Confocal Microscopy List

Olympus TIRF

Classic

List

Threaded

8 messages Options

Ekaterina PAPUSHEVA-2

Olympus TIRF

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

Dear all,

We got a new Olympus TIRF delivered three weeks ago, and could not bring it to running.
Problem: we observe strong background, seemingly coming from laser, in a TIRF mode, when we use 405 or 488 nm laser for excitation. Increasing laser power does not improve signal-to-noise ratio 561 nm is seemingly ok.
Also, when we set 100 nm penetration depths through the software, and focus through the volume, 488 (and 405) nm background seems to shift from one side to the other very strongly. I know this is to be expected to some extent in objective-based TIRF, but we do not have it so bad on our other TIRF system with 100x. My feeling is, software calibration of the beam angle is wrong, also because when I set it to 'critical angle' or '100 nm penetration depth' beam still exits at about 40 degree angle. But I could not get good TIRF also by manually adjusting the slider undtil it looks like what is critical angle for me! The background stays just the same.
TIRF dischroics, emission filters and laser cleanup filters are correct and positioned correctly. Laser spectra are clean. PSF of the objective seems to be ok. It also cannot be a wrong cover glass- ours are highly corrected, cleaned and sonicated coverglasses, and I see the background even when I focus up from the glass
What else could I check? Is there anyone out who has Olympus TIRF and knows these problems?

I put few images here: widefield and TIRF images of Tetraspeck beads in water and a Zstack in TIRF
(to show the background shift) obtained with 488 nm excitation, just to illustrate what I am talking about.

http://pub.ist.ac.at/~papusheva/olympus_TIRF_30102013

Would be grateful for any tips!
Best regards,
Ekaterina

John Oreopoulos

Re: Olympus TIRF

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

Ekaterina,

I'm having trouble accessing the images you posted in your link. My browser says the following:

The requested URL /~papusheva/olympus_TIRF_30102013 was not found on this server.

Can you try reposting another way? Try sending them to me directly offline from the confocal listserver.
Can you also list here exactly what dichroic mirror, emission filters, and excitation filters (if any) that are present in your system? This sounds like it could be a case of a bad filter with poor out-of-band rejection of your laser line. Without seeing the images and knowing what filters you're dealing with, it's hard to say.

John Oreopoulos
Staff Scientist
Spectral Applied Research
Richmond Hill, Ontario
Canada
www.spectral.ca

On 2013-10-31, at 5:10 AM, Ekaterina PAPUSHEVA wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear all,
>
> We got a new Olympus TIRF delivered three weeks ago, and could not bring it to running.
> Problem: we observe strong background, seemingly coming from laser, in a TIRF mode, when we use 405 or 488 nm laser for excitation. Increasing laser power does not improve signal-to-noise ratio 561 nm is seemingly ok.
> Also, when we set 100 nm penetration depths through the software, and focus through the volume, 488 (and 405) nm background seems to shift from one side to the other very strongly. I know this is to be expected to some extent in objective-based TIRF, but we do not have it so bad on our other TIRF system with 100x. My feeling is, software calibration of the beam angle is wrong, also because when I set it to 'critical angle' or '100 nm penetration depth' beam still exits at about 40 degree angle. But I could not get good TIRF also by manually adjusting the slider undtil it looks like what is critical angle for me! The background stays just the same.
> TIRF dischroics, emission filters and laser cleanup filters are correct and positioned correctly. Laser spectra are clean. PSF of the objective seems to be ok. It also cannot be a wrong cover glass- ours are highly corrected, cleaned and sonicated coverglasses, and I see the background even when I focus up from the glass
> What else could I check? Is there anyone out who has Olympus TIRF and knows these problems?
>
> I put few images here: widefield and TIRF images of Tetraspeck beads in water and a Zstack in TIRF
> (to show the background shift) obtained with 488 nm excitation, just to illustrate what I am talking about.
>
> http://pub.ist.ac.at/~papusheva/olympus_TIRF_30102013
>
> Would be grateful for any tips!
> Best regards,
> Ekaterina

Unruh, Jay

Re: Olympus TIRF

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

A couple of thoughts:

Firstly, I have seen this sort of problem due to back-reflection of the TIRF beam from an excitation filter in the cube. Make sure the TIRF cube you are using doesn't have an excitation filter and that the laser is filtered independently prior to entering the TIRF launch (and more efficiently than simply an aotf alone).

Secondly, the first thing I do on every TIRF scope is to move the beam straight up out of the objective and tilt back the condenser (you will need to defeat the interlock and take appropriate laser safety precautions). That way you will see the beam on the ceiling. It should be as small as possible (probably less than 1 inch in diameter depending on the height of your ceiling). There should be some sort of divergence alignment within the system that you can use to make the beam smaller.

Once these two things are done, there are very few other reasons why TIRF wouldn't work (other than the catastrophic like a bad objective or a bad emission filter). Your system should allow for alignment of the laser to full blocking position. Then simply back off from that position until you see signal and it should be in TIRF. Of course, getting the angle calibrated for depth is a whole other issue which has been discussed on this list before.

Jay

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of John Oreopoulos
Sent: Thursday, October 31, 2013 8:13 AM
To: [hidden email]
Subject: Re: Olympus TIRF

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

Ekaterina,

I'm having trouble accessing the images you posted in your link. My browser says the following:

The requested URL /~papusheva/olympus_TIRF_30102013 was not found on this server.

Can you try reposting another way? Try sending them to me directly offline from the confocal listserver.
Can you also list here exactly what dichroic mirror, emission filters, and excitation filters (if any) that are present in your system? This sounds like it could be a case of a bad filter with poor out-of-band rejection of your laser line. Without seeing the images and knowing what filters you're dealing with, it's hard to say.

John Oreopoulos
Staff Scientist
Spectral Applied Research
Richmond Hill, Ontario
Canada
www.spectral.ca

On 2013-10-31, at 5:10 AM, Ekaterina PAPUSHEVA wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear all,
>
> We got a new Olympus TIRF delivered three weeks ago, and could not bring it to running.
> Problem: we observe strong background, seemingly coming from laser, in a TIRF mode, when we use 405 or 488 nm laser for excitation. Increasing laser power does not improve signal-to-noise ratio 561 nm is seemingly ok.
> Also, when we set 100 nm penetration depths through the software, and focus through the volume, 488 (and 405) nm background seems to shift from one side to the other very strongly. I know this is to be expected to some extent in objective-based TIRF, but we do not have it so bad on our other TIRF system with 100x. My feeling is, software calibration of the beam angle is wrong, also because when I set it to 'critical angle' or '100 nm penetration depth' beam still exits at about 40 degree angle. But I could not get good TIRF also by manually adjusting the slider undtil it looks like what is critical angle for me! The background stays just the same.
> TIRF dischroics, emission filters and laser cleanup filters are
> correct and positioned correctly. Laser spectra are clean. PSF of the objective seems to be ok. It also cannot be a wrong cover glass- ours are highly corrected, cleaned and sonicated coverglasses, and I see the background even when I focus up from the glass What else could I check? Is there anyone out who has Olympus TIRF and knows these problems?
>
> I put few images here: widefield and TIRF images of Tetraspeck beads
> in water and a Zstack in TIRF (to show the background shift) obtained with 488 nm excitation, just to illustrate what I am talking about.
>
> http://pub.ist.ac.at/~papusheva/olympus_TIRF_30102013
>
> Would be grateful for any tips!
> Best regards,
> Ekaterina

phil laissue-2

Re: Olympus TIRF

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

adding to Jay's comment - for getting the beam spot as small as possible on
the ceiling, I also recommend putting a coverslip (with immersion oil) on
top of the objective for best results, as the coverslip is effectively the
last lens in the setup.

_____________________________________
Philippe Laissue, PhD, Bioimaging Manager
School of Biological Sciences, Room 4.17
University of Essex, Colchester CO4 3SQ, UK
(0044) 01206 872246 / (0044) 07842 676 456
[hidden email]
privatewww.essex.ac.uk/~plaissue

On 31 October 2013 14:42, Unruh, Jay <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> A couple of thoughts:
>
> Firstly, I have seen this sort of problem due to back-reflection of the
> TIRF beam from an excitation filter in the cube. Make sure the TIRF cube
> you are using doesn't have an excitation filter and that the laser is
> filtered independently prior to entering the TIRF launch (and more
> efficiently than simply an aotf alone).
>
> Secondly, the first thing I do on every TIRF scope is to move the beam
> straight up out of the objective and tilt back the condenser (you will need
> to defeat the interlock and take appropriate laser safety precautions).
> That way you will see the beam on the ceiling. It should be as small as
> possible (probably less than 1 inch in diameter depending on the height of
> your ceiling). There should be some sort of divergence alignment within
> the system that you can use to make the beam smaller.
>
> Once these two things are done, there are very few other reasons why TIRF
> wouldn't work (other than the catastrophic like a bad objective or a bad
> emission filter). Your system should allow for alignment of the laser to
> full blocking position. Then simply back off from that position until you
> see signal and it should be in TIRF. Of course, getting the angle
> calibrated for depth is a whole other issue which has been discussed on
> this list before.
>
> Jay
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of John Oreopoulos
> Sent: Thursday, October 31, 2013 8:13 AM
> To: [hidden email]
> Subject: Re: Olympus TIRF
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Ekaterina,
>
> I'm having trouble accessing the images you posted in your link. My
> browser says the following:
>
> The requested URL /~papusheva/olympus_TIRF_30102013 was not found on this
> server.
>
> Can you try reposting another way? Try sending them to me directly offline
> from the confocal listserver.
> Can you also list here exactly what dichroic mirror, emission filters, and
> excitation filters (if any) that are present in your system? This sounds
> like it could be a case of a bad filter with poor out-of-band rejection of
> your laser line. Without seeing the images and knowing what filters you're
> dealing with, it's hard to say.
>
> John Oreopoulos
> Staff Scientist
> Spectral Applied Research
> Richmond Hill, Ontario
> Canada
> www.spectral.ca
>
>
>
> On 2013-10-31, at 5:10 AM, Ekaterina PAPUSHEVA wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Dear all,
> >
> > We got a new Olympus TIRF delivered three weeks ago, and could not bring
> it to running.
> > Problem: we observe strong background, seemingly coming from laser, in a
> TIRF mode, when we use 405 or 488 nm laser for excitation. Increasing laser
> power does not improve signal-to-noise ratio 561 nm is seemingly ok.
> > Also, when we set 100 nm penetration depths through the software, and
> focus through the volume, 488 (and 405) nm background seems to shift from
> one side to the other very strongly. I know this is to be expected to some
> extent in objective-based TIRF, but we do not have it so bad on our other
> TIRF system with 100x. My feeling is, software calibration of the beam
> angle is wrong, also because when I set it to 'critical angle' or '100 nm
> penetration depth' beam still exits at about 40 degree angle. But I could
> not get good TIRF also by manually adjusting the slider undtil it looks
> like what is critical angle for me! The background stays just the same.
> > TIRF dischroics, emission filters and laser cleanup filters are
> > correct and positioned correctly. Laser spectra are clean. PSF of the
> objective seems to be ok. It also cannot be a wrong cover glass- ours are
> highly corrected, cleaned and sonicated coverglasses, and I see the
> background even when I focus up from the glass What else could I check? Is
> there anyone out who has Olympus TIRF and knows these problems?
> >
> > I put few images here: widefield and TIRF images of Tetraspeck beads
> > in water and a Zstack in TIRF (to show the background shift) obtained
> with 488 nm excitation, just to illustrate what I am talking about.
> >
> > http://pub.ist.ac.at/~papusheva/olympus_TIRF_30102013
> >
> > Would be grateful for any tips!
> > Best regards,
> > Ekaterina
>

Paul Paroutis

Re: Olympus TIRF

In reply to this post by Ekaterina PAPUSHEVA-2

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

Hi Ekaterina,

We have an Olympus CellTIRF system and have observed that choosing a good
quality dichroic can make or break your system. In fact, we went through 3
'identical' dichroics before settling on one that gave the best images and the
lowest background/light scatter. You may want to try a few different dichroics and
see if that helps...

Thanks,
Paul

Neil Anthony

Re: Olympus TIRF

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

I agree with Paul. We just changed a Semrock for a Chroma filter cube
set, which rectified a huge optical issue we were having that manifested
as a large distinct region of non-TIR. There was no visual signs that
the Semrock had anything wrong with it, and remounting (gently) didn't
affect the problem.
The new Chroma line filter set works perfectly as far as I can tell.

see here:
http://chroma.com/product/complete-filter-sets/laser-applications/dual--TRF59904-EM--ET-488-561nm-laser-Dual-Band-set-for-TIRF-applications

Neil

On 10/31/2013 4:18 PM, Paul Paroutis wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Ekaterina,
>
> We have an Olympus CellTIRF system and have observed that choosing a good
> quality dichroic can make or break your system. In fact, we went through 3
> 'identical' dichroics before settling on one that gave the best images and the
> lowest background/light scatter. You may want to try a few different dichroics and
> see if that helps...
>
> Thanks,
> Paul
>

Prabhat, Prashant

Re: Olympus TIRF

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

*** Commercial Response ***

Hi Neil,

Our popular TIRF filters have been in use by the microscopy community for several years and we have not come across issues with our filters, as you may have experienced. Therefore in a separate (off the Confocal List) email, I would like to connect with you to learn from your recent experience with Semrock filters for TIRF.

Sincerely,
Prashant

Prashant Prabhat, Ph.D.
Applications Scientist

Semrock, Inc., A Unit of IDEX Corporation
3625 Buffalo Road, Suite 6
Rochester, NY 14624
Direct: (585) 594-7064
Cell: (585) 520-8602
FAX: (585) 594-7095
[hidden email]

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Neil Anthony
Sent: Monday, November 04, 2013 8:49 AM
To: [hidden email]
Subject: Re: Olympus TIRF

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

I agree with Paul. We just changed a Semrock for a Chroma filter cube set, which rectified a huge optical issue we were having that manifested as a large distinct region of non-TIR. There was no visual signs that the Semrock had anything wrong with it, and remounting (gently) didn't affect the problem.
The new Chroma line filter set works perfectly as far as I can tell.

see here:
http://chroma.com/product/complete-filter-sets/laser-applications/dual--TRF59904-EM--ET-488-561nm-laser-Dual-Band-set-for-TIRF-applications

Neil

On 10/31/2013 4:18 PM, Paul Paroutis wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Ekaterina,
>
> We have an Olympus CellTIRF system and have observed that choosing a
> good quality dichroic can make or break your system. In fact, we went
> through 3 'identical' dichroics before settling on one that gave the
> best images and the lowest background/light scatter. You may want to
> try a few different dichroics and see if that helps...
>
> Thanks,
> Paul
>

**********************************************************************
This e-mail is intended solely for the intended recipient or recipients. If this e-mail is addressed to you in error or you otherwise receive this e-mail in error, please advise the sender, do not read, print, forward or save this e-mail, and promptly delete and destroy all copies of this e-mail.
This email may contain information that is confidential, proprietary or secret and should be treated as confidential by all recipients. This e-mail may also be a confidential attorney-client communication, contain attorney work product, or otherwise be privileged and exempt from disclosure. If there is a confidentiality or non-disclosure agreement or protective order covering any information contained in this e-mail, such information shall be treated as confidential and subject to restriction on disclosure and use in accordance with such agreement or order, and this notice shall constitute identification, labeling or marking of such information as confidential, proprietary or secret in accordance with such agreement or order.
The term 'this e-mail' includes any and all attachments.
**********************************************************************

Ekaterina PAPUSHEVA-2

Re: Olympus TIRF

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

Dear all,

Thanks a lot for the helpful advice! The problem was, indeed, wrong filters in the delivery, plus some dirt in the microscope.
Solved and running now.

All best,
Katja

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Prabhat, Prashant
Sent: Monday, November 04, 2013 11:28 PM
To: [hidden email]
Subject: Re: Olympus TIRF

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

*** Commercial Response ***

Hi Neil,

Our popular TIRF filters have been in use by the microscopy community for several years and we have not come across issues with our filters, as you may have experienced. Therefore in a separate (off the Confocal List) email, I would like to connect with you to learn from your recent experience with Semrock filters for TIRF.

Sincerely,
Prashant

Prashant Prabhat, Ph.D.
Applications Scientist

Semrock, Inc., A Unit of IDEX Corporation
3625 Buffalo Road, Suite 6
Rochester, NY 14624
Direct: (585) 594-7064
Cell: (585) 520-8602
FAX: (585) 594-7095
[hidden email]

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Neil Anthony
Sent: Monday, November 04, 2013 8:49 AM
To: [hidden email]
Subject: Re: Olympus TIRF

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

I agree with Paul. We just changed a Semrock for a Chroma filter cube set, which rectified a huge optical issue we were having that manifested as a large distinct region of non-TIR. There was no visual signs that the Semrock had anything wrong with it, and remounting (gently) didn't affect the problem.
The new Chroma line filter set works perfectly as far as I can tell.

see here:
http://chroma.com/product/complete-filter-sets/laser-applications/dual--TRF59904-EM--ET-488-561nm-laser-Dual-Band-set-for-TIRF-applications

Neil

On 10/31/2013 4:18 PM, Paul Paroutis wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Ekaterina,
>
> We have an Olympus CellTIRF system and have observed that choosing a
> good quality dichroic can make or break your system. In fact, we went
> through 3 'identical' dichroics before settling on one that gave the
> best images and the lowest background/light scatter. You may want to
> try a few different dichroics and see if that helps...
>
> Thanks,
> Paul
>