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TIRF objective for routine imaging

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Charu Tanwar

TIRF objective for routine imaging

Dear List

Anybody please let me know whether we can use TIRF 100XH (N.A.
1.49,Working Distance 0.12mm,Coverglass correction 0.13-.20mm - this is a
special TIRF objective) for routine confocal imaging of bacteria and RBC's with
low flourescence signal.
Can it be mounted on a confocal microscope from TIRF microscope if both the
systems are from same company.

Thanks in advance

Charu Tanwar
Imaging Specialist
Advanced Instrumentation Research Facility
Jawaharlal Nehru University
New Delhi
India.

John Oreopoulos

Re: TIRF objective for routine imaging

There's no reason why you can't do this, and the images will be fine,
but if you're looking to do 3D sectional imaging of the RBCs, you
might be better off with a high NA water immersion objective instead
which will have lower spherical aberrations - you likely will get
better signal with that type objective. Try comparing both types of
objectives if possible.

John Oreopoulos

On 22-Mar-10, at 5:29 AM, Charu Tanwar wrote:

> Dear List
>
> Anybody please let me know whether we can use TIRF 100XH (N.A.
> 1.49,Working Distance 0.12mm,Coverglass correction 0.13-.20mm -
> this is a
> special TIRF objective) for routine confocal imaging of bacteria
> and RBC's with
> low flourescence signal.
> Can it be mounted on a confocal microscope from TIRF microscope if
> both the
> systems are from same company.
>
> Thanks in advance
>
> Charu Tanwar
> Imaging Specialist
> Advanced Instrumentation Research Facility
> Jawaharlal Nehru University
> New Delhi
> India.

Marco Dal Maschio-2

Re: TIRF objective for routine imaging

In reply to this post by Charu Tanwar

Dear Charu,

I had the opportunity to test from the same company 60x 1.45tirf // 60x VC // 60x 1.49 tirf.

I remember that probably 60x 1.49 is not plan, related to the flat field correction.

But this objective is currently used by colleagues performing tracking and imaging in neuronal cultures

with optimal image quality. If you want I can send you images comparing 60x VC and 60x 1.49 tirf.

Sorry but no experience about 100x.

best

Marco

On Mon, Mar 22, 2010 at 10:29 AM, Charu Tanwar <[hidden email]> wrote:

Dear List

Anybody please let me know whether we can use TIRF 100XH (N.A.
1.49,Working Distance 0.12mm,Coverglass correction 0.13-.20mm - this is a
special TIRF objective) for routine confocal imaging of bacteria and RBC's with
low flourescence signal.
Can it be mounted on a confocal microscope from TIRF microscope if both the
systems are from same company.

Thanks in advance

Charu Tanwar
Imaging Specialist
Advanced Instrumentation Research Facility
Jawaharlal Nehru University
New Delhi
India.

Neeraj Gohad-2

Re: TIRF objective for routine imaging

Hi Charu,

We have A Nikon TIRF module on our Nikon TiE so we have the Apo 60X 1.49 NA TIRF Objective, I have used this for regular confocal imaging with great success.

Best,

Neeraj.

Neeraj V. Gohad, Ph.D.

Postdoctoral Fellow

Okeanos Research Group

Department of Biological Sciences

132 Long Hall

Clemson University

Clemson,SC-29634

Phone: 864-656-3597

Fax: 864-656-0435

Website: http://www.clemson.edu/okeanos

Please note my new email address: [hidden email]

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Marco Dal Maschio
Sent: Monday, March 22, 2010 3:56 PM
To: [hidden email]
Subject: Re: TIRF objective for routine imaging

Dear Charu,

I had the opportunity to test from the same company 60x 1.45tirf // 60x VC // 60x 1.49 tirf.

I remember that probably 60x 1.49 is not plan, related to the flat field correction.

But this objective is currently used by colleagues performing tracking and imaging in neuronal cultures

with optimal image quality. If you want I can send you images comparing 60x VC and 60x 1.49 tirf.

Sorry but no experience about 100x.

best

Marco

On Mon, Mar 22, 2010 at 10:29 AM, Charu Tanwar <[hidden email]> wrote:

Tim Feinstein-2

Re: TIRF objective for routine imaging

<base href="x-msg://295/">We use the same objective as Neeraj on a A1/TiE for confocal, widefield and TIRF. No complaints.

cheers,

Tim Feinstein

On Mar 22, 2010, at 7:39 PM, Neeraj Gohad wrote:

Hi Charu,

We have A Nikon TIRF module on our Nikon TiE so we have the Apo 60X 1.49 NA TIRF Objective, I have used this for regular confocal imaging with great success.

Best,

Neeraj.

Neeraj V. Gohad, Ph.D.
Postdoctoral Fellow
Okeanos Research Group
Department of Biological Sciences
132 Long Hall
Clemson University
Clemson,SC-29634
Phone: 864-656-3597
Fax: 864-656-0435

Website: http://www.clemson.edu/okeanos

Please note my new email address: [hidden email]

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Marco Dal Maschio
Sent: Monday, March 22, 2010 3:56 PM
To: [hidden email]
Subject: Re: TIRF objective for routine imaging

Dear Charu,
I had the opportunity to test from the same company 60x 1.45tirf // 60x VC // 60x 1.49 tirf.
I remember that probably 60x 1.49 is not plan, related to the flat field correction.
But this objective is currently used by colleagues performing tracking and imaging in neuronal cultures
with optimal image quality. If you want I can send you images comparing 60x VC and 60x 1.49 tirf.
Sorry but no experience about 100x.

best
Marco

On Mon, Mar 22, 2010 at 10:29 AM, Charu Tanwar <[hidden email]> wrote:
Dear List

Anybody please let me know whether we can use TIRF 100XH (N.A.
1.49,Working Distance 0.12mm,Coverglass correction 0.13-.20mm - this is a
special TIRF objective) for routine confocal imaging of bacteria and RBC's with
low flourescence signal.
Can it be mounted on a confocal microscope from TIRF microscope if both the
systems are from same company.

Thanks in advance

Charu Tanwar
Imaging Specialist
Advanced Instrumentation Research Facility
Jawaharlal Nehru University
New Delhi
India.

Charu Tanwar

Re: TIRF objective for routine imaging

Thank you all for your valuable inputs.

Charu

CHARU TANWAR
Imaging Specialist
Advanced Instrumentation Research Facility
Jawaharlal Nehru University
New Delhi 110067
India.

--- On Tue, 23/3/10, Tim Feinstein <[hidden email]> wrote:

From: Tim Feinstein <[hidden email]>
Subject: Re: TIRF objective for routine imaging
To: [hidden email]
Date: Tuesday, 23 March, 2010, 5:55 AM

We use the same objective as Neeraj on a A1/TiE for confocal, widefield and TIRF. No complaints.

cheers,

Tim Feinstein

On Mar 22, 2010, at 7:39 PM, Neeraj Gohad wrote:

Hi Charu,

We have A Nikon TIRF module on our Nikon TiE so we have the Apo 60X 1.49 NA TIRF Objective, I have used this for regular confocal imaging with great success.

Best,

Neeraj.

Neeraj V. Gohad, Ph.D.

Postdoctoral Fellow

Okeanos Research Group

Department of Biological Sciences

132 Long Hall

Clemson University

Clemson,SC-29634

Phone: 864-656-3597

Fax: 864-656-0435

Website: http://www.clemson.edu/okeanos

Please note my new email address: http://in.mc83.mail.yahoo.com/mc/compose?to=neerajg@...

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Marco Dal Maschio
Sent: Monday, March 22, 2010 3:56 PM
To: http://in.mc83.mail.yahoo.com/mc/compose?to=CONFOCALMICROSCOPY@...
Subject: Re: TIRF objective for routine imaging

Dear Charu,

I had the opportunity to test from the same company 60x 1.45tirf // 60x VC // 60x 1.49 tirf.

I remember that probably 60x 1.49 is not plan, related to the flat field correction.

But this objective is currently used by colleagues performing tracking and imaging in neuronal cultures

with optimal image quality. If you want I can send you images comparing 60x VC and 60x 1.49 tirf.

Sorry but no experience about 100x.

best

Marco

On Mon, Mar 22, 2010 at 10:29 AM, Charu Tanwar <http://in.mc83.mail.yahoo.com/mc/compose?to=tanwar_charu@...> wrote:

Dear List

Anybody please let me know whether we can use TIRF 100XH (N.A.
1.49,Working Distance 0.12mm,Coverglass correction 0.13-.20mm - this is a
special TIRF objective) for routine confocal imaging of bacteria and RBC's with
low flourescence signal.
Can it be mounted on a confocal microscope from TIRF microscope if both the
systems are from same company.

Thanks in advance

Charu Tanwar
Imaging Specialist
Advanced Instrumentation Research Facility
Jawaharlal Nehru University
New Delhi
India.

Your Mail works best with the New Yahoo Optimized IE8. Get it NOW!.

Peter Gabriel Pitrone

Re: TIRF objective for routine imaging

Hello Charu,

If using this objective on a CLSM is your plan, then I would go for a lower magnification (the 60x/1.49 instead of the 100x/1.49 for example) due to transmission efficiencies. Objective with the same N.A. but with lower magnifications are almost always a better choice when working with point scanners. Just "zoom" in to meet your Nyquist!

Pete

On Mar 23, 2010, at 04:55 AM, charu tanwar wrote:

Thank you all for your valuable inputs.

Charu

CHARU TANWAR
Imaging Specialist
Advanced Instrumentation Research Facility
Jawaharlal Nehru University
New Delhi 110067
India.

--- On Tue, 23/3/10, Tim Feinstein <[hidden email]> wrote:

From: Tim Feinstein <[hidden email]>
Subject: Re: TIRF objective for routine imaging
To: [hidden email]
Date: Tuesday, 23 March, 2010, 5:55 AM

We use the same objective as Neeraj on a A1/TiE for confocal, widefield and TIRF. No complaints.

cheers,

Tim Feinstein

On Mar 22, 2010, at 7:39 PM, Neeraj Gohad wrote:

Hi Charu,

We have A Nikon TIRF module on our Nikon TiE so we have the Apo 60X 1.49 NA TIRF Objective, I have used this for regular confocal imaging with great success.

Best,

Neeraj.

Neeraj V. Gohad, Ph.D.

Postdoctoral Fellow

Okeanos Research Group

Department of Biological Sciences

132 Long Hall

Clemson University

Clemson,SC-29634

Phone: 864-656-3597

Fax: 864-656-0435

Website: http://www.clemson.edu/okeanos

Please note my new email address: http://in.mc83.mail.yahoo.com/mc/compose?to=neerajg@...

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Marco Dal Maschio
Sent: Monday, March 22, 2010 3:56 PM
To: http://in.mc83.mail.yahoo.com/mc/compose?to=CONFOCALMICROSCOPY@...
Subject: Re: TIRF objective for routine imaging

Dear Charu,

I had the opportunity to test from the same company 60x 1.45tirf // 60x VC // 60x 1.49 tirf.

I remember that probably 60x 1.49 is not plan, related to the flat field correction.

But this objective is currently used by colleagues performing tracking and imaging in neuronal cultures

with optimal image quality. If you want I can send you images comparing 60x VC and 60x 1.49 tirf.

Sorry but no experience about 100x.

best

Marco

On Mon, Mar 22, 2010 at 10:29 AM, Charu Tanwar <http://in.mc83.mail.yahoo.com/mc/compose?to=tanwar_charu@...> wrote:

Dear List

Anybody please let me know whether we can use TIRF 100XH (N.A.
1.49,Working Distance 0.12mm,Coverglass correction 0.13-.20mm - this is a
special TIRF objective) for routine confocal imaging of bacteria and RBC's with
low flourescence signal.
Can it be mounted on a confocal microscope from TIRF microscope if both the
systems are from same company.

Thanks in advance

Charu Tanwar
Imaging Specialist
Advanced Instrumentation Research Facility
Jawaharlal Nehru University
New Delhi
India.

Your Mail works best with the New Yahoo Optimized IE8. Get it NOW!.

James Pawley

Re: TIRF objective for routine imaging

Hello Charu,

If using this objective on a CLSM is your plan, then I would go for a lower magnification (the 60x/1.49 instead of the 100x/1.49 for example) due to transmission efficiencies. Objective with the same N.A. but with lower magnifications are almost always a better choice when working with point scanners. Just "zoom" in to meet your Nyquist!

Pete

Hi Charu and Pete,

"Efficiency" is a tricky thing. Everyone is in favor of it but it often means different things to different people.

In the days of widefield systems with Hg arcs and excitation optics that made a large image of the arc in the objective BFP, there was a rumor that the "brightness" of a fluorescent image would vary with the fourth power of the NA and inversely with the square of the magnification. So high NA, 40x (and now 20x!) objectives became popular. The problem was that this brightness was related to visibility by eye. Once you wanted to record the low-mag image on film, you had to use slower film with finer grain to preserve the details (Ok. Many people didn't care about the details and used fast film just to see if there was some stain, but this approach lost the details.)

When we got CCD cameras, we all became a bit more conscious of Nyquist sampling and realized that lower mag meant smaller camera pixels and, in the end, we collected the same number of photons/pixel no matter what objective mag you choose. Assuming that you had settled on a camera built around one of the ubiquitous SONY chips designed for the Japanese HDTV standard, the pixels were 6.7µm and you would just have to choose the right coupling tube mag to allow you to meet Nyquist.

So that is the mag part: In terms of image intensity, mag isn't important with laser instruments (including laser TIRF), unless you need low lag to cover a larger field and are willing to scan slowly, with a huge raster and tolerate some off-axis image degradation.

What about the NA part? As a mercury arc is about the same dimensions as the BFP of the objective (say 3-5 mm. It varies with arc power: 50w, 100w, 200w), any efficient illumination system will focus this into the BFP at about 1:1 because 1:1 optics have the highest potential light throughput.

Therefore, the objective that accepts light from the largest fraction of this arc-image and conveys it to the specimen will excite the most fluorescence. As the diameter of the BFP varies directly with NA, its area varies with (NA)*2. As the same should follow for the efficiency with which the objective collects the excited fluorescence, it will also vary with (NA)*2 and the total overall brightness will vary with (NA)*4.

Some BFP background

All other things being equal, a 100x objective has a focal length that is 2.5x shorter than a 40x lens. If, for a moment, you can just imagine the simplest lens diagram with on-axis, parallel illumination striking the lens and then converging to form a focused spot, one focal length away from the optical center of the lens (the optical center is the plane about which it seems to act). No matter what the objective magnification, the boundary of the angle of convergence is set only by the NA. Therefore, as the 40x lens has a 2.5x longer focal length, its principle plane must be 2.5x farther from the focal plane compared to that of the 100x lens. And as the divergence is the same, from similar triangles, the diameter of the parallel ray bundled needed to fill the 40x lens must be 2.5x larger than that needed to fill the 100x lens.

Put simply, the diameter of the clear aperture that you see when you look at a 100x NA 1.3 is about only about 40% as large as that seen when looking at the back of a 40x NA 1.3, and if you focus a more-or-less uniform image of the arc onto this plane, and it is big enough to "fill" the BFP of the 40x, then you will find that about (2.5)*2 = 6.25x more light comes out of the 40x, compared to the 100x. (On the other hand, if your excitation optics make an image of the arc in the BFP that is much smaller than that of the 40x BFP, this effect will be proportionally less. Assuming Kohler illumination, you should be able to see the relative size of the arc and the BFP if you just let the illumination proceed from the objective and fall onto a piece of lens tissue placed a cm or two on the far side of the focus plane.)

However, if you have a laser-based illumination (such as is found on many laser confocals), things are a little different. First all all, on the illumination side, excitation intensity itself is not a usually a problem. In fact we have to adjust the laser power down to prevent singlet-state saturation and undue photodamage. So one of the two (NA)*2 intensity terms become irrelevant. (On the imaging side, of course, larger NA will collect more light and, in the absence of spherical aberration etc, will deliver more photons to the detector.)

On the other hand, in laser-based systems, the diameter of the diffraction-limited spot is inversely proportional not to the NA engraved on the objective barrel but to the fraction of this NA actually filled by the laser beam. A 1mm diam unexpanded laser beam filling a 60x 1.3NA lens, with a BFP about 8mm in diameter will form a focused spot that is 8x LARGER than it should be. (The confocal image may still look sort of OK as the NA on the imaging side will still be 1.3, but the X,Y and Z resolution will still be lower.)

Now, confocal manufacturers know all this and they try to set things up so that the laser beam is large enough to "fill the BFP" of the most common lenses with more-or-less uniform laser intensity. However, you can see their problem, unless the illumination optics actually adjusts to match each lens, then we are back to the widefield system: if it is adjusted so that the laser "fills" the 40x BFP, then only 1/6th of that light will be used with the 100x. The rest will be bouncing around causing troublesome reflections. So they compromise, with the result that, in practice, one will often record slightly better resolution in X, Y and Z with the higher mag objective because the illumination will better fill its aperture.

So wasn't that a long story. I hope you are not all asleep (but I will be checking!)

Sorry. Must be Spring! And sorry for the typos...

Jim P.

**********************************************

Prof. James B. Pawley,

                    Ph. 608-263-3147
Room 223, Zoology Research Building,                                  FAX 608-265-5315
1117 Johnson Ave., Madison, WI, 53706                                [hidden email]
3D Microscopy of Living Cells Course, June 12-24, 2010, UBC, Vancouver Canada

Info: http://www.3dcourse.ubc.ca/ Applications still being accepted

"If it ain't diffraction, it must be statistics." Anon.

On Mar 23, 2010, at 04:55 AM, charu tanwar wrote:

Thank you all for your valuable inputs.

Charu

CHARU TANWAR
Imaging Specialist
Advanced Instrumentation Research Facility
Jawaharlal Nehru University
New Delhi 110067
India.

--- On Tue, 23/3/10, Tim Feinstein <[hidden email]> wrote:

From: Tim Feinstein <[hidden email]>
Subject: Re: TIRF objective for routine imaging
To: [hidden email]
Date: Tuesday, 23 March, 2010, 5:55 AM

We use the same objective as Neeraj on a A1/TiE for confocal, widefield and TIRF. No complaints.

cheers,

Tim Feinstein

On Mar 22, 2010, at 7:39 PM, Neeraj Gohad wrote:

Hi Charu,

We have A Nikon TIRF module on our Nikon TiE so we have the Apo 60X 1.49 NA TIRF Objective, I have used this for regular confocal imaging with great success.

Best,

Neeraj.

Neeraj V. Gohad, Ph.D.

Postdoctoral Fellow

Okeanos Research Group

Department of Biological Sciences

132 Long Hall

Clemson University

Clemson,SC-29634

Phone: 864-656-3597

Fax: 864-656-0435

Website: http://www.clemson.edu/okeanos

Please note my new email address: http://in.mc83.mail.yahoo.com/mc/compose?to=neerajg@...

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Marco Dal Maschio
Sent: Monday, March 22, 2010 3:56 PM
To: http://in.mc83.mail.yahoo.com/mc/compose?to=CONFOCALMICROSCOPY@LISTS.UMN.EDU
Subject: Re: TIRF objective for routine imaging

Dear Charu,

I had the opportunity to test from the same company 60x 1.45tirf // 60x VC // 60x 1.49 tirf.

I remember that probably 60x 1.49 is not plan, related to the flat field correction.

But this objective is currently used by colleagues performing tracking and imaging in neuronal cultures

with optimal image quality. If you want I can send you images comparing 60x VC and 60x 1.49 tirf.

Sorry but no experience about 100x.

best

Marco

On Mon, Mar 22, 2010 at 10:29 AM, Charu Tanwar <http://in.mc83.mail.yahoo.com/mc/compose?to=tanwar_charu@...> wrote:

Dear List

Anybody please let me know whether we can use TIRF 100XH (N.A.
1.49,Working Distance 0.12mm,Coverglass correction 0.13-.20mm - this is a
special TIRF objective) for routine confocal imaging of bacteria and RBC's with
low flourescence signal.
Can it be mounted on a confocal microscope from TIRF microscope if both the
systems are from same company.

Thanks in advance

Charu Tanwar
Imaging Specialist
Advanced Instrumentation Research Facility
Jawaharlal Nehru University
New Delhi
India.

Your Mail works best with the New Yahoo Optimized IE8. Get it NOW!.

--

**********************************************
Prof. James B. Pawley,

"If it ain't diffraction, it must be statistics." Anon.

Guy Cox-2

Re: TIRF objective for routine imaging

I’m not sure about other manufacturers but Leica adjust the beam expander according to the objective selected.

Guy

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of James Pawley
Sent: Wednesday, 24 March 2010 5:38 AM
To: [hidden email]
Subject: Re: TIRF objective for routine imaging

Hello Charu,

If using this objective on a CLSM is your plan, then I would go for a lower magnification (the 60x/1.49 instead of the 100x/1.49 for example) due to transmission efficiencies. Objective with the same N.A. but with lower magnifications are almost always a better choice when working with point scanners. Just "zoom" in to meet your Nyquist!

Pete

Hi Charu and Pete,

"Efficiency" is a tricky thing. Everyone is in favor of it but it often means different things to different people.

Some BFP background

So wasn't that a long story. I hope you are not all asleep (but I will be checking!)

Sorry. Must be Spring! And sorry for the typos...

Jim P.

**********************************************

Prof. James B. Pawley,                                      Ph. 608-263-3147
Room 223, Zoology Research Building,                                  FAX 608-265-5315
1117 Johnson Ave., Madison, WI, 53706                                [hidden email]
3D Microscopy of Living Cells Course, June 12-24, 2010, UBC, Vancouver Canada

Info: http://www.3dcourse.ubc.ca/ Applications still being accepted

"If it ain't diffraction, it must be statistics." Anon.

On Mar 23, 2010, at 04:55 AM, charu tanwar wrote:

Thank you all for your valuable inputs.

Charu

CHARU TANWAR
Imaging Specialist
Advanced Instrumentation Research Facility
Jawaharlal Nehru University
New Delhi 110067
India.

--- On Tue, 23/3/10, Tim Feinstein <[hidden email]> wrote:

From: Tim Feinstein <[hidden email]>
Subject: Re: TIRF objective for routine imaging
To: [hidden email]
Date: Tuesday, 23 March, 2010, 5:55 AM

We use the same objective as Neeraj on a A1/TiE for confocal, widefield and TIRF. No complaints.

cheers,

Tim Feinstein

On Mar 22, 2010, at 7:39 PM, Neeraj Gohad wrote:

Hi Charu,

We have A Nikon TIRF module on our Nikon TiE so we have the Apo 60X 1.49 NA TIRF Objective, I have used this for regular confocal imaging with great success.

Best,

Neeraj.

Neeraj V. Gohad, Ph.D.

Postdoctoral Fellow

Okeanos Research Group

Department of Biological Sciences

132 Long Hall

Clemson University

Clemson,SC-29634

Phone: 864-656-3597

Fax: 864-656-0435

Website: http://www.clemson.edu/okeanos

Please note my new email address: http://in.mc83.mail.yahoo.com/mc/compose?to=neerajg@...

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Marco Dal Maschio
Sent: Monday, March 22, 2010 3:56 PM
To: http://in.mc83.mail.yahoo.com/mc/compose?to=CONFOCALMICROSCOPY@...
Subject: Re: TIRF objective for routine imaging

Dear Charu,

I had the opportunity to test from the same company 60x 1.45tirf // 60x VC // 60x 1.49 tirf.

I remember that probably 60x 1.49 is not plan, related to the flat field correction.

But this objective is currently used by colleagues performing tracking and imaging in neuronal cultures

with optimal image quality. If you want I can send you images comparing 60x VC and 60x 1.49 tirf.

Sorry but no experience about 100x.

best

Marco

On Mon, Mar 22, 2010 at 10:29 AM, Charu Tanwar <http://in.mc83.mail.yahoo.com/mc/compose?to=tanwar_charu@...> wrote:

Dear List

Anybody please let me know whether we can use TIRF 100XH (N.A.
1.49,Working Distance 0.12mm,Coverglass correction 0.13-.20mm - this is a
special TIRF objective) for routine confocal imaging of bacteria and RBC's with
low flourescence signal.
Can it be mounted on a confocal microscope from TIRF microscope if both the
systems are from same company.

Thanks in advance

Charu Tanwar
Imaging Specialist
Advanced Instrumentation Research Facility
Jawaharlal Nehru University
New Delhi
India.

Your Mail works best with the New Yahoo Optimized IE8. Get it NOW!.

--

              **********************************************
Prof. James B. Pawley,                                          Ph. 608-263-3147
Room 223, Zoology Research Building,                                  FAX 608-265-5315
1117 Johnson Ave., Madison, WI, 53706                                [hidden email]
3D Microscopy of Living Cells Course, June 12-24, 2010, UBC, Vancouver Canada
Info: http://www.3dcourse.ubc.ca/            Applications due by March 15, 2010

"If it ain't diffraction, it must be statistics." Anon.

Peter Gabriel Pitrone

Re: TIRF objective for routine imaging

In reply to this post by James Pawley

Hello Jim,

Thanks for filling me in on the subject, it was as clear and concise as I could have asked for. Could you give an example of the loss in spacial resolution the 40x would have compared to the 60x or 100x assuming the N.A (which being 1.3 theoretically should have a optical resolution of 0.2115 um at 0.550 um) is the same.

Thank again!

Pete

On Mar 23, 2010, at 19:37 PM, James Pawley wrote:

Hello Charu,

If using this objective on a CLSM is your plan, then I would go for a lower magnification (the 60x/1.49 instead of the 100x/1.49 for example) due to transmission efficiencies. Objective with the same N.A. but with lower magnifications are almost always a better choice when working with point scanners. Just "zoom" in to meet your Nyquist!

Pete

Hi Charu and Pete,

"Efficiency" is a tricky thing. Everyone is in favor of it but it often means different things to different people.

In the days of widefield systems with Hg arcs and excitation optics that made a large image of the arc in the objective BFP, there was a rumor that the "brightness" of a fluorescent image would vary with the fourth power of the NA and inversely with the square of the magnification. So high NA, 40x (and now 20x!) objectives became popular. The problem was that this brightness was related to visibility by eye. Once you wanted to record the low-mag image on film, you had to use slower film with finer grain to preserve the details (Ok. Many people didn't care about the details and used fast film just to see if there was some stain, but this approach lost the details.)

When we got CCD cameras, we all became a bit more conscious of Nyquist sampling and realized that lower mag meant smaller camera pixels and, in the end, we collected the same number of photons/pixel no matter what objective mag you choose. Assuming that you had settled on a camera built around one of the ubiquitous SONY chips designed for the Japanese HDTV standard, the pixels were 6.7µm and you would just have to choose the right coupling tube mag to allow you to meet Nyquist.

So that is the mag part: In terms of image intensity, mag isn't important with laser instruments (including laser TIRF), unless you need low lag to cover a larger field and are willing to scan slowly, with a huge raster and tolerate some off-axis image degradation.

What about the NA part? As a mercury arc is about the same dimensions as the BFP of the objective (say 3-5 mm. It varies with arc power: 50w, 100w, 200w), any efficient illumination system will focus this into the BFP at about 1:1 because 1:1 optics have the highest potential light throughput.

Therefore, the objective that accepts light from the largest fraction of this arc-image and conveys it to the specimen will excite the most fluorescence. As the diameter of the BFP varies directly with NA, its area varies with (NA)*2. As the same should follow for the efficiency with which the objective collects the excited fluorescence, it will also vary with (NA)*2 and the total overall brightness will vary with (NA)*4.

Some BFP background

All other things being equal, a 100x objective has a focal length that is 2.5x shorter than a 40x lens. If, for a moment, you can just imagine the simplest lens diagram with on-axis, parallel illumination striking the lens and then converging to form a focused spot, one focal length away from the optical center of the lens (the optical center is the plane about which it seems to act). No matter what the objective magnification, the boundary of the angle of convergence is set only by the NA. Therefore, as the 40x lens has a 2.5x longer focal length, its principle plane must be 2.5x farther from the focal plane compared to that of the 100x lens. And as the divergence is the same, from similar triangles, the diameter of the parallel ray bundled needed to fill the 40x lens must be 2.5x larger than that needed to fill the 100x lens.

Put simply, the diameter of the clear aperture that you see when you look at a 100x NA 1.3 is about only about 40% as large as that seen when looking at the back of a 40x NA 1.3, and if you focus a more-or-less uniform image of the arc onto this plane, and it is big enough to "fill" the BFP of the 40x, then you will find that about (2.5)*2 = 6.25x more light comes out of the 40x, compared to the 100x. (On the other hand, if your excitation optics make an image of the arc in the BFP that is much smaller than that of the 40x BFP, this effect will be proportionally less. Assuming Kohler illumination, you should be able to see the relative size of the arc and the BFP if you just let the illumination proceed from the objective and fall onto a piece of lens tissue placed a cm or two on the far side of the focus plane.)

However, if you have a laser-based illumination (such as is found on many laser confocals), things are a little different. First all all, on the illumination side, excitation intensity itself is not a usually a problem. In fact we have to adjust the laser power down to prevent singlet-state saturation and undue photodamage. So one of the two (NA)*2 intensity terms become irrelevant. (On the imaging side, of course, larger NA will collect more light and, in the absence of spherical aberration etc, will deliver more photons to the detector.)

On the other hand, in laser-based systems, the diameter of the diffraction-limited spot is inversely proportional not to the NA engraved on the objective barrel but to the fraction of this NA actually filled by the laser beam. A 1mm diam unexpanded laser beam filling a 60x 1.3NA lens, with a BFP about 8mm in diameter will form a focused spot that is 8x LARGER than it should be. (The confocal image may still look sort of OK as the NA on the imaging side will still be 1.3, but the X,Y and Z resolution will still be lower.)

Now, confocal manufacturers know all this and they try to set things up so that the laser beam is large enough to "fill the BFP" of the most common lenses with more-or-less uniform laser intensity. However, you can see their problem, unless the illumination optics actually adjusts to match each lens, then we are back to the widefield system: if it is adjusted so that the laser "fills" the 40x BFP, then only 1/6th of that light will be used with the 100x. The rest will be bouncing around causing troublesome reflections. So they compromise, with the result that, in practice, one will often record slightly better resolution in X, Y and Z with the higher mag objective because the illumination will better fill its aperture.

So wasn't that a long story. I hope you are not all asleep (but I will be checking!)

Sorry. Must be Spring! And sorry for the typos...

Jim P.

              **********************************************
Prof. James B. Pawley,                                      Ph. 608-263-3147
Room 223, Zoology Research Building,                                  FAX 608-265-5315
1117 Johnson Ave., Madison, WI, 53706                                [hidden email]
3D Microscopy of Living Cells Course, June 12-24, 2010, UBC, Vancouver Canada
Info: http://www.3dcourse.ubc.ca/                Applications still being accepted
               "If it ain't diffraction, it must be statistics." Anon.

On Mar 23, 2010, at 04:55 AM, charu tanwar wrote:

Thank you all for your valuable inputs.
Charu

CHARU TANWAR
Imaging Specialist
Advanced Instrumentation Research Facility
Jawaharlal Nehru University
New Delhi 110067
India.

--- On Tue, 23/3/10, Tim Feinstein <[hidden email]> wrote:

From: Tim Feinstein <[hidden email]>
Subject: Re: TIRF objective for routine imaging
To: [hidden email]
Date: Tuesday, 23 March, 2010, 5:55 AM
We use the same objective as Neeraj on a A1/TiE for confocal, widefield and TIRF. No complaints.

cheers,

Tim Feinstein

On Mar 22, 2010, at 7:39 PM, Neeraj Gohad wrote:

Hi Charu,

We have A Nikon TIRF module on our Nikon TiE so we have the Apo 60X 1.49 NA TIRF Objective, I have used this for regular confocal imaging with great success.

Best,

Neeraj.

Neeraj V. Gohad, Ph.D.
Postdoctoral Fellow
Okeanos Research Group
Department of Biological Sciences
132 Long Hall
Clemson University
Clemson,SC-29634
Phone: 864-656-3597
Fax: 864-656-0435

Website: http://www.clemson.edu/okeanos

Please note my new email address: http://in.mc83.mail.yahoo.com/mc/compose?to=neerajg@...

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Marco Dal Maschio
Sent: Monday, March 22, 2010 3:56 PM
To: http://in.mc83.mail.yahoo.com/mc/compose?to=CONFOCALMICROSCOPY@LISTS.UMN.EDU
Subject: Re: TIRF objective for routine imaging

Dear Charu,
I had the opportunity to test from the same company 60x 1.45tirf // 60x VC // 60x 1.49 tirf.
I remember that probably 60x 1.49 is not plan, related to the flat field correction.
But this objective is currently used by colleagues performing tracking and imaging in neuronal cultures
with optimal image quality. If you want I can send you images comparing 60x VC and 60x 1.49 tirf.
Sorry but no experience about 100x.

best
Marco

On Mon, Mar 22, 2010 at 10:29 AM, Charu Tanwar <http://in.mc83.mail.yahoo.com/mc/compose?to=tanwar_charu@...> wrote:
Dear List

Anybody please let me know whether we can use TIRF 100XH (N.A.
1.49,Working Distance 0.12mm,Coverglass correction 0.13-.20mm - this is a
special TIRF objective) for routine confocal imaging of bacteria and RBC's with
low flourescence signal.
Can it be mounted on a confocal microscope from TIRF microscope if both the
systems are from same company.

Thanks in advance

Charu Tanwar
Imaging Specialist
Advanced Instrumentation Research Facility
Jawaharlal Nehru University
New Delhi
India.



Your Mail works best with the New Yahoo Optimized IE8. Get it NOW!.

--
              **********************************************
Prof. James B. Pawley,                                          Ph. 608-263-3147
Room 223, Zoology Research Building,                                  FAX 608-265-5315
1117 Johnson Ave., Madison, WI, 53706                                [hidden email]
3D Microscopy of Living Cells Course, June 12-24, 2010, UBC, Vancouver Canada
Info: http://www.3dcourse.ubc.ca/            Applications due by March 15, 2010
               "If it ain't diffraction, it must be statistics." Anon.

Barbara Foster

Re: TIRF objective for routine imaging

In reply to this post by Charu Tanwar

Hi, all

Just remember that the 1.49 NA only works with special, high refractive index liquids.

Barbara Foster, President and Sr. Consultant

Microscopy/Microscopy Education
7101 Royal Glen Trail, Suite A
McKinney TX 75070
P: (972)924-5310 Skype: fostermme
W: www.MicroscopyEducation.com

NEWS! Visit the NEW and IMPROVED www.MicroscopyEducation.com! And don't forget: MME is now scheduling customized, on-site courses for 2010. Call me for a free assessment and quote.

At 05:39 PM 3/22/2010, you wrote:

Hi Charu,

We have A Nikon TIRF module on our Nikon TiE so we have the Apo 60X 1.49 NA TIRF Objective, I have used this for regular confocal imaging with great success.

Best,

Neeraj.

Neeraj V. Gohad, Ph.D.
Postdoctoral Fellow
Okeanos Research Group
Department of Biological Sciences
132 Long Hall
Clemson University
Clemson,SC-29634
Phone: 864-656-3597
Fax: 864-656-0435

Website: http://www.clemson.edu/okeanos

Please note my new email address: [hidden email]

From: Confocal Microscopy List [[hidden email]] On Behalf Of Marco Dal Maschio
Sent: Monday, March 22, 2010 3:56 PM
To: [hidden email]
Subject: Re: TIRF objective for routine imaging

Dear Charu,
I had the opportunity to test from the same company 60x 1.45tirf // 60x VC // 60x 1.49 tirf.
I remember that probably 60x 1.49 is not plan, related to the flat field correction.
But this objective is currently used by colleagues performing tracking and imaging in neuronal cultures
with optimal image quality. If you want I can send you images comparing 60x VC and 60x 1.49 tirf.
Sorry but no experience about 100x.

best
Marco

On Mon, Mar 22, 2010 at 10:29 AM, Charu Tanwar <[hidden email] > wrote:
Dear List

Anybody please let me know whether we can use TIRF 100XH (N.A.
1.49,Working Distance 0.12mm,Coverglass correction 0.13-.20mm - this is a
special TIRF objective) for routine confocal imaging of bacteria and RBC's with
low flourescence signal.
Can it be mounted on a confocal microscope from TIRF microscope if both the
systems are from same company.

Thanks in advance

Charu Tanwar
Imaging Specialist
Advanced Instrumentation Research Facility
Jawaharlal Nehru University
New Delhi
India.

Kurt Thorn

Re: TIRF objective for routine imaging

This isn't true - our Nikon 1.49 TIRF lens works with the standard
immersion oil.

Barbara Foster wrote:

> Hi, all
>
> Just remember that the 1.49 NA only works with special, high
> refractive index liquids.
>
> *Barbara Foster, President and Sr. Consultant
>
> Microscopy/Microscopy Education
> *7101 Royal Glen Trail, Suite A
> McKinney TX 75070
> *P: *(972)924-5310 *Skype: *fostermme
> *W: *www.MicroscopyEducation.com
>
> <http://www.microscopyeducation.com/>*NEWS! Visit the NEW and IMPROVED
> www.MicroscopyEducation.com <http://www.microscopyeducation.com/>! And
> don't forget: MME is now scheduling customized, on-site courses for
> 2010. Call me for a free assessment and quote.
>
> *

Cameron, Lisa

Re: TIRF objective for routine imaging

Yes, I concur with Kurt -

Nikon 60x and 100x 1.49 NA TIRF objectives work well with the standard 1.515
Nikon oil.

---------------------------------------
Lisa Cameron, Ph.D.
Director of Confocal and Light Microscopy
Dana Farber Cancer Institute

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Kurt Thorn
Sent: Wednesday, March 24, 2010 7:28 PM
To: [hidden email]
Subject: Re: [CONFOCALMICROSCOPY] TIRF objective for routine imaging

This isn't true - our Nikon 1.49 TIRF lens works with the standard
immersion oil.

Barbara Foster wrote:

The information in this e-mail is intended only for the person to whom it is
addressed. If you believe this e-mail was sent to you in error and the e-mail
contains patient information, please contact the Partners Compliance HelpLine at
http://www.partners.org/complianceline . If the e-mail was sent to you in error
but does not contain patient information, please contact the sender and properly
dispose of the e-mail.

Tim Feinstein-2

Re: TIRF objective for routine imaging

Acknowledging in advance that I am hardly a deconvolution expert, our Nikon 60x 1.49 provides a sharper PSF than our 40x plan fluor (hardly an apples-to-apples comparison, I know). We measured the PSF because our blind deconvolution software (Huygens) complained about the NA/medium mismatch. It still appeared to work fairly well.

cheers,

Tim

Timothy Feinstein, PhD
Postdoctoral Associate
University of Pittsburgh Dept. of Pharmacology
Pittsburgh, PA

On Mar 24, 2010, at 7:33 PM, Cameron, Lisa wrote:

> Yes, I concur with Kurt -
>
> Nikon 60x and 100x 1.49 NA TIRF objectives work well with the standard 1.515
> Nikon oil.
>
>
> ---------------------------------------
> Lisa Cameron, Ph.D.
> Director of Confocal and Light Microscopy
> Dana Farber Cancer Institute
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On
> Behalf Of Kurt Thorn
> Sent: Wednesday, March 24, 2010 7:28 PM
> To: [hidden email]
> Subject: Re: [CONFOCALMICROSCOPY] TIRF objective for routine imaging
>
> This isn't true - our Nikon 1.49 TIRF lens works with the standard
> immersion oil.
>
> Barbara Foster wrote:
>> Hi, all
>>
>> Just remember that the 1.49 NA only works with special, high
>> refractive index liquids.
>>
>> *Barbara Foster, President and Sr. Consultant
>>
>> Microscopy/Microscopy Education
>> *7101 Royal Glen Trail, Suite A
>> McKinney TX 75070
>> *P: *(972)924-5310 *Skype: *fostermme
>> *W: *www.MicroscopyEducation.com
>>
>> <http://www.microscopyeducation.com/>*NEWS! Visit the NEW and IMPROVED
>> www.MicroscopyEducation.com <http://www.microscopyeducation.com/>! And
>> don't forget: MME is now scheduling customized, on-site courses for
>> 2010. Call me for a free assessment and quote.
>>
>> *
>
>
> The information in this e-mail is intended only for the person to whom it is
> addressed. If you believe this e-mail was sent to you in error and the e-mail
> contains patient information, please contact the Partners Compliance HelpLine at
> http://www.partners.org/complianceline . If the e-mail was sent to you in error
> but does not contain patient information, please contact the sender and properly
> dispose of the e-mail.

John Oreopoulos

Re: TIRF objective for routine imaging

In reply to this post by Cameron, Lisa

I think maybe Barbara was referring to the Olympus special 1.65 NA
TIRF objective which does require special (toxic and reactive)
immersion oil and quartz substrates. But the objective in question
was a Nikon one which I'm not familiar with.

John Oreopoulos

On 24-Mar-10, at 7:33 PM, Cameron, Lisa wrote:

> Yes, I concur with Kurt -
>
> Nikon 60x and 100x 1.49 NA TIRF objectives work well with the
> standard 1.515
> Nikon oil.
>
>
> ---------------------------------------
> Lisa Cameron, Ph.D.
> Director of Confocal and Light Microscopy
> Dana Farber Cancer Institute
>
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]] On
> Behalf Of Kurt Thorn
> Sent: Wednesday, March 24, 2010 7:28 PM
> To: [hidden email]
> Subject: Re: [CONFOCALMICROSCOPY] TIRF objective for routine imaging
>
> This isn't true - our Nikon 1.49 TIRF lens works with the standard
> immersion oil.
>
> Barbara Foster wrote:
>> Hi, all
>>
>> Just remember that the 1.49 NA only works with special, high
>> refractive index liquids.
>>
>> *Barbara Foster, President and Sr. Consultant
>>
>> Microscopy/Microscopy Education
>> *7101 Royal Glen Trail, Suite A
>> McKinney TX 75070
>> *P: *(972)924-5310 *Skype: *fostermme
>> *W: *www.MicroscopyEducation.com
>>
>> <http://www.microscopyeducation.com/>*NEWS! Visit the NEW and
>> IMPROVED
>> www.MicroscopyEducation.com <http://www.microscopyeducation.com/>!
>> And
>> don't forget: MME is now scheduling customized, on-site courses for
>> 2010. Call me for a free assessment and quote.
>>
>> *
>
>
> The information in this e-mail is intended only for the person to
> whom it is
> addressed. If you believe this e-mail was sent to you in error and
> the e-mail
> contains patient information, please contact the Partners
> Compliance HelpLine at
> http://www.partners.org/complianceline . If the e-mail was sent to
> you in error
> but does not contain patient information, please contact the sender
> and properly
> dispose of the e-mail.

Stephen Cody-2

Re: TIRF objective for routine imaging

John is on the right track,

The Nikon TIRF Objectives including the 60 and 100X 1.49 NA, TIRF
objective use conventional oil and coverslips. Several years ago I
purchase a 60X 1.45 NA objective from Nikon to use on our C1 confocal,
for high resolution applications. It worked very but I did notice it
was not as flat as our 1.4 Plan Apo oil form the same company. Horses
for courses. BTW later we bought their White light Arc TIRF system to
go with the lens!

Cheers
Steve

On 25 March 2010 10:52, John Oreopoulos <[hidden email]> wrote:

> I think maybe Barbara was referring to the Olympus special 1.65 NA TIRF
> objective which does require special (toxic and reactive) immersion oil and
> quartz substrates. But the objective in question was a Nikon one which I'm
> not familiar with.
>
> John Oreopoulos
>
> On 24-Mar-10, at 7:33 PM, Cameron, Lisa wrote:
>
>> Yes, I concur with Kurt -
>>
>> Nikon 60x and 100x 1.49 NA TIRF objectives work well with the standard
>> 1.515
>> Nikon oil.
>>
>>
>> ---------------------------------------
>> Lisa Cameron, Ph.D.
>> Director of Confocal and Light Microscopy
>> Dana Farber Cancer Institute
>>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:[hidden email]]
>> On
>> Behalf Of Kurt Thorn
>> Sent: Wednesday, March 24, 2010 7:28 PM
>> To: [hidden email]
>> Subject: Re: [CONFOCALMICROSCOPY] TIRF objective for routine imaging
>>
>> This isn't true - our Nikon 1.49 TIRF lens works with the standard
>> immersion oil.
>>
>> Barbara Foster wrote:
>>>
>>> Hi, all
>>>
>>> Just remember that the 1.49 NA only works with special, high
>>> refractive index liquids.
>>>
>>> *Barbara Foster, President and Sr. Consultant
>>>
>>> Microscopy/Microscopy Education
>>> *7101 Royal Glen Trail, Suite A
>>> McKinney TX 75070
>>> *P: *(972)924-5310 *Skype: *fostermme
>>> *W: *www.MicroscopyEducation.com
>>>
>>> <http://www.microscopyeducation.com/>*NEWS! Visit the NEW and IMPROVED
>>> www.MicroscopyEducation.com <http://www.microscopyeducation.com/>! And
>>> don't forget: MME is now scheduling customized, on-site courses for
>>> 2010. Call me for a free assessment and quote.
>>>
>>> *
>>
>>
>> The information in this e-mail is intended only for the person to whom it
>> is
>> addressed. If you believe this e-mail was sent to you in error and the
>> e-mail
>> contains patient information, please contact the Partners Compliance
>> HelpLine at
>> http://www.partners.org/complianceline . If the e-mail was sent to you in
>> error
>> but does not contain patient information, please contact the sender and
>> properly
>> dispose of the e-mail.
>

--
Stephen H. Cody

James Pawley

Re: TIRF objective for routine imaging

In reply to this post by Tim Feinstein-2

Re: TIRF objective for routine imaging

Acknowledging in advance that I am hardly a deconvolution expert, our Nikon 60x 1.49 provides a sharper PSF than our 40x plan fluor (hardly an apples-to-apples comparison, I know). We measured the PSF because our blind deconvolution software (Huygens) complained about the NA/medium mismatch. It still appeared to work fairly well.

cheers,

Tim

Timothy Feinstein, PhD
Postdoctoral Associate
University of Pittsburgh Dept. of Pharmacology
Pittsburgh, PA

Hi Tim,

You don't mention the NA of the 40x (or if we are talking widefield or laser confocal, where underfilling might be less of a factor at 60x than at 40x) but I think that your comment about the useful suggestion that the Huygens software made about mismatch might be the nub of the argument.

I am assuming that Hans van der Voort's clever program makes this warning when it detects that the PSF is asymmetrical in z. Such asymmetry implies spherical aberration and this is usually caused by a mismatch between the refractive index (RI) that the lens is designed for and the actual RI of the specimen or using a coverslip of the incorrect thickness.

None of the high NA 40x Fluor lenses I can think of include an SA corection collar, while the Nikon NA 1.49 does do so (did you use it?). So, if your specimen RI is not the same as that of oil or your coverslip is the wrong thickness, you can correct for this on the 1.49 but not on the other 40x lens.

SA correction being equal, and assuming that your 40x is NA 1.3, I would expect that the on-axis x-y resolution of the NA 1.49 would be about 14% better (smaller spacing) than the 40x.

Cheers,

Jim P.

*********************************************************************************
Prof. James B. Pawley,

Info: http://www.3dcourse.ubc.ca/ Applications still being accepted

"If it ain't diffraction, it must be statistics." Anon.

On Mar 24, 2010, at 7:33 PM, Cameron, Lisa wrote:

> Yes, I concur with Kurt -
>
> Nikon 60x and 100x 1.49 NA TIRF objectives work well with the standard 1.515
> Nikon oil.
>
>
> ---------------------------------------
> Lisa Cameron, Ph.D.
> Director of Confocal and Light Microscopy
> Dana Farber Cancer Institute
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On
> Behalf Of Kurt Thorn
> Sent: Wednesday, March 24, 2010 7:28 PM
> To: [hidden email]
> Subject: Re: [CONFOCALMICROSCOPY] TIRF objective for routine imaging
>
> This isn't true - our Nikon 1.49 TIRF lens works with the standard
> immersion oil.
>
> Barbara Foster wrote:
>> Hi, all
>>
>> Just remember that the 1.49 NA only works with special, high
>> refractive index liquids.
>>
>> *Barbara Foster, President and Sr. Consultant
>>
>> Microscopy/Microscopy Education
>> *7101 Royal Glen Trail, Suite A
>> McKinney TX 75070
>> *P: *(972)924-5310 *Skype: *fostermme
>> *W: *www.MicroscopyEducation.com
>>
>> <http://www.microscopyeducation.com/>*NEWS! Visit the NEW and IMPROVED
>> www.MicroscopyEducation.com <http://www.microscopyeducation.com/>! And
>> don't forget: MME is now scheduling customized, on-site courses for
>> 2010. Call me for a free assessment and quote.
>>
>> *
>
>
> The information in this e-mail is intended only for the person to whom it is
> addressed. If you believe this e-mail was sent to you in error and the e-mail
> contains patient information, please contact the Partners Compliance HelpLine at
> http://www.partners.org/complianceline . If the e-mail was sent to you in error
> but does not contain patient information, please contact the sender and properly
> dispose of the e-mail.

--

*********************************************************************************
Prof. James B. Pawley,

"If it ain't diffraction, it must be statistics." Anon.

Michal Opas

TIRF objective for routine imaging

In reply to this post by John Oreopoulos

John is correct: it is the Olympus 100X 1.65NA that requires special coverslips and special immersion liquid. The rest of lenses use standard immersion oil.
Michal

      Dr. Michal Opas
     Professor
     Department of Laboratory Medicine and Pathobiology
     University of Toronto
     1 King's College Circle
     Medical Sciences Building, room 6326
     Toronto, Ontario, M5S 1A8 Canada

°°°°°°°°°°°°°
phone: (416) 978-8947 (laboratory)
         (416) 971-2140 (office)
   fax: (416) 978-5959
e-mail: [hidden email]

WWW: http://www.utoronto.ca/mocell

Tim O'Brien Sr.

Carolina Workshop on Force Microscopy-Free Registration Notice

In reply to this post by Stephen Cody-2

Dear Colleagues,

We have decided to reduce the registration fee for this years workshop to zero for NIH-supported investigators, and I wanted to get the word out. The workshop is a very good introduction to using and analyzing how cells-and materials made by cells- respond to applied forces. It thus would likely be of interest to a wide range of Biologists. Most of the techniques can be used with confocal microscopes, especially spinning disk systems. The announcement is as follows:

The sixth annual Carolina Workshop on Force Measurement and Manipulation in Biological Microscopy, scheduled for May 18-21, 2010, announces its own “stimulus package.” Due to tight budgets everywhere, we are offering the workshop with FREE registration (a $775 value) for researchers with NIH funding. Presented by Computer Integrated Systems for Microscopy and Manipulation (CISMM), an NIH/NIBIB supported resource, we demonstrate the theory and practical side of magnetic tweezers, AFM imaging and manipulation combined with fluorescence light microscopy, and microfluidics. This year’s workshop will also feature an optical tweezers station from JPK Instruments (http://www.jpk.com/nanotracker-tm.387.html), and a keynote lecture from Dennis Discher, U. Penn, entitled "Cell Mechanics with AFM and other small tools." Senior investigators to beginning grad students have found this four day, hands-on introduction to the theory and practice of applying forces to biological specimens to be very useful. Attendees are invited to bring (or send ahead of time) their own cells or biological material of interest to probe during the workshop. We provide supplies, breakfasts, and two dinners, but not lodging or travel. Space is limited! Please contact Cassandra Houston at (919)-962-4057 ([hidden email]) to register or to find out more about the workshop, or go to http://cismm.cs.unc.edu/resources/events/.

Thanks for your time,
Tim O'Brien
UNC Chapel Hill

Tim Feinstein-2

Re: TIRF objective for routine imaging

In reply to this post by James Pawley

<base href="x-msg://415/">Hi James,

Many thanks for your explanation. Apologies for my hastily written message - the NA of the 40x objective is 1.3 and we indeed use the correction collar on our 60x 1.49 to account for refractive indices. Another correction, Huygens is not "blind" per se but uses equipment parameters to predict a PSF.

It is 'reassuring' to know that the index mismatch produces an asymmetrical PSF in the Z axis and that the decon software accounts for it. I wondered what was causing that.

All the best,

Tim

Thank you

On Mar 25, 2010, at 10:55 AM, James Pawley wrote:

Acknowledging in advance that I am hardly a deconvolution expert, our Nikon 60x 1.49 provides a sharper PSF than our 40x plan fluor (hardly an apples-to-apples comparison, I know). We measured the PSF because our blind deconvolution software (Huygens) complained about the NA/medium mismatch. It still appeared to work fairly well.

cheers,

Tim

Timothy Feinstein, PhD
Postdoctoral Associate
University of Pittsburgh Dept. of Pharmacology
Pittsburgh, PA

Hi Tim,

You don't mention the NA of the 40x (or if we are talking widefield or laser confocal, where underfilling might be less of a factor at 60x than at 40x) but I think that your comment about the useful suggestion that the Huygens software made about mismatch might be the nub of the argument.

I am assuming that Hans van der Voort's clever program makes this warning when it detects that the PSF is asymmetrical in z. Such asymmetry implies spherical aberration and this is usually caused by a mismatch between the refractive index (RI) that the lens is designed for and the actual RI of the specimen or using a coverslip of the incorrect thickness.

None of the high NA 40x Fluor lenses I can think of include an SA corection collar, while the Nikon NA 1.49 does do so (did you use it?). So, if your specimen RI is not the same as that of oil or your coverslip is the wrong thickness, you can correct for this on the 1.49 but not on the other 40x lens.

SA correction being equal, and assuming that your 40x is NA 1.3, I would expect that the on-axis x-y resolution of the NA 1.49 would be about 14% better (smaller spacing) than the 40x.

Cheers,

Jim P.

*********************************************************************************
Prof. James B. Pawley,                                     Ph. 608-263-3147
Room 223, Zoology Research Building,                                  FAX 608-265-5315
1117 Johnson Ave., Madison, WI, 53706                                [hidden email]
3D Microscopy of Living Cells Course, June 12-24, 2010, UBC, Vancouver Canada
Info: http://www.3dcourse.ubc.ca/                Applications still being accepted
               "If it ain't diffraction, it must be statistics." Anon.

On Mar 24, 2010, at 7:33 PM, Cameron, Lisa wrote:

> Yes, I concur with Kurt -
>
> Nikon 60x and 100x 1.49 NA TIRF objectives work well with the standard 1.515
> Nikon oil.
>
>
> ---------------------------------------
> Lisa Cameron, Ph.D.
> Director of Confocal and Light Microscopy
> Dana Farber Cancer Institute
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On
> Behalf Of Kurt Thorn
> Sent: Wednesday, March 24, 2010 7:28 PM
> To: [hidden email]
> Subject: Re: [CONFOCALMICROSCOPY] TIRF objective for routine imaging
>
> This isn't true - our Nikon 1.49 TIRF lens works with the standard
> immersion oil.
>
> Barbara Foster wrote:
>> Hi, all
>>
>> Just remember that the 1.49 NA only works with special, high
>> refractive index liquids.
>>
>> *Barbara Foster, President and Sr. Consultant
>>
>> Microscopy/Microscopy Education
>> *7101 Royal Glen Trail, Suite A
>> McKinney TX 75070
>> *P: *(972)924-5310 *Skype: *fostermme
>> *W: *www.MicroscopyEducation.com
>>
>> <http://www.microscopyeducation.com/>*NEWS! Visit the NEW and IMPROVED
>> www.MicroscopyEducation.com <http://www.microscopyeducation.com/>! And
>> don't forget: MME is now scheduling customized, on-site courses for
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>
>
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--
*********************************************************************************
Prof. James B. Pawley,                                     Ph. 608-263-3147
Room 223, Zoology Research Building,                                  FAX 608-265-5315
1117 Johnson Ave., Madison, WI, 53706                                [hidden email]
3D Microscopy of Living Cells Course, June 12-24, 2010, UBC, Vancouver Canada
Info: http://www.3dcourse.ubc.ca/                Applications still being accepted
               "If it ain't diffraction, it must be statistics." Anon.