Spacers for Inverted Microscop

[Sam Albers]

Hello all,

My institution's confocal set up uses an inverted microscope. So when I view some biofilm samples I am collecting, I need to also invert my slide and place it in a NUNC coverslip chamber. Because I do not want to crush my biofilm sample, I need thin spacers to separate the slide from the coverslip bottom. Thin glass microscope slide have been suggested as a spacer. I was wondering if any one here had any other suggestions.

I am looking for a convenient inert object that can be be easily placed inside the coverslip chamber that will separate the slide from the coverslip bottom. Ideally, this would some type of material that I could vary the length to account for different thicknesses of biofilms.

Any suggestions would be greatly appreciated. I guess I am just wondering what other people do.

Thanks!

Sam

[John Oreopoulos]

You could try double sided tape layered on each other or wax. Grace  
Biolabs also makes imaging chamber spacers (no commercial interest):

http://www.gracebio.com/Products/Imaging__Microscopy/ 
SecureSeal_Imaging_Spacers/

They make spacers with a variety of thicknesses, and I recall using  
one version that was very thin.
John Oreopoulos


On 9-Jul-09, at 3:27 PM, Sam Albers wrote:

> Hello all,
>
> My institution's confocal set up uses an inverted microscope. So  
> when I view
> some biofilm samples I am collecting, I need to also invert my  
> slide and
> place it in a NUNC coverslip chamber. Because I do not want to  
> crush my
> biofilm sample, I need thin spacers to separate the slide from the  
> coverslip
> bottom. Thin glass microscope slide have been suggested as a  
> spacer. I was
> wondering if any one here had any other suggestions.
>
> I am looking for a convenient inert object that can be be easily  
> placed
> inside the coverslip chamber that will separate the slide from the  
> coverslip
> bottom. Ideally, this would some type of material that I could vary  
> the
> length to account for different thicknesses of biofilms.
>
> Any suggestions would be greatly appreciated. I guess I am just  
> wondering
> what other people do.
>
> Thanks!
>
> Sam
> --
> View this message in context: http://n2.nabble.com/Spacers-for- 
> Inverted-Microscop-tp3233585p3233585.html
> Sent from the Confocal Microscopy List mailing list archive at  
> Nabble.com.

[Glen MacDonald-2]

You can get a diamond pencil and score coverslips into strips.  
Coverslips may be purchased with thicknesses ranging from about 90  
microns to 250 microns.  Each thickness type has a 30 um range.  If  
you would need greater accuracy, a micrometer would be required to  
measure the thickness.

Regards,
Glen
On Jul 9, 2009, at 12:27 PM, Sam Albers wrote:

> Hello all,
>
> My institution's confocal set up uses an inverted microscope. So  
> when I view
> some biofilm samples I am collecting, I need to also invert my slide  
> and
> place it in a NUNC coverslip chamber. Because I do not want to crush  
> my
> biofilm sample, I need thin spacers to separate the slide from the  
> coverslip
> bottom. Thin glass microscope slide have been suggested as a spacer.  
> I was
> wondering if any one here had any other suggestions.
>
> I am looking for a convenient inert object that can be be easily  
> placed
> inside the coverslip chamber that will separate the slide from the  
> coverslip
> bottom. Ideally, this would some type of material that I could vary  
> the
> length to account for different thicknesses of biofilms.
>
> Any suggestions would be greatly appreciated. I guess I am just  
> wondering
> what other people do.
>
> Thanks!
>
> Sam
> --
> View this message in context: http://n2.nabble.com/Spacers-for-Inverted-Microscop-tp3233585p3233585.html
> Sent from the Confocal Microscopy List mailing list archive at  
> Nabble.com.

[Deanne Veronica Catmull]

Why can't you grow your biofilm in a flow cell or a slide chamber? These
can be easily imaged via the Confocal without all the fuss of designing
and using these "spacers"? This is just a thought.

Kind regards,
Deanne.


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Sam Albers
Sent: Friday, 10 July 2009 5:27 AM
To: [hidden email]
Subject: Spacers for Inverted Microscop

Hello all,

My institution's confocal set up uses an inverted microscope. So when I
view
some biofilm samples I am collecting, I need to also invert my slide and
place it in a NUNC coverslip chamber. Because I do not want to crush my
biofilm sample, I need thin spacers to separate the slide from the
coverslip
bottom. Thin glass microscope slide have been suggested as a spacer. I
was
wondering if any one here had any other suggestions.

I am looking for a convenient inert object that can be be easily placed
inside the coverslip chamber that will separate the slide from the
coverslip
bottom. Ideally, this would some type of material that I could vary the
length to account for different thicknesses of biofilms.

Any suggestions would be greatly appreciated. I guess I am just
wondering
what other people do.

Thanks!

Sam
--
View this message in context:
http://n2.nabble.com/Spacers-for-Inverted-Microscop-tp3233585p3233585.ht
ml
Sent from the Confocal Microscopy List mailing list archive at
Nabble.com.

[Raman Rajagopal]

Why don't you try glass wool. They are inert and come in different sizes.

They have been routinely used in observing nematodes and other fragile invertebrates.

Alternatively, you could use thin wall Petri-plates used for live cell imaging, to prepare your samples. This can viewed directly ie without inverting.

Best wishes

RAJAGOPAL

University of Delhi

 

On Fri, Jul 10, 2009 at 12:57 AM, Sam Albers <[hidden email]> wrote:

Hello all,

My institution's confocal set up uses an inverted microscope. So when I view
some biofilm samples I am collecting, I need to also invert my slide and
place it in a NUNC coverslip chamber. Because I do not want to crush my
biofilm sample, I need thin spacers to separate the slide from the coverslip
bottom. Thin glass microscope slide have been suggested as a spacer. I was
wondering if any one here had any other suggestions.

I am looking for a convenient inert object that can be be easily placed
inside the coverslip chamber that will separate the slide from the coverslip
bottom. Ideally, this would some type of material that I could vary the
length to account for different thicknesses of biofilms.

Any suggestions would be greatly appreciated. I guess I am just wondering
what other people do.

Thanks!

Sam
--
View this message in context: http://n2.nabble.com/Spacers-for-Inverted-Microscop-tp3233585p3233585.html
Sent from the Confocal Microscopy List mailing list archive at Nabble.com.

[Sam Albers]

Thanks everyone for your suggestions! I think this is a good amount of material to go on.

[Evelyn Ralston]

Sorry, I was not following the listserv for a while. We use Secure Seal Spacers from Electron Microscopy Sciences. They are 0.12 mm thick, different sizes & opening diameters, and stick to glass perfectly.

Evelyn Ralston, Ph.D.

Head, Light imaging Section,

Office of Science and Technology, NIAMS, NIH

Rm 1535, Bldg 50

Bethesda MD 20892-8023

tel [phone]; FAX [phone]

On Jul 10, 2009, at 4:36 PM, Sam Albers wrote:

Thanks everyone for your suggestions! I think this is a good amount of

material to go on.

-- 

View this message in context: http://n2.nabble.com/Spacers-for-Inverted-Microscop-tp3233585p3237553.html

Sent from the Confocal Microscopy List mailing list archive at Nabble.com.

[Matthew J. Gastinger]

We have someone who wants to use ProSense 750 (a protease activatable fluorescent in vivo imaging agent) from VisEN.  He wants to image in vivo using confocal microscopy.  The alternative is ProSense 680, but 750 is likely to produce less autofluorescence.  ProSense 680 will likely work with 633 laser or whitelight laser at 670nm.

 

Has anyone imaged VisEN ProSense 750 using multiphoton confocal microscopy? If so, what are equipment specs generating images.

 

Thanks.

[Craig Brideau]

For multiphoton (in this case 2 photon) won't you need a very long
wavelength?  Probably >1200nm?  Ti:Saphs don't go beyond 1100nm so you
won't be able to use multiphoton to stimulate it unless you have a
Cr:Forsterite laser lying around or an OPA/OPO system strapped to your
Ti:Saph.  If you want to use single photon you can just tune your
Ti:Saph to 750 and you should be good to go.

Craig


On Wed, Jul 15, 2009 at 1:30 PM, Gastinger, Matthew (NIH/NIAID)
[C]<[hidden email]> wrote:

> We have someone who wants to use ProSense 750 (a protease activatable
> fluorescent in vivo imaging agent) from VisEN.  He wants to image in vivo
> using confocal microscopy.  The alternative is ProSense 680, but 750 is
> likely to produce less autofluorescence.  ProSense 680 will likely work with
> 633 laser or whitelight laser at 670nm.
>
>
>
> Has anyone imaged VisEN ProSense 750 using multiphoton confocal microscopy?
> If so, what are equipment specs generating images.
>
>
>
> Thanks.

[Andreas Bruckbauer]

>If you want to use single photon you can just tune your Ti:Saph to 750 and you should be good to go

You would need a suitable dichroic for direct excitation at 750 nm, the multiphoton dichroics usually reflect everything above 680 or so, your fluorescence signal would not reach the detector. Furthermore the normal photomultiplier detectors are almost blind in the far red, laser power would need to be reduced quite a lot... Has anyone tried direct 1P excitation of these dyes?

Andreas

-----Original Message-----
From: Craig Brideau <[hidden email]>
To: [hidden email]
Sent: Wed, 15 Jul 2009 22:37
Subject: Re: VisEn Prosense 750 on multiphoton?

For multiphoton (in this case 2 photon) won't you need a very long

wavelength?  Probably >1200nm?  Ti:Saphs don't go beyond 1100nm so you

won't be able to use multiphoton to stimulate it unless you have a

Cr:Forsterite laser lying around or an OPA/OPO system strapped to your

Ti:Saph.  If you want to use single photon you can just tune your

Ti:Saph to 750 and you should be good to go.



Craig





On Wed, Jul 15, 2009 at 1:30 PM, Gastinger, Matthew (NIH/NIAID)

[C]<[hidden email]> wrote:

> We have someone who wants to use ProSense 750 (a protease activatable

> fluorescent in vivo imaging agent) from VisEN.  He wants to image in vivo

> using confocal microscopy.  The alternative is ProSense 680, but 750 is

> likely to produce less autofluorescence.  ProSense 680 will likely work with

> 633 laser or whitelight laser at 670nm.

>

>

>

> Has anyone imaged VisEN ProSense 750 using multiphoton confocal microscopy?

> If so, what are equipment specs generating images.

>

>

>

> Thanks.

---

Download AOL Toolbar and get access to all of your favourite websites and Google powered Search in an instant. Download AOL Toolbar for FREE.

[Craig Brideau]

You can get extended wavelength PMTs, but your system may or may not
include them.  Many of Hamamatsu's (major PMT manufacturer) PMTs are
good out to 900nm or so which should hopefully work.  The one problem
with these longer IR dyes is that your detection system needs to be
capable of picking up the NIR signal from them.  I was assuming the
questioner had already taken this into account but I suppose it should
be brought up given that off-the-shelf systems won't have the
necessary detectors or filters.

Some Hamamatsu PMTs out to 900nm:
http://jp.hamamatsu.com/products/sensor-etd/pd002/pd395/index_en.html
(scroll down to see the spectral response curves)

Craig


On Wed, Jul 15, 2009 at 4:03 PM, Andreas Bruckbauer<[hidden email]> wrote:

>>If you want to use single photon you can just tune your Ti:Saph to 750 and
>> you should be good to go
>
> You would need a suitable dichroic for direct excitation at 750 nm, the
> multiphoton dichroics usually reflect everything above 680 or so, your
> fluorescence signal would not reach the detector. Furthermore the normal
> photomultiplier detectors are almost blind in the far red, laser power would
> need to be reduced quite a lot... Has anyone tried direct 1P excitation of
> these dyes?
>
> Andreas
>
>
> -----Original Message-----
> From: Craig Brideau <[hidden email]>
> To: [hidden email]
> Sent: Wed, 15 Jul 2009 22:37
> Subject: Re: VisEn Prosense 750 on multiphoton?
>
> For multiphoton (in this case 2 photon) won't you need a very long
>
> wavelength?  Probably >1200nm?  Ti:Saphs don't go beyond 1100nm so you
>
> won't be able to use multiphoton to stimulate it unless you have a
>
> Cr:Forsterite laser lying around or an OPA/OPO system strapped to your
>
> Ti:Saph.  If you want to use single photon you can just tune your
>
> Ti:Saph to 750 and you should be good to go.
>
>
>
> Craig
>
>
>
>
>
> On Wed, Jul 15, 2009 at 1:30 PM, Gastinger, Matthew (NIH/NIAID)
>
> [C]<[hidden email]> wrote:
>
>> We have someone who wants to use ProSense 750 (a protease activatable
>
>> fluorescent in vivo imaging agent) from VisEN.  He wants to image in vivo
>
>> using confocal microscopy.  The alternative is ProSense 680, but 750 is
>
>> likely to produce less autofluorescence.  ProSense 680 will likely work
>> with
>
>> 633 laser or whitelight laser at 670nm.
>
>>
>
>>
>
>>
>
>> Has anyone imaged VisEN ProSense 750 using multiphoton confocal
>> microscopy?
>
>> If so, what are equipment specs generating images.
>
>>
>
>>
>
>>
>
>> Thanks.
>
>
> ________________________________
> Download AOL Toolbar and get access to all of your favourite websites and
> Google powered Search in an instant. Download AOL Toolbar for FREE.

[Rainer Kohler-2]

HI,

we use VIsen probes such as Prosense and Angiosense 680 and 750 a lot in laser scanning microscopy with the Olympus IV100.

The IV100 has a laser for 748 nm excitation and appropriate filters.

I have not tried multi photon imaging of these probes yet.

Rainer

At 6:03 PM -0400 7/15/09, Andreas Bruckbauer wrote:

>If you want to use single photon you can just tune your Ti:Saph to 750 and you should be good to go

You would need a suitable dichroic for direct excitation at 750 nm, the multiphoton dichroics usually reflect everything above 680 or so, your fluorescence signal would not reach the detector. Furthermore the normal photomultiplier detectors are almost blind in the far red, laser power would need to be reduced quite a lot... Has anyone tried direct 1P excitation of these dyes?

Andreas

-----Original Message-----
From: Craig Brideau <[hidden email]>
To: [hidden email]
Sent: Wed, 15 Jul 2009 22:37
Subject: Re: VisEn Prosense 750 on multiphoton?

For multiphoton (in this case 2 photon) won't you need a very long

wavelength?  Probably >1200nm?  Ti:Saphs don't go beyond 1100nm so you

won't be able to use multiphoton to stimulate it unless you have a

Cr:Forsterite laser lying around or an OPA/OPO system strapped to your

Ti:Saph.  If you want to use single photon you can just tune your

Ti:Saph to 750 and you should be good to go.



Craig





On Wed, Jul 15, 2009 at 1:30 PM, Gastinger, Matthew (NIH/NIAID)

[C]<[hidden email]> wrote:

> We have someone who wants to use ProSense 750 (a protease activatable

> fluorescent in vivo imaging agent) from VisEN.  He wants to image in vivo

> using confocal microscopy.  The alternative is ProSense 680, but 750 is

> likely to produce less autofluorescence.  ProSense 680 will likely work with

> 633 laser or whitelight laser at 670nm.

>

>

>

> Has anyone imaged VisEN ProSense 750 using multiphoton confocal microscopy?

> If so, what are equipment specs generating images.

>

>

>

> Thanks.

 

---

Download AOL Toolbar and get access to all of your favourite websites and Google powered Search in an instant. Download AOL Toolbar for FREE.

-- 

Rainer Kohler, Ph.D
Assistant in Research
Cell Phone: [phone]
E.mail: [hidden email]

Center for Systems Biology
Massachusetts General Hospital
Richard B. Simches Research Center
185 Cambridge Street
Suite 5.210
Boston, MA 02114
Phone: [phone]

The information in this e-mail is intended only for the person to whom it is
addressed. If you believe this e-mail was sent to you in error and the e-mail
contains patient information, please contact the Partners Compliance HelpLine at
http://www.partners.org/complianceline . If the e-mail was sent to you in error
but does not contain patient information, please contact the sender and properly
dispose of the e-mail.