Measuring PSFs using water dipping objective

[Stephan Junek]

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Dear all,

I am looking for a good protocol to measure PSFs on a confocal microscope
using water dipping objectives (objectives used for physiology, i.e. no
cover glass!).
I tried using fluorescent beads embedded in 5 % low melting point agarose,
but it appears that the beads are moving, probably due to slight swelling of
the agarose which is immersed in the water.
I would prefer not to use a cover glass, as this probably introduces
aberrations. Using beads dried on a glass surface is not ideal either due to
the reflection at the water/glass interface.

Thanks in advance for any suggestions!

Best,
Stephan.


--
________________________________________
Dr. Stephan Junek
Max Planck Institute for Brain Research
Deutschordenstr. 46
60528 Frankfurt am Main
Germany

[hidden email]
T: [phone] – 2008
F: [phone] – 2002

[mmodel]

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Hi Stephan,

You can try polyvinyl acetate. It is soluble in methanol (1% w/v) but not in water, and if you mix its solution with beads and dry it on a glass slide, some beads may get trapped in PVA and stay there even after you add water.

Mike

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Stephan Junek
Sent: Wednesday, June 13, 2012 12:35 PM
To: [hidden email]
Subject: Measuring PSFs using water dipping objective

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Dear all,

I am looking for a good protocol to measure PSFs on a confocal microscope
using water dipping objectives (objectives used for physiology, i.e. no
cover glass!).
I tried using fluorescent beads embedded in 5 % low melting point agarose,
but it appears that the beads are moving, probably due to slight swelling of
the agarose which is immersed in the water.
I would prefer not to use a cover glass, as this probably introduces
aberrations. Using beads dried on a glass surface is not ideal either due to
the reflection at the water/glass interface.

Thanks in advance for any suggestions!

Best,
Stephan.


--
________________________________________
Dr. Stephan Junek
Max Planck Institute for Brain Research
Deutschordenstr. 46
60528 Frankfurt am Main
Germany

[hidden email]
T: [phone] - 2008
F: [phone] - 2002

[Michael Doube]

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Hi Stephan,

> I am looking for a good protocol to measure PSFs on a confocal microscope
> using water dipping objectives (objectives used for physiology, i.e. no
> cover glass!).
> I tried using fluorescent beads embedded in 5 % low melting point agarose,
> but it appears that the beads are moving, probably due to slight swelling of
> the agarose which is immersed in the water.

I did something like that recently, using 80 nm diameter nanogold
particles embedded in low-melt agarose and imaged in reflected light
mode on a Zeiss LSM 780 NLO with dip-in lenses. The advantage over using
a fluorescent bead is that the reflected signal is bright and of
narrowly defined spectral range (we wanted to define chromatic
aberration of our objectives, not get an experimental PSF for
deconvolution). Particle movement didn't seem to be too much of an
issue, perhaps because I could scan fast thanks to the high signal. Or
possibly, the agarose mixture was a bit different to yours. Or, it might
not have mattered for our question and I have forgotten. If I scrape my
neurons a bit harder I recall that the near-IR laser made the particles
wobble a bit, maybe because of local heating. Let me know if you want
details of the protocol.

Michael

[Zac Arrac Atelaz]

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I can think of two possible issues afecting the result, very thick layer of agar being cracked by the objective, very high power output from the laser setting free the particles, if you keep the power low and change the wavelength you can avoid that one. I hope this can help you. Regards Gabriel OH
 > Date: Wed, 13 Jun 2012 20:30:29 +0200

> From: [hidden email]
> Subject: Re: Measuring PSFs using water dipping objective
> To: [hidden email]
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Stephan,
>
> > I am looking for a good protocol to measure PSFs on a confocal microscope
> > using water dipping objectives (objectives used for physiology, i.e. no
> > cover glass!).
> > I tried using fluorescent beads embedded in 5 % low melting point agarose,
> > but it appears that the beads are moving, probably due to slight swelling of
> > the agarose which is immersed in the water.
>
> I did something like that recently, using 80 nm diameter nanogold
> particles embedded in low-melt agarose and imaged in reflected light
> mode on a Zeiss LSM 780 NLO with dip-in lenses. The advantage over using
> a fluorescent bead is that the reflected signal is bright and of
> narrowly defined spectral range (we wanted to define chromatic
> aberration of our objectives, not get an experimental PSF for
> deconvolution). Particle movement didn't seem to be too much of an
> issue, perhaps because I could scan fast thanks to the high signal. Or
> possibly, the agarose mixture was a bit different to yours. Or, it might
> not have mattered for our question and I have forgotten. If I scrape my
> neurons a bit harder I recall that the near-IR laser made the particles
> wobble a bit, maybe because of local heating. Let me know if you want
> details of the protocol.
>
> Michael

     

[Peng Xi-2]

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Hi Stephan,
     To measure the PSF you should learn from Stefan --- Stefan Hell
who invented STED nanoscopy. They measure PSF on a daily basis, as
they need to overlap confocal PSF with a doughnut shape PSF in X, Y,
and Z direction. Now that I have also stepped in this field, I can
tell you how to do it.
     Basically, the confocal PSF can be measured with  fluorescent
bead. Attach the bead on the surface of coverglass with poly-L-Lysin.
Use Mowoil as an embedding medium. Add antifade reagent such as DABCO,
because during PSF measurement you often stick to a very small region,
therefore the sample undergoes large photobleaching. Also, if you want
PSF more close to real situation, you may try smaller beads, as in
fluorescent mode your measured PSF is a convolution of real PSF with a
nanopartile with a certain size. You have to deduct the particle size
from it
(Diameter of real PSF)^2=(Diameter of measured PSF)^2 -(Diameter of bead)^2.
     If you are able to remove your bandpass filter (I assume you are
using fluorescent confocal) before the detector pinhole, you can also
try to see whether you are able to use gold nanoparticle reflection to
get PSF. Then you don't have photobleaching, and as long as your gold
nanoparticle is smaller than your theoretical resolution, you don't
need to count the size of nanopartile, because the reflection is only
on the tip of the bead, not the waist. :)
     A good reference for such sample preparation:
http://www.springerlink.com/content/t8ntp60137050851/#section=614830&page=1

     Good luck!

Sincerely,
Peng Xi
Ph. D.    Associate Professor
Dept. of Biomedical Engineering, College of Engineering
Peking University, Beijing, China
Tel: [phone]
Email: [hidden email]
http://www.opticsinfobase.org/oe/abstract.cfm?uri=oe-20-13-14100
On Thu, Jun 14, 2012 at 3:28 AM, Zac Arrac Atelaz <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
>
> I can think of two possible issues afecting the result, very thick layer of agar being cracked by the objective, very high power output from the laser setting free the particles, if you keep the power low and change the wavelength you can avoid that one. I hope this can help you. Regards Gabriel OH
>  > Date: Wed, 13 Jun 2012 20:30:29 +0200
>> From: [hidden email]
>> Subject: Re: Measuring PSFs using water dipping objective
>> To: [hidden email]
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi Stephan,
>>
>> > I am looking for a good protocol to measure PSFs on a confocal microscope
>> > using water dipping objectives (objectives used for physiology, i.e. no
>> > cover glass!).
>> > I tried using fluorescent beads embedded in 5 % low melting point agarose,
>> > but it appears that the beads are moving, probably due to slight swelling of
>> > the agarose which is immersed in the water.
>>
>> I did something like that recently, using 80 nm diameter nanogold
>> particles embedded in low-melt agarose and imaged in reflected light
>> mode on a Zeiss LSM 780 NLO with dip-in lenses. The advantage over using
>> a fluorescent bead is that the reflected signal is bright and of
>> narrowly defined spectral range (we wanted to define chromatic
>> aberration of our objectives, not get an experimental PSF for
>> deconvolution). Particle movement didn't seem to be too much of an
>> issue, perhaps because I could scan fast thanks to the high signal. Or
>> possibly, the agarose mixture was a bit different to yours. Or, it might
>> not have mattered for our question and I have forgotten. If I scrape my
>> neurons a bit harder I recall that the near-IR laser made the particles
>> wobble a bit, maybe because of local heating. Let me know if you want
>> details of the protocol.
>>
>> Michael
>

[David Baddeley]

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You could try either a higher agarose concentration, or think about using collagen (aka gelatin) or possibly even pluronic instead (because of its weird phase behaviour pluronic might be more resistant to local melting due to laser heating). I've successfully taken PSFs in an admittedly fairly concentrated (~ 30%) gelatin gel without any bead movement. The downside of gelatin is that it has some background fluorescence, but this is likely to be more of a problem in widefield. A second caveat is that a high concentration gel of either gelatin or agarose will have a refractive index higher than that of water. 

cheers,
David

________________________________
 From: Stephan Junek <[hidden email]>
To: [hidden email]
Sent: Thursday, 14 June 2012 4:35 AM
Subject: Measuring PSFs using water dipping objective
 
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Dear all,

I am looking for a good protocol to measure PSFs on a confocal microscope
using water dipping objectives (objectives used for physiology, i.e. no
cover glass!).
I tried using fluorescent beads embedded in 5 % low melting point agarose,
but it appears that the beads are moving, probably due to slight swelling of
the agarose which is immersed in the water.
I would prefer not to use a cover glass, as this probably introduces
aberrations. Using beads dried on a glass surface is not ideal either due to
the reflection at the water/glass interface.

Thanks in advance for any suggestions!

Best,
Stephan.


--
________________________________________
Dr. Stephan Junek
Max Planck Institute for Brain Research
Deutschordenstr. 46
60528 Frankfurt am Main
Germany

[hidden email]
T: [phone] – 2008
F: [phone] – 2002

[Stuart McIntyre]

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Hi Stephan

I have been taking measurements from our 2-photon system, I have taken to
using a chroma test slide to take edge spread functions in the Z-axis.
Obviously this will not give me the lateral resolution but it is very simple
to set up there is lots of signal and bleaching doesn’t seem to be an issue.
I am able to quickly measure the resolution at any point in the field of
view, it will also show how well my system is aligned.

Hope that helps.

Stuart

[Jeremy Adler-4]

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The Refractive index of Mowiol is that of oil, which will alter the  
PSF of a microsphere embedded in it and imaged with a water dipping  
objective. Is there a way of correcting the PSF for use with a dipping  
objective ?
Quoting Peng Xi <[hidden email]>:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Stephan,
>      To measure the PSF you should learn from Stefan --- Stefan Hell
> who invented STED nanoscopy. They measure PSF on a daily basis, as
> they need to overlap confocal PSF with a doughnut shape PSF in X, Y,
> and Z direction. Now that I have also stepped in this field, I can
> tell you how to do it.
>      Basically, the confocal PSF can be measured with  fluorescent
> bead. Attach the bead on the surface of coverglass with poly-L-Lysin.
> Use Mowoil as an embedding medium. Add antifade reagent such as DABCO,
> because during PSF measurement you often stick to a very small region,
> therefore the sample undergoes large photobleaching. Also, if you want
> PSF more close to real situation, you may try smaller beads, as in
> fluorescent mode your measured PSF is a convolution of real PSF with a
> nanopartile with a certain size. You have to deduct the particle size
> from it
> (Diameter of real PSF)^2=(Diameter of measured PSF)^2 -(Diameter of bead)^2.
>      If you are able to remove your bandpass filter (I assume you are
> using fluorescent confocal) before the detector pinhole, you can also
> try to see whether you are able to use gold nanoparticle reflection to
> get PSF. Then you don't have photobleaching, and as long as your gold
> nanoparticle is smaller than your theoretical resolution, you don't
> need to count the size of nanopartile, because the reflection is only
> on the tip of the bead, not the waist. :)
>      A good reference for such sample preparation:
> http://www.springerlink.com/content/t8ntp60137050851/#section=614830&page=1
>
>      Good luck!
>
> Sincerely,
> Peng Xi
> Ph. D.    Associate Professor
> Dept. of Biomedical Engineering, College of Engineering
> Peking University, Beijing, China
> Tel: [phone]
> Email: [hidden email]
> http://www.opticsinfobase.org/oe/abstract.cfm?uri=oe-20-13-14100
> On Thu, Jun 14, 2012 at 3:28 AM, Zac Arrac Atelaz  
> <[hidden email]> wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>>
>> I can think of two possible issues afecting the result, very thick  
>> layer of agar being cracked by the objective, very high power  
>> output from the laser setting free the particles, if you keep the  
>> power low and change the wavelength you can avoid that one. I hope  
>> this can help you. Regards Gabriel OH
>>  > Date: Wed, 13 Jun 2012 20:30:29 +0200
>>> From: [hidden email]
>>> Subject: Re: Measuring PSFs using water dipping objective
>>> To: [hidden email]
>>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Hi Stephan,
>>>
>>> > I am looking for a good protocol to measure PSFs on a confocal microscope
>>> > using water dipping objectives (objectives used for physiology, i.e. no
>>> > cover glass!).
>>> > I tried using fluorescent beads embedded in 5 % low melting  
>>> point agarose,
>>> > but it appears that the beads are moving, probably due to slight  
>>> swelling of
>>> > the agarose which is immersed in the water.
>>>
>>> I did something like that recently, using 80 nm diameter nanogold
>>> particles embedded in low-melt agarose and imaged in reflected light
>>> mode on a Zeiss LSM 780 NLO with dip-in lenses. The advantage over using
>>> a fluorescent bead is that the reflected signal is bright and of
>>> narrowly defined spectral range (we wanted to define chromatic
>>> aberration of our objectives, not get an experimental PSF for
>>> deconvolution). Particle movement didn't seem to be too much of an
>>> issue, perhaps because I could scan fast thanks to the high signal. Or
>>> possibly, the agarose mixture was a bit different to yours. Or, it might
>>> not have mattered for our question and I have forgotten. If I scrape my
>>> neurons a bit harder I recall that the near-IR laser made the particles
>>> wobble a bit, maybe because of local heating. Let me know if you want
>>> details of the protocol.
>>>
>>> Michael
>>
>

Jeremy Adler
IGP
Rudbeckslaboratoriet
Daghammersköljdsväg 20
751 85 Uppsala
Sweden

0046 (0)18 471 4607

[Alex Brown]

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Stephan,

I tried this precise thing recently - measuring the PSF of a water dipping
objective (in a two-photon microscope). I used tetraspeck beads, as they are
bright and perfect for my application.

I found that the beads move a lot. To prevent this:

Embed the beads in a thin layer of agar in a small petri dish. Leave the dish
overnight (in the dark). The beads will tend to move to the bottom of the layer,
so make sure it is thin - though I presume you're using a fairly long WD
objective?

Use agar instead of agarose, and use a high (5%) concentration. Plain agar
contains agarose as well as agaropectins, which are a collection of smaller
molecules. This seems to really cut down the movement. I haven't tried gelatin,
but that may well be a good alternative.

You could also image the (settled) beads at the bottom of the dish by flipping it
over. Obviously this may introduce some aberration, as you are now imaging
through a layer of plastic, but it seems to work pretty well.

[Michael Weber-4]

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Stephan,

I am surprised to read that beads still move in 5% agarose. We do mount beads in 1.5% low-melting agarose for PSF measurements and dual-channel alignment on a regular base and have no problems with movements. For an upright system with water dipping lenses I would try a two-layer approach. First coat a plastic dish with a layer of agarose without beads, let it solidify, and put a second layer of agarose with beads on top. This would avoid beads accumulating at the agarose-plastic interface. Make sure to keep the second layer thin, to avoid touching/pressing the agarose with your objective while trying to find the beads. Based on our experience I would also recommend to not go beyond 2% agarose concentration since this decreases optical quality, apparently due to the change in refractive index. If you cover the agarose with the same medium you used for preparing the agarose, shrinking should not be an issue either. And the agarose will not dry.

Good luck,
Michael


On Jun 13, 2012, at 6:35 PM, Stephan Junek wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear all,
>
> I am looking for a good protocol to measure PSFs on a confocal microscope
> using water dipping objectives (objectives used for physiology, i.e. no
> cover glass!).
> I tried using fluorescent beads embedded in 5 % low melting point agarose,
> but it appears that the beads are moving, probably due to slight swelling of
> the agarose which is immersed in the water.
> I would prefer not to use a cover glass, as this probably introduces
> aberrations. Using beads dried on a glass surface is not ideal either due to
> the reflection at the water/glass interface.
>
> Thanks in advance for any suggestions!
>
> Best,
> Stephan.
>
>
> --
> ________________________________________
> Dr. Stephan Junek
> Max Planck Institute for Brain Research
> Deutschordenstr. 46
> 60528 Frankfurt am Main
> Germany
>
> [hidden email]
> T: [phone] – 2008
> F: [phone] – 2002

_____________

Michael Weber
PhD Student, Huisken lab
Max Planck Institute of Molecular Cell Biology and Genetics
Pfotenhauerstrasse 108, 01307 Dresden
Tel. [phone]

http://www.mpi-cbg.de/research/research-groups/jan-huisken