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Concentrating bacteria cells for microscope visualization.

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Tom Lawson

Concentrating bacteria cells for microscope visualization.

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Dear Confocalists,

I am having a few problems separating and concentrating bacteria cells for
microscope visualization.

I have a 1 ml solution in an aliquot that contains some eukaryote debris and
10 to 100 bacteria cells. After spotting the centrifuge concentrate to a
slide-well, I can see the bacteria with a nucleic stain. But the debris in
the concentrate makes it hard to pipette and blocks seeing the bacteria. In
addition, the concentrate is spread over a 6 mm slide-well which makes it
hard to locate the bacteria.

I would be grateful for any suggestions.

Regards,

--
Tom Lawson
[hidden email]
PhD Student,
Macquarie University
NSW, 2109
Australia

Kilgore, Jason-2

Re: Concentrating bacteria cells for microscope visualization.

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Tom, though it won't help with microscopic examination, have you considered using a microplate reader format? You could use a nucleic acid stain, such as Hoechst 33342, and compare the signal to a standard curve generated from wells with known bacterial densities.

Jason

** vendor association acknowledged **

Jason A. Kilgore
Technical Application Scientist
Molecular Probes Labeling and Detection Technologies
Cells Systems Division

T 1 800 955 6288 then option 5 or 541 335 0353 . F 541 335 0238
29851 Willow Creek Rd . Eugene . OR . 97402-9132 . United States
www.invitrogen.com/technicalsupport

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Tom Lawson
Sent: Sunday, June 26, 2011 9:45 PM
To: [hidden email]
Subject: Concentrating bacteria cells for microscope visualization.

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

Dear Confocalists,

I am having a few problems separating and concentrating bacteria cells for
microscope visualization.

I have a 1 ml solution in an aliquot that contains some eukaryote debris and
10 to 100 bacteria cells. After spotting the centrifuge concentrate to a
slide-well, I can see the bacteria with a nucleic stain. But the debris in
the concentrate makes it hard to pipette and blocks seeing the bacteria. In
addition, the concentrate is spread over a 6 mm slide-well which makes it
hard to locate the bacteria.

I would be grateful for any suggestions.

Regards,

--
Tom Lawson
[hidden email]
PhD Student,
Macquarie University
NSW, 2109
Australia

Tom Lawson

Re: Concentrating bacteria cells for microscope visualization.

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

Hi Jason, Thank you for the good idea. Tom

On Mon, Jun 27, 2011 at 4:13 PM, Kilgore, Jason
<[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Tom, though it won't help with microscopic examination, have you considered
> using a microplate reader format? You could use a nucleic acid stain, such
> as Hoechst 33342, and compare the signal to a standard curve generated from
> wells with known bacterial densities.
>
> Jason
>
> ** vendor association acknowledged **
>
> Jason A. Kilgore
> Technical Application Scientist
> Molecular Probes Labeling and Detection Technologies
> Cells Systems Division
>
> T 1 800 955 6288 then option 5 or 541 335 0353 . F 541 335 0238
> 29851 Willow Creek Rd . Eugene . OR . 97402-9132 . United States
> www.invitrogen.com/technicalsupport
>
>
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Tom Lawson
> Sent: Sunday, June 26, 2011 9:45 PM
> To: [hidden email]
> Subject: Concentrating bacteria cells for microscope visualization.
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Confocalists,
>
> I am having a few problems separating and concentrating bacteria cells for
> microscope visualization.
>
> I have a 1 ml solution in an aliquot that contains some eukaryote debris
> and
> 10 to 100 bacteria cells. After spotting the centrifuge concentrate to a
> slide-well, I can see the bacteria with a nucleic stain. But the debris in
> the concentrate makes it hard to pipette and blocks seeing the bacteria. In
> addition, the concentrate is spread over a 6 mm slide-well which makes it
> hard to locate the bacteria.
>
> I would be grateful for any suggestions.
>
> Regards,
>
> --
> Tom Lawson
> [hidden email]
> PhD Student,
> Macquarie University
> NSW, 2109
> Australia
>

--
Tom Lawson
[hidden email]
PhD Student,
Macquarie University
NSW, 2109
Australia

Tobias Baskin

endocytosis for cell biology lab

In reply to this post by Tom Lawson

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

Hi all,
This is not a "confocal" question but rather a cell
bio/microscopy related one. Nevertheless, I am hoping someone will be
able to answer.

I co-teach a cell biology lab course where students learn how
to use conventional epi fluorescence and video microscopy. We have a
variety of "cells in action" labs for them to do, one of these being
endocytosis. As markers, e have used labeled-transferrin and also DiI
labeled LDL, with the idea being that the transferrin is recycled
back to the plasma membrane whereas the LDL moves into lysosomes.
While the transferrin labeling seems to work more or less as
expected, results with the DiI LDL are erratic. Sometimes there is no
labeling at all, and we never have seen what looks like deep internal
labeling in putative lysosomes.

We plan to troubleshoot later this summer but as none of us
have studied endocytosis, we have no first hand experience. I am
wondering if anyone out there knows of a tried and true endocytosis
cell bio lab? Or perhaps knows about some tricks for handling DiI
LDL. Or perhaps it is a matter of a good cell line -- we work
typically with a 3T3 fibroblasts or LLCPK epithelial cells (with
equally dismal results for the DiI LDL endocytosis).

Thanks in advance for any suggestions.

As ever
Tobias Baskin
--
_ ____ __ ____
/ \ / / \ / \ \ Tobias I. Baskin
/ / / / \ \ \ Biology Department
/_ / __ /__ \ \ \__ 611 N. Pleasant St.
/ / / \ \ \ University of Massachusetts
/ / / \ \ \ Amherst, MA, 01003
/ / ___ / \ \__/ \ ____
www.bio.umass.edu/biology/baskin
Voice: 413 - 545 - 1533 Fax: 413 - 545 - 3243

G. Esteban Fernandez

Re: endocytosis for cell biology lab

In reply to this post by Tom Lawson

*****
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*****

Lucifer yellow reaches yeast cell (S. cerevisiae) vacuoles by fluid-phase
endocytosis in an hour. Might work with mammalian cells too.
http://jcs.biologists.org/content/117/14/2983/F6.expansion.html

-Esteban

On Tue, Jun 28, 2011 at 11:25 AM, Tobias Baskin <[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
> *****
>
> Hi all,
> This is not a "confocal" question but rather a cell bio/microscopy
> related one. Nevertheless, I am hoping someone will be able to answer.
>
> I co-teach a cell biology lab course where students learn how to use
> conventional epi fluorescence and video microscopy. We have a variety of
> "cells in action" labs for them to do, one of these being endocytosis. As
> markers, e have used labeled-transferrin and also DiI labeled LDL, with the
> idea being that the transferrin is recycled back to the plasma membrane
> whereas the LDL moves into lysosomes. While the transferrin labeling seems
> to work more or less as expected, results with the DiI LDL are erratic.
> Sometimes there is no labeling at all, and we never have seen what looks
> like deep internal labeling in putative lysosomes.
>
> We plan to troubleshoot later this summer but as none of us have
> studied endocytosis, we have no first hand experience. I am wondering if
> anyone out there knows of a tried and true endocytosis cell bio lab? Or
> perhaps knows about some tricks for handling DiI LDL. Or perhaps it is a
> matter of a good cell line -- we work typically with a 3T3 fibroblasts or
> LLCPK epithelial cells (with equally dismal results for the DiI LDL
> endocytosis).
>
> Thanks in advance for any suggestions.
>
> As ever
> Tobias Baskin
> --
> _ ____ __ ____
> / \ / / \ / \ \ Tobias I. Baskin
> / / / / \ \ \ Biology Department
> /_ / __ /__ \ \ \__ 611 N. Pleasant St.
> / / / \ \ \ University of Massachusetts
> / / / \ \ \ Amherst, MA, 01003
> / / ___ / \ \__/ \ ____
> www.bio.umass.edu/biology/**baskin<http://www.bio.umass.edu/biology/baskin>
> Voice: 413 - 545 - 1533 Fax: 413 - 545 - 3243
>

Gudrun Ihrke

Re: endocytosis for cell biology lab

In reply to this post by Tobias Baskin

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

Tobias,

Unless you preincubate with lipoprotein-deficient serum, the LDL
receptor will be downregulated. It would be much easier to use a small
fluorescent dextran to label the endocytic pathway towards lysosomes:
~3 min incubation will give you early endosomal staining, ~15-45 min
late endosomal (best use a pulse of 10-15 min pulse and chase 15-30
min for bright staining, >1h late endosomal/lysosomal staining (use
also a pulse if you want to chase the dextran out of earlier
compartments).

Gudrun

Gudrun Ihrke, Ph.D.
Research Assistant Professor
Department of Pharmacology (C2025)
Uniformed Services University School of Medicine
4301 Jones Bridge Road
Bethesda, MD 20814

On Jun 28, 2011, at 2:25 PM, Tobias Baskin wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi all,
> This is not a "confocal" question but rather a cell bio/microscopy
> related one. Nevertheless, I am hoping someone will be able to answer.
>
> I co-teach a cell biology lab course where students learn how to
> use conventional epi fluorescence and video microscopy. We have a
> variety of "cells in action" labs for them to do, one of these being
> endocytosis. As markers, e have used labeled-transferrin and also
> DiI labeled LDL, with the idea being that the transferrin is
> recycled back to the plasma membrane whereas the LDL moves into
> lysosomes. While the transferrin labeling seems to work more or less
> as expected, results with the DiI LDL are erratic. Sometimes there
> is no labeling at all, and we never have seen what looks like deep
> internal labeling in putative lysosomes.
>
> We plan to troubleshoot later this summer but as none of us have
> studied endocytosis, we have no first hand experience. I am
> wondering if anyone out there knows of a tried and true endocytosis
> cell bio lab? Or perhaps knows about some tricks for handling DiI
> LDL. Or perhaps it is a matter of a good cell line -- we work
> typically with a 3T3 fibroblasts or LLCPK epithelial cells (with
> equally dismal results for the DiI LDL endocytosis).
>
> Thanks in advance for any suggestions.
>
> As ever
> Tobias Baskin
> --
> _ ____ __ ____
> / \ / / \ / \ \ Tobias I. Baskin
> / / / / \ \ \ Biology Department
> /_ / __ /__ \ \ \__ 611 N. Pleasant St.
> / / / \ \ \ University of
> Massachusetts
> / / / \ \ \ Amherst, MA, 01003
> / / ___ / \ \__/ \ ____
> www.bio.umass.edu/biology/baskin
> Voice: 413 - 545 - 1533 Fax: 413 - 545 - 3243

email: [hidden email]

Classification: UNCLASSIFIED
Caveats: None

Kilgore, Jason-2

Re: endocytosis for cell biology lab

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

I second that advice, though I imagine the timing would depend upon the cell line and environmental conditions.

Gudrun, what was the cell line and media you used?

Jason

** vendor association acknowledged **

Jason A. Kilgore
Technical Application Scientist
Molecular Probes Labeling and Detection Technologies
Cells Systems Division

T 1 800 955 6288 then option 5 or 541 335 0353 . F 541 335 0238
29851 Willow Creek Rd . Eugene . OR . 97402-9132 . United States
www.invitrogen.com/technicalsupport

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Gudrun Ihrke
Sent: Tuesday, June 28, 2011 12:26 PM
To: [hidden email]
Subject: Re: endocytosis for cell biology lab

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

Tobias,

Unless you preincubate with lipoprotein-deficient serum, the LDL
receptor will be downregulated. It would be much easier to use a small
fluorescent dextran to label the endocytic pathway towards lysosomes:
~3 min incubation will give you early endosomal staining, ~15-45 min
late endosomal (best use a pulse of 10-15 min pulse and chase 15-30
min for bright staining, >1h late endosomal/lysosomal staining (use
also a pulse if you want to chase the dextran out of earlier
compartments).

Gudrun

Gudrun Ihrke, Ph.D.
Research Assistant Professor
Department of Pharmacology (C2025)
Uniformed Services University School of Medicine
4301 Jones Bridge Road
Bethesda, MD 20814

On Jun 28, 2011, at 2:25 PM, Tobias Baskin wrote:

email: [hidden email]

Classification: UNCLASSIFIED
Caveats: None

"Brüne Venus"

Re: Concentrating bacteria cells for microscope visualization.

In reply to this post by Tom Lawson

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

Hi Tom,

Since a couple of years now Jakob Pernthaler concentrates bacteria using a filter approach and afterwards detects them using fluorescent markers like DAPI and even does FISH. For data acquisition he uses an upright fluorescence microscope with scanning stage. He is now based in Zuerich Switzerland www.limnology.ch - or pubmed.

Best regards

Bruene Venus

Company affiliation:
Carl Zeiss MicroImaging Germany
Mailto: [hidden email]

-------- Original-Nachricht --------
> Datum: Mon, 27 Jun 2011 14:44:51 +1000
> Von: Tom Lawson <[hidden email]>
> An: [hidden email]
> Betreff: Concentrating bacteria cells for microscope visualization.

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Confocalists,
>
> I am having a few problems separating and concentrating bacteria cells for
> microscope visualization.
>
> I have a 1 ml solution in an aliquot that contains some eukaryote debris
> and
> 10 to 100 bacteria cells. After spotting the centrifuge concentrate to a
> slide-well, I can see the bacteria with a nucleic stain. But the debris in
> the concentrate makes it hard to pipette and blocks seeing the bacteria.
> In
> addition, the concentrate is spread over a 6 mm slide-well which makes it
> hard to locate the bacteria.
>
> I would be grateful for any suggestions.
>
> Regards,
>
> --
> Tom Lawson
> [hidden email]
> PhD Student,
> Macquarie University
> NSW, 2109
> Australia

--
Empfehlen Sie GMX DSL Ihren Freunden und Bekannten und wir
belohnen Sie mit bis zu 50,- Euro! https://freundschaftswerbung.gmx.de

Gudrun Ihrke

Re: endocytosis for cell biology lab

In reply to this post by Kilgore, Jason-2

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

Jason,
The times should hold up pretty well for many mammalian cell lines,
provided the cells are incubated at 37C and have prominent clathrin-
mediated endocytosis. We have used fibroblasts or epithelial cells.
Fluid phase uptake is of course through all endocytic pathways, but I
have little experience with cells that use mostly other uptake
mechanisms. We have used either normal serum-containing tissue
culture medium (buffered with HEPES if pH is a concern due to small
volumes, and/or preequilibrate the dextran-containing medium in CO2
incubator) or BSA-containing medium for the pulse.

Regards,
Gudrun

On Jun 28, 2011, at 4:05 PM, Kilgore, Jason wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I second that advice, though I imagine the timing would depend upon
> the cell line and environmental conditions.
>
> Gudrun, what was the cell line and media you used?
>
> Jason
>
> ** vendor association acknowledged **
>
> Jason A. Kilgore
> Technical Application Scientist
> Molecular Probes Labeling and Detection Technologies
> Cells Systems Division
>
> T 1 800 955 6288 then option 5 or 541 335 0353 . F 541 335 0238
> 29851 Willow Creek Rd . Eugene . OR . 97402-9132 . United States
> www.invitrogen.com/technicalsupport
>
>
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]
> ] On Behalf Of Gudrun Ihrke
> Sent: Tuesday, June 28, 2011 12:26 PM
> To: [hidden email]
> Subject: Re: endocytosis for cell biology lab
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Tobias,
>
> Unless you preincubate with lipoprotein-deficient serum, the LDL
> receptor will be downregulated. It would be much easier to use a small
> fluorescent dextran to label the endocytic pathway towards lysosomes:
> ~3 min incubation will give you early endosomal staining, ~15-45 min
> late endosomal (best use a pulse of 10-15 min pulse and chase 15-30
> min for bright staining, >1h late endosomal/lysosomal staining (use
> also a pulse if you want to chase the dextran out of earlier
> compartments).
>
> Gudrun
>
> Gudrun Ihrke, Ph.D.
> Research Assistant Professor
> Department of Pharmacology (C2025)
> Uniformed Services University School of Medicine
> 4301 Jones Bridge Road
> Bethesda, MD 20814
>
>
> On Jun 28, 2011, at 2:25 PM, Tobias Baskin wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi all,
>> This is not a "confocal" question but rather a cell bio/microscopy
>> related one. Nevertheless, I am hoping someone will be able to
>> answer.
>>
>> I co-teach a cell biology lab course where students learn how to
>> use conventional epi fluorescence and video microscopy. We have a
>> variety of "cells in action" labs for them to do, one of these being
>> endocytosis. As markers, e have used labeled-transferrin and also
>> DiI labeled LDL, with the idea being that the transferrin is
>> recycled back to the plasma membrane whereas the LDL moves into
>> lysosomes. While the transferrin labeling seems to work more or less
>> as expected, results with the DiI LDL are erratic. Sometimes there
>> is no labeling at all, and we never have seen what looks like deep
>> internal labeling in putative lysosomes.
>>
>> We plan to troubleshoot later this summer but as none of us have
>> studied endocytosis, we have no first hand experience. I am
>> wondering if anyone out there knows of a tried and true endocytosis
>> cell bio lab? Or perhaps knows about some tricks for handling DiI
>> LDL. Or perhaps it is a matter of a good cell line -- we work
>> typically with a 3T3 fibroblasts or LLCPK epithelial cells (with
>> equally dismal results for the DiI LDL endocytosis).
>>
>> Thanks in advance for any suggestions.
>>
>> As ever
>> Tobias Baskin
>> --
>> _ ____ __ ____
>> / \ / / \ / \ \ Tobias I. Baskin
>> / / / / \ \ \ Biology Department
>> /_ / __ /__ \ \ \__ 611 N. Pleasant St.
>> / / / \ \ \ University of
>> Massachusetts
>> / / / \ \ \ Amherst, MA, 01003
>> / / ___ / \ \__/ \ ____
>> www.bio.umass.edu/biology/baskin
>> Voice: 413 - 545 - 1533 Fax: 413 - 545 - 3243
>
>
>
>
> email: [hidden email]
>
>
> Classification: UNCLASSIFIED
> Caveats: None

Classification: UNCLASSIFIED
Caveats: None

JOEL B. SHEFFIELD

Re: endocytosis for cell biology lab

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

Hi,

Gudrun, by "small", what mw dextran are you suggesting?

Joel

On Wed, Jun 29, 2011 at 11:21 AM, Gudrun Ihrke <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
> *****
>
> Jason,
> The times should hold up pretty well for many mammalian cell lines,
> provided the cells are incubated at 37C and have prominent clathrin-mediated
> endocytosis. We have used fibroblasts or epithelial cells. Fluid phase
> uptake is of course through all endocytic pathways, but I have little
> experience with cells that use mostly other uptake mechanisms. We have used
> either normal serum-containing tissue culture medium (buffered with HEPES if
> pH is a concern due to small volumes, and/or preequilibrate the
> dextran-containing medium in CO2 incubator) or BSA-containing medium for
> the pulse.
>
> Regards,
> Gudrun
>
>
> On Jun 28, 2011, at 4:05 PM, Kilgore, Jason wrote:
>
> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
>> *****
>>
>> I second that advice, though I imagine the timing would depend upon the
>> cell line and environmental conditions.
>>
>> Gudrun, what was the cell line and media you used?
>>
>> Jason
>>
>> ** vendor association acknowledged **
>>
>> Jason A. Kilgore
>> Technical Application Scientist
>> Molecular Probes Labeling and Detection Technologies
>> Cells Systems Division
>>
>> T 1 800 955 6288 then option 5 or 541 335 0353 . F 541 335 0238
>> 29851 Willow Creek Rd . Eugene . OR . 97402-9132 . United States
>> www.invitrogen.com/**technicalsupport<http://www.invitrogen.com/technicalsupport>
>>
>>
>>
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:CONFOCALMICROSCOPY@**LISTS.UMN.EDU<[hidden email]>]
>> On Behalf Of Gudrun Ihrke
>> Sent: Tuesday, June 28, 2011 12:26 PM
>> To: [hidden email].**EDU <[hidden email]>
>> Subject: Re: endocytosis for cell biology lab
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
>> *****
>>
>> Tobias,
>>
>> Unless you preincubate with lipoprotein-deficient serum, the LDL
>> receptor will be downregulated. It would be much easier to use a small
>> fluorescent dextran to label the endocytic pathway towards lysosomes:
>> ~3 min incubation will give you early endosomal staining, ~15-45 min
>> late endosomal (best use a pulse of 10-15 min pulse and chase 15-30
>> min for bright staining, >1h late endosomal/lysosomal staining (use
>> also a pulse if you want to chase the dextran out of earlier
>> compartments).
>>
>> Gudrun
>>
>> Gudrun Ihrke, Ph.D.
>> Research Assistant Professor
>> Department of Pharmacology (C2025)
>> Uniformed Services University School of Medicine
>> 4301 Jones Bridge Road
>> Bethesda, MD 20814
>>
>>
>> On Jun 28, 2011, at 2:25 PM, Tobias Baskin wrote:
>>
>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
>>> *****
>>>
>>> Hi all,
>>> This is not a "confocal" question but rather a cell bio/microscopy
>>> related one. Nevertheless, I am hoping someone will be able to answer.
>>>
>>> I co-teach a cell biology lab course where students learn how to
>>> use conventional epi fluorescence and video microscopy. We have a
>>> variety of "cells in action" labs for them to do, one of these being
>>> endocytosis. As markers, e have used labeled-transferrin and also
>>> DiI labeled LDL, with the idea being that the transferrin is
>>> recycled back to the plasma membrane whereas the LDL moves into
>>> lysosomes. While the transferrin labeling seems to work more or less
>>> as expected, results with the DiI LDL are erratic. Sometimes there
>>> is no labeling at all, and we never have seen what looks like deep
>>> internal labeling in putative lysosomes.
>>>
>>> We plan to troubleshoot later this summer but as none of us have
>>> studied endocytosis, we have no first hand experience. I am
>>> wondering if anyone out there knows of a tried and true endocytosis
>>> cell bio lab? Or perhaps knows about some tricks for handling DiI
>>> LDL. Or perhaps it is a matter of a good cell line -- we work
>>> typically with a 3T3 fibroblasts or LLCPK epithelial cells (with
>>> equally dismal results for the DiI LDL endocytosis).
>>>
>>> Thanks in advance for any suggestions.
>>>
>>> As ever
>>> Tobias Baskin
>>> --
>>> _ ____ __ ____
>>> / \ / / \ / \ \ Tobias I. Baskin
>>> / / / / \ \ \ Biology Department
>>> /_ / __ /__ \ \ \__ 611 N. Pleasant St.
>>> / / / \ \ \ University of
>>> Massachusetts
>>> / / / \ \ \ Amherst, MA, 01003
>>> / / ___ / \ \__/ \ ____
>>> www.bio.umass.edu/biology/**baskin<http://www.bio.umass.edu/biology/baskin>
>>> Voice: 413 - 545 - 1533 Fax: 413 - 545 - 3243
>>>
>>
>>
>>
>>
>> email: [hidden email]
>>
>>
>> Classification: UNCLASSIFIED
>> Caveats: None
>>
>
>
> Classification: UNCLASSIFIEDCaveats: None
>

--

Joel B. Sheffield, Ph.D
Department of Biology
Temple University
Philadelphia, PA 19122
Voice: 215 204 8839
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Gudrun Ihrke

Re: endocytosis for cell biology lab

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We have used both 10K and 3K successfully (the lysine-fixable
dextrans), but the 3K is preferable.

On Jun 29, 2011, at 11:46 AM, JOEL B. SHEFFIELD wrote:

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> *****
>
> Hi,
>
> Gudrun, by "small", what mw dextran are you suggesting?
>
> Joel
>
>
> On Wed, Jun 29, 2011 at 11:21 AM, Gudrun Ihrke <[hidden email]>
> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> >
>> *****
>>
>> Jason,
>> The times should hold up pretty well for many mammalian cell lines,
>> provided the cells are incubated at 37C and have prominent clathrin-
>> mediated
>> endocytosis. We have used fibroblasts or epithelial cells. Fluid
>> phase
>> uptake is of course through all endocytic pathways, but I have little
>> experience with cells that use mostly other uptake mechanisms. We
>> have used
>> either normal serum-containing tissue culture medium (buffered with
>> HEPES if
>> pH is a concern due to small volumes, and/or preequilibrate the
>> dextran-containing medium in CO2 incubator) or BSA-containing
>> medium for
>> the pulse.
>>
>> Regards,
>> Gudrun
>>
>>
>> On Jun 28, 2011, at 4:05 PM, Kilgore, Jason wrote:
>>
>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> >
>>> *****
>>>
>>> I second that advice, though I imagine the timing would depend
>>> upon the
>>> cell line and environmental conditions.
>>>
>>> Gudrun, what was the cell line and media you used?
>>>
>>> Jason
>>>
>>> ** vendor association acknowledged **
>>>
>>> Jason A. Kilgore
>>> Technical Application Scientist
>>> Molecular Probes Labeling and Detection Technologies
>>> Cells Systems Division
>>>
>>> T 1 800 955 6288 then option 5 or 541 335 0353 . F 541 335 0238
>>> 29851 Willow Creek Rd . Eugene . OR . 97402-9132 . United States
>>> www.invitrogen.com/**technicalsupport<http://www.invitrogen.com/technicalsupport
>>> >
>>>
>>>
>>>
>>>
>>>
>>> -----Original Message-----
>>> From: Confocal Microscopy List [mailto:CONFOCALMICROSCOPY@**LISTS.UMN.EDU
>>> <[hidden email]>]
>>> On Behalf Of Gudrun Ihrke
>>> Sent: Tuesday, June 28, 2011 12:26 PM
>>> To: [hidden email].**EDU <[hidden email]
>>> >
>>> Subject: Re: endocytosis for cell biology lab
>>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> >
>>> *****
>>>
>>> Tobias,
>>>
>>> Unless you preincubate with lipoprotein-deficient serum, the LDL
>>> receptor will be downregulated. It would be much easier to use a
>>> small
>>> fluorescent dextran to label the endocytic pathway towards
>>> lysosomes:
>>> ~3 min incubation will give you early endosomal staining, ~15-45 min
>>> late endosomal (best use a pulse of 10-15 min pulse and chase 15-30
>>> min for bright staining, >1h late endosomal/lysosomal staining (use
>>> also a pulse if you want to chase the dextran out of earlier
>>> compartments).
>>>
>>> Gudrun
>>>
>>> Gudrun Ihrke, Ph.D.
>>> Research Assistant Professor
>>> Department of Pharmacology (C2025)
>>> Uniformed Services University School of Medicine
>>> 4301 Jones Bridge Road
>>> Bethesda, MD 20814
>>>
>>>
>>> On Jun 28, 2011, at 2:25 PM, Tobias Baskin wrote:
>>>
>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> >
>>>> *****
>>>>
>>>> Hi all,
>>>> This is not a "confocal" question but rather a cell bio/
>>>> microscopy
>>>> related one. Nevertheless, I am hoping someone will be able to
>>>> answer.
>>>>
>>>> I co-teach a cell biology lab course where students learn
>>>> how to
>>>> use conventional epi fluorescence and video microscopy. We have a
>>>> variety of "cells in action" labs for them to do, one of these
>>>> being
>>>> endocytosis. As markers, e have used labeled-transferrin and also
>>>> DiI labeled LDL, with the idea being that the transferrin is
>>>> recycled back to the plasma membrane whereas the LDL moves into
>>>> lysosomes. While the transferrin labeling seems to work more or
>>>> less
>>>> as expected, results with the DiI LDL are erratic. Sometimes there
>>>> is no labeling at all, and we never have seen what looks like deep
>>>> internal labeling in putative lysosomes.
>>>>
>>>> We plan to troubleshoot later this summer but as none of us
>>>> have
>>>> studied endocytosis, we have no first hand experience. I am
>>>> wondering if anyone out there knows of a tried and true endocytosis
>>>> cell bio lab? Or perhaps knows about some tricks for handling DiI
>>>> LDL. Or perhaps it is a matter of a good cell line -- we work
>>>> typically with a 3T3 fibroblasts or LLCPK epithelial cells (with
>>>> equally dismal results for the DiI LDL endocytosis).
>>>>
>>>> Thanks in advance for any suggestions.
>>>>
>>>> As ever
>>>> Tobias Baskin
>>>> --
>>>> _ ____ __ ____
>>>> / \ / / \ / \ \ Tobias I. Baskin
>>>> / / / / \ \ \ Biology Department
>>>> /_ / __ /__ \ \ \__ 611 N. Pleasant St.
>>>> / / / \ \ \ University of
>>>> Massachusetts
>>>> / / / \ \ \ Amherst, MA,
>>>> 01003
>>>> / / ___ / \ \__/ \ ____
>>>> www.bio.umass.edu/biology/**baskin<http://www.bio.umass.edu/biology/baskin
>>>> >
>>>> Voice: 413 - 545 - 1533 Fax: 413 - 545 - 3243
>>>>
>>>
>>>
>>>
>>>
>>> email: [hidden email]
>>>
>>>
>>> Classification: UNCLASSIFIED
>>> Caveats: None
>>>
>>
>>
>> Classification: UNCLASSIFIEDCaveats: None
>>
>
>
>
> --
>
>
> Joel B. Sheffield, Ph.D
> Department of Biology
> Temple University
> Philadelphia, PA 19122
> Voice: 215 204 8839
> e-mail: [hidden email]
> URL: http://astro.temple.edu/~jbs

Gudrun Ihrke, Ph.D.
Research Assistant Professor
Department of Pharmacology (C2025)
Uniformed Services University School of Medicine
4301 Jones Bridge Road
Bethesda, MD 20814

phone office: (301) 295 3225
phone lab: (301) 295 3823
FAX: (301) 295 3220
email: [hidden email]

Classification: UNCLASSIFIED
Caveats: None

mmodel

chromatic aberration

In reply to this post by Tobias Baskin

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Hello and happy holidays to everyone!

Both of our 10/0.4 planapo objectives have a bad longitudinal chromatic aberration between blue and far red (2.5 um axial shift with correct coverslip). Is this normal for low-power objectives?

Mike

Martin Wessendorf-2

Re: chromatic aberration

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Hey, Mike--

On 12/28/2011 2:01 PM, MODEL, MICHAEL wrote:

> Both of our 10/0.4 planapo objectives have a bad longitudinal chromatic aberration between blue and far red (2.5 um axial shift with correct coverslip). Is this normal for low-power objectives?

What manufacturer? Not to trash any particular brand but I've seen
problems that bad on 60x oil objectives from some makers and would
expect a 10x objective to be even worse.

Martin
--
Martin Wessendorf, Ph.D. office: (612) 626-0145
Assoc Prof, Dept Neuroscience lab: (612) 624-2991
University of Minnesota Preferred FAX: (612) 624-8118
6-145 Jackson Hall, 321 Church St. SE Dept Fax: (612) 626-5009
Minneapolis, MN 55455 e-mail: [hidden email]

mmodel

Re: chromatic aberration

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Olympus. Their higher-NA objectives are fine

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Martin Wessendorf
Sent: Wednesday, December 28, 2011 3:25 PM
To: [hidden email]
Subject: Re: chromatic aberration

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Hey, Mike--

On 12/28/2011 2:01 PM, MODEL, MICHAEL wrote:

> Both of our 10/0.4 planapo objectives have a bad longitudinal chromatic aberration between blue and far red (2.5 um axial shift with correct coverslip). Is this normal for low-power objectives?

What manufacturer? Not to trash any particular brand but I've seen
problems that bad on 60x oil objectives from some makers and would
expect a 10x objective to be even worse.

Martin
--
Martin Wessendorf, Ph.D. office: (612) 626-0145
Assoc Prof, Dept Neuroscience lab: (612) 624-2991
University of Minnesota Preferred FAX: (612) 624-8118
6-145 Jackson Hall, 321 Church St. SE Dept Fax: (612) 626-5009
Minneapolis, MN 55455 e-mail: [hidden email]

Martin Wessendorf-2

Re: chromatic aberration

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How are you measuring the aberration? Beads? Reflectance?

On 12/28/2011 2:46 PM, MODEL, MICHAEL wrote:

> Olympus. Their higher-NA objectives are fine
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Martin Wessendorf
> Sent: Wednesday, December 28, 2011 3:25 PM
> To: [hidden email]
> Subject: Re: chromatic aberration
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hey, Mike--
>
> On 12/28/2011 2:01 PM, MODEL, MICHAEL wrote:
>
>> Both of our 10/0.4 planapo objectives have a bad longitudinal chromatic aberration between blue and far red (2.5 um axial shift with correct coverslip). Is this normal for low-power objectives?
>
> What manufacturer? Not to trash any particular brand but I've seen
> problems that bad on 60x oil objectives from some makers and would
> expect a 10x objective to be even worse.
>
> Martin

--
Martin Wessendorf, Ph.D. office: (612) 626-0145
Assoc Prof, Dept Neuroscience lab: (612) 624-2991
University of Minnesota Preferred FAX: (612) 624-8118
6-145 Jackson Hall, 321 Church St. SE Dept Fax: (612) 626-5009
Minneapolis, MN 55455 e-mail: [hidden email]

mmodel

Re: chromatic aberration

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I use fluorescent multicolor beads on a coverslip. But even before, I have noticed that when I am looking at cells in transmission, I see different things depending on the filter. You may argue that with cells, there is spherical aberration, etc but with beads there shouldn't be any. So I thought that perhaps low-NA objectives are generally less well corrected for longitudinal chromatic aberration? Even when of the planapo type. Olympus does not provide any numbers, just says that the objectives are 'excellent".

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Martin Wessendorf
Sent: Wednesday, December 28, 2011 4:26 PM
To: [hidden email]
Subject: Re: chromatic aberration

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How are you measuring the aberration? Beads? Reflectance?

On 12/28/2011 2:46 PM, MODEL, MICHAEL wrote:

Guy Cox-2

Re: chromatic aberration

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The CA you mention seems quite extreme for an NA 0.4 objective, but you need to define what you mean by far red. I think the red wavelength usually used for the third correction point is 646nm. Some apochromats can diverge quite sharply beyond this point, so if your 'far-red' is 700nm then you are beyond the correction range of the objective and have no grounds for complaint. Some modern apos are violet corrected and will therefore probably be even worse at long wavelengths - but this should be marked on the lens. Some apos are marked IR but I'm not sure if that implies chromatic correction into the IR or just good transmission.

If you get a 2.5µm focal shift between 486 (blue) and 646 (red) then you should demand a replacement from the manufacturer.

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)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone +61 2 9351 3176 Fax +61 2 9351 7682
Mobile 0413 281 861
______________________________________________
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-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of MODEL, MICHAEL
Sent: Thursday, 29 December 2011 8:36 AM
To: [hidden email]
Subject: Re: chromatic aberration

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I use fluorescent multicolor beads on a coverslip. But even before, I have noticed that when I am looking at cells in transmission, I see different things depending on the filter. You may argue that with cells, there is spherical aberration, etc but with beads there shouldn't be any. So I thought that perhaps low-NA objectives are generally less well corrected for longitudinal chromatic aberration? Even when of the planapo type. Olympus does not provide any numbers, just says that the objectives are 'excellent".

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Martin Wessendorf
Sent: Wednesday, December 28, 2011 4:26 PM
To: [hidden email]
Subject: Re: chromatic aberration

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How are you measuring the aberration? Beads? Reflectance?

On 12/28/2011 2:46 PM, MODEL, MICHAEL wrote:

mcammer

Re: chromatic aberration

In reply to this post by mmodel

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Every objective needs to be tested with beads with the wavelengths you are using. Any time we need precise XY or Z axis registration we test with beads or fluorescent coatings on the surfaces in the same media as the biological imaging, sometimes even on the cells. Years ago we found out the hard way, when doing CFP/YFP ratio FRET, that there is no substitute for characterizing each objective as close to the real imaging conditions as practical. Generalizations such as, "High NA brand X planapo objective are fine," just are not true.
_________________________________________
Michael Cammer, Assistant Research Scientist
Skirball Institute of Biomolecular Medicine
Lab: (212) 263-3208 Cell: (914) 309-3270

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Johannes Helm

Re: chromatic aberration

In reply to this post by mmodel

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Dear Michael,

it possibly depends on the generation of objective.

I recall that, during the early 90s, when I was a PhD student at Aaslund's
and Carlsson's at Physics IV at KTH in Stockholm, we worked intensively on
UV CLSM techniques. We measured the axial chromatic aberrations of a large
number of lenses, including "exotic" objectives like Ealing mirror
objectives, a Bausch & Lomb catadioptric water immersion lens and the
Zeiss 32x/0,4 Glyc Ultrafluar, all of which behaved excellently.
At the time, a local Olympus engineer wanted to learn more about UV vs
visible chromatic aberrations of the lenses he sold, called us and asked
us to measure the aberrations of the objectives he had, among which a 10X
apochromat, which, however, never had been designed for UV wavelengths or
confocal microscopy. He just wanted to "get an idea about the aberrations"
and whether the lenses would be suitable for UV, even if they had not been
designed neither for confocal, nor for UV microscopy.
PLEASE NOTE! THESE WERE OLD STYLE LENSES (160mm tubelength), and at the
time considerably cheaper than lenses by other manufacturers!
If I recall correctly, we experienced at the time a more than 10 microns
axial shift between 365nm and 514nm, while there were, if I recall
correctly, a little bit more than 1 micron between 488nm and 514nm. The
measurements were done using a plane mirror as reflective specimen and the
mentioned aberrations were in the center of the field of view.
At the time, we did not have any deep blue laser line available, neither a
red one.

In order to not be misunderstood here:
These old Olympus lenses most probably cannot be compared to modern.
However, if you have an old lens, the values you indicate might be
absolutely correct.

Best wishes,
Johannes

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello and happy holidays to everyone!
>
> Both of our 10/0.4 planapo objectives have a bad longitudinal chromatic
> aberration between blue and far red (2.5 um axial shift with correct
> coverslip). Is this normal for low-power objectives?
>
> Mike
>

--
P. Johannes Helm

Voice: (+47) 228 51159 (office)
Fax: (+47) 228 51499 (office)

Richard Cole

Re: chromatic aberration

In reply to this post by mmodel

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Aberrations within our optical systems are more common that most images
think. This belief was confirmed in an international image performance
study recently completed (results: Stack, R., Bayles, C., Girard, A.,
Martin, K., Opansky, C., Schulz, K., and Cole, R. (2011) Quality Assurance
Testing for Modern Optical Imaging Systems. Microscopy & Microanalysis
17(4):598-606).

Not wanting to be redundant but, check all the objective that you image with
regularly. It is well worth the time and energy!

Richard Cole
Research Scientist V
Director: Advanced Light Microscopy & Image Analysis Core
Wadsworth Center
P.O. Box 509 Albany N.Y. 12201-0509

Research Assistant Professor

Dept. of Biomedical Sciences

School of Public Health State University of New York Albany, New York

( 518-474-7048
Ê 518-474-4430

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