timeseries, large images and focus

classic Classic list List threaded Threaded
16 messages Options
Sarah Chacko Sarah Chacko
Reply | Threaded
Open this post in threaded view
|

timeseries, large images and focus

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

Hello,

I am using a Nikon A1 confocal and I am trying to take a time series over several hours,  scanning across channels in a microfluidic chamber - my channels are 20 fields of view across, there are three channels.

I am finding that even with one long (20 fields of view) image across one channel the focus is lost half way across, and moving to another region nearly always loses focus.

I could increase the pinhole or take a small z stack so that being out of focus would matter less - I'm looking at bacteria ~1 micron in size, but don't need to resolve anything smaller, just distinguish between them using fluorescent labels.

I wondered whether in principle this should work, using long image and PFS with several starting points, and if anyone has something similar working well, or whether I am expecting too much.

Sarah
(student)
Tim Feinstein-2 Tim Feinstein-2
Reply | Threaded
Open this post in threaded view
|

Re: timeseries, large images and focus

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

Hi Sarah,

If PFS is engaged throughout your movie, your microfluidic chamber almost certainly has irregularities in the 'coverslip' width.  If so then you can semi-solve the problem with a wide open pinhole and low NA objective.   Alternatively, you could set up the 20 fields of view as two or three independent image series with their own set PFS position, and then stitch them into one long time series/image after using an ImageJ plugin like Stack Combiner.  This could take a bit more work on your part (I do a lot of that with our lab's A1s) but it uses less imaging time and memory than a z stack.  

Moving between regions could be more problematic.  As I recall PFS depends on the glass/water interface to work.  In between fluid channels you most likely have a material whose index of refraction does not allow the infrared PFS beam to return useful information to its sensor. The other possibility is less dire: we have found that PFS can lose its 'lock' when you have the stage move too far too fast, most likely due to slight tilt/curvature of the coverslip.  We solved this problem a couple of ways.  If we have to make a long jump between XY positions, then we register a 'dummy' position midway along the jump that lets the PFS catch up.  It's a slight waste of time and memory but it's better than losing a movie.  We also found that the slowing down the automatic stage movement speed can help, sometimes quite a bit.  I believe that most systems are set on max speed, and slowing ours a bit (you may need your service rep to do that) has not slowed down our time series imaging noticeably.  

Good luck and all the best,


Tim

__________________
Timothy Feinstein, PhD
Postdoctoral Associate, Vilardaga lab
University of Pittsburgh Dept. of Pharmacology and Chemical Biology
BST W1301, 200 Lothrop St.
Pgh, PA  15261


On Nov 17, 2010, at 8:29 AM, Sarah Chacko wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello,
>
> I am using a Nikon A1 confocal and I am trying to take a time series over several hours,  scanning across channels in a microfluidic chamber - my channels are 20 fields of view across, there are three channels.
>
> I am finding that even with one long (20 fields of view) image across one channel the focus is lost half way across, and moving to another region nearly always loses focus.
>
> I could increase the pinhole or take a small z stack so that being out of focus would matter less - I'm looking at bacteria ~1 micron in size, but don't need to resolve anything smaller, just distinguish between them using fluorescent labels.
>
> I wondered whether in principle this should work, using long image and PFS with several starting points, and if anyone has something similar working well, or whether I am expecting too much.
>
> Sarah
> (student)
Axel Kurt Preuss Axel Kurt Preuss
Reply | Threaded
Open this post in threaded view
|

Re: timeseries, large images and focus

Dear Sarah, Tim
Is there any stronger fluid movement in the microfluid chamber?

Thanks
   Cheers

Axel
—————
Axel K Preuss, PhD,
Central Imaging, IMCB, A*Star, 61 Biopolis Dr, 6-19B, Singapore 138673,  sent from 9271.5622


On Nov 17, 2010, at 10:07 PM, Tim Feinstein <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Sarah,
>
> If PFS is engaged throughout your movie, your microfluidic chamber almost certainly has irregularities in the 'coverslip' width.  If so then you can semi-solve the problem with a wide open pinhole and low NA objective.   Alternatively, you could set up the 20 fields of view as two or three independent image series with their own set PFS position, and then stitch them into one long time series/image after using an ImageJ plugin like Stack Combiner.  This could take a bit more work on your part (I do a lot of that with our lab's A1s) but it uses less imaging time and memory than a z stack.
>
> Moving between regions could be more problematic.  As I recall PFS depends on the glass/water interface to work.  In between fluid channels you most likely have a material whose index of refraction does not allow the infrared PFS beam to return useful information to its sensor. The other possibility is less dire: we have found that PFS can lose its 'lock' when you have the stage move too far too fast, most likely due to slight tilt/curvature of the coverslip.  We solved this problem a couple of ways.  If we have to make a long jump between XY positions, then we register a 'dummy' position midway along the jump that lets the PFS catch up.  It's a slight waste of time and memory but it's better than losing a movie.  We also found that the slowing down the automatic stage movement speed can help, sometimes quite a bit.  I believe that most systems are set on max speed, and slowing ours a bit (you may need your service rep to do that) has not slowed down our time series imaging noticeably.
>
> Good luck and all the best,
>
>
> Tim
>
> __________________
> Timothy Feinstein, PhD
> Postdoctoral Associate, Vilardaga lab
> University of Pittsburgh Dept. of Pharmacology and Chemical Biology
> BST W1301, 200 Lothrop St.
> Pgh, PA  15261
>
>
> On Nov 17, 2010, at 8:29 AM, Sarah Chacko wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hello,
>>
>> I am using a Nikon A1 confocal and I am trying to take a time series over several hours,  scanning across channels in a microfluidic chamber - my channels are 20 fields of view across, there are three channels.
>>
>> I am finding that even with one long (20 fields of view) image across one channel the focus is lost half way across, and moving to another region nearly always loses focus.
>>
>> I could increase the pinhole or take a small z stack so that being out of focus would matter less - I'm looking at bacteria ~1 micron in size, but don't need to resolve anything smaller, just distinguish between them using fluorescent labels.
>>
>> I wondered whether in principle this should work, using long image and PFS with several starting points, and if anyone has something similar working well, or whether I am expecting too much.
>>
>> Sarah
>> (student)

Note: This message may contain confidential information. If this Email/Fax has been sent to you by mistake, please notify the sender and delete it immediately. Thank you.
Csúcs  Gábor Csúcs Gábor
Reply | Threaded
Open this post in threaded view
|

Re: timeseries, large images and focus

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

Dear Sarah,

I'd suggest that your PI invest in the Perfect Focus System (PFS) and
that would solve these problems (given that you are using an inverted
stage).

Cheers   Gabor

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello,
>
> I am using a Nikon A1 confocal and I am trying to take a time series over several hours,  scanning across channels in a microfluidic chamber - my channels are 20 fields of view across, there are three channels.
>
> I am finding that even with one long (20 fields of view) image across one channel the focus is lost half way across, and moving to another region nearly always loses focus.
>
> I could increase the pinhole or take a small z stack so that being out of focus would matter less - I'm looking at bacteria ~1 micron in size, but don't need to resolve anything smaller, just distinguish between them using fluorescent labels.
>
> I wondered whether in principle this should work, using long image and PFS with several starting points, and if anyone has something similar working well, or whether I am expecting too much.
>
> Sarah
> (student)


--
Gabor Csucs
Light Microscopy Centre, ETH Zurich
Schafmattstrasse 18, HPM F16
CH-8093, Zurich, Switzerland

Web: www.lmc.ethz.ch
Phone: +41 44 633 6221
Mobile: +41 79 758 21 58
Fax: +41 44 632 1298
e-mail: [hidden email]
Adams,Henry P Adams,Henry P
Reply | Threaded
Open this post in threaded view
|

Re: time series, large images and focus

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

Sarah,
I have a Ti with PFS and we do long timelapse experiments quite a bit with multiwell plates and also running 4 culture dishes.
It is very important to make sure that your slide, plate or dishes are level as possible. I have had similar problems with PFS failing but by adjusting the set screws on the stage insert while using a small level tool on the plate or dishes I have been able to get PFS to work.
Good luck,
Hank Adams
Microscopy Core
Genetics
U.T.M.D.Anderson Cancer Center
Houston, Tx


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Gabor Csucs
Sent: Friday, November 19, 2010 5:03 AM
To: [hidden email]
Subject: Re: timeseries, large images and focus

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

Dear Sarah,

I'd suggest that your PI invest in the Perfect Focus System (PFS) and
that would solve these problems (given that you are using an inverted
stage).

Cheers   Gabor

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello,
>
> I am using a Nikon A1 confocal and I am trying to take a time series over several hours,  scanning across channels in a microfluidic chamber - my channels are 20 fields of view across, there are three channels.
>
> I am finding that even with one long (20 fields of view) image across one channel the focus is lost half way across, and moving to another region nearly always loses focus.
>
> I could increase the pinhole or take a small z stack so that being out of focus would matter less - I'm looking at bacteria ~1 micron in size, but don't need to resolve anything smaller, just distinguish between them using fluorescent labels.
>
> I wondered whether in principle this should work, using long image and PFS with several starting points, and if anyone has something similar working well, or whether I am expecting too much.
>
> Sarah
> (student)


--
Gabor Csucs
Light Microscopy Centre, ETH Zurich
Schafmattstrasse 18, HPM F16
CH-8093, Zurich, Switzerland

Web: www.lmc.ethz.ch
Phone: +41 44 633 6221
Mobile: +41 79 758 21 58
Fax: +41 44 632 1298
e-mail: [hidden email]
Sylvie Le Guyader-2 Sylvie Le Guyader-2
Reply | Threaded
Open this post in threaded view
|

Re: time series, large images and focus

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

Hi Hank

I suppose you use a dry objective for your multiwell experiments is that
right?

Med vänlig hälsning / Best regards
 
Sylvie
 
@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader
Live Cell Imaging Unit
Dept of Biosciences and Nutrition
Karolinska Institutet
14183 Huddinge
Sweden
office: +46 (0)8 608 9240
This number will change to +46 (0) 8 5248 1107 on the 13th of December 2010
LCI room: +46 (0)8 608 9248
mobile: +46 (0) 73 733 5008
 

> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of
> Adams,Henry P
> Sent: 19 November 2010 17:01
> To: [hidden email]
> Subject: Re: time series, large images and focus
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Sarah,
> I have a Ti with PFS and we do long timelapse experiments quite a bit with
> multiwell plates and also running 4 culture dishes.
> It is very important to make sure that your slide, plate or dishes are
level as
> possible. I have had similar problems with PFS failing but by adjusting
the set
> screws on the stage insert while using a small level tool on the plate or
dishes I

> have been able to get PFS to work.
> Good luck,
> Hank Adams
> Microscopy Core
> Genetics
> U.T.M.D.Anderson Cancer Center
> Houston, Tx
>
>
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of Gabor Csucs
> Sent: Friday, November 19, 2010 5:03 AM
> To: [hidden email]
> Subject: Re: timeseries, large images and focus
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Sarah,
>
> I'd suggest that your PI invest in the Perfect Focus System (PFS) and
> that would solve these problems (given that you are using an inverted
> stage).
>
> Cheers   Gabor
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Hello,
> >
> > I am using a Nikon A1 confocal and I am trying to take a time series
over several
> hours,  scanning across channels in a microfluidic chamber - my channels
are 20
> fields of view across, there are three channels.
> >
> > I am finding that even with one long (20 fields of view) image across
one channel
> the focus is lost half way across, and moving to another region nearly
always loses
> focus.
> >
> > I could increase the pinhole or take a small z stack so that being out
of focus
> would matter less - I'm looking at bacteria ~1 micron in size, but don't
need to
> resolve anything smaller, just distinguish between them using fluorescent
labels.
> >
> > I wondered whether in principle this should work, using long image and
PFS with
> several starting points, and if anyone has something similar working well,
or

> whether I am expecting too much.
> >
> > Sarah
> > (student)
>
>
> --
> Gabor Csucs
> Light Microscopy Centre, ETH Zurich
> Schafmattstrasse 18, HPM F16
> CH-8093, Zurich, Switzerland
>
> Web: www.lmc.ethz.ch
> Phone: +41 44 633 6221
> Mobile: +41 79 758 21 58
> Fax: +41 44 632 1298
> e-mail: [hidden email]
Adams,Henry P Adams,Henry P
Reply | Threaded
Open this post in threaded view
|

Re: time series, large images and focus

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

Sylvie,
It depends. For multiwell plates we use dry. But I have used a 40x oil on more than one dish. Though now I plan to switch my users to the Lab-tek II chambered coverglass bottom slides which should make the task for using oil immersion objectives les challenging. I should also mention that I am using a piezo stage with Oko Lab inserts.
hank

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Sylvie Le Guyader
Sent: Friday, November 19, 2010 10:17 AM
To: [hidden email]
Subject: Re: time series, large images and focus

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

Hi Hank

I suppose you use a dry objective for your multiwell experiments is that
right?

Med vänlig hälsning / Best regards
 
Sylvie
 
@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader
Live Cell Imaging Unit
Dept of Biosciences and Nutrition
Karolinska Institutet
14183 Huddinge
Sweden
office: +46 (0)8 608 9240
This number will change to +46 (0) 8 5248 1107 on the 13th of December 2010
LCI room: +46 (0)8 608 9248
mobile: +46 (0) 73 733 5008
 

> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of
> Adams,Henry P
> Sent: 19 November 2010 17:01
> To: [hidden email]
> Subject: Re: time series, large images and focus
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Sarah,
> I have a Ti with PFS and we do long timelapse experiments quite a bit with
> multiwell plates and also running 4 culture dishes.
> It is very important to make sure that your slide, plate or dishes are
level as
> possible. I have had similar problems with PFS failing but by adjusting
the set
> screws on the stage insert while using a small level tool on the plate or
dishes I

> have been able to get PFS to work.
> Good luck,
> Hank Adams
> Microscopy Core
> Genetics
> U.T.M.D.Anderson Cancer Center
> Houston, Tx
>
>
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of Gabor Csucs
> Sent: Friday, November 19, 2010 5:03 AM
> To: [hidden email]
> Subject: Re: timeseries, large images and focus
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Sarah,
>
> I'd suggest that your PI invest in the Perfect Focus System (PFS) and
> that would solve these problems (given that you are using an inverted
> stage).
>
> Cheers   Gabor
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Hello,
> >
> > I am using a Nikon A1 confocal and I am trying to take a time series
over several
> hours,  scanning across channels in a microfluidic chamber - my channels
are 20
> fields of view across, there are three channels.
> >
> > I am finding that even with one long (20 fields of view) image across
one channel
> the focus is lost half way across, and moving to another region nearly
always loses
> focus.
> >
> > I could increase the pinhole or take a small z stack so that being out
of focus
> would matter less - I'm looking at bacteria ~1 micron in size, but don't
need to
> resolve anything smaller, just distinguish between them using fluorescent
labels.
> >
> > I wondered whether in principle this should work, using long image and
PFS with
> several starting points, and if anyone has something similar working well,
or

> whether I am expecting too much.
> >
> > Sarah
> > (student)
>
>
> --
> Gabor Csucs
> Light Microscopy Centre, ETH Zurich
> Schafmattstrasse 18, HPM F16
> CH-8093, Zurich, Switzerland
>
> Web: www.lmc.ethz.ch
> Phone: +41 44 633 6221
> Mobile: +41 79 758 21 58
> Fax: +41 44 632 1298
> e-mail: [hidden email]
Sarah Chacko Sarah Chacko
Reply | Threaded
Open this post in threaded view
|

Re: time series, large images and focus

In reply to this post by Adams,Henry P
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Many thanks for such useful answers, I have a lot of ideas to work though.

I have media flowing through the channels, and my channels are 4mm by 35mm by 100 microns deep, three side by side, made of PDMS bonded to a coverslip.. I was using x60 oil immersion lens but I think from what you say that the oil may be a problem and I will try a x40 lens without oil. And Nikon have told me how to slow down the stage.

 It is a real help also to know that I should be able to do this, and that other people have made it work.

Thank you, Sarah


________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of Adams,Henry P [[hidden email]]
Sent: 19 November 2010 16:01
To: [hidden email]
Subject: Re: time series, large images and focus

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

Sarah,
I have a Ti with PFS and we do long timelapse experiments quite a bit with multiwell plates and also running 4 culture dishes.
It is very important to make sure that your slide, plate or dishes are level as possible. I have had similar problems with PFS failing but by adjusting the set screws on the stage insert while using a small level tool on the plate or dishes I have been able to get PFS to work.
Good luck,
Hank Adams
Microscopy Core
Genetics
U.T.M.D.Anderson Cancer Center
Houston, Tx


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Gabor Csucs
Sent: Friday, November 19, 2010 5:03 AM
To: [hidden email]
Subject: Re: timeseries, large images and focus

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

Dear Sarah,

I'd suggest that your PI invest in the Perfect Focus System (PFS) and
that would solve these problems (given that you are using an inverted
stage).

Cheers   Gabor

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello,
>
> I am using a Nikon A1 confocal and I am trying to take a time series over several hours,  scanning across channels in a microfluidic chamber - my channels are 20 fields of view across, there are three channels.
>
> I am finding that even with one long (20 fields of view) image across one channel the focus is lost half way across, and moving to another region nearly always loses focus.
>
> I could increase the pinhole or take a small z stack so that being out of focus would matter less - I'm looking at bacteria ~1 micron in size, but don't need to resolve anything smaller, just distinguish between them using fluorescent labels.
>
> I wondered whether in principle this should work, using long image and PFS with several starting points, and if anyone has something similar working well, or whether I am expecting too much.
>
> Sarah
> (student)


--
Gabor Csucs
Light Microscopy Centre, ETH Zurich
Schafmattstrasse 18, HPM F16
CH-8093, Zurich, Switzerland

Web: www.lmc.ethz.ch
Phone: +41 44 633 6221
Mobile: +41 79 758 21 58
Fax: +41 44 632 1298
e-mail: [hidden email]
Knecht, David Knecht, David
Reply | Threaded
Open this post in threaded view
|

Re: time series, large images and focus

In reply to this post by Adams,Henry P
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Hank- PFS is not supposed to work with plastic.  Are you saying that you got it to work with a standard plastic multiwell plate or were you using a glass bottom dish?  Dave

On Nov 19, 2010, at 11:01 AM, Adams,Henry P wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Sarah,
> I have a Ti with PFS and we do long timelapse experiments quite a bit with multiwell plates and also running 4 culture dishes.
> It is very important to make sure that your slide, plate or dishes are level as possible. I have had similar problems with PFS failing but by adjusting the set screws on the stage insert while using a small level tool on the plate or dishes I have been able to get PFS to work.
> Good luck,
> Hank Adams
> Microscopy Core
> Genetics
> U.T.M.D.Anderson Cancer Center
> Houston, Tx
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Gabor Csucs
> Sent: Friday, November 19, 2010 5:03 AM
> To: [hidden email]
> Subject: Re: timeseries, large images and focus
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Sarah,
>
> I'd suggest that your PI invest in the Perfect Focus System (PFS) and
> that would solve these problems (given that you are using an inverted
> stage).
>
> Cheers   Gabor
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hello,
>>
>> I am using a Nikon A1 confocal and I am trying to take a time series over several hours,  scanning across channels in a microfluidic chamber - my channels are 20 fields of view across, there are three channels.
>>
>> I am finding that even with one long (20 fields of view) image across one channel the focus is lost half way across, and moving to another region nearly always loses focus.
>>
>> I could increase the pinhole or take a small z stack so that being out of focus would matter less - I'm looking at bacteria ~1 micron in size, but don't need to resolve anything smaller, just distinguish between them using fluorescent labels.
>>
>> I wondered whether in principle this should work, using long image and PFS with several starting points, and if anyone has something similar working well, or whether I am expecting too much.
>>
>> Sarah
>> (student)
>
>
> --
> Gabor Csucs
> Light Microscopy Centre, ETH Zurich
> Schafmattstrasse 18, HPM F16
> CH-8093, Zurich, Switzerland
>
> Web: www.lmc.ethz.ch
> Phone: +41 44 633 6221
> Mobile: +41 79 758 21 58
> Fax: +41 44 632 1298
> e-mail: [hidden email]

Dr. David Knecht    
Department of Molecular and Cell Biology
Co-head Flow Cytometry and Confocal Microscopy Facility
U-3125
91 N. Eagleville Rd.
University of Connecticut
Storrs, CT 06269
860-486-2200
860-486-4331 (fax)
Adams,Henry P Adams,Henry P
Reply | Threaded
Open this post in threaded view
|

Re: time series, large images and focus

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

Dave,
We were using glass bottom plates, although I believe, but I could be wrong, one of my users was able to get PFS to work using a 10x phase on an all plastic multiwell plate. I will have to check, though it will be easy to try. I will get back to you tomorrow or later this afternoon if I can get PSF to work.

Hank

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of David Knecht charter
Sent: Tuesday, November 23, 2010 12:12 PM
To: [hidden email]
Subject: Re: time series, large images and focus

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

Hi Hank- PFS is not supposed to work with plastic.  Are you saying that you got it to work with a standard plastic multiwell plate or were you using a glass bottom dish?  Dave

On Nov 19, 2010, at 11:01 AM, Adams,Henry P wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Sarah,
> I have a Ti with PFS and we do long timelapse experiments quite a bit with multiwell plates and also running 4 culture dishes.
> It is very important to make sure that your slide, plate or dishes are level as possible. I have had similar problems with PFS failing but by adjusting the set screws on the stage insert while using a small level tool on the plate or dishes I have been able to get PFS to work.
> Good luck,
> Hank Adams
> Microscopy Core
> Genetics
> U.T.M.D.Anderson Cancer Center
> Houston, Tx
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Gabor Csucs
> Sent: Friday, November 19, 2010 5:03 AM
> To: [hidden email]
> Subject: Re: timeseries, large images and focus
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Sarah,
>
> I'd suggest that your PI invest in the Perfect Focus System (PFS) and
> that would solve these problems (given that you are using an inverted
> stage).
>
> Cheers   Gabor
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hello,
>>
>> I am using a Nikon A1 confocal and I am trying to take a time series over several hours,  scanning across channels in a microfluidic chamber - my channels are 20 fields of view across, there are three channels.
>>
>> I am finding that even with one long (20 fields of view) image across one channel the focus is lost half way across, and moving to another region nearly always loses focus.
>>
>> I could increase the pinhole or take a small z stack so that being out of focus would matter less - I'm looking at bacteria ~1 micron in size, but don't need to resolve anything smaller, just distinguish between them using fluorescent labels.
>>
>> I wondered whether in principle this should work, using long image and PFS with several starting points, and if anyone has something similar working well, or whether I am expecting too much.
>>
>> Sarah
>> (student)
>
>
> --
> Gabor Csucs
> Light Microscopy Centre, ETH Zurich
> Schafmattstrasse 18, HPM F16
> CH-8093, Zurich, Switzerland
>
> Web: www.lmc.ethz.ch
> Phone: +41 44 633 6221
> Mobile: +41 79 758 21 58
> Fax: +41 44 632 1298
> e-mail: [hidden email]

Dr. David Knecht    
Department of Molecular and Cell Biology
Co-head Flow Cytometry and Confocal Microscopy Facility
U-3125
91 N. Eagleville Rd.
University of Connecticut
Storrs, CT 06269
860-486-2200
860-486-4331 (fax)
Adams,Henry P Adams,Henry P
Reply | Threaded
Open this post in threaded view
|

Re: time series, large images and focus

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

Hi Dave,
I just tried PFS using a plastic 48-well plate and a 10x phase. I setup 5 positions with offsets in 4 different wells with water. It worked PERFECTly. I then rotated in my 20x/0.75. Five different positions within one well (similar to 35mm plastic dish) and it worked ok. Although, I am not sure what practical use the 20x would be for imaging.  However, PFS did not work when I attempted to setup positions in two adjacent wells. Also, this was not a phase contrast objective. I don't believe their 20x phase is compatible with their PFSystem.
Also, phase contrast is tricky in multiwell dishes as most of you know. Unless you are near the center of the well, in my experience, it is impossible to get a phase contrast image.  Fyi, all my users who were using the 10x phase in multiwell dishes were conducting scratch assays. The images away from the center of wells made me cringe. But they claim to get what they needed. Ugh.

hank

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of David Knecht charter
Sent: Tuesday, November 23, 2010 12:12 PM
To: [hidden email]
Subject: Re: time series, large images and focus

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

Hi Hank- PFS is not supposed to work with plastic.  Are you saying that you got it to work with a standard plastic multiwell plate or were you using a glass bottom dish?  Dave

On Nov 19, 2010, at 11:01 AM, Adams,Henry P wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Sarah,
> I have a Ti with PFS and we do long timelapse experiments quite a bit with multiwell plates and also running 4 culture dishes.
> It is very important to make sure that your slide, plate or dishes are level as possible. I have had similar problems with PFS failing but by adjusting the set screws on the stage insert while using a small level tool on the plate or dishes I have been able to get PFS to work.
> Good luck,
> Hank Adams
> Microscopy Core
> Genetics
> U.T.M.D.Anderson Cancer Center
> Houston, Tx
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Gabor Csucs
> Sent: Friday, November 19, 2010 5:03 AM
> To: [hidden email]
> Subject: Re: timeseries, large images and focus
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Sarah,
>
> I'd suggest that your PI invest in the Perfect Focus System (PFS) and
> that would solve these problems (given that you are using an inverted
> stage).
>
> Cheers   Gabor
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hello,
>>
>> I am using a Nikon A1 confocal and I am trying to take a time series over several hours,  scanning across channels in a microfluidic chamber - my channels are 20 fields of view across, there are three channels.
>>
>> I am finding that even with one long (20 fields of view) image across one channel the focus is lost half way across, and moving to another region nearly always loses focus.
>>
>> I could increase the pinhole or take a small z stack so that being out of focus would matter less - I'm looking at bacteria ~1 micron in size, but don't need to resolve anything smaller, just distinguish between them using fluorescent labels.
>>
>> I wondered whether in principle this should work, using long image and PFS with several starting points, and if anyone has something similar working well, or whether I am expecting too much.
>>
>> Sarah
>> (student)
>
>
> --
> Gabor Csucs
> Light Microscopy Centre, ETH Zurich
> Schafmattstrasse 18, HPM F16
> CH-8093, Zurich, Switzerland
>
> Web: www.lmc.ethz.ch
> Phone: +41 44 633 6221
> Mobile: +41 79 758 21 58
> Fax: +41 44 632 1298
> e-mail: [hidden email]

Dr. David Knecht    
Department of Molecular and Cell Biology
Co-head Flow Cytometry and Confocal Microscopy Facility
U-3125
91 N. Eagleville Rd.
University of Connecticut
Storrs, CT 06269
860-486-2200
860-486-4331 (fax)
Adams,Henry P Adams,Henry P
Reply | Threaded
Open this post in threaded view
|

Re: time series, large images and focus

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

To all,
I should qualify my statement about phase problems using multiwell dishes. This is universal when using low mag objectives like the 10x/0.3 phase in the case I was describing.

hank

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Adams,Henry P
Sent: Tuesday, November 23, 2010 5:25 PM
To: [hidden email]
Subject: Re: time series, large images and focus

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

Hi Dave,
I just tried PFS using a plastic 48-well plate and a 10x phase. I setup 5 positions with offsets in 4 different wells with water. It worked PERFECTly. I then rotated in my 20x/0.75. Five different positions within one well (similar to 35mm plastic dish) and it worked ok. Although, I am not sure what practical use the 20x would be for imaging.  However, PFS did not work when I attempted to setup positions in two adjacent wells. Also, this was not a phase contrast objective. I don't believe their 20x phase is compatible with their PFSystem.
Also, phase contrast is tricky in multiwell dishes as most of you know. Unless you are near the center of the well, in my experience, it is impossible to get a phase contrast image.  Fyi, all my users who were using the 10x phase in multiwell dishes were conducting scratch assays. The images away from the center of wells made me cringe. But they claim to get what they needed. Ugh.

hank

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of David Knecht charter
Sent: Tuesday, November 23, 2010 12:12 PM
To: [hidden email]
Subject: Re: time series, large images and focus

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

Hi Hank- PFS is not supposed to work with plastic.  Are you saying that you got it to work with a standard plastic multiwell plate or were you using a glass bottom dish?  Dave

On Nov 19, 2010, at 11:01 AM, Adams,Henry P wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Sarah,
> I have a Ti with PFS and we do long timelapse experiments quite a bit with multiwell plates and also running 4 culture dishes.
> It is very important to make sure that your slide, plate or dishes are level as possible. I have had similar problems with PFS failing but by adjusting the set screws on the stage insert while using a small level tool on the plate or dishes I have been able to get PFS to work.
> Good luck,
> Hank Adams
> Microscopy Core
> Genetics
> U.T.M.D.Anderson Cancer Center
> Houston, Tx
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Gabor Csucs
> Sent: Friday, November 19, 2010 5:03 AM
> To: [hidden email]
> Subject: Re: timeseries, large images and focus
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Sarah,
>
> I'd suggest that your PI invest in the Perfect Focus System (PFS) and
> that would solve these problems (given that you are using an inverted
> stage).
>
> Cheers   Gabor
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hello,
>>
>> I am using a Nikon A1 confocal and I am trying to take a time series over several hours,  scanning across channels in a microfluidic chamber - my channels are 20 fields of view across, there are three channels.
>>
>> I am finding that even with one long (20 fields of view) image across one channel the focus is lost half way across, and moving to another region nearly always loses focus.
>>
>> I could increase the pinhole or take a small z stack so that being out of focus would matter less - I'm looking at bacteria ~1 micron in size, but don't need to resolve anything smaller, just distinguish between them using fluorescent labels.
>>
>> I wondered whether in principle this should work, using long image and PFS with several starting points, and if anyone has something similar working well, or whether I am expecting too much.
>>
>> Sarah
>> (student)
>
>
> --
> Gabor Csucs
> Light Microscopy Centre, ETH Zurich
> Schafmattstrasse 18, HPM F16
> CH-8093, Zurich, Switzerland
>
> Web: www.lmc.ethz.ch
> Phone: +41 44 633 6221
> Mobile: +41 79 758 21 58
> Fax: +41 44 632 1298
> e-mail: [hidden email]

Dr. David Knecht    
Department of Molecular and Cell Biology
Co-head Flow Cytometry and Confocal Microscopy Facility
U-3125
91 N. Eagleville Rd.
University of Connecticut
Storrs, CT 06269
860-486-2200
860-486-4331 (fax)
Koo, Lily (NIH/NIAID) [E] Koo, Lily (NIH/NIAID) [E]
Reply | Threaded
Open this post in threaded view
|

Far red FP for 2P imaging

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

Dear List,

Is there a far red FP you'd recommend for 2-photon imaging in addition to green/red pairs such as GFP/dsRED?

Thanks,

Lily
Deepak nair Deepak nair
Reply | Threaded
Open this post in threaded view
|

Re: Far red FP for 2P imaging

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

Hi,

I do not know much about the 2photon behaviour but what about the far red
proteins IFP from Tsien lab and Crimson from the lab of Ben Glick

Deepak

On Wed, Nov 24, 2010 at 4:28 PM, Koo, Lily (NIH/NIAID) [E] <
[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear List,
>
> Is there a far red FP you'd recommend for 2-photon imaging in addition to
> green/red pairs such as GFP/dsRED?
>
> Thanks,
>
> Lily
>



--
 Deepak Nair, PhD
"Physiologie Cellulaire de la Synapse"
UMR 5091 CNRS
Université Bordeaux 2
Institut François Magendie
33077 Bordeaux Cedex
Tél. : 05 57 57 40 81
Fax : 05 57 57 40 82
e-mail : [hidden email]
Craig Brideau Craig Brideau
Reply | Threaded
Open this post in threaded view
|

Re: Far red FP for 2P imaging

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

I seem to recall some preliminary work with Chromium:Forsterite lasers with
longer dyes.  Cr:Forsterite picks up at about 1200-1300nm so apparently it
is a good wavelength range to stimulate the really red dyes ~700-800nm alexa
dyes and the like.  You'd need red extended detectors to actually pick up
the signal from the dye though.

Craig

On Wed, Nov 24, 2010 at 8:45 AM, Deepak nair <[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi,
>
> I do not know much about the 2photon behaviour but what about the far red
> proteins IFP from Tsien lab and Crimson from the lab of Ben Glick
>
> Deepak
>
> On Wed, Nov 24, 2010 at 4:28 PM, Koo, Lily (NIH/NIAID) [E] <
> [hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Dear List,
> >
> > Is there a far red FP you'd recommend for 2-photon imaging in addition to
> > green/red pairs such as GFP/dsRED?
> >
> > Thanks,
> >
> > Lily
> >
>
>
>
> --
>  Deepak Nair, PhD
> "Physiologie Cellulaire de la Synapse"
> UMR 5091 CNRS
> Université Bordeaux 2
> Institut François Magendie
> 33077 Bordeaux Cedex
> Tél. : 05 57 57 40 81
> Fax : 05 57 57 40 82
> e-mail : [hidden email]
>
Guy Cox-2 Guy Cox-2
Reply | Threaded
Open this post in threaded view
|

Re: Far red FP for 2P imaging

In reply to this post by Koo, Lily (NIH/NIAID) [E]
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

There is a problem with doing 2P imaging within the tunable range of
your excitation laser, since the microscope will have dichroics and
blocking filters to cut out that part of the spectrum.  You'd need to
change some filters - and, for safety's sake, probably put in some
interlock to prevent your laser being tuned into the danger range.

                                            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
______________________________________________
      http://www.guycox.net
 

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Koo, Lily (NIH/NIAID) [E]
Sent: Thursday, 25 November 2010 2:28 AM
To: [hidden email]
Subject: Far red FP for 2P imaging

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

Dear List,

Is there a far red FP you'd recommend for 2-photon imaging in addition
to green/red pairs such as GFP/dsRED?

Thanks,

Lily