Alternative immersion media for a water immersion lens

[Farid Jalali]

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Hello all,
Is anyone aware of a suitable alternative immersion media for a water
immersion lens? This lab is doing experiments at room temperature and the
scope is not enclosed therefore no humidity control.
Thanks very much all.

Farid Jalali

[Guy Cox-2]

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Yes, you can get a silicone (I think) immersion fluid with the same
refractive index as water.  I don't know a supplier off hand but I'm
sure someone here does.

                                                    Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
______________________________________________
Phone [phone]     Fax [phone]
Mobile [phone]
______________________________________________
      http://www.guycox.net
 

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Farid Jalali
Sent: Friday, 22 July 2011 2:43 PM
To: [hidden email]
Subject: Alternative immersion media for a water immersion lens

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Hello all,
Is anyone aware of a suitable alternative immersion media for a water
immersion lens? This lab is doing experiments at room temperature and
the
scope is not enclosed therefore no humidity control.
Thanks very much all.

Farid Jalali

-----
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Checked by AVG - www.avg.com
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[George McNamara]

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  Zeiss Immersol W immersion oil with refractive index 1.33. Should be
available through the Zeiss micro-shop web site.

On 7/22/2011 12:42 AM, Farid Jalali wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello all,
> Is anyone aware of a suitable alternative immersion media for a water
> immersion lens? This lab is doing experiments at room temperature and the
> scope is not enclosed therefore no humidity control.
> Thanks very much all.
>
> Farid Jalali
>
>    

--


George McNamara, PhD
Analytical Imaging Core Facility
University of Miami

[Cameron Nowell]

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Hi Farid,

For small volumes we use the Zeiss Immersol W. But for large scale stuff
like using the large 20x water objective on our multiphoton system we
use a 1:5 dilution of ultrasound gel (make sure you use the clear one,
not the blue tinted stuff).


Cheers

Cam



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Farid Jalali
Sent: Friday, 22 July 2011 2:43 PM
To: [hidden email]
Subject: Alternative immersion media for a water immersion lens

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Hello all,
Is anyone aware of a suitable alternative immersion media for a water
immersion lens? This lab is doing experiments at room temperature and
the
scope is not enclosed therefore no humidity control.
Thanks very much all.

Farid Jalali


This communication is intended only for the named recipient and may contain information that is confidential, legally privileged or subject to copyright; the Ludwig Institute for Cancer Research Ltd does not waive any rights if you have received this communication in error.
The views expressed in this communication are those of the sender and do not necessarily reflect the views of the Ludwig Institute for Cancer Research Ltd.

[Oshel, Philip Eugene]

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Cargille makes water-RI immersion oils for both 37 deg C and 25 deg
C. (The 37 deg is 1.3350 and the 25 deg is 1.330 - by their labels.)
These are in their "AAA" series of immersion oils.

Phil

>*****
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>*****
>
>Hello all,
>Is anyone aware of a suitable alternative immersion media for a water
>immersion lens? This lab is doing experiments at room temperature and the
>scope is not enclosed therefore no humidity control.
>Thanks very much all.
>
>Farid Jalali

--
Philip Oshel
Microscopy Facility Supervisor
Biology Department
024C Brooks Hall
Central Michigan University
Mt. Pleasant, MI 48859
[phone]

[Steffen Dietzel]

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Hi there,

I have used 1:5 ultrasound gel too in the past. The Ri is not identical
to water but close enough for many applications. Just make sure you use
water for dilution. With NaCl solution, somehow the gel structure breaks
down and you get an aqueous liquid with a precipitate, at least with our
ultrasound gel.

I don't know though what the 1:5 dilution in water does to exposed
tissues in terms of physiology, as opposed to superfusion with buffer.
Did anybody investigate?

Steffen


On 22.07.2011 09:08, Cameron Nowell wrote:
>
> Hi Farid,
>
> For small volumes we use the Zeiss Immersol W. But for large scale stuff
> like using the large 20x water objective on our multiphoton system we
> use a 1:5 dilution of ultrasound gel (make sure you use the clear one,
> not the blue tinted stuff).
>


--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
Head of light microscopy

Mail room:
Marchioninistr. 15, D-81377 München

Building location:
Marchioninistr. 27,  München-Großhadern

[Farid Jalali]

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Hello All. Thank you very much to everyone for your replies and insightful
suggestions. It never ceases to amaze me what a wonderful community this is.
Have a great day all.
Cheers
Farid

On Thu, Jul 21, 2011 at 9:42 PM, Farid Jalali <[hidden email]> wrote:

> Hello all,
> Is anyone aware of a suitable alternative immersion media for a water
> immersion lens? This lab is doing experiments at room temperature and the
> scope is not enclosed therefore no humidity control.
> Thanks very much all.
>
> Farid Jalali
>

[Stephen Firth (Med)]

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The other way to do it is a continual water source.
We have the Leica 63x with computer controlled water pump (no commercial
interest), but also use a lab-made system using a pipette, reservoir and
pump (designed by Steve Cody).

We also use the Zeiss immersol and Cargille water RI oils but find they dry
over time and the quality of the image deteriorates. The water pump also
allow imaging multipoint experiments where there is considerable movement of
the objective.
Cheers,
Stephen

On 23 July 2011 03:19, Farid Jalali <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello All. Thank you very much to everyone for your replies and insightful
> suggestions. It never ceases to amaze me what a wonderful community this
> is.
> Have a great day all.
> Cheers
> Farid
>
> On Thu, Jul 21, 2011 at 9:42 PM, Farid Jalali <[hidden email]>
> wrote:
>
> > Hello all,
> > Is anyone aware of a suitable alternative immersion media for a water
> > immersion lens? This lab is doing experiments at room temperature and the
> > scope is not enclosed therefore no humidity control.
> > Thanks very much all.
> >
> > Farid Jalali
> >
>

--
Stephen Firth
Manager of Advanced Optical Microscopy
Monash Micro Imaging
Monash University
Room G58 Building 75
Phone [phone]
Mob [phone]

[Cameron Nowell]

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Hi Steffen

For any live animal work we dilute in PBS or 0.9% saline without any problems.

Cheers

Cam

Sent from my iPhone

On 23/07/2011, at 12:58 AM, "Steffen Dietzel" <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi there,
>
> I have used 1:5 ultrasound gel too in the past. The Ri is not identical to water but close enough for many applications. Just make sure you use water for dilution. With NaCl solution, somehow the gel structure breaks down and you get an aqueous liquid with a precipitate, at least with our ultrasound gel.
>
> I don't know though what the 1:5 dilution in water does to exposed tissues in terms of physiology, as opposed to superfusion with buffer. Did anybody investigate?
>
> Steffen
>
>
> On 22.07.2011 09:08, Cameron Nowell wrote:
>>
>> Hi Farid,
>>
>> For small volumes we use the Zeiss Immersol W. But for large scale stuff
>> like using the large 20x water objective on our multiphoton system we
>> use a 1:5 dilution of ultrasound gel (make sure you use the clear one,
>> not the blue tinted stuff).
>>
>
>
> --
> ------------------------------------------------------------
> Steffen Dietzel, PD Dr. rer. nat
> Ludwig-Maximilians-Universität München
> Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
> Head of light microscopy
>
> Mail room:
> Marchioninistr. 15, D-81377 München
>
> Building location:
> Marchioninistr. 27,  München-Großhadern

This communication is intended only for the named recipient and may contain information that is confidential, legally privileged or subject to copyright; the Ludwig Institute for Cancer Research Ltd does not waive any rights if you have received this communication in error.
The views expressed in this communication are those of the sender and do not necessarily reflect the views of the Ludwig Institute for Cancer Research Ltd.

[Zac Arrac Atelaz]

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We have the Olympus FV10i with automatic water pump, it works fine, it keeps water continuosly on the lens tip in multipoint, time lapse, z-stack acquisitions with no trouble, even combining all of them, it also controls temperature and allows CO2 input working with a external perfussion pump in 35mm dish. I am not sure it can use something different to water but I also feel there is no need
 
Gabriel OH

 

> Date: Sat, 23 Jul 2011 08:25:40 +1000
> From: [hidden email]
> Subject: Re: Alternative immersion media for a water immersion lens
> To: [hidden email]
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> The other way to do it is a continual water source.
> We have the Leica 63x with computer controlled water pump (no commercial
> interest), but also use a lab-made system using a pipette, reservoir and
> pump (designed by Steve Cody).
>
> We also use the Zeiss immersol and Cargille water RI oils but find they dry
> over time and the quality of the image deteriorates. The water pump also
> allow imaging multipoint experiments where there is considerable movement of
> the objective.
> Cheers,
> Stephen
>
> On 23 July 2011 03:19, Farid Jalali <[hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Hello All. Thank you very much to everyone for your replies and insightful
> > suggestions. It never ceases to amaze me what a wonderful community this
> > is.
> > Have a great day all.
> > Cheers
> > Farid
> >
> > On Thu, Jul 21, 2011 at 9:42 PM, Farid Jalali <[hidden email]>
> > wrote:
> >
> > > Hello all,
> > > Is anyone aware of a suitable alternative immersion media for a water
> > > immersion lens? This lab is doing experiments at room temperature and the
> > > scope is not enclosed therefore no humidity control.
> > > Thanks very much all.
> > >
> > > Farid Jalali
> > >
> >
>
>
>
> --
> Stephen Firth
> Manager of Advanced Optical Microscopy
> Monash Micro Imaging
> Monash University
> Room G58 Building 75
> Phone [phone]
> Mob [phone]

     

[Lathrop, Kira L]

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Hi Farid,
You can use Genteal (Novartis Ophthalmics) RI 1.3. It's the media we use to couple a corneal confocal system to the patient's eye. It has a thick, gel-like consistency and so is easy to use on inverted scopes. The Cargille series AAA oil RI 1.335 works well but tends to run.
Hope all is well,
Kira

[Glen MacDonald-2]

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Kira,
How long will Genteal last on a microscope before drying out or changing RI?

regards,
Glen
Glen MacDonald
Core for Communication Research
Virginia Merrill Bloedel Hearing Research Center
Box 357923
University of Washington
Seattle, WA 98195-7923  USA
[phone]
[hidden email]








On Jul 25, 2011, at 4:40 AM, Lathrop, Kira L wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Farid,
> You can use Genteal (Novartis Ophthalmics) RI 1.3. It's the media we use to couple a corneal confocal system to the patient's eye. It has a thick, gel-like consistency and so is easy to use on inverted scopes. The Cargille series AAA oil RI 1.335 works well but tends to run.
> Hope all is well,
> Kira

[leoncio vergara]

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I was wondering if there is any non toxic alternative help in segmentation of nucleus and cytosol compartments to measure protein translocation dynamics in 12-24 hrs time lapse experiments in live cultured cells.

We are working with cell lines stably expressing both GFP and Strawberry fluorescent protein constructs. We want to follow the single cell translocation process and correlate the two signals over a period of 12-24 hrs. We are looking for a tool to aid in segmentation between both compartments for automated image analysis. The problem is that all the DNA binding dyes we have tried are cytotoxic. We have tried DRAQ5, DAPI, HOESCHT and several SITO dyes from molecular probes. We have not tried the Vybrant(r) DyeCycle stains from Invitrogen, violet and Ruby could be compatible with GFP and Strawberry, but a call to their technical support was not very encouraging.

I am wondering if there are any long term live cell tracking dyes that can label the cytosol and give a "negative" image of the nucleus, be non toxic and be compatible with GFP and Strawberry.

Invitrogen also has the CellLight reagents but they are base either on GFP or RFP so won't be a solution either since we are using those channels. I guess we could develop a far-red construct for triple FP labeling, but I was hoping for an easier solution... :)

For additional reference: the microscope we are using is a Prairie Technologies Swept Field Confocal, we have 4 channels available (typical DAPI/FITC/TRITC/Cy5) to work. This is not an spectral imaging system.  

Thanks in advance, any suggestion would be greatly appreciated

Leoncio Vergara
Technical Director
Optical Microscopy Core
Galveston
Texas

[David Baddeley]

One option you could look at is using replication labellin
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Hi Leoncio,

One option you could look at is using replication labelling with a labelled
nuclotide as available from e.g:

http://www.jenabioscience.com/cms/en/1/catalog/1001 (no commercial interest -
was just the first site I hit on in  google. They seem to have conjugates
covering the whole spectral range.)

I'm not too sure on cytotoxicity, but know that cells labelled in this way can
be observed through multiple divisions. The other option that comes to mind is
fluorescent protein tagged histone, although you'd need to find something which
worked spectrally.

Both of these will give you a nuclear labelling rather than a cytoplasmic one.

cheers,
David


----- Original Message ----
From: "Vergara, Leoncio A." <[hidden email]>
To: [hidden email]
Sent: Tue, 26 July, 2011 9:20:01 AM
Subject: Long term Nuclear labeling in live cells

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I was wondering if there is any non toxic alternative help in segmentation of
nucleus and cytosol compartments to measure protein translocation dynamics in
12-24 hrs time lapse experiments in live cultured cells.


We are working with cell lines stably expressing both GFP and Strawberry
fluorescent protein constructs. We want to follow the single cell translocation
process and correlate the two signals over a period of 12-24 hrs. We are looking
for a tool to aid in segmentation between both compartments for automated image
analysis. The problem is that all the DNA binding dyes we have tried are
cytotoxic. We have tried DRAQ5, DAPI, HOESCHT and several SITO dyes from
molecular probes. We have not tried the Vybrant(r) DyeCycle stains from
Invitrogen, violet and Ruby could be compatible with GFP and Strawberry, but a
call to their technical support was not very encouraging.


I am wondering if there are any long term live cell tracking dyes that can label
the cytosol and give a "negative" image of the nucleus, be non toxic and be
compatible with GFP and Strawberry.

Invitrogen also has the CellLight reagents but they are base either on GFP or
RFP so won't be a solution either since we are using those channels. I guess we
could develop a far-red construct for triple FP labeling, but I was hoping for
an easier solution... :)

For additional reference: the microscope we are using is a Prairie Technologies
Swept Field Confocal, we have 4 channels available (typical DAPI/FITC/TRITC/Cy5)
to work. This is not an spectral imaging system.  


Thanks in advance, any suggestion would be greatly appreciated

Leoncio Vergara
Technical Director
Optical Microscopy Core
Galveston
Texas

[Paul Rigby-2]

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Hi Leoncio,
While I can't suggest a solution, I am interested in your comments on the dyes you have tried for your live cell imaging, specifically, the UV/violet excited dyes Hoechst and DAPI.

My experience has been that Hoechst 33342 will stain nuclei in living cells and has not proved toxic over 24 hours of imaging, albeit using very low laser powers and only infrequent imaging. Were you using Hoechst 33342 or Hoechst 33258 (or the slightly longer wavelength excited Hoechst 34580)? Also, what concentration were you using? Frigault et al (J Cell Sci, 122, 753-767, 2009) suggest that to avoid toxicity effects in live cell imaging, these dyes may need to be used at 10-100x more dilute concentrations than usually recommended.

Also you mentioned using DAPI for nuclear staining. This probe is relatively live cell membrane impermeant and usually requires quite high concentrations to get significant nuclear labelling in living cells. At high concentrations DAPI is also toxic so I am not surprised you had problems with this dye.

As an aside, has anyone seen inhibition of cell division when using these DNA intercalating dyes? Some people suggest that cell division is inhibited, but others have not reported any problems. What is the concensus?

Regards
Paul

Assoc. Prof. Paul Rigby
Centre for Microscopy, Characterisation & Analysis (M510)
The University of Western Australia
35 Stirling Highway
Crawley  WA  6007
Australia



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Vergara, Leoncio A.
Sent: Tuesday, 26 July 2011 5:20 AM
To: [hidden email]
Subject: Long term Nuclear labeling in live cells

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I was wondering if there is any non toxic alternative help in segmentation of nucleus and cytosol compartments to measure protein translocation dynamics in 12-24 hrs time lapse experiments in live cultured cells.

We are working with cell lines stably expressing both GFP and Strawberry fluorescent protein constructs. We want to follow the single cell translocation process and correlate the two signals over a period of 12-24 hrs. We are looking for a tool to aid in segmentation between both compartments for automated image analysis. The problem is that all the DNA binding dyes we have tried are cytotoxic. We have tried DRAQ5, DAPI, HOESCHT and several SITO dyes from molecular probes. We have not tried the Vybrant(r) DyeCycle stains from Invitrogen, violet and Ruby could be compatible with GFP and Strawberry, but a call to their technical support was not very encouraging.

I am wondering if there are any long term live cell tracking dyes that can label the cytosol and give a "negative" image of the nucleus, be non toxic and be compatible with GFP and Strawberry.

Invitrogen also has the CellLight reagents but they are base either on GFP or RFP so won't be a solution either since we are using those channels. I guess we could develop a far-red construct for triple FP labeling, but I was hoping for an easier solution... :)

For additional reference: the microscope we are using is a Prairie Technologies Swept Field Confocal, we have 4 channels available (typical DAPI/FITC/TRITC/Cy5) to work. This is not an spectral imaging system.  

Thanks in advance, any suggestion would be greatly appreciated

Leoncio Vergara
Technical Director
Optical Microscopy Core
Galveston
Texas

[Cameron Nowell]

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Hi Paul,

I have used Hoechst 33342 in the past for live imaging both primary and
cultured cells. After about 24 hours they will dye (usually quite
spectacularly). But i have managed to image cells divinging with
Hoechst, but the best is only ever one round of division.

Cheers

Cam



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Paul Rigby
Sent: Tuesday, 26 July 2011 11:46 AM
To: [hidden email]
Subject: Re: Long term Nuclear labeling in live cells

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Hi Leoncio,
While I can't suggest a solution, I am interested in your comments on
the dyes you have tried for your live cell imaging, specifically, the
UV/violet excited dyes Hoechst and DAPI.

My experience has been that Hoechst 33342 will stain nuclei in living
cells and has not proved toxic over 24 hours of imaging, albeit using
very low laser powers and only infrequent imaging. Were you using
Hoechst 33342 or Hoechst 33258 (or the slightly longer wavelength
excited Hoechst 34580)? Also, what concentration were you using?
Frigault et al (J Cell Sci, 122, 753-767, 2009) suggest that to avoid
toxicity effects in live cell imaging, these dyes may need to be used at
10-100x more dilute concentrations than usually recommended.

Also you mentioned using DAPI for nuclear staining. This probe is
relatively live cell membrane impermeant and usually requires quite high
concentrations to get significant nuclear labelling in living cells. At
high concentrations DAPI is also toxic so I am not surprised you had
problems with this dye.

As an aside, has anyone seen inhibition of cell division when using
these DNA intercalating dyes? Some people suggest that cell division is
inhibited, but others have not reported any problems. What is the
concensus?

Regards
Paul

Assoc. Prof. Paul Rigby
Centre for Microscopy, Characterisation & Analysis (M510)
The University of Western Australia
35 Stirling Highway
Crawley  WA  6007
Australia



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Vergara, Leoncio A.
Sent: Tuesday, 26 July 2011 5:20 AM
To: [hidden email]
Subject: Long term Nuclear labeling in live cells

*****
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I was wondering if there is any non toxic alternative help in
segmentation of nucleus and cytosol compartments to measure protein
translocation dynamics in 12-24 hrs time lapse experiments in live
cultured cells.

We are working with cell lines stably expressing both GFP and Strawberry
fluorescent protein constructs. We want to follow the single cell
translocation process and correlate the two signals over a period of
12-24 hrs. We are looking for a tool to aid in segmentation between both
compartments for automated image analysis. The problem is that all the
DNA binding dyes we have tried are cytotoxic. We have tried DRAQ5, DAPI,
HOESCHT and several SITO dyes from molecular probes. We have not tried
the Vybrant(r) DyeCycle stains from Invitrogen, violet and Ruby could be
compatible with GFP and Strawberry, but a call to their technical
support was not very encouraging.

I am wondering if there are any long term live cell tracking dyes that
can label the cytosol and give a "negative" image of the nucleus, be non
toxic and be compatible with GFP and Strawberry.

Invitrogen also has the CellLight reagents but they are base either on
GFP or RFP so won't be a solution either since we are using those
channels. I guess we could develop a far-red construct for triple FP
labeling, but I was hoping for an easier solution... :)

For additional reference: the microscope we are using is a Prairie
Technologies Swept Field Confocal, we have 4 channels available (typical
DAPI/FITC/TRITC/Cy5) to work. This is not an spectral imaging system.  

Thanks in advance, any suggestion would be greatly appreciated

Leoncio Vergara
Technical Director
Optical Microscopy Core
Galveston
Texas


This communication is intended only for the named recipient and may contain information that is confidential, legally privileged or subject to copyright; the Ludwig Institute for Cancer Research Ltd does not waive any rights if you have received this communication in error.
The views expressed in this communication are those of the sender and do not necessarily reflect the views of the Ludwig Institute for Cancer Research Ltd.

[George McNamara]

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Hi Paul,

Hoechst 33### are minor groove binders, not intercalators (at least at
usual concentrations).

With respect to Frigault et al review - they pan Leibowitz media. Not
all mammalian cells behave like CHO-K1 (their Fig 1). One of my users
found Leibowitz works well for her lymphoma cells (and possibly normal
B-cells). I recommend not taking reviews as gospel or any single cell
line as representative for all mammalian (or other) cells.


Enjoy,

George


On 7/25/2011 9:46 PM, Paul Rigby wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Leoncio,
> While I can't suggest a solution, I am interested in your comments on the dyes you have tried for your live cell imaging, specifically, the UV/violet excited dyes Hoechst and DAPI.
>
> My experience has been that Hoechst 33342 will stain nuclei in living cells and has not proved toxic over 24 hours of imaging, albeit using very low laser powers and only infrequent imaging. Were you using Hoechst 33342 or Hoechst 33258 (or the slightly longer wavelength excited Hoechst 34580)? Also, what concentration were you using? Frigault et al (J Cell Sci, 122, 753-767, 2009) suggest that to avoid toxicity effects in live cell imaging, these dyes may need to be used at 10-100x more dilute concentrations than usually recommended.
>
> Also you mentioned using DAPI for nuclear staining. This probe is relatively live cell membrane impermeant and usually requires quite high concentrations to get significant nuclear labelling in living cells. At high concentrations DAPI is also toxic so I am not surprised you had problems with this dye.
>
> As an aside, has anyone seen inhibition of cell division when using these

> DNA intercalating dyes?

> Some people suggest that cell division is inhibited, but others have not reported any problems. What is the concensus?
>
> Regards
> Paul
>
> Assoc. Prof. Paul Rigby
> Centre for Microscopy, Characterisation&  Analysis (M510)
> The University of Western Australia
> 35 Stirling Highway
> Crawley  WA  6007
> Australia
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Vergara, Leoncio A.
> Sent: Tuesday, 26 July 2011 5:20 AM
> To: [hidden email]
> Subject: Long term Nuclear labeling in live cells
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I was wondering if there is any non toxic alternative help in segmentation of nucleus and cytosol compartments to measure protein translocation dynamics in 12-24 hrs time lapse experiments in live cultured cells.
>
> We are working with cell lines stably expressing both GFP and Strawberry fluorescent protein constructs. We want to follow the single cell translocation process and correlate the two signals over a period of 12-24 hrs. We are looking for a tool to aid in segmentation between both compartments for automated image analysis. The problem is that all the DNA binding dyes we have tried are cytotoxic. We have tried DRAQ5, DAPI, HOESCHT and several SITO dyes from molecular probes. We have not tried the Vybrant(r) DyeCycle stains from Invitrogen, violet and Ruby could be compatible with GFP and Strawberry, but a call to their technical support was not very encouraging.
>
> I am wondering if there are any long term live cell tracking dyes that can label the cytosol and give a "negative" image of the nucleus, be non toxic and be compatible with GFP and Strawberry.
>
> Invitrogen also has the CellLight reagents but they are base either on GFP or RFP so won't be a solution either since we are using those channels. I guess we could develop a far-red construct for triple FP labeling, but I was hoping for an easier solution... :)
>
> For additional reference: the microscope we are using is a Prairie Technologies Swept Field Confocal, we have 4 channels available (typical DAPI/FITC/TRITC/Cy5) to work. This is not an spectral imaging system.
>
> Thanks in advance, any suggestion would be greatly appreciated
>
> Leoncio Vergara
> Technical Director
> Optical Microscopy Core
> Galveston
> Texas
>
>    

--


George McNamara, PhD
Analytical Imaging Core Facility
University of Miami

[Claudia Florindo]

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Hi Leoncio

Dapi is probably not good because you use UV light for imaging, and cells do
dye under UVs.

I have imaged cells for 24h (time points every 3 min) labeled with
GFP-Histone 2B. If you can get histone 2B with a in fusion with a
fluorescent protein that goes to the CY5 channel it should work.

You could also try Histone GFP and do a bright field /DIC image where you
can clearly see the nucleus and the cytoplasm. Or DIC alone merged with the
other 2 florescent channels images that you have can also help you to
distinguish between the nucleus and the cytoplasm.

Good luck

Claudia.  

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Vergara, Leoncio A.
Sent: segunda-feira, 25 de Julho de 2011 22:20
To: [hidden email]
Subject: Long term Nuclear labeling in live cells

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

I was wondering if there is any non toxic alternative help in segmentation
of nucleus and cytosol compartments to measure protein translocation
dynamics in 12-24 hrs time lapse experiments in live cultured cells.

We are working with cell lines stably expressing both GFP and Strawberry
fluorescent protein constructs. We want to follow the single cell
translocation process and correlate the two signals over a period of 12-24
hrs. We are looking for a tool to aid in segmentation between both
compartments for automated image analysis. The problem is that all the DNA
binding dyes we have tried are cytotoxic. We have tried DRAQ5, DAPI, HOESCHT
and several SITO dyes from molecular probes. We have not tried the
Vybrant(r) DyeCycle stains from Invitrogen, violet and Ruby could be
compatible with GFP and Strawberry, but a call to their technical support
was not very encouraging.

I am wondering if there are any long term live cell tracking dyes that can
label the cytosol and give a "negative" image of the nucleus, be non toxic
and be compatible with GFP and Strawberry.

Invitrogen also has the CellLight reagents but they are base either on GFP
or RFP so won't be a solution either since we are using those channels. I
guess we could develop a far-red construct for triple FP labeling, but I was
hoping for an easier solution... :)

For additional reference: the microscope we are using is a Prairie
Technologies Swept Field Confocal, we have 4 channels available (typical
DAPI/FITC/TRITC/Cy5) to work. This is not an spectral imaging system.  

Thanks in advance, any suggestion would be greatly appreciated

Leoncio Vergara
Technical Director
Optical Microscopy Core
Galveston
Texas

-----
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07/25/11

[Confocal2011]

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While our work is with budding yeast and I have virtually no experience
outside of yeast, the fact that a fluorescent protein tag was mentioned by
someone else suggests that maybe it would work for you. In that case I will
just add that we use histone 2B with a C-terminal mCherry tag with amazing
results.

On Tue, Jul 26, 2011 at 3:53 AM, Claudia Florindo <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Leoncio
>
> Dapi is probably not good because you use UV light for imaging, and cells
> do
> dye under UVs.
>
> I have imaged cells for 24h (time points every 3 min) labeled with
> GFP-Histone 2B. If you can get histone 2B with a in fusion with a
> fluorescent protein that goes to the CY5 channel it should work.
>
> You could also try Histone GFP and do a bright field /DIC image where you
> can clearly see the nucleus and the cytoplasm. Or DIC alone merged with the
> other 2 florescent channels images that you have can also help you to
> distinguish between the nucleus and the cytoplasm.
>
> Good luck
>
> Claudia.
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On
> Behalf Of Vergara, Leoncio A.
> Sent: segunda-feira, 25 de Julho de 2011 22:20
> To: [hidden email]
> Subject: Long term Nuclear labeling in live cells
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I was wondering if there is any non toxic alternative help in segmentation
> of nucleus and cytosol compartments to measure protein translocation
> dynamics in 12-24 hrs time lapse experiments in live cultured cells.
>
> We are working with cell lines stably expressing both GFP and Strawberry
> fluorescent protein constructs. We want to follow the single cell
> translocation process and correlate the two signals over a period of 12-24
> hrs. We are looking for a tool to aid in segmentation between both
> compartments for automated image analysis. The problem is that all the DNA
> binding dyes we have tried are cytotoxic. We have tried DRAQ5, DAPI,
> HOESCHT
> and several SITO dyes from molecular probes. We have not tried the
> Vybrant(r) DyeCycle stains from Invitrogen, violet and Ruby could be
> compatible with GFP and Strawberry, but a call to their technical support
> was not very encouraging.
>
> I am wondering if there are any long term live cell tracking dyes that can
> label the cytosol and give a "negative" image of the nucleus, be non toxic
> and be compatible with GFP and Strawberry.
>
> Invitrogen also has the CellLight reagents but they are base either on GFP
> or RFP so won't be a solution either since we are using those channels. I
> guess we could develop a far-red construct for triple FP labeling, but I
> was
> hoping for an easier solution... :)
>
> For additional reference: the microscope we are using is a Prairie
> Technologies Swept Field Confocal, we have 4 channels available (typical
> DAPI/FITC/TRITC/Cy5) to work. This is not an spectral imaging system.
>
> Thanks in advance, any suggestion would be greatly appreciated
>
> Leoncio Vergara
> Technical Director
> Optical Microscopy Core
> Galveston
> Texas
>
> -----
> Nenhum vírus encontrado nessa mensagem.
> Verificado por AVG - www.avgbrasil.com.br
> Versão: 10.0.1390 / Banco de dados de vírus: 1518/3788 - Data de
> Lançamento:
> 07/25/11
>

[Masur, Sandra]

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

Dear all who indicate that various nuclear dyes will cause

> cultured cells....to... dye (usually quite
> spectacularly)

You mean that the "dye" (addition of a color) will cause the cells to "die" (cease living).

English spelling can be very confusing!

On Jul 25, 2011, at 10:24 PM, Cameron Nowell wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Paul,
>
> I have used Hoechst 33342 in the past for live imaging both primary and
> cultured cells. After about 24 hours they will dye (usually quite
> spectacularly). But i have managed to image cells divinging with
> Hoechst, but the best is only ever one round of division.
>
> Cheers
>
> Cam
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Paul Rigby
> Sent: Tuesday, 26 July 2011 11:46 AM
> To: [hidden email]
> Subject: Re: Long term Nuclear labeling in live cells
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Leoncio,
> While I can't suggest a solution, I am interested in your comments on
> the dyes you have tried for your live cell imaging, specifically, the
> UV/violet excited dyes Hoechst and DAPI.
>
> My experience has been that Hoechst 33342 will stain nuclei in living
> cells and has not proved toxic over 24 hours of imaging, albeit using
> very low laser powers and only infrequent imaging. Were you using
> Hoechst 33342 or Hoechst 33258 (or the slightly longer wavelength
> excited Hoechst 34580)? Also, what concentration were you using?
> Frigault et al (J Cell Sci, 122, 753-767, 2009) suggest that to avoid
> toxicity effects in live cell imaging, these dyes may need to be used at
> 10-100x more dilute concentrations than usually recommended.
>
> Also you mentioned using DAPI for nuclear staining. This probe is
> relatively live cell membrane impermeant and usually requires quite high
> concentrations to get significant nuclear labelling in living cells. At
> high concentrations DAPI is also toxic so I am not surprised you had
> problems with this dye.
>
> As an aside, has anyone seen inhibition of cell division when using
> these DNA intercalating dyes? Some people suggest that cell division is
> inhibited, but others have not reported any problems. What is the
> concensus?
>
> Regards
> Paul
>
> Assoc. Prof. Paul Rigby
> Centre for Microscopy, Characterisation & Analysis (M510)
> The University of Western Australia
> 35 Stirling Highway
> Crawley  WA  6007
> Australia
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Vergara, Leoncio A.
> Sent: Tuesday, 26 July 2011 5:20 AM
> To: [hidden email]
> Subject: Long term Nuclear labeling in live cells
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I was wondering if there is any non toxic alternative help in
> segmentation of nucleus and cytosol compartments to measure protein
> translocation dynamics in 12-24 hrs time lapse experiments in live
> cultured cells.
>
> We are working with cell lines stably expressing both GFP and Strawberry
> fluorescent protein constructs. We want to follow the single cell
> translocation process and correlate the two signals over a period of
> 12-24 hrs. We are looking for a tool to aid in segmentation between both
> compartments for automated image analysis. The problem is that all the
> DNA binding dyes we have tried are cytotoxic. We have tried DRAQ5, DAPI,
> HOESCHT and several SITO dyes from molecular probes. We have not tried
> the Vybrant(r) DyeCycle stains from Invitrogen, violet and Ruby could be
> compatible with GFP and Strawberry, but a call to their technical
> support was not very encouraging.
>
> I am wondering if there are any long term live cell tracking dyes that
> can label the cytosol and give a "negative" image of the nucleus, be non
> toxic and be compatible with GFP and Strawberry.
>
> Invitrogen also has the CellLight reagents but they are base either on
> GFP or RFP so won't be a solution either since we are using those
> channels. I guess we could develop a far-red construct for triple FP
> labeling, but I was hoping for an easier solution... :)
>
> For additional reference: the microscope we are using is a Prairie
> Technologies Swept Field Confocal, we have 4 channels available (typical
> DAPI/FITC/TRITC/Cy5) to work. This is not an spectral imaging system.  
>
> Thanks in advance, any suggestion would be greatly appreciated
>
> Leoncio Vergara
> Technical Director
> Optical Microscopy Core
> Galveston
> Texas
>
>
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