Upright dipping lenses - artefacts during solution changes

[Christof Schwiening]

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

Dear All,
I wonder if someone could throw some light on a problem we have been having
for the past few years! We use a Leica SP5 confocal on an upright microscope
with dipping lenses to image pH and calcium-sensitive fluorescent dyes. During
solution changes - most notably the addition and removal of organic
compounds such as propionate (20 mM)- we get severe image distortion and
changes in focal depth. I attempted to attach a graph of a ROI from a fixed
sealed specimen which we have imaged during solution changes with 3
different objectives - however, the ListServer rejected every image format I
tried...I can send it by email if anyone is interested. It is impossible that the
propionate is actually getting to the specimen itself - I suspect it must be as a
result of refractive index changes.

My questions are: How do others image small structures (i.e. dendritic spines
or NMJs) during changes of solution with differing refractive indicies? Is there
some well know trick? Or, am I using the wrong objectives? Does wide field
microscopy have the same problems?
Many thanks,
Christof

[Vladimir Ghukasyan-2]

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

Hello Christof,

As you mentioned, one of the most prominent reasons for the
distortions you see is change of the refractive index. Lenses are
usually corrected for chromatic and spherical aberrations for some
defined refractive index. The more you deviate from it, the more will
be the extent of the aberrations affecting your image. One way of
overcoming this is using lenses with adjustable refractive index
correction collar.

With regards,
Vladimir

On Mon, Mar 18, 2013 at 11:29 AM, Christof Schwiening <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear All,
> I wonder if someone could throw some light on a problem we have been having
> for the past few years! We use a Leica SP5 confocal on an upright microscope
> with dipping lenses to image pH and calcium-sensitive fluorescent dyes. During
> solution changes - most notably the addition and removal of organic
> compounds such as propionate (20 mM)- we get severe image distortion and
> changes in focal depth. I attempted to attach a graph of a ROI from a fixed
> sealed specimen which we have imaged during solution changes with 3
> different objectives - however, the ListServer rejected every image format I
> tried...I can send it by email if anyone is interested. It is impossible that the
> propionate is actually getting to the specimen itself - I suspect it must be as a
> result of refractive index changes.
>
> My questions are: How do others image small structures (i.e. dendritic spines
> or NMJs) during changes of solution with differing refractive indicies? Is there
> some well know trick? Or, am I using the wrong objectives? Does wide field
> microscopy have the same problems?
> Many thanks,
> Christof

[Julio Vazquez]

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

Christof,

I don't actually have an answer to your problem. However, my feeling is that going from let's say water to 20 mM propionate should have only a very minimal effect on refractive index (RI water = 1.333, 100% propionate: 1.386; 20 mM propionate maybe ~ 1.335; check tables/charts listing RI as a function of concentration for propionate or another similar substance). Adjusting the refractive index collar as suggested by Vladimir is one good idea, if you have one; those however are found most likely on high NA immersion objectives, not so much on dipping objectives. I guess what may be worth knowing is which specific objectives you are using, and also what the distortions look like. Is it possible that you are getting your cells dry and wet again (in which case maybe the cells themselves may be doing something funky); or they may not like to be in propionic acid; or is it possible you are trapping air bubbles under your objective as you replace medium?


Julio Vazquez,
Fred Hutchinson Cancer Research Center

http://www.fhcrc.org

==

On Mar 18, 2013, at 9:24 AM, Vladimir Ghukasyan wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello Christof,
>
> As you mentioned, one of the most prominent reasons for the
> distortions you see is change of the refractive index. Lenses are
> usually corrected for chromatic and spherical aberrations for some
> defined refractive index. The more you deviate from it, the more will
> be the extent of the aberrations affecting your image. One way of
> overcoming this is using lenses with adjustable refractive index
> correction collar.
>
> With regards,
> Vladimir
>
> On Mon, Mar 18, 2013 at 11:29 AM, Christof Schwiening <[hidden email]> wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear All,
>> I wonder if someone could throw some light on a problem we have been having
>> for the past few years! We use a Leica SP5 confocal on an upright microscope
>> with dipping lenses to image pH and calcium-sensitive fluorescent dyes. During
>> solution changes - most notably the addition and removal of organic
>> compounds such as propionate (20 mM)- we get severe image distortion and
>> changes in focal depth. I attempted to attach a graph of a ROI from a fixed
>> sealed specimen which we have imaged during solution changes with 3
>> different objectives - however, the ListServer rejected every image format I
>> tried...I can send it by email if anyone is interested. It is impossible that the
>> propionate is actually getting to the specimen itself - I suspect it must be as a
>> result of refractive index changes.
>>
>> My questions are: How do others image small structures (i.e. dendritic spines
>> or NMJs) during changes of solution with differing refractive indicies? Is there
>> some well know trick? Or, am I using the wrong objectives? Does wide field
>> microscopy have the same problems?
>> Many thanks,
>> Christof

[Craig Brideau]

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

The list doesn't allow image attachments as everyone's inbox would quickly
fill up.  Your best bet is to host the image on dropbox or the like and
include the link to the image in your email.

Craig


On Mon, Mar 18, 2013 at 9:29 AM, Christof Schwiening <[hidden email]>wrote:

> *****
>  I attempted to attach a graph of a ROI from a fixed
> sealed specimen which we have imaged during solution changes with 3
> different objectives - however, the ListServer rejected every image format
> I
> tried...I can send it by email if anyone is interested.

Christof
>

[Arvydas Matiukas]

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

Hi Christof,
 
More quantitative description of your imaging geometry/optics
would be useful. I have been observing similar effects when  imaging
a thick sample  on
inverted scope using 40x water dipping lens in immersion mode.  
When water drop substantially shrinks after 2-3 hrs
focus starts shifting until disappears completely (from the set range).
This corresponds to ultimate refractive index change from 1.333 to 1.0.
The possible solution is to set big enough thickness of z-stack so that
your specimen still fits into imaged  focal range (taking into account the
z-range shift due to refractive index change).
 
I would like to see your graph and the distorted image. I wonder
if your chemicals are not dissolving well or recrystalizing thus creating
nonhomogenous layers distorting  your image. Or maybe some vibrations
create inhomogeneities in the immersion/dipping medium. Do you observe this
type of distortion with fixed specimens as well?
 
Best regards,
Arvydas
--------------------------------


 
 
Arvydas Matiukas, Ph.D.
Director of Confocal&Two-Photon Core
Department of Neurosci& Physiology
SUNY Upstate Medical University
766 Irving Ave., WH 3167
Syracuse, NY 13210
tel.: [phone]
fax: [phone]
email: [hidden email]
>>> Christof Schwiening <[hidden email]> 3/18/2013 10:29 AM >>>
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Dear All,
I wonder if someone could throw some light on a problem we have been having
for the past few years! We use a Leica SP5 confocal on an upright microscope
with dipping lenses to image pH and calcium-sensitive fluorescent dyes. During
solution changes - most notably the addition and removal of organic
compounds such as propionate (20 mM)- we get severe image distortion and
changes in focal depth. I attempted to attach a graph of a ROI from a fixed
sealed specimen which we have imaged during solution changes with 3
different objectives - however, the ListServer rejected every image format I
tried...I can send it by email if anyone is interested. It is impossible that the
propionate is actually getting to the specimen itself - I suspect it must be as a
result of refractive index changes.

My questions are: How do others image small structures (i.e. dendritic spines
or NMJs) during changes of solution with differing refractive indicies? Is there
some well know trick? Or, am I using the wrong objectives? Does wide field
microscopy have the same problems?
Many thanks,
Christof

[Christof Schwiening]

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

On Mon, 18 Mar 2013 10:13:51 -0700, Julio Vazquez <[hidden email]>
wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
Dear Julio,
There is no adjustable collar on the objectives - the link below shows the list
of dipping objectives I have used and what a ROI plot looks like.
https://dl.dropbox.com/u/24087924/Convallaria%20slide.jpg
The collar adjustment would be of little use since I need to record changes
during solution switches. My cells are definitely always staying wet. The effect
is nothing to do with the effect of propionate on the cells since I see the
distortions when I run the solution over a fixed glass slide with a preparation
beneath a coverslip (embeded and sealed).
I agree that the refractive index change is small...
There are no air bubbles beneath the objective. I think it must be a Schlieren
type of effect.
Greetings,
Christof

[Christof Schwiening]

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

Dear Craig,
Here is the link:
https://dl.dropbox.com/u/24087924/Convallaria%20slide.jpg
Greetings,
Christof

[Christof Schwiening]

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

Dear Vladimir,
Yes, but I need to record during the solution changes - on a second by
second basis - and there is no correction collar....
Greetings,
Christof

On Mon, 18 Mar 2013 12:24:01 -0400, Vladimir Ghukasyan
<[hidden email]> wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Hello Christof,
>
>As you mentioned, one of the most prominent reasons for the
>distortions you see is change of the refractive index. Lenses are
>usually corrected for chromatic and spherical aberrations for some
>defined refractive index. The more you deviate from it, the more will
>be the extent of the aberrations affecting your image. One way of
>overcoming this is using lenses with adjustable refractive index
>correction collar.
>
>With regards,
>Vladimir
>
>On Mon, Mar 18, 2013 at 11:29 AM, Christof Schwiening <[hidden email]>

wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear All,
>> I wonder if someone could throw some light on a problem we have been
having
>> for the past few years! We use a Leica SP5 confocal on an upright
microscope
>> with dipping lenses to image pH and calcium-sensitive fluorescent dyes.
During
>> solution changes - most notably the addition and removal of organic
>> compounds such as propionate (20 mM)- we get severe image distortion
and
>> changes in focal depth. I attempted to attach a graph of a ROI from a fixed
>> sealed specimen which we have imaged during solution changes with 3
>> different objectives - however, the ListServer rejected every image format I
>> tried...I can send it by email if anyone is interested. It is impossible that the
>> propionate is actually getting to the specimen itself - I suspect it must be
as a
>> result of refractive index changes.
>>
>> My questions are: How do others image small structures (i.e. dendritic
spines
>> or NMJs) during changes of solution with differing refractive indicies? Is
there
>> some well know trick? Or, am I using the wrong objectives? Does wide field
>> microscopy have the same problems?
>> Many thanks,
>> Christof

[Christof Schwiening]

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

Dear Arvydas,
The graph is here: https://dl.dropbox.com/u/24087924/Convallaria%20slide.jpg
I have tried three long working distance objectives (20-63X). I don't have a
good example of the distorted image since it shifts faster than my resonnant
scanner can capture it. However, the ROI plot shows the magnitude of the
shifts on the fluorescence signal. We do see the same effect on fixed samples
(which is what the image above shows). I am sure that it is some kind of
Schlieren type of effect (refractive index). I think that in areas of high
contrast even small optical deviations cause quite marked intensity changes.....
Greetings,
Christof

 On Mon, 18 Mar 2013 16:25:51 -0400, Arvydas Matiukas
<[hidden email]> wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Hi Christof,
>
>More quantitative description of your imaging geometry/optics
>would be useful. I have been observing similar effects when  imaging
>a thick sample  on
>inverted scope using 40x water dipping lens in immersion mode.  
>When water drop substantially shrinks after 2-3 hrs
>focus starts shifting until disappears completely (from the set range).
>This corresponds to ultimate refractive index change from 1.333 to 1.0.
>The possible solution is to set big enough thickness of z-stack so that
>your specimen still fits into imaged  focal range (taking into account the
>z-range shift due to refractive index change).
>
>I would like to see your graph and the distorted image. I wonder
>if your chemicals are not dissolving well or recrystalizing thus creating
>nonhomogenous layers distorting  your image. Or maybe some vibrations
>create inhomogeneities in the immersion/dipping medium. Do you observe this
>type of distortion with fixed specimens as well?
>
>Best regards,
>Arvydas
>--------------------------------
>
>
>
>
>Arvydas Matiukas, Ph.D.
>Director of Confocal&Two-Photon Core
>Department of Neurosci& Physiology
>SUNY Upstate Medical University
>766 Irving Ave., WH 3167
>Syracuse, NY 13210
>tel.: [phone]
>fax: [phone]
>email: [hidden email]
>>>> Christof Schwiening <[hidden email]> 3/18/2013 10:29 AM >>>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Dear All,
>I wonder if someone could throw some light on a problem we have been

having
>for the past few years! We use a Leica SP5 confocal on an upright
microscope
>with dipping lenses to image pH and calcium-sensitive fluorescent dyes.
During
>solution changes - most notably the addition and removal of organic
>compounds such as propionate (20 mM)- we get severe image distortion and
>changes in focal depth. I attempted to attach a graph of a ROI from a fixed
>sealed specimen which we have imaged during solution changes with 3
>different objectives - however, the ListServer rejected every image format I
>tried...I can send it by email if anyone is interested. It is impossible that the
>propionate is actually getting to the specimen itself - I suspect it must be as
a
>result of refractive index changes.
>
>My questions are: How do others image small structures (i.e. dendritic spines
>or NMJs) during changes of solution with differing refractive indicies? Is there
>some well know trick? Or, am I using the wrong objectives? Does wide field
>microscopy have the same problems?
>Many thanks,
>Christof

[Mark Cannell-2]

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

It looks like a movement problem to me…

Cheers

On 19/03/2013, at 3:02 PM, Christof Schwiening <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Arvydas,
> The graph is here: https://dl.dropbox.com/u/24087924/Convallaria%20slide.jpg
> I have tried three long working distance objectives (20-63X). I don't have a
> good example of the distorted image since it shifts faster than my resonnant
> scanner can capture it. However, the ROI plot shows the magnitude of the
> shifts on the fluorescence signal. We do see the same effect on fixed samples
> (which is what the image above shows). I am sure that it is some kind of
> Schlieren type of effect (refractive index). I think that in areas of high
> contrast even small optical deviations cause quite marked intensity changes.....
> Greetings,
> Christof
>
> On Mon, 18 Mar 2013 16:25:51 -0400, Arvydas Matiukas
> <[hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi Christof,
>>
>> More quantitative description of your imaging geometry/optics
>> would be useful. I have been observing similar effects when  imaging
>> a thick sample  on
>> inverted scope using 40x water dipping lens in immersion mode.  
>> When water drop substantially shrinks after 2-3 hrs
>> focus starts shifting until disappears completely (from the set range).
>> This corresponds to ultimate refractive index change from 1.333 to 1.0.
>> The possible solution is to set big enough thickness of z-stack so that
>> your specimen still fits into imaged  focal range (taking into account the
>> z-range shift due to refractive index change).
>>
>> I would like to see your graph and the distorted image. I wonder
>> if your chemicals are not dissolving well or recrystalizing thus creating
>> nonhomogenous layers distorting  your image. Or maybe some vibrations
>> create inhomogeneities in the immersion/dipping medium. Do you observe this
>> type of distortion with fixed specimens as well?
>>
>> Best regards,
>> Arvydas
>> --------------------------------
>>
>>
>>
>>
>> Arvydas Matiukas, Ph.D.
>> Director of Confocal&Two-Photon Core
>> Department of Neurosci& Physiology
>> SUNY Upstate Medical University
>> 766 Irving Ave., WH 3167
>> Syracuse, NY 13210
>> tel.: [phone]
>> fax: [phone]
>> email: [hidden email]
>>>>> Christof Schwiening <[hidden email]> 3/18/2013 10:29 AM >>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear All,
>> I wonder if someone could throw some light on a problem we have been
> having
>> for the past few years! We use a Leica SP5 confocal on an upright
> microscope
>> with dipping lenses to image pH and calcium-sensitive fluorescent dyes.
> During
>> solution changes - most notably the addition and removal of organic
>> compounds such as propionate (20 mM)- we get severe image distortion and
>> changes in focal depth. I attempted to attach a graph of a ROI from a fixed
>> sealed specimen which we have imaged during solution changes with 3
>> different objectives - however, the ListServer rejected every image format I
>> tried...I can send it by email if anyone is interested. It is impossible that the
>> propionate is actually getting to the specimen itself - I suspect it must be as
> a
>> result of refractive index changes.
>>
>> My questions are: How do others image small structures (i.e. dendritic spines
>> or NMJs) during changes of solution with differing refractive indicies? Is there
>> some well know trick? Or, am I using the wrong objectives? Does wide field
>> microscopy have the same problems?
>> Many thanks,
>> Christof

Mark  B. Cannell Ph.D. FRSNZ
Professor of Cardiac Cell Biology
School of Physiology&  Pharmacology
Medical Sciences Building
University of Bristol
Bristol
BS8 1TD UK

[hidden email]

[Glen MacDonald-2]

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

I second Mark's statement.  
Glen MacDonald
        Core for Communication Research
Virginia Merrill Bloedel Hearing Research Center
        Cellular Morphology Core
Center on Human Development and Disability
Box 357923
University of Washington
Seattle, WA 98195-7923  USA
[phone]
[hidden email]
[hidden email]





On Mar 19, 2013, at 8:08 AM, Mark Cannell <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> It looks like a movement problem to me…
>
> Cheers
>
> On 19/03/2013, at 3:02 PM, Christof Schwiening <[hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear Arvydas,
>> The graph is here: https://dl.dropbox.com/u/24087924/Convallaria%20slide.jpg
>> I have tried three long working distance objectives (20-63X). I don't have a
>> good example of the distorted image since it shifts faster than my resonnant
>> scanner can capture it. However, the ROI plot shows the magnitude of the
>> shifts on the fluorescence signal. We do see the same effect on fixed samples
>> (which is what the image above shows). I am sure that it is some kind of
>> Schlieren type of effect (refractive index). I think that in areas of high
>> contrast even small optical deviations cause quite marked intensity changes.....
>> Greetings,
>> Christof
>>
>> On Mon, 18 Mar 2013 16:25:51 -0400, Arvydas Matiukas
>> <[hidden email]> wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Hi Christof,
>>>
>>> More quantitative description of your imaging geometry/optics
>>> would be useful. I have been observing similar effects when  imaging
>>> a thick sample  on
>>> inverted scope using 40x water dipping lens in immersion mode.  
>>> When water drop substantially shrinks after 2-3 hrs
>>> focus starts shifting until disappears completely (from the set range).
>>> This corresponds to ultimate refractive index change from 1.333 to 1.0.
>>> The possible solution is to set big enough thickness of z-stack so that
>>> your specimen still fits into imaged  focal range (taking into account the
>>> z-range shift due to refractive index change).
>>>
>>> I would like to see your graph and the distorted image. I wonder
>>> if your chemicals are not dissolving well or recrystalizing thus creating
>>> nonhomogenous layers distorting  your image. Or maybe some vibrations
>>> create inhomogeneities in the immersion/dipping medium. Do you observe this
>>> type of distortion with fixed specimens as well?
>>>
>>> Best regards,
>>> Arvydas
>>> --------------------------------
>>>
>>>
>>>
>>>
>>> Arvydas Matiukas, Ph.D.
>>> Director of Confocal&Two-Photon Core
>>> Department of Neurosci& Physiology
>>> SUNY Upstate Medical University
>>> 766 Irving Ave., WH 3167
>>> Syracuse, NY 13210
>>> tel.: [phone]
>>> fax: [phone]
>>> email: [hidden email]
>>>>>> Christof Schwiening <[hidden email]> 3/18/2013 10:29 AM >>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Dear All,
>>> I wonder if someone could throw some light on a problem we have been
>> having
>>> for the past few years! We use a Leica SP5 confocal on an upright
>> microscope
>>> with dipping lenses to image pH and calcium-sensitive fluorescent dyes.
>> During
>>> solution changes - most notably the addition and removal of organic
>>> compounds such as propionate (20 mM)- we get severe image distortion and
>>> changes in focal depth. I attempted to attach a graph of a ROI from a fixed
>>> sealed specimen which we have imaged during solution changes with 3
>>> different objectives - however, the ListServer rejected every image format I
>>> tried...I can send it by email if anyone is interested. It is impossible that the
>>> propionate is actually getting to the specimen itself - I suspect it must be as
>> a
>>> result of refractive index changes.
>>>
>>> My questions are: How do others image small structures (i.e. dendritic spines
>>> or NMJs) during changes of solution with differing refractive indicies? Is there
>>> some well know trick? Or, am I using the wrong objectives? Does wide field
>>> microscopy have the same problems?
>>> Many thanks,
>>> Christof
>
> Mark  B. Cannell Ph.D. FRSNZ
> Professor of Cardiac Cell Biology
> School of Physiology&  Pharmacology
> Medical Sciences Building
> University of Bristol
> Bristol
> BS8 1TD UK
>
> [hidden email]

[Arvydas Matiukas]

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

Hi Christof ,
 
whatever is the reason for image distortion (Schlieren  or mechanical movement)
why don't you exclude the changing solution from the optical path, i.e. image your
cells from below while adding solution from the top. Any confocal on an inverted
stand would allow you to do this (you may need to switch to a dish with a coverslip
window at the center).
 
If switching to the inverted scope is not feasable/practical then try to improve your
injection/perfusion. I recall my cell perfusion experiments on an inverted scope
 (some with simultaneous patch clamp), and the perfusion practically never was
a problem both for image or patch clamp under condition that perfusion is properly
designed ( flow <1ml/min, opening diameter ~0.5 mm and located few mm apart
from the imaged/clamped cells. observing).
 
Moreover, some organic compounds do not dissolve well (eg. when dissolving BA
or BB in ethanol you can see Schlieren effect by a naked eye for several minutes).
Try to very well dissolve (vortex?)  your compound at elevated temperature, or try
more gradual (slower) addition and replacement of the compound. Regarding possible
formation of bubbles try cool the solution below room temperature ( at elevated temperatures
gas solubility in liquid decreases and bubbles are not produced. Maybe your compound
releases a lot of heat during dissolution, or changes the surfaces tension - if you introduce
it more gradually  all spatial inhomogeneties should be reduced.
 
My first choice in your situation would be to try an inverted configuration.
 
Best,
Arvydas


>>> Christof Schwiening <[hidden email]> 3/19/2013 10:02 AM >>>
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Dear Arvydas,
The graph is here: https://dl.dropbox.com/u/24087924/Convallaria%20slide.jpg
I have tried three long working distance objectives (20-63X). I don't have a
good example of the distorted image since it shifts faster than my resonnant
scanner can capture it. However, the ROI plot shows the magnitude of the
shifts on the fluorescence signal. We do see the same effect on fixed samples
(which is what the image above shows). I am sure that it is some kind of
Schlieren type of effect (refractive index). I think that in areas of high
contrast even small optical deviations cause quite marked intensity changes.....
Greetings,
Christof

On Mon, 18 Mar 2013 16:25:51 -0400, Arvydas Matiukas
<[hidden email]> wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Hi Christof,
>
>More quantitative description of your imaging geometry/optics
>would be useful. I have been observing similar effects when  imaging
>a thick sample  on
>inverted scope using 40x water dipping lens in immersion mode.  
>When water drop substantially shrinks after 2-3 hrs
>focus starts shifting until disappears completely (from the set range).
>This corresponds to ultimate refractive index change from 1.333 to 1.0.
>The possible solution is to set big enough thickness of z-stack so that
>your specimen still fits into imaged  focal range (taking into account the
>z-range shift due to refractive index change).
>
>I would like to see your graph and the distorted image. I wonder
>if your chemicals are not dissolving well or recrystalizing thus creating
>nonhomogenous layers distorting  your image. Or maybe some vibrations
>create inhomogeneities in the immersion/dipping medium. Do you observe this
>type of distortion with fixed specimens as well?
>
>Best regards,
>Arvydas
>--------------------------------
>
>
>
>
>Arvydas Matiukas, Ph.D.
>Director of Confocal&Two-Photon Core
>Department of Neurosci& Physiology
>SUNY Upstate Medical University
>766 Irving Ave., WH 3167
>Syracuse, NY 13210
>tel.: [phone]
>fax: [phone]
>email: [hidden email]
>>>> Christof Schwiening <[hidden email]> 3/18/2013 10:29 AM >>>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Dear All,
>I wonder if someone could throw some light on a problem we have been

having
>for the past few years! We use a Leica SP5 confocal on an upright
microscope
>with dipping lenses to image pH and calcium-sensitive fluorescent dyes.
During
>solution changes - most notably the addition and removal of organic
>compounds such as propionate (20 mM)- we get severe image distortion and
>changes in focal depth. I attempted to attach a graph of a ROI from a fixed
>sealed specimen which we have imaged during solution changes with 3
>different objectives - however, the ListServer rejected every image format I
>tried...I can send it by email if anyone is interested. It is impossible that the
>propionate is actually getting to the specimen itself - I suspect it must be as
a
>result of refractive index changes.
>
>My questions are: How do others image small structures (i.e. dendritic spines
>or NMJs) during changes of solution with differing refractive indicies? Is there
>some well know trick? Or, am I using the wrong objectives? Does wide field
>microscopy have the same problems?
>Many thanks,
>Christof

[Christof Schwiening]

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

I really do doubt that it is real physical movement, but I will test it. If it is
movement then there are only two things that could be moving: the objective
or the glass slide. There is not much I can do about the objective but I can
take greater steps to make sure that the glass slide does not move. It is
already clamped into a standard microscope X-Y stage - but the glass slide
might flex or the whole stage might move. I will attempt to test this by
directly attaching the slide to the microscope. Although why this might only
happen when 20 mM propionate (or other organic) is washed into, or out of
the bath I have no idea. The movement is not coincident with the valves
switching solutions but occurs with the appropriate delay for the arrival of the
new solution from the manifold. There are no air bubbles and the movement
does not occur when I switch between solution lines containing the same
solution....

[Christof Schwiening]

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

Dear Arvydas,
I agree that the problem would not be visible on an invert because the light
does not travel through the mixing solution. Unfortunately we are imaging both
NMJs in vivo (Drosophila larvae) and brain slices - both require good optics
(both for patching and for imaging) on the top side of a relatively thick
preparation.
I can see that speeding up the solution flow would restrict the time the
artefacts occur for, but we are already flowing at over 1 ml/min with a bath
volume that is no greater than the bubble of solution that spans the
preparation and objective (i.e. minimum volume, probably less than 100 uL). I
think the problem is that we are not adequately mixing the whole solution
space between the objective and preparation such that even with good flow
rates a full solution change takes ~30s (about 5 bath volumes?).
We want to image the pH changes during the instantaneous addition of weak
acids and bases. The best we have managed so far is to add the solution
directly to the site of interest using a puffer pipette.
Greetings,
Christof

On Tue, 19 Mar 2013 14:33:08 -0400, Arvydas Matiukas
<[hidden email]> wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Hi Christof ,
>
>whatever is the reason for image distortion (Schlieren  or mechanical
movement)
>why don't you exclude the changing solution from the optical path, i.e. image
your
>cells from below while adding solution from the top. Any confocal on an
inverted
>stand would allow you to do this (you may need to switch to a dish with a
coverslip
>window at the center).
>
>If switching to the inverted scope is not feasable/practical then try to
improve your
>injection/perfusion. I recall my cell perfusion experiments on an inverted scope
> (some with simultaneous patch clamp), and the perfusion practically never
was
>a problem both for image or patch clamp under condition that perfusion is
properly
>designed ( flow <1ml/min, opening diameter ~0.5 mm and located few mm
apart
>from the imaged/clamped cells. observing).
>
>Moreover, some organic compounds do not dissolve well (eg. when dissolving
BA
>or BB in ethanol you can see Schlieren effect by a naked eye for several
minutes).
>Try to very well dissolve (vortex?)  your compound at elevated temperature,
or try
>more gradual (slower) addition and replacement of the compound. Regarding
possible
>formation of bubbles try cool the solution below room temperature ( at
elevated temperatures
>gas solubility in liquid decreases and bubbles are not produced. Maybe your
compound
>releases a lot of heat during dissolution, or changes the surfaces tension - if
you introduce

>it more gradually  all spatial inhomogeneties should be reduced.
>
>My first choice in your situation would be to try an inverted configuration.
>
>Best,
>Arvydas
>
>
>>>> Christof Schwiening <[hidden email]> 3/19/2013 10:02 AM >>>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Dear Arvydas,
>The graph is here: <a href="https://dl.dropbox.com/u/24087924/Convallaria%">https://dl.dropbox.com/u/24087924/Convallaria%

20slide.jpg
>I have tried three long working distance objectives (20-63X). I don't have a
>good example of the distorted image since it shifts faster than my resonnant
>scanner can capture it. However, the ROI plot shows the magnitude of the
>shifts on the fluorescence signal. We do see the same effect on fixed
samples
>(which is what the image above shows). I am sure that it is some kind of
>Schlieren type of effect (refractive index). I think that in areas of high
>contrast even small optical deviations cause quite marked intensity
changes.....

>Greetings,
>Christof
>
>On Mon, 18 Mar 2013 16:25:51 -0400, Arvydas Matiukas
><[hidden email]> wrote:
>
>>*****
>>To join, leave or search the confocal microscopy listserv, go to:
>>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>*****
>>
>>Hi Christof,
>>
>>More quantitative description of your imaging geometry/optics
>>would be useful. I have been observing similar effects when  imaging
>>a thick sample  on
>>inverted scope using 40x water dipping lens in immersion mode.  
>>When water drop substantially shrinks after 2-3 hrs
>>focus starts shifting until disappears completely (from the set range).
>>This corresponds to ultimate refractive index change from 1.333 to 1.0.
>>The possible solution is to set big enough thickness of z-stack so that
>>your specimen still fits into imaged  focal range (taking into account the
>>z-range shift due to refractive index change).
>>
>>I would like to see your graph and the distorted image. I wonder
>>if your chemicals are not dissolving well or recrystalizing thus creating
>>nonhomogenous layers distorting  your image. Or maybe some vibrations
>>create inhomogeneities in the immersion/dipping medium. Do you observe this
>>type of distortion with fixed specimens as well?
>>
>>Best regards,
>>Arvydas
>>--------------------------------
>>
>>
>>
>>
>>Arvydas Matiukas, Ph.D.
>>Director of Confocal&Two-Photon Core
>>Department of Neurosci& Physiology
>>SUNY Upstate Medical University
>>766 Irving Ave., WH 3167
>>Syracuse, NY 13210
>>tel.: [phone]
>>fax: [phone]
>>email: [hidden email]
>>>>> Christof Schwiening <[hidden email]> 3/18/2013 10:29 AM >>>
>>*****
>>To join, leave or search the confocal microscopy listserv, go to:
>>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>*****
>>
>>Dear All,
>>I wonder if someone could throw some light on a problem we have been
>having
>>for the past few years! We use a Leica SP5 confocal on an upright
>microscope
>>with dipping lenses to image pH and calcium-sensitive fluorescent dyes.
>During
>>solution changes - most notably the addition and removal of organic
>>compounds such as propionate (20 mM)- we get severe image distortion

and
>>changes in focal depth. I attempted to attach a graph of a ROI from a fixed
>>sealed specimen which we have imaged during solution changes with 3
>>different objectives - however, the ListServer rejected every image format I
>>tried...I can send it by email if anyone is interested. It is impossible that the
>>propionate is actually getting to the specimen itself - I suspect it must be
as
>a
>>result of refractive index changes.
>>
>>My questions are: How do others image small structures (i.e. dendritic
spines
>>or NMJs) during changes of solution with differing refractive indicies? Is
there
>>some well know trick? Or, am I using the wrong objectives? Does wide field
>>microscopy have the same problems?
>>Many thanks,
>>Christof

[Christof Schwiening]

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

Dear Mark,
I attached the glass slide to the base of my stage and tried the solution
change again. This time I attempted to setup the optics so that I can video
the effects (DIC). I have uploaded a video here:
https://dl.dropbox.com/u/24087924/propionate.wmv
A few seconds into the video I switch the perfusion valve to add the 20 mM
propionate - it does cause a small instantaneous jump which is only just
visible. The propionate then washes in and the effect can most clearly be seen
from 23s after which I repeatedly stop and start the solution flow.
I think the phenomenon that is visible under DIC must be the same one
causing the confocal distortions. Does it look like you would expect for
physical movement?
Greetings,
Christof

On Tue, 19 Mar 2013 15:08:25 +0000, Mark Cannell
<[hidden email]> wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>It looks like a movement problem to me…
>
>Cheers
>
>On 19/03/2013, at 3:02 PM, Christof Schwiening <[hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear Arvydas,
>> The graph is here: <a href="https://dl.dropbox.com/u/24087924/Convallaria%">https://dl.dropbox.com/u/24087924/Convallaria%

20slide.jpg
>> I have tried three long working distance objectives (20-63X). I don't have
a
>> good example of the distorted image since it shifts faster than my
resonnant
>> scanner can capture it. However, the ROI plot shows the magnitude of the
>> shifts on the fluorescence signal. We do see the same effect on fixed
samples
>> (which is what the image above shows). I am sure that it is some kind of
>> Schlieren type of effect (refractive index). I think that in areas of high
>> contrast even small optical deviations cause quite marked intensity
changes.....

>> Greetings,
>> Christof
>>
>> On Mon, 18 Mar 2013 16:25:51 -0400, Arvydas Matiukas
>> <[hidden email]> wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Hi Christof,
>>>
>>> More quantitative description of your imaging geometry/optics
>>> would be useful. I have been observing similar effects when  imaging
>>> a thick sample  on
>>> inverted scope using 40x water dipping lens in immersion mode.  
>>> When water drop substantially shrinks after 2-3 hrs
>>> focus starts shifting until disappears completely (from the set range).
>>> This corresponds to ultimate refractive index change from 1.333 to 1.0.
>>> The possible solution is to set big enough thickness of z-stack so that
>>> your specimen still fits into imaged  focal range (taking into account the
>>> z-range shift due to refractive index change).
>>>
>>> I would like to see your graph and the distorted image. I wonder
>>> if your chemicals are not dissolving well or recrystalizing thus creating
>>> nonhomogenous layers distorting  your image. Or maybe some vibrations
>>> create inhomogeneities in the immersion/dipping medium. Do you observe

this

>>> type of distortion with fixed specimens as well?
>>>
>>> Best regards,
>>> Arvydas
>>> --------------------------------
>>>
>>>
>>>
>>>
>>> Arvydas Matiukas, Ph.D.
>>> Director of Confocal&Two-Photon Core
>>> Department of Neurosci& Physiology
>>> SUNY Upstate Medical University
>>> 766 Irving Ave., WH 3167
>>> Syracuse, NY 13210
>>> tel.: [phone]
>>> fax: [phone]
>>> email: [hidden email]
>>>>>> Christof Schwiening <[hidden email]> 3/18/2013 10:29 AM >>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Dear All,
>>> I wonder if someone could throw some light on a problem we have been
>> having
>>> for the past few years! We use a Leica SP5 confocal on an upright
>> microscope
>>> with dipping lenses to image pH and calcium-sensitive fluorescent dyes.
>> During
>>> solution changes - most notably the addition and removal of organic
>>> compounds such as propionate (20 mM)- we get severe image distortion

and
>>> changes in focal depth. I attempted to attach a graph of a ROI from a
fixed
>>> sealed specimen which we have imaged during solution changes with 3
>>> different objectives - however, the ListServer rejected every image
format I
>>> tried...I can send it by email if anyone is interested. It is impossible that
the
>>> propionate is actually getting to the specimen itself - I suspect it must be
as
>> a
>>> result of refractive index changes.
>>>
>>> My questions are: How do others image small structures (i.e. dendritic
spines
>>> or NMJs) during changes of solution with differing refractive indicies? Is
there
>>> some well know trick? Or, am I using the wrong objectives? Does wide
field

>>> microscopy have the same problems?
>>> Many thanks,
>>> Christof
>
>Mark  B. Cannell Ph.D. FRSNZ
>Professor of Cardiac Cell Biology
>School of Physiology&  Pharmacology
>Medical Sciences Building
>University of Bristol
>Bristol
>BS8 1TD UK
>
>[hidden email]

[Glen MacDonald-2]

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

Dear Christof,
That movie looked like motion and it seemed oscillatory.  Is that a peristaltic pump with a stepper motor?  Other questions would be whether there is a temperature difference between the propionate solution and the other solutions;  size of tubing or differences in tubing adapters and couplers that may affect velocity or pressure, or differences in how the tubing is looped?  what is the shape of your chamber?  Diamond shaped chambers seem to have better mixing than round or square.  Improved mixing may allow a reduction in the flow rate.  Maybe test a reduced flow rate and see how that alters the shift, and compare to gravity flow.  
Glen MacDonald
        Core for Communication Research
Virginia Merrill Bloedel Hearing Research Center
        Cellular Morphology Core
Center on Human Development and Disability
Box 357923
University of Washington
Seattle, WA 98195-7923  USA
[phone]
[hidden email]
[hidden email]





On Mar 20, 2013, at 4:17 AM, Christof Schwiening <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I really do doubt that it is real physical movement, but I will test it. If it is
> movement then there are only two things that could be moving: the objective
> or the glass slide. There is not much I can do about the objective but I can
> take greater steps to make sure that the glass slide does not move. It is
> already clamped into a standard microscope X-Y stage - but the glass slide
> might flex or the whole stage might move. I will attempt to test this by
> directly attaching the slide to the microscope. Although why this might only
> happen when 20 mM propionate (or other organic) is washed into, or out of
> the bath I have no idea. The movement is not coincident with the valves
> switching solutions but occurs with the appropriate delay for the arrival of the
> new solution from the manifold. There are no air bubbles and the movement
> does not occur when I switch between solution lines containing the same
> solution....

[Christof Schwiening]

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

Dear Glen,
The solutions are gravity-fed via independent tubes from the base of 50 ml
syringes suspended about 50cm above the microscope stage. The tubes are
identical and are connected to electrical on-off valves (General Valves). The
tubes meet at a manifold about 15 cm from the output. In the case of the
video the output is directly onto the top of a fixed-preparation (Convallaria)
slide which was mounted directly onto the fixed platform of the microscope.
The output tube was about 2 mm from the objective at the level of the slide.
The suction was similarly sighted about 2 mm from the objective at an angle of
180 degrees to the input. The suction was from a bottle (2 L) maintained a
low pressure with a high frequency low volume pump. The only oscillations that
occur within the system are surface tension effects on the suction when air
mixes with the water in the output lines - this occurs all of the time and
sounds like a child sucking through a straw from an empty glass of lemonade!
I doubt that it is movement. I guess I would need a force transducer mounted
on the slide to prove that.
Greetings,
Christof

[Mark Cannell-2]

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

You could use reflection mode from the surface of the slide to see if it's moving?

Cheers

On 21/03/2013, at 10:40 AM, Christof Schwiening <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Glen,
> The solutions are gravity-fed via independent tubes from the base of 50 ml
> syringes suspended about 50cm above the microscope stage. The tubes are
> identical and are connected to electrical on-off valves (General Valves). The
> tubes meet at a manifold about 15 cm from the output. In the case of the
> video the output is directly onto the top of a fixed-preparation (Convallaria)
> slide which was mounted directly onto the fixed platform of the microscope.
> The output tube was about 2 mm from the objective at the level of the slide.
> The suction was similarly sighted about 2 mm from the objective at an angle of
> 180 degrees to the input. The suction was from a bottle (2 L) maintained a
> low pressure with a high frequency low volume pump. The only oscillations that
> occur within the system are surface tension effects on the suction when air
> mixes with the water in the output lines - this occurs all of the time and
> sounds like a child sucking through a straw from an empty glass of lemonade!
> I doubt that it is movement. I guess I would need a force transducer mounted
> on the slide to prove that.
> Greetings,
> Christof

Mark  B. Cannell Ph.D. FRSNZ
Professor of Cardiac Cell Biology
School of Physiology&  Pharmacology
Medical Sciences Building
University of Bristol
Bristol
BS8 1TD UK

[hidden email]

[Stanislav Vitha-2]

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

After looking at the video, the "movements" look to me like a Schlieren effect;
A wave of higher-RI solution rolls over the sample from left to right. Do you
know what is the difference (if any) in refractive indices between the two
solutions that you are switching?
If that is indeed the effect of RI, you should not see any movements when
you use two identical solutions and perform the same valve switching.  If the
movement is still there,

A change in RI will cause Z-axis compression/expansion (focus shift);
Perhaps you could tune the first buffer to the same RI as the propionate
solution by some "inert" additive.  BSA, gelatine, or some other non-osmotic
agent comes to mind (but I know nothing about what might interfere with your
samples).  

I am just guessing here, but  another explanation may be that switching to the
propionate buffer ('open-system buffer') enables osmotic effects of other
media components and leads to cell shrinkage and intracellular pH changes.  
Something like in this paper: Thomas A. Heming, Gregory Boyarsky, Divina M.
Tuazon, and Akhil Bidani:  pHi responses to osmotic cell shrinkage in the
presence of open-system buffers J Appl Physiol October 1, 2000 89:(4) 1543-
1552

Stan Vitha
Microscopy and Imaging Center
Texas A&M University

[Glen MacDonald-2]

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

From your description, it seems to be a well designed flow system.  But, the motion seemed to be oscillatory, which made me think it had a hydraulic cause.  If Schlieren effects, an eddy in the chamber may be causing the higher RI solution to move back and forth as it mixes.  Switching between similar solutions, as proposed by Stan, (including 2 propionates)  would be a good test for motion.  


Glen MacDonald
        Core for Communication Research
Virginia Merrill Bloedel Hearing Research Center
        Cellular Morphology Core
Center on Human Development and Disability
Box 357923
University of Washington
Seattle, WA 98195-7923  USA
[phone]
[hidden email]
[hidden email]





On Mar 21, 2013, at 3:40 AM, Christof Schwiening <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Glen,
> The solutions are gravity-fed via independent tubes from the base of 50 ml
> syringes suspended about 50cm above the microscope stage. The tubes are
> identical and are connected to electrical on-off valves (General Valves). The
> tubes meet at a manifold about 15 cm from the output. In the case of the
> video the output is directly onto the top of a fixed-preparation (Convallaria)
> slide which was mounted directly onto the fixed platform of the microscope.
> The output tube was about 2 mm from the objective at the level of the slide.
> The suction was similarly sighted about 2 mm from the objective at an angle of
> 180 degrees to the input. The suction was from a bottle (2 L) maintained a
> low pressure with a high frequency low volume pump. The only oscillations that
> occur within the system are surface tension effects on the suction when air
> mixes with the water in the output lines - this occurs all of the time and
> sounds like a child sucking through a straw from an empty glass of lemonade!
> I doubt that it is movement. I guess I would need a force transducer mounted
> on the slide to prove that.
> Greetings,
> Christof