solution is to spin a wedge in the light source and try to have multiple spins per exposure time.
>
> I recall back in the early 1990s reading a paper on Nomarski optics to image microtubule dynamics in vitro (maybe M Sheetz or RD Vale?) which used a green laser for illumination and spinning diffusion filter in the light path to remove coherence and, thus, speckle.
>
> Does anybody have such a modification to the light path and would you please share your results?
>
>
> On the axial resolution issue, about six years ago to estimate depth penetration of the TIRF field in the simple case of a glass, thin protein coating, and PBS interface with the Olympus TIRF system of the time, we stuck 4 um Tetraspeck beads to a 1.5 coverslip with polylysine.  As we adjusted the micrometer the beads appeared smaller and larger.  Using simple trig, we estimated the depth of the field.  Of course, this was far from perfect since the beads were big and tend to squish down where they stick, but we got a rough idea.
>
> -Michael
>
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of John Oreopoulos
> Sent: Wednesday, November 24, 2010 10:33 AM
> To: [hidden email]
> Subject: Re: TIRF depth calibration
>
>
>
> ------------------------------------------------------------
> This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email.
> =================================

[lechristophe]

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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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Sorry for my previous email, I didn't see the Axelrod paper was
precisely what you pointed in you email...

Christophe

On Wed, Nov 24, 2010 at 17:37, Christophe Leterrier
<[hidden email]> wrote:

> Michael, no direct experience with what you suggest but these two
> papers may help :
>
> The article I linked in my previous post (Fiolka Microsc Res Tech
> 2007) is primarily about uniformizing the field of view in TIRF by
> azimuthal rotation of the laser beam (that circles around the edge of
> the objective) :
> http://www.neuro.nano-optics.ethz.ch/publications/fiolka.pdf
>
> The rotating prism method is used and details provided in this paper
> by the Axelrod lab : Mattheyses J Microsc Res 2006
> http://deepblue.lib.umich.edu/bitstream/2027.42/55799/1/20334_ftp.pdf
>
> Christophe
>
>
> On Wed, Nov 24, 2010 at 17:24, Cammer, Michael
> <[hidden email]> wrote:
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Related to the axial resolution issue (which I'll briefly address at the end), I have a question regarding the uniformity of excitation over the TIRF field.
>>
>> At the end of http://lists.umn.edu/cgi-bin/wa?A3=ind0702&L=CONFOCALMICROSCOPY&E=quoted-printable&P=827770&B=--Apple-Mail-181-588547976&T=text%2Fhtml;%20charset=ISO-8859-1
>> there is a reference to a solution to non-uniformity of excitation across the TIRF field due to the coherent light source.  The solution is to spin a wedge in the light source and try to have multiple spins per exposure time.
>>
>> I recall back in the early 1990s reading a paper on Nomarski optics to image microtubule dynamics in vitro (maybe M Sheetz or RD Vale?) which used a green laser for illumination and spinning diffusion filter in the light path to remove coherence and, thus, speckle.
>>
>> Does anybody have such a modification to the light path and would you please share your results?
>>
>>
>> On the axial resolution issue, about six years ago to estimate depth penetration of the TIRF field in the simple case of a glass, thin protein coating, and PBS interface with the Olympus TIRF system of the time, we stuck 4 um Tetraspeck beads to a 1.5 coverslip with polylysine.  As we adjusted the micrometer the beads appeared smaller and larger.  Using simple trig, we estimated the depth of the field.  Of course, this was far from perfect since the beads were big and tend to squish down where they stick, but we got a rough idea.
>>
>> -Michael
>>
>>
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of John Oreopoulos
>> Sent: Wednesday, November 24, 2010 10:33 AM
>> To: [hidden email]
>> Subject: Re: TIRF depth calibration
>>
>>
>>
>> ------------------------------------------------------------
>> This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email.
>> =================================
>

[Mark Cannell]

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To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi All,

FCS might be a good method to solve what may be a very difficult  
measurement. I'm not sure how methods that actually alter the local  
refractive index' (e.g. beads that are of significant to size to the  
TIRF field or inclined coverslips) can give the 'correct' answer  
though. Furthermore, measuring the distance is of little value unless  
the optical properties of  your testing sample are exactly the same as  
the experimental. For example, measuring TIRF depth in water does not  
give the TIRF depth inside attached cells (because their refractive  
indexes are different). Since the TIRF field is determined by  
Maxwell's equations, could one not just calculate the TIRF field since  
you can can measure the incident angle of the input light and it's  
divergence quite accurately and *may* know the refractive indices in  
the system?

My 2c

Cheers Mark


On 25/11/2010, at 5:20 AM, John Oreopoulos wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> And for the more adventurous types out there, you can measure your  
> penetration depth using fluorescence correlation spectroscopy (FCS)  
> if you have the right hardware:
>
> Harlepp, S., et al., Subnanometric measurements of evanescent wave  
> penetration depth using total internal reflection microscopy  
> combined with fluorescent correlation spectroscopy. Applied Physics  
> Letters, 2004. 85(17): p. 3917-3919.
>
> John Oreopoulos
> Research Assistant
> Spectral Applied Research
> 9078 Leslie Street, Unit 11
> Richmond Hill
> Ontario, Canada
>
>
> On 2010-11-24, at 10:37 AM, Christophe Leterrier wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> There is also a simple method in Fiolka et al. Miocroscoc. res Tech  
>> 2007 :
>> http://www.neuro.nano-optics.ethz.ch/publications/fiolka.pdf
>>
>> <http://www.neuro.nano-optics.ethz.ch/publications/ 
>> fiolka.pdf>involving an
>> inclined coverslip with attached fluorescent beads. I didn't try it  
>> myself
>> so I can't tell if it works, but seems simpler than AFM or  
>> microtubules
>> method.
>>
>> Christophe
>>
>> On Wed, Nov 24, 2010 at 16:32, John Oreopoulos
>> <[hidden email]>wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Sebastian, I remember reading a publication from about ten years  
>>> ago that
>>> talked about mounting a pencil in a piezoelectric micro-
>>> manipulator and
>>> sticking a fluorescent bead on the end of the pencil tip. This was  
>>> very
>>> similar to the AFM method. Is it the same thing?
>>>
>>> Graham, there is a fairly detailed discussion on this topic from a  
>>> few
>>> years ago on the archive that talks about a few other ways to  
>>> measure the
>>> actual TIRF penetration depth (as opposed to calculating it based  
>>> on an
>>> assumed refractive index and crudely measured incident angle):
>>>
>>>
>>> http://lists.umn.edu/cgi-bin/wa?A3=ind0702&L=CONFOCALMICROSCOPY&E=quoted-printable&P=827770&B=--Apple-Mail-181-588547976&T=text%2Fhtml;%20charset=ISO-8859-1
>>>
>>> I've tried the AFM method as well - it works, but my main  
>>> complaint with
>>> this and some of the other protocols is that they require  
>>> complicated and
>>> expensive equipment, and can be difficult to get right. The method  
>>> involving
>>> fluorescent microtubles by Jorg Enderlein's group is a fairly new  
>>> one that
>>> is elegantly simple, but again requires you to have access to some  
>>> fairly
>>> special reagents that might not be found in every lab. A few  
>>> months ago I
>>> came across yet another older method that had evaded my previous  
>>> searches on
>>> the topic. This one is similar to the others that involve imaging
>>> fluorescent microbeads, but I like this because all it requires is a
>>> microscope with a motorized drive on the z-axis:
>>>
>>> Steyer, J.A. and W. Almers, Tracking single secretory granules in  
>>> live
>>> chromaffin cells by evanescent-field fluorescence microscopy.  
>>> Biophysical
>>> Journal, 1999. 76(4): p. 2262-2271.
>>>
>>> John Oreopoulos
>>> Research Assistant
>>> Spectral Applied Research
>>> 9078 Leslie Street, Unit 11
>>> Richmond Hill
>>> Ontario, Canada
>>>
>>>
>>> On 2010-11-24, at 6:54 AM, Sebastian Rhode wrote:
>>>
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> Hi Graham,
>>>>
>>>> one method which definitly works (I tried it out by myself) is  
>>>> the use of
>>> an
>>>> combined TIRF-AFM setup. You just have to couple fluorescent  
>>>> beads to the
>>>> tip of your AFM an record pictures while approaching/or moving  
>>>> away the
>>>> coverslip surface. Unfortunately an AFM is really expensive.
>>>>
>>>> So I found some other methods, which might work as well --> see
>>>> TIRF_Introduction.pdf, which I send to you directly (LIST server  
>>>> does not
>>>> accepted this pdf-file).
>>>>
>>>> One methodes uses an objective piezo-drive and a pencil and the  
>>>> second
>>> one
>>>> stained beads or a stained solution with intransparent beads.
>>>>
>>>> I case of questions, feel free to contact me directly.
>>>>
>>>> Cheers,
>>>> Sebastian
>>>>
>>>>
>>>> Dr. Sebastian Rhode
>>>> Project Manager
>>>> Research & Development
>>>>
>>>> TILL Photonics GmbH
>>>> Lochhamer Schlag 21
>>>> D- 82166 Gräfelfing, Germany
>>>> Phone   [phone]
>>>> Fax     [phone]
>>>> www.till-photonics.com
>>>

[Andreas Bruckbauer]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****


 Hi all,
it would be good to have some calibrated sample e.g. n = 1.4 with a fluorescent particles (Q dots?) at exact distances from the cover slip so that one could adjust the angle and reach a given penetration.

best wishes

Andreas

 


 

 

-----Original Message-----
From: Mark Cannell <[hidden email]>
To: [hidden email]
Sent: Wed, 24 Nov 2010 19:37
Subject: Re: TIRF depth calibration


*****
To join, leave or search the confocal microscopy listserv, go to:
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*****
 
Hi All,
 
FCS might be a good method to solve what may be a very difficult measurement. I'm not sure how methods that actually alter the local refractive index' (e.g. beads that are of significant to size to the TIRF field or inclined coverslips) can give the 'correct' answer though. Furthermore, measuring the distance is of little value unless the optical properties of  your testing sample are exactly the same as the experimental. For example, measuring TIRF depth in water does not give the TIRF depth inside attached cells (because their refractive indexes are different). Since the TIRF field is determined by Maxwell's equations, could one not just calculate the TIRF field since you can can measure the incident angle of the input light and it's divergence quite accurately and *may* know the refractive indices in the system?
 
My 2c
 
Cheers Mark
 
On 25/11/2010, at 5:20 AM, John Oreopoulos wrote:
 

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy 
> *****
>
> And for the more adventurous types out there, you can measure your > penetration depth using fluorescence correlation spectroscopy (FCS) > if you have the right hardware:
>
> Harlepp, S., et al., Subnanometric measurements of evanescent wave > penetration depth using total internal reflection microscopy > combined with fluorescent correlation spectroscopy. Applied Physics > Letters, 2004. 85(17): p. 3917-3919.
>
> John Oreopoulos
> Research Assistant
> Spectral Applied Research
> 9078 Leslie Street, Unit 11
> Richmond Hill
> Ontario, Canada
>
>
> On 2010-11-24, at 10:37 AM, Christophe Leterrier wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy 
>> *****
>>
>> There is also a simple method in Fiolka et al. Miocroscoc. res Tech >> 2007 :
>> http://www.neuro.nano-optics.ethz.ch/publications/fiolka.pdf 
>>
>> <http://www.neuro.nano-optics.ethz.ch/publications/>> fiolka.pdf>involving an
>> inclined coverslip with attached fluorescent beads. I didn't try it >> myself
>> so I can't tell if it works, but seems simpler than AFM or >> microtubules
>> method.
>>
>> Christophe
>>
>> On Wed, Nov 24, 2010 at 16:32, John Oreopoulos
>> <[hidden email]>wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy 
>>> *****
>>>
>>> Sebastian, I remember reading a publication from about ten years >>> ago that
>>> talked about mounting a pencil in a piezoelectric micro->>> manipulator and
>>> sticking a fluorescent bead on the end of the pencil tip. This was >>> very
>>> similar to the AFM method. Is it the same thing?
>>>
>>> Graham, there is a fairly detailed discussion on this topic from a >>> few
>>> years ago on the archive that talks about a few other ways to >>> measure the
>>> actual TIRF penetration depth (as opposed to calculating it based >>> on an
>>> assumed refractive index and crudely measured incident angle):
>>>
>>>
>>> http://lists.umn.edu/cgi-bin/wa?A3=ind0702&L=CONFOCALMICROSCOPY&E=quoted-printable&P=827770&B=--Apple-Mail-181-588547976&T=text%2Fhtml;%20charset=ISO-8859-1 
>>>
>>> I've tried the AFM method as well - it works, but my main >>> complaint with
>>> this and some of the other protocols is that they require >>> complicated and
>>> expensive equipment, and can be difficult to get right. The method >>> involving
>>> fluorescent microtubles by Jorg Enderlein's group is a fairly new >>> one that
>>> is elegantly simple, but again requires you to have access to some >>> fairly
>>> special reagents that might not be found in every lab. A few >>> months ago I
>>> came across yet another older method that had evaded my previous >>> searches on
>>> the topic. This one is similar to the others that involve imaging
>>> fluorescent microbeads, but I like this because all it requires is a
>>> microscope with a motorized drive on the z-axis:
>>>
>>> Steyer, J.A. and W. Almers, Tracking single secretory granules in >>> live
>>> chromaffin cells by evanescent-field fluorescence microscopy. >>> Biophysical
>>> Journal, 1999. 76(4): p. 2262-2271.
>>>
>>> John Oreopoulos
>>> Research Assistant
>>> Spectral Applied Research
>>> 9078 Leslie Street, Unit 11
>>> Richmond Hill
>>> Ontario, Canada
>>>
>>>
>>> On 2010-11-24, at 6:54 AM, Sebastian Rhode wrote:
>>>
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy 
>>>> *****
>>>>
>>>> Hi Graham,
>>>>
>>>> one method which definitly works (I tried it out by myself) is >>>> the use of
>>> an
>>>> combined TIRF-AFM setup. You just have to couple fluorescent >>>> beads to the
>>>> tip of your AFM an record pictures while approaching/or moving >>>> away the
>>>> coverslip surface. Unfortunately an AFM is really expensive.
>>>>
>>>> So I found some other methods, which might work as well --> see
>>>> TIRF_Introduction.pdf, which I send to you directly (LIST server >>>> does not
>>>> accepted this pdf-file).
>>>>
>>>> One methodes uses an objective piezo-drive and a pencil and the >>>> second
>>> one
>>>> stained beads or a stained solution with intransparent beads.
>>>>
>>>> I case of questions, feel free to contact me directly.
>>>>
>>>> Cheers,
>>>> Sebastian
>>>>
>>>>
>>>> Dr. Sebastian Rhode
>>>> Project Manager
>>>> Research & Development
>>>>
>>>> TILL Photonics GmbH
>>>> Lochhamer Schlag 21
>>>> D- 82166 Gräfelfing, Germany
>>>> Phone   [phone]
>>>> Fax     [phone]
>>>> www.till-photonics.com
>>>

 

[Lauran Oomen]

*****
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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Another publication related to this topic is this one:
http://spiedl.aip.org/getabs/servlet/GetabsServlet?prog=normal&id=JBOPFO000011000001014006000001&idtype=cvips&gifs=yes&ref=no
Mattheyses, A.L. and Axelrod, D.
Direct measurement of the evanescent field profile produced by objective-based total internal reflection fluorescence
J. Biomed. Opt., Vol. 11, 014006 (2006)

Did not try it (yet), however.

Best wishes

Lauran Oomen
--------------------------------------------------------------
Lauran Oomen
Manager Digital Microscopy Facility (C.2.023)
NKI-AVL
Plesmanlaan 121
PO Box 90203
1006 BE Amsterdam
The Netherlands
 
phone [phone]
-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of John Oreopoulos
Sent: woensdag 24 november 2010 17:21
To: [hidden email]
Subject: Re: TIRF depth calibration

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

And for the more adventurous types out there, you can measure your penetration depth using fluorescence correlation spectroscopy (FCS) if you have the right hardware:

Harlepp, S., et al., Subnanometric measurements of evanescent wave penetration depth using total internal reflection microscopy combined with fluorescent correlation spectroscopy. Applied Physics Letters, 2004. 85(17): p. 3917-3919.

John Oreopoulos
Research Assistant
Spectral Applied Research
9078 Leslie Street, Unit 11
Richmond Hill
Ontario, Canada


On 2010-11-24, at 10:37 AM, Christophe Leterrier wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> There is also a simple method in Fiolka et al. Miocroscoc. res Tech 2007 :
> http://www.neuro.nano-optics.ethz.ch/publications/fiolka.pdf
>
> <http://www.neuro.nano-optics.ethz.ch/publications/fiolka.pdf>involving an
> inclined coverslip with attached fluorescent beads. I didn't try it myself
> so I can't tell if it works, but seems simpler than AFM or microtubules
> method.
>
> Christophe
>
> On Wed, Nov 24, 2010 at 16:32, John Oreopoulos
> <[hidden email]>wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Sebastian, I remember reading a publication from about ten years ago that
>> talked about mounting a pencil in a piezoelectric micro-manipulator and
>> sticking a fluorescent bead on the end of the pencil tip. This was very
>> similar to the AFM method. Is it the same thing?
>>
>> Graham, there is a fairly detailed discussion on this topic from a few
>> years ago on the archive that talks about a few other ways to measure the
>> actual TIRF penetration depth (as opposed to calculating it based on an
>> assumed refractive index and crudely measured incident angle):
>>
>>
>> http://lists.umn.edu/cgi-bin/wa?A3=ind0702&L=CONFOCALMICROSCOPY&E=quoted-printable&P=827770&B=--Apple-Mail-181-588547976&T=text%2Fhtml;%20charset=ISO-8859-1
>>
>> I've tried the AFM method as well - it works, but my main complaint with
>> this and some of the other protocols is that they require complicated and
>> expensive equipment, and can be difficult to get right. The method involving
>> fluorescent microtubles by Jorg Enderlein's group is a fairly new one that
>> is elegantly simple, but again requires you to have access to some fairly
>> special reagents that might not be found in every lab. A few months ago I
>> came across yet another older method that had evaded my previous searches on
>> the topic. This one is similar to the others that involve imaging
>> fluorescent microbeads, but I like this because all it requires is a
>> microscope with a motorized drive on the z-axis:
>>
>> Steyer, J.A. and W. Almers, Tracking single secretory granules in live
>> chromaffin cells by evanescent-field fluorescence microscopy. Biophysical
>> Journal, 1999. 76(4): p. 2262-2271.
>>
>> John Oreopoulos
>> Research Assistant
>> Spectral Applied Research
>> 9078 Leslie Street, Unit 11
>> Richmond Hill
>> Ontario, Canada
>>
>>
>> On 2010-11-24, at 6:54 AM, Sebastian Rhode wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Hi Graham,
>>>
>>> one method which definitly works (I tried it out by myself) is the use of
>> an
>>> combined TIRF-AFM setup. You just have to couple fluorescent beads to the
>>> tip of your AFM an record pictures while approaching/or moving away the
>>> coverslip surface. Unfortunately an AFM is really expensive.
>>>
>>> So I found some other methods, which might work as well --> see
>>> TIRF_Introduction.pdf, which I send to you directly (LIST server does not
>>> accepted this pdf-file).
>>>
>>> One methodes uses an objective piezo-drive and a pencil and the second
>> one
>>> stained beads or a stained solution with intransparent beads.
>>>
>>> I case of questions, feel free to contact me directly.
>>>
>>> Cheers,
>>> Sebastian
>>>
>>>
>>> Dr. Sebastian Rhode
>>> Project Manager
>>> Research & Development
>>>
>>> TILL Photonics GmbH
>>> Lochhamer Schlag 21
>>> D- 82166 Gräfelfing, Germany
>>> Phone   [phone]
>>> Fax     [phone]
>>> www.till-photonics.com
>>

[Graham Wright]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Thanks to everyone for the input and apologies for missing the earlier
discussion in my search (wrong search terms).

So, for the people that have tried any of the varied methods, how different
are the empirical results to the calculated depth, based on the angle, given
by some of the commercial systems?

Thanks again,
Graham

---
Graham Wright, PhD

Microscopy Unit Manager
Institute of Medical Biology
8A Biomedical Grove, #06-06 Immunos, Singapore 138648

W:  http://www.imb.a-star.edu.sg/imu/

On 25 November 2010 18:04, Lauran Oomen <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Another publication related to this topic is this one:
>
> http://spiedl.aip.org/getabs/servlet/GetabsServlet?prog=normal&id=JBOPFO000011000001014006000001&idtype=cvips&gifs=yes&ref=no
> Mattheyses, A.L. and Axelrod, D.
> Direct measurement of the evanescent field profile produced by
> objective-based total internal reflection fluorescence
> J. Biomed. Opt., Vol. 11, 014006 (2006)
>
> Did not try it (yet), however.
>
> Best wishes
>
> Lauran Oomen
> --------------------------------------------------------------
> Lauran Oomen
> Manager Digital Microscopy Facility (C.2.023)
> NKI-AVL
> Plesmanlaan 121
> PO Box 90203
> 1006 BE Amsterdam
> The Netherlands
>
> phone [phone]
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of John Oreopoulos
> Sent: woensdag 24 november 2010 17:21
> To: [hidden email]
> Subject: Re: TIRF depth calibration
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> And for the more adventurous types out there, you can measure your
> penetration depth using fluorescence correlation spectroscopy (FCS) if you
> have the right hardware:
>
> Harlepp, S., et al., Subnanometric measurements of evanescent wave
> penetration depth using total internal reflection microscopy combined with
> fluorescent correlation spectroscopy. Applied Physics Letters, 2004. 85(17):
> p. 3917-3919.
>
> John Oreopoulos
> Research Assistant
> Spectral Applied Research
> 9078 Leslie Street, Unit 11
> Richmond Hill
> Ontario, Canada
>
>
> On 2010-11-24, at 10:37 AM, Christophe Leterrier wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > There is also a simple method in Fiolka et al. Miocroscoc. res Tech 2007
> :
> > http://www.neuro.nano-optics.ethz.ch/publications/fiolka.pdf
> >
> > <http://www.neuro.nano-optics.ethz.ch/publications/fiolka.pdf>involving
> an
> > inclined coverslip with attached fluorescent beads. I didn't try it
> myself
> > so I can't tell if it works, but seems simpler than AFM or microtubules
> > method.
> >
> > Christophe
> >
> > On Wed, Nov 24, 2010 at 16:32, John Oreopoulos
> > <[hidden email]>wrote:
> >
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> *****
> >>
> >> Sebastian, I remember reading a publication from about ten years ago
> that
> >> talked about mounting a pencil in a piezoelectric micro-manipulator and
> >> sticking a fluorescent bead on the end of the pencil tip. This was very
> >> similar to the AFM method. Is it the same thing?
> >>
> >> Graham, there is a fairly detailed discussion on this topic from a few
> >> years ago on the archive that talks about a few other ways to measure
> the
> >> actual TIRF penetration depth (as opposed to calculating it based on an
> >> assumed refractive index and crudely measured incident angle):
> >>
> >>
> >>
> http://lists.umn.edu/cgi-bin/wa?A3=ind0702&L=CONFOCALMICROSCOPY&E=quoted-printable&P=827770&B=--Apple-Mail-181-588547976&T=text%2Fhtml;%20charset=ISO-8859-1
> >>
> >> I've tried the AFM method as well - it works, but my main complaint with
> >> this and some of the other protocols is that they require complicated
> and
> >> expensive equipment, and can be difficult to get right. The method
> involving
> >> fluorescent microtubles by Jorg Enderlein's group is a fairly new one
> that
> >> is elegantly simple, but again requires you to have access to some
> fairly
> >> special reagents that might not be found in every lab. A few months ago
> I
> >> came across yet another older method that had evaded my previous
> searches on
> >> the topic. This one is similar to the others that involve imaging
> >> fluorescent microbeads, but I like this because all it requires is a
> >> microscope with a motorized drive on the z-axis:
> >>
> >> Steyer, J.A. and W. Almers, Tracking single secretory granules in live
> >> chromaffin cells by evanescent-field fluorescence microscopy.
> Biophysical
> >> Journal, 1999. 76(4): p. 2262-2271.
> >>
> >> John Oreopoulos
> >> Research Assistant
> >> Spectral Applied Research
> >> 9078 Leslie Street, Unit 11
> >> Richmond Hill
> >> Ontario, Canada
> >>
> >>
> >> On 2010-11-24, at 6:54 AM, Sebastian Rhode wrote:
> >>
> >>> *****
> >>> To join, leave or search the confocal microscopy listserv, go to:
> >>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >>> *****
> >>>
> >>> Hi Graham,
> >>>
> >>> one method which definitly works (I tried it out by myself) is the use
> of
> >> an
> >>> combined TIRF-AFM setup. You just have to couple fluorescent beads to
> the
> >>> tip of your AFM an record pictures while approaching/or moving away the
> >>> coverslip surface. Unfortunately an AFM is really expensive.
> >>>
> >>> So I found some other methods, which might work as well --> see
> >>> TIRF_Introduction.pdf, which I send to you directly (LIST server does
> not
> >>> accepted this pdf-file).
> >>>
> >>> One methodes uses an objective piezo-drive and a pencil and the second
> >> one
> >>> stained beads or a stained solution with intransparent beads.
> >>>
> >>> I case of questions, feel free to contact me directly.
> >>>
> >>> Cheers,
> >>> Sebastian
> >>>
> >>>
> >>> Dr. Sebastian Rhode
> >>> Project Manager
> >>> Research & Development
> >>>
> >>> TILL Photonics GmbH
> >>> Lochhamer Schlag 21
> >>> D- 82166 Gräfelfing, Germany
> >>> Phone   [phone]
> >>> Fax     [phone]
> >>> www.till-photonics.com
> >>
>

[Louis Villeneuve]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Bojour à tous,

I would like some information/feellings about immersion (water/oil)
dispenser for the use  high resolution objective in the context of High
content screening microscopy.

I would like to know if those device works well for imaging a lot of
plates? Is it reliable?

Would it be more realistic to use lower NA objective (without immersion)
for HCS microscopy?

Thanks for your help,

Louis

[Emmanuel Gustin]

*****
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Hi Louis,

We have such a system, on a PerkinElmer Opera. We find that it works reliably if you take basic, sensible precautions: Make sure all connectors are tight, sonicate the immersion water to degas it and reduce the formation of bubbles in the fluid path, clean the collection channels regularly to prevent them getting clogged by dust, flush the system a few minutes before every run. It is suitable for long runs.

As for it being 'realistic', it all depends on what your application needs. There is no single best option for HCS experiments... Almost all HCS labs have several different instruments to choose from, dependent on the application, and having a low-resolution and a high-resolution system side-by-side is fairly typical.

High-NA water immersion objectives are a good tool if you are looking at small cells, want to see sub-cellular structural detail, have weak signals, or require good Z-sectioning performance on a confocal system. But they have disadvantages as well, such as short working distances, a requirement to have high-quality plate bottoms, and a relatively small field of view as they are all (relatively) high magnification.

Best Regards,

Emmanuel

--
 Emmanuel Gustin,    Tel. [phone],    e-mail: [hidden email]     ! telephone number changed !


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of [hidden email]
Sent: maandag 6 december 2010 16:26
To: [hidden email]
Subject: HCS microscopy-immersion (water/oil) dispenser

Bojour à tous,

I would like some information/feellings about immersion (water/oil)
dispenser for the use  high resolution objective in the context of High
content screening microscopy.

I would like to know if those device works well for imaging a lot of
plates? Is it reliable?

Would it be more realistic to use lower NA objective (without immersion)
for HCS microscopy?

Thanks for your help,

Louis

[Michal Opas]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Dear Listers,
I'm not sure if my question was posted or not, so I'd like to ask again:
did someone trued and succeeded in running Todd Brelje's Confocal
Assistant under Win7 somehow?
Thanks,
Michal


--

* Dr. Michal Opas
      Professor
      Department of Laboratory Medicine and Pathobiology
      University of Toronto
      1 King's College Circle
      Medical Sciences Building, room 6326
      Toronto, Ontario, M5S 1A8 Canada

**°°°°°°°°°°°°°**
  phone: [phone] (laboratory)
         [phone] (office)
    fax: [phone]
e*-*mail: **[hidden email] <mailto:[hidden email]>***

*WWW**:** **http://www.utoronto.ca/mocell
<http://www.utoronto.ca/mocell>*

[Dariusz Maluchnik]

*****
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*****

Confocal Assistant runs under 32-bit Windows 7. But not under 64-bit Windows, as it is an old 16-bit application. Try virtualization (Virtual PC or VirtualBox).
http://microsoft.com/windows/virtual-pc
Darek Maluchnik

On 2011  Aug 22,, at 15:19, Michal Opas wrote:

> Dear Listers,
> I'm not sure if my question was posted or not, so I'd like to ask again: did someone trued and succeeded in running Todd Brelje's Confocal Assistant under Win7 somehow?
> Thanks,
> Michal

[Michal Opas]

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*****

Dear Listers,

My search of archives leads me nowhere hence this question:

we need to mount spherical cell aggregates (mouse EBs) circa 300 µm in
diameter in a mountant that would let them wiggle while focused up and
down and, importantly, prevent coverslip from moving and shearing them
to shreds. Our commercial mountant (DAKO) does not cure. Nail polish
remedies this a bit but I'd hope for an improvement with a mountant that
would gel (solidify) in a decent time.

Thank you very much in advance!

Michal*
<http://www.utoronto.ca/mocell>*

[Sarang Kulkarni]

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*****

Just a guess, but you could try a very low percentage low melt agarose spread very thinly.  This should set in a decent amount of time.  Not sure about the wiggle room that you desire.

Sarang Kulkarni

Sent from my iPhone

On 2012-11-01, at 5:33 PM, "Michal Opas" <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Listers,
>
> My search of archives leads me nowhere hence this question:
>
> we need to mount spherical cell aggregates (mouse EBs) circa 300 µm in diameter in a mountant that would let them wiggle while focused up and down and, importantly, prevent coverslip from moving and shearing them to shreds. Our commercial mountant (DAKO) does not cure. Nail polish remedies this a bit but I'd hope for an improvement with a mountant that would gel (solidify) in a decent time.
>
> Thank you very much in advance!
>
> Michal*
> <http://www.utoronto.ca/mocell>*

[Glen MacDonald-2]

*****
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*****

Hi,

Have you tried ~1.5% low melting point agarose?  Maybe even lower concentration for your purposes.  Use a coverslip bridge to create a spacer - use cyanoacrylate glue to stack 22 mm square coverslips to a desired depth onto a 50 or 60 mm coverslip. the top coverslip usually stays in place by capillary action.

 Regards,
Glen

Glen MacDonald
Cellular Morphology Core
Center for Human Development and Disability
Box 357920
University of Washington
Seattle, WA 98195-7920  USA
[phone]
[hidden email]


On Nov 1, 2012, at 2:28 PM, Michal Opas wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Listers,
>
> My search of archives leads me nowhere hence this question:
>
> we need to mount spherical cell aggregates (mouse EBs) circa 300 µm in diameter in a mountant that would let them wiggle while focused up and down and, importantly, prevent coverslip from moving and shearing them to shreds. Our commercial mountant (DAKO) does not cure. Nail polish remedies this a bit but I'd hope for an improvement with a mountant that would gel (solidify) in a decent time.
>
> Thank you very much in advance!
>
> Michal*
> <http://www.utoronto.ca/mocell>*

[Armstrong, Brian]

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I am not sure how much wiggle you want, but PuraMatrix and Matrigel will both provide room for large colonies to form. You can obtain good 3D structure information using these in your set-up.
Cheers,

Brian Armstrong PhD
Assistant Research Professor
Director, Light Microscopy Core Facility
Beckman Research Institute
City of Hope
626-256-4673 x62872

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Glen MacDonald
Sent: Thursday, November 01, 2012 2:52 PM
To: [hidden email]
Subject: Re: TIRF depth calibration

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Hi,

Have you tried ~1.5% low melting point agarose?  Maybe even lower concentration for your purposes.  Use a coverslip bridge to create a spacer - use cyanoacrylate glue to stack 22 mm square coverslips to a desired depth onto a 50 or 60 mm coverslip. the top coverslip usually stays in place by capillary action.

 Regards,
Glen

Glen MacDonald
Cellular Morphology Core
Center for Human Development and Disability Box 357920 University of Washington Seattle, WA 98195-7920  USA
[phone]
[hidden email]


On Nov 1, 2012, at 2:28 PM, Michal Opas wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Listers,
>
> My search of archives leads me nowhere hence this question:
>
> we need to mount spherical cell aggregates (mouse EBs) circa 300 µm in diameter in a mountant that would let them wiggle while focused up and down and, importantly, prevent coverslip from moving and shearing them to shreds. Our commercial mountant (DAKO) does not cure. Nail polish remedies this a bit but I'd hope for an improvement with a mountant that would gel (solidify) in a decent time.
>
> Thank you very much in advance!
>
> Michal*
> <http://www.utoronto.ca/mocell>*

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