Comparing objective lens performance

[Eric Shelden]

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All:
I recently had a chance to look at a 40X water immersion 1.1NA Corr lens
on our Leica SP5 using both conventional and multiphoton illumination.
Theoretically, it should have given better resolution and better brightness
than our 20X 0.7 Corr multi-immersion lens. re, with two different
specimens and preparations it provided only slightly better resolution and
prequired higher gain settings to achieve saturation with the same laser
power settings. I was wondering if there was a reasonable explanation, and
if anyone has had experience with the 40X lens in question.
Thanks,
Eric

[John Oreopoulos]

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Eric,

When you did your comparison, did you change the digital sampling rate (number of pixels 512x512, 1024x1024, etc.) or the scan zoom settings between the two objectives, or were the settings all the same for both objective tests?

How are you judging resolution? Are you looking at the FWHM of sub-diffraction sized fluorescent beads in XY and Z?

What are the test specimens? What medium are they embedded in (dry/air, water, oil mounting, etc.). Is there a coverslip present or not?

Answers to these questions might point out some key factors that might explain the result you got.

John Oreopoulos
Staff Scientist
Spectral Applied Research Inc.
A Division of Andor Technology
Richmond Hill, Ontario
Canada
www.spectral.ca


On 2015-03-13, at 1:19 PM, Eric Shelden wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> All:
> I recently had a chance to look at a 40X water immersion 1.1NA Corr lens
> on our Leica SP5 using both conventional and multiphoton illumination.
> Theoretically, it should have given better resolution and better brightness
> than our 20X 0.7 Corr multi-immersion lens. re, with two different
> specimens and preparations it provided only slightly better resolution and
> prequired higher gain settings to achieve saturation with the same laser
> power settings. I was wondering if there was a reasonable explanation, and
> if anyone has had experience with the 40X lens in question.
> Thanks,
> Eric

[Paul Rigby-2]

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Hi Eric,
Unless you have a significant problem with your test objective (someone has dropped it), I would suspect that the differences you observed were more likely due to incorrect specimen preparation.
If you image a single fluorescent bead, is there evidence of any spherical aberration? (Is the shape of the PSF identical above and below the bead focal plane?) If you are using a coverslip, how thick is that coverslip (is it = 0.17mm thick?). Have you adjusted any correction collar on your objectives to achieve optimal imaging? For the WI objective, is your sample embedded in water (or does the mounting medium have a RI = 1.33)? These issues of correct specimen preparation are critical, especially for higher NA objectives.
Hope this helps.
Cheers
Paul

Assoc. Prof. Paul Rigby
Optical Microscopy Specialist
Centre for Microscopy, Characterisation & Analysis (M519)
The University of Western Australia
35 Stirling Highway
Crawley  WA  6007
Australia

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of John Oreopoulos
Sent: Saturday, 14 March 2015 2:01 AM
To: [hidden email]
Subject: Re: Comparing objective lens performance

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Eric,

When you did your comparison, did you change the digital sampling rate (number of pixels 512x512, 1024x1024, etc.) or the scan zoom settings between the two objectives, or were the settings all the same for both objective tests?

How are you judging resolution? Are you looking at the FWHM of sub-diffraction sized fluorescent beads in XY and Z?

What are the test specimens? What medium are they embedded in (dry/air, water, oil mounting, etc.). Is there a coverslip present or not?

Answers to these questions might point out some key factors that might explain the result you got.

John Oreopoulos
Staff Scientist
Spectral Applied Research Inc.
A Division of Andor Technology
Richmond Hill, Ontario
Canada
www.spectral.ca


On 2015-03-13, at 1:19 PM, Eric Shelden wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> All:
> I recently had a chance to look at a 40X water immersion 1.1NA Corr
> lens on our Leica SP5 using both conventional and multiphoton illumination.
> Theoretically, it should have given better resolution and better
> brightness than our 20X 0.7 Corr multi-immersion lens. re, with two
> different specimens and preparations it provided only slightly better
> resolution and prequired higher gain settings to achieve saturation
> with the same laser power settings. I was wondering if there was a
> reasonable explanation, and if anyone has had experience with the 40X lens in question.
> Thanks,
> Eric

[Zdenek Svindrych-2]

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Hi Eric,
bear in mind, that the same power settings do not mean the same excitation
powers. The two objectives have very different diameters of the apertures at
the back focal plane...

Best, zdenek


---------- Původní zpráva ----------
Od: Eric Shelden <[hidden email]>
Komu: [hidden email]
Datum: 13. 3. 2015 13:21:55
Předmět: Comparing objective lens performance

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All:
I recently had a chance to look at a 40X water immersion 1.1NA Corr lens
on our Leica SP5 using both conventional and multiphoton illumination.
Theoretically, it should have given better resolution and better brightness
than our 20X 0.7 Corr multi-immersion lens. re, with two different
specimens and preparations it provided only slightly better resolution and
prequired higher gain settings to achieve saturation with the same laser
power settings. I was wondering if there was a reasonable explanation, and
if anyone has had experience with the 40X lens in question.
Thanks,
Eric"

[jerie]

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Am 13.03.2015 14:21 schrieb "Eric Shelden" <[hidden email]>:
>40X water immersion 1.1NA Corr lens
> should have given better resolution and better brightness
> than our 20X 0.7 Corr multi-immersion lens.
> it provided only slightly better resolution and
> prequired higher gain settings to achieve saturation with the same laser
> power settings. I was wondering if there was a reasonable explanation

Eric,

you can measure the angle of light emitted from small but bright objects in
your specimen or of beads. xz scan through a couple of beads or objects.
The sinus of the full angle multiplied with the refractive index of your
media gives the NA of the detection system. To make sure to achieve full
resolution you also need to make sure to overfill the back aperture of the
objective. There is a beam expander that should be set to the largest
number.
Make sure to also adapt the magnification to integrate photons from the
same area. Without that you would expect a lower intensity for the 40x,
because you integrate from a smaller area.

Hope it helps,  Jens

Visiting Scientist @ Center for Technological Development in Health (CDTS),
Oswaldo Cruz Foundation (Fiocruz), Ministry of Health, Rio de Janeiro,
Brazil