another TDE experience

[Oky]

Hello listers,

We have been working on refractive index matching following the
direction from Staudt, etc 2007.
[Reference] Thorsten Staudt, Marion C. Lang, Rebecca Medda, Johann
Engelhardt, and Stefan W. Hell. 2,2'-Thiodiethanol: A New Water Soluble
Mounting Medium for High Resolution Optical Microscopy. Microscopy
Research and Technique 70:1-9 (2007).

We prepared 10 %, 25 %, 50 %, and 97 % of TDE solution as stated in the
above reference. Linear relationship between the refractive index and
TDE concentration was confirmed by several measurements. However, we
have encountered two issues.

1. During the preparation of pure TDE (Thiodiethylene glycol from Fluka
88559) at pH 7.5 it was found out that the titration process is very
difficult. As stated in the above literature, we put 1 micro liter of
HCl (1.2 N). The pH was significantly changed to close to 2. Thus, we
dilute the HCl solution to 0.6 N. However, the changes of pH was also
significant. To compensate the pH level, 1 micro liter of NaOH (1 N) was
put into the very acidic solution. Then the pH went up to 9. From this
point, we prepared a very dilute HCl solution (0.015 N). Through series
of titration with 5 micro liter at each time the pH of the TDE solution
was adjusted to 7.42.

2. Sections of mouse brain with DNA stain or fluorescent Nissl were
prepared and kept in a refrigerator. After overnight storage,  crystal
was formed in the slide. So we prepared another sample and kept in the
air to see whether temperature affects. Still, crystal was formed. Yet,
we don't know the reason for formation of crystals in the slides.

If you have any similar experience and overcame it, please let us know.
Thanks very much.

Best regards,
Ohkyung Kwon

* I resend this message due to my first posting was rejected.

[regan m]

Can you decrease the dye concentraton for dna staining as i think that the Dye is phase separating or crystalising out of samples (ensure that there is no microbial contamination by adding smal amounts of Sodium azide to the DNA if stored for long periods) and let me know at what temperature did you store the DNa samples overnight 4deg c or in the freezer at -10 deg.

Ideally store samples at 4deg cen under controlled humidity conditions.  

 

Rgds

On Tue, Oct 14, 2008 at 3:46 AM, Ohkyung Kwon <[hidden email]> wrote:

Hello listers,

We have been working on refractive index matching following the direction from Staudt, etc 2007.
[Reference] Thorsten Staudt, Marion C. Lang, Rebecca Medda, Johann Engelhardt, and Stefan W. Hell. 2,2'-Thiodiethanol: A New Water Soluble Mounting Medium for High Resolution Optical Microscopy. Microscopy Research and Technique 70:1-9 (2007).

We prepared 10 %, 25 %, 50 %, and 97 % of TDE solution as stated in the above reference. Linear relationship between the refractive index and TDE concentration was confirmed by several measurements. However, we have encountered two issues.

1. During the preparation of pure TDE (Thiodiethylene glycol from Fluka 88559) at pH 7.5 it was found out that the titration process is very difficult. As stated in the above literature, we put 1 micro liter of HCl (1.2 N). The pH was significantly changed to close to 2. Thus, we dilute the HCl solution to 0.6 N. However, the changes of pH was also significant. To compensate the pH level, 1 micro liter of NaOH (1 N) was put into the very acidic solution. Then the pH went up to 9. From this point, we prepared a very dilute HCl solution (0.015 N). Through series of titration with 5 micro liter at each time the pH of the TDE solution was adjusted to 7.42.

2. Sections of mouse brain with DNA stain or fluorescent Nissl were prepared and kept in a refrigerator. After overnight storage,  crystal was formed in the slide. So we prepared another sample and kept in the air to see whether temperature affects. Still, crystal was formed. Yet, we don't know the reason for formation of crystals in the slides.

If you have any similar experience and overcame it, please let us know. Thanks very much.

Best regards,
Ohkyung Kwon

* I resend this message due to my first posting was rejected.

[Carol Bayles]

I have a request from someone in industry who is not on the confocal
list.  He wants to look at a
4uM x 3,000uM sample with a confocal microscope using a laser with a
wavelength of 980 nm and
image the light emitted at 1060 nm.   does anyone have this
capability and would like to collaborate?  Please contact me offline.
thanks,
Carol Bayles
--
<><><><><><><><><><><><><><><><><><><><><>
Carol Bayles, Manager
Microscopy & Imaging Facility (MIF)
Life Sciences Core Lab Center
160a Biotech Bldg
607-254-4860
607-254-6379 fax
http://cores.lifesciences.cornell.edu

Confocal and Multiphoton Microscopy
Nanobiotechnology Center
www.nbtc.cornell.edu

Cornell University
Ithaca NY 14853

[SUBSCRIBE CONFOCALMICROSCOPY Anonymous]

hello kwon,

Did you also measure the pH of the TDE before adding HCl (It´s  a bit
difficult because if no water is present, H3O+ are not built affecting the
potential on the glas elektrode; but you can add a small amount of pure
water to measure it)?
The pH of the commercially available TDE varies from batch to batch ranging
from around 5.5 to 11. The lower the purity the lower the pH. Maybe there is
an acidic contamination in the lower purity batches. But this should not be
an issue since the concentration of impurities are very low (not detectable
by GCMS) and the pH can be adjusted to around 7 if you consider one
important fact: The liquid is quite viscous which hinders the establishment
of the equilibrium. You should measure the pH for at least one hour to
equilibrate the electrode. If you start to adjust the pH with a batch
exhibiting a pH of around 6 i can imagine that you end up at a pH of around
2 or three adding HCl. For each individual batch you have to preadjust the pH.

The crystallisation problem.

Maybe NaCl is precipitating since you added 1µl 1N HCl and 1µl 1N NaOH to
the TDE (unfortunately i don´t know the volume) and PBS 1x is also added to
match the refractive index. I would just replace the buffer by pure water
(The performance should not change dramatically) and search for crystals
inside the sample. I doubt that the TDE itself causes the problem. I think
it is a salt from the used buffer system.
 

[Patrick Van Oostveldt]

Dear,

Jus a small remark,

If it is the PBS, try to use Kalium instead of sodium-hydroxyde  
because KH2PO4 has a better solibity at lower temperature. About 167  
to 59.

Bye

PatrickQuoting SUBSCRIBE CONFOCALMICROSCOPY Anonymous  
<[hidden email]>:

> hello kwon,
>
> Did you also measure the pH of the TDE before adding HCl (It´s  a bit
> difficult because if no water is present, H3O+ are not built affecting the
> potential on the glas elektrode; but you can add a small amount of pure
> water to measure it)?
> The pH of the commercially available TDE varies from batch to batch ranging
> from around 5.5 to 11. The lower the purity the lower the pH. Maybe there is
> an acidic contamination in the lower purity batches. But this should not be
> an issue since the concentration of impurities are very low (not detectable
> by GCMS) and the pH can be adjusted to around 7 if you consider one
> important fact: The liquid is quite viscous which hinders the establishment
> of the equilibrium. You should measure the pH for at least one hour to
> equilibrate the electrode. If you start to adjust the pH with a batch
> exhibiting a pH of around 6 i can imagine that you end up at a pH of around
> 2 or three adding HCl. For each individual batch you have to  
> preadjust the pH.
>
> The crystallisation problem.
>
> Maybe NaCl is precipitating since you added 1µl 1N HCl and 1µl 1N NaOH to
> the TDE (unfortunately i don´t know the volume) and PBS 1x is also added to
> match the refractive index. I would just replace the buffer by pure water
> (The performance should not change dramatically) and search for crystals
> inside the sample. I doubt that the TDE itself causes the problem. I think
> it is a salt from the used buffer system.
>
>

--
Dep. Moleculaire Biotechnologie
Coupure links 653
B 9000 GENT

tel [phone]
fax [phone]

[regan m]

Hii

 

If you think that these could be salts .... then the best deal would be to directly pin point at what salt it could be .... to avoid it in the buffer..

 

The most easiest way is to load the sample into an Electron microscope and analyse what salt it is through EDX maping ..........If there is no Electron microscpy facility with EDX I can help you out with analysis.

 

.. Let me know if you need any help.

On Tue, Oct 14, 2008 at 2:44 PM, Patrick Van Oostveldt <[hidden email]> wrote:

Dear,

Jus a small remark,

If it is the PBS, try to use Kalium instead of sodium-hydroxyde because KH2PO4 has a better solibity at lower temperature. About 167 to 59.

Bye

PatrickQuoting SUBSCRIBE CONFOCALMICROSCOPY Anonymous <[hidden email]>:

hello kwon,

Did you also measure the pH of the TDE before adding HCl (It´s  a bit
difficult because if no water is present, H3O+ are not built affecting the
potential on the glas elektrode; but you can add a small amount of pure
water to measure it)?
The pH of the commercially available TDE varies from batch to batch ranging
from around 5.5 to 11. The lower the purity the lower the pH. Maybe there is
an acidic contamination in the lower purity batches. But this should not be
an issue since the concentration of impurities are very low (not detectable
by GCMS) and the pH can be adjusted to around 7 if you consider one
important fact: The liquid is quite viscous which hinders the establishment
of the equilibrium. You should measure the pH for at least one hour to
equilibrate the electrode. If you start to adjust the pH with a batch
exhibiting a pH of around 6 i can imagine that you end up at a pH of around
2 or three adding HCl. For each individual batch you have to  preadjust the pH.

The crystallisation problem.

Maybe NaCl is precipitating since you added 1µl 1N HCl and 1µl 1N NaOH to
the TDE (unfortunately i don´t know the volume) and PBS 1x is also added to
match the refractive index. I would just replace the buffer by pure water
(The performance should not change dramatically) and search for crystals
inside the sample. I doubt that the TDE itself causes the problem. I think
it is a salt from the used buffer system.

--
Dep. Moleculaire Biotechnologie
Coupure links 653
B 9000 GENT

tel [phone]
fax [phone]

[Julio Vazquez]

-

Greetings!

The Northwest Regional Cytometry Association and the Fred Hutchinson Cancer Research Center are sponsoring a seminar devoted to Laser Scanning Cytometry on Tuesday, November 18, entitled “Quantitative Imaging Cytometry in Biomedical Research, Drug Discovery and Biomarker Development.” 

 

The presentations include:

 

·       “Regulation of Cell Cycle Transitions” – James Jacobberger, Professor of Oncology, Case Western Reserve University and Director, Cell Analysis Core, Case Comprehensive Cancer Center, Cleveland, OH

·       “Assessment of DNA Damage Response in Relation to Cell Cycle and Induction of Apoptosis by Laser Scanning Cytometry” - Zbigniew Darzynkiewicz, Director, Brander Cancer Research Institute, New York Medical College, Valhalla, NY

 

We will also offer an opportunity on November 18 and 19 to see a demonstration of the iCys^® Research Imaging Cytometer.  There will be an opportunity as well to have your samples evaluated on the instrument.

 

The meeting takes place in the Weintraub Building at the Fred Hutchinson Cancer Research Center in Seattle.  The meeting, demonstration and sample evaluation are free of charge; however, advance registration for the seminar, demonstration, and/or sample evaluation would be appreciated.  Please email Raffi Manoukian at CompuCyte Corporation ([hidden email]) if you wish to attend.

 

--

Julio Vazquez, PhD

Director of Scientific Imaging

Fred Hutchinson Cancer Research Center

1100 Fairview Ave. N.,  mailstop DE-512

Seattle, WA 98109-1024

Tel: Office: 206-667-1215/ Lab: 206-667-4205

FAX: [phone]

[hidden email]

http://www.fhcrc.org/science/shared_resources/imaging/

for directions to the FHCRC:

http://www.fhcrc.org/about/maps/