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Oil vs water objectives

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Gabriel Lapointe-4 Gabriel Lapointe-4
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Oil vs water objectives

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Hi,

I have a user who insist that using a 1,27NA water immersion objective is
brighter and would give better images than using a 1,4NA oil immersion. I
understand that deeper into the media that would be true. But, in that
particular case, we are talking about imaging GFP at less than 100 micron
away with a spinning disk.

So, I was wondering at which distance from the coverslips do we start
seeing benefits of using a water immersion objective over an oil objective
in aqueous media.

Thanks for your help.

Sincerely
*Gabriel Lapointe, M.Sc.*
Lab Manager / Microscopy Specialist
Concordia University, Biology Department
7141 Sherbrooke St. West SP 534
Montréal QC H4B 1R6 Canada
[hidden email]
cmac.concordia.ca
http://gabriellapointe.ca
Cammer, Michael Cammer, Michael
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Re: Oil vs water objectives


This is the type of question that can result in a big theoretical discussion, but in my experience dealing with a wide variety of (mostly) biological material prepared in many different ways, trying each optic for an empirical comparison is the way to go.  

________________________________________________________
Michael Cammer, Assistant Research Scientist Skirball Institute of Biomolecular Medicine
Lab: (212) 263-3208  Cell: (914) 309-3270


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Gabriel Lapointe
Sent: Thursday, October 04, 2012 9:15 AM
Subject: Oil vs water objectives

Hi,

I have a user who insist that using a 1,27NA water immersion objective is brighter and would give better images than using a 1,4NA oil immersion. I understand that deeper into the media that would be true. But, in that particular case, we are talking about imaging GFP at less than 100 micron away with a spinning disk.

So, I was wondering at which distance from the coverslips do we start seeing benefits of using a water immersion objective over an oil objective in aqueous media.

Thanks for your help.

Sincerely
*Gabriel Lapointe, M.Sc.*
Lab Manager / Microscopy Specialist
Concordia University, Biology Department
7141 Sherbrooke St. West SP 534
Montréal QC H4B 1R6 Canada
[hidden email]
cmac.concordia.ca
http://gabriellapointe.ca


Steffen Dietzel Steffen Dietzel
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Re: Oil vs water objectives

In reply to this post by Gabriel Lapointe-4
*****
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*****

I second Michael's comment.

Apart from that, when you have an aqueous(!) preparation, spherical
aberration decreases image quality faster for the oil objective. So,
directly behind the coverslip the oil objective (1.4) will give you the
better image due to the higher NA, at some depth both objectives will
give you similar quality and beyond that, the water immersion will be
better.

I seem to remember that for oil 1.4 and water 1.2 the crossing point is
10-20 µm into the sample.

If you have the Handbook laying around, check the chapter on Lens
aberrations. Chapter 20 by Hell and Stelzer in the second edition,
chapter 20 by Egner and Hell in the 3rd edition.

Needles to say that all above considerations assume that both objectives
have the same transparency for excitation and emission wavelengths -
which they probably don't. So we are back at Michael's comment: You've
got to test it. Beads attached to the coverslip and the slide (various
preparations with various distances between the two) give good test
objects.

Steffen

On 04.10.2012 15:15, Gabriel Lapointe wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi,
>
> I have a user who insist that using a 1,27NA water immersion objective is
> brighter and would give better images than using a 1,4NA oil immersion. I
> understand that deeper into the media that would be true. But, in that
> particular case, we are talking about imaging GFP at less than 100 micron
> away with a spinning disk.
>
> So, I was wondering at which distance from the coverslips do we start
> seeing benefits of using a water immersion objective over an oil objective
> in aqueous media.
>
> Thanks for your help.
>
> Sincerely
> *Gabriel Lapointe, M.Sc.*
> Lab Manager / Microscopy Specialist
> Concordia University, Biology Department
> 7141 Sherbrooke St. West SP 534
> Montréal QC H4B 1R6 Canada
> [hidden email]
> cmac.concordia.ca
> http://gabriellapointe.ca
>


--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
Head of light microscopy

Mail room:
Marchioninistr. 15, D-81377 München

Building location:
Marchioninistr. 27,  München-Großhadern
Mark Cannell-2 Mark Cannell-2
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Re: Oil vs water objectives

In reply to this post by Cammer, Michael
*****
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*****

Do they have the same magnification?

Mark
On 4/10/2012, at 2:59 PM, "Cammer, Michael" <[hidden email]> wrote:

>
> This is the type of question that can result in a big theoretical discussion, but in my experience dealing with a wide variety of (mostly) biological material prepared in many different ways, trying each optic for an empirical comparison is the way to go.  
>
> ________________________________________________________
> Michael Cammer, Assistant Research Scientist Skirball Institute of Biomolecular Medicine
> Lab: (212) 263-3208  Cell: (914) 309-3270
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Gabriel Lapointe
> Sent: Thursday, October 04, 2012 9:15 AM
> Subject: Oil vs water objectives
>
> Hi,
>
> I have a user who insist that using a 1,27NA water immersion objective is brighter and would give better images than using a 1,4NA oil immersion. I understand that deeper into the media that would be true. But, in that particular case, we are talking about imaging GFP at less than 100 micron away with a spinning disk.
>
> So, I was wondering at which distance from the coverslips do we start seeing benefits of using a water immersion objective over an oil objective in aqueous media.
>
> Thanks for your help.
>
> Sincerely
> *Gabriel Lapointe, M.Sc.*
> Lab Manager / Microscopy Specialist
> Concordia University, Biology Department
> 7141 Sherbrooke St. West SP 534
> Montréal QC H4B 1R6 Canada
> [hidden email]
> cmac.concordia.ca
> http://gabriellapointe.ca
>
>

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

[hidden email]
Cammer, Michael Cammer, Michael
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Re: Oil vs water objectives

In reply to this post by Steffen Dietzel
Also, while people have generated various test samples of collagen and latex and acrylic and fat globules and glass beads and carrageen gum and dissolved Scotch tape glue etc., the only test sample that really answers the question is the sample itself.  
________________________________________________________
Michael Cammer, Assistant Research Scientist
Skirball Institute of Biomolecular Medicine
Lab: (212) 263-3208  Cell: (914) 309-3270


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Steffen Dietzel
Sent: Thursday, October 04, 2012 10:30 AM
To: [hidden email]
Subject: Re: Oil vs water objectives

So we are back at Michael's comment: You've got to test it. Beads attached to the coverslip and the slide (various preparations with various distances between the two) give good test objects.

Steffen


Scot C Kuo Scot C Kuo
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Re: Oil vs water objectives

In reply to this post by Gabriel Lapointe-4
*****
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*****

I've measured it quantitatively and the performance difference flips surprisingly close to the coverslip (see supplemental info, Fisher & Kuo 2009 PNAS 106, 133-138).  For an Olympus 60x U-PlanApoS lenses, comparing 1.2 and 1.4 NA, the flip happens ~8 microns into an aqueous sample.  For fluorescence closer than ~8um, oil is brighter, whereas for objects further, water immersion is brighter.  If lenses aren't matched, then the cross-over can happen elsewhere, but the relative shapes of the curves are the same.  Oil lenses (1.4NA) will have half the brightness by ~50um.

For the information you've provided (higher NA on water lens), I'd expect the cross-over to be closer to the coverslip surface.

-- Scot

============================================================================
...............Scot C. Kuo (410) 955-4536; email:[hidden email]...............
...Director, Microscope Facility, JHU-SOM, www.hopkinsmedicine.org/micfac...
..Assoc Professor, Biomedical Engineering & Cell Biology, www.jhu.edu/cmml..


----- Original Message -----
From: Gabriel Lapointe <[hidden email]>
Date: Thursday, October 4, 2012 9:16 am
Subject: [CONFOCALMICROSCOPY] Oil vs water objectives
To: [hidden email]


> *****
>  To join, leave or search the confocal microscopy listserv, go to:
>  
>  *****
>  
>  Hi,
>  
>  I have a user who insist that using a 1,27NA water immersion
> objective is
>  brighter and would give better images than using a 1,4NA oil
> immersion. I
>  understand that deeper into the media that would be true. But, in that
>  particular case, we are talking about imaging GFP at less than 100 micron
>  away with a spinning disk.
>  
>  So, I was wondering at which distance from the coverslips do we start
>  seeing benefits of using a water immersion objective over an oil objective
>  in aqueous media.
>  
>  Thanks for your help.
>  
>  Sincerely
>  *Gabriel Lapointe, M.Sc.*
>  Lab Manager / Microscopy Specialist
>  Concordia University, Biology Department
>  7141 Sherbrooke St. West SP 534
>  Montréal QC H4B 1R6 Canada
>  [hidden email]
>  cmac.concordia.ca
>
Cromey, Douglas W - (dcromey) Cromey, Douglas W - (dcromey)
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Re: Oil vs water objectives

In reply to this post by Steffen Dietzel
Make sure the coverslip is orthogonal to the image axis with your water objective.

A common aberration with water-immersion objective lenses.
J Microsc. 2004 Oct;216(Pt 1):49-51.
http://www.ncbi.nlm.nih.gov/pubmed/15369482

Doug

^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
Douglas W. Cromey, M.S. - Associate Scientific Investigator
Dept. of Cellular & Molecular Medicine, University of Arizona
1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA

office:  AHSC 4212         email: [hidden email]
voice:  520-626-2824       fax:  520-626-2097

http://swehsc.pharmacy.arizona.edu/exppath/
Home of: "Microscopy and Imaging Resources on the WWW"


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Steffen Dietzel
Sent: Thursday, October 04, 2012 7:30 AM
To: [hidden email]
Subject: Re: Oil vs water objectives

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

I second Michael's comment.

Apart from that, when you have an aqueous(!) preparation, spherical aberration decreases image quality faster for the oil objective. So, directly behind the coverslip the oil objective (1.4) will give you the better image due to the higher NA, at some depth both objectives will give you similar quality and beyond that, the water immersion will be better.

I seem to remember that for oil 1.4 and water 1.2 the crossing point is
10-20 µm into the sample.

If you have the Handbook laying around, check the chapter on Lens aberrations. Chapter 20 by Hell and Stelzer in the second edition, chapter 20 by Egner and Hell in the 3rd edition.

Needles to say that all above considerations assume that both objectives have the same transparency for excitation and emission wavelengths - which they probably don't. So we are back at Michael's comment: You've got to test it. Beads attached to the coverslip and the slide (various preparations with various distances between the two) give good test objects.

Steffen

On 04.10.2012 15:15, Gabriel Lapointe wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi,
>
> I have a user who insist that using a 1,27NA water immersion objective
> is brighter and would give better images than using a 1,4NA oil
> immersion. I understand that deeper into the media that would be true.
> But, in that particular case, we are talking about imaging GFP at less
> than 100 micron away with a spinning disk.
>
> So, I was wondering at which distance from the coverslips do we start
> seeing benefits of using a water immersion objective over an oil
> objective in aqueous media.
>
> Thanks for your help.
>
> Sincerely
> *Gabriel Lapointe, M.Sc.*
> Lab Manager / Microscopy Specialist
> Concordia University, Biology Department
> 7141 Sherbrooke St. West SP 534
> Montréal QC H4B 1R6 Canada
> [hidden email]
> cmac.concordia.ca
> http://gabriellapointe.ca
>


--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex) Head of light microscopy

Mail room:
Marchioninistr. 15, D-81377 München

Building location:
Marchioninistr. 27,  München-Großhadern
James Pawley James Pawley
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Re: Oil vs water objectives

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

Hi all,

Looks like you have received a lot of good
advice, but I would like to add one thing.

Yes, do a test BUT don't forget to be very
careful in adjusting the SA correction collar on
the water lens. )Find a point object: focus up
and down and adjust for symmetry (i.e., for the
same image a little above focus as a little below
focus. DON'T just try to "make it sharper!" The
collar changes the focus plane.)

The collars usually have markings in terms of the
coverslip thickness that they are designed to
correct for. One can easily see the difference
created by mis-adjusting a 1.2 NA objective by 2
µm-of-glass-replaced-by-water (my favorite unit
for "measuring" SA).

When modern water objectives first emerged in the
early 1990s, many folk bought them and then went
back to their oil objectives and I am convinced
that it was because they just didn't want to take
the time to adjust the collar properly.

Because, if you do adjust it properly, you will
get better (brighter? higher resolution?) images
of aqeous specimens beyond about 3 µm

Best,

Jim Pawley,

Near Harbin, China.



>Make sure the coverslip is orthogonal to the
>image axis with your water objective.
>
>A common aberration with water-immersion objective lenses.
>J Microsc. 2004 Oct;216(Pt 1):49-51.
>http://www.ncbi.nlm.nih.gov/pubmed/15369482
>
>Doug
>
>^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
>Douglas W. Cromey, M.S. - Associate Scientific Investigator
>Dept. of Cellular & Molecular Medicine, University of Arizona
>1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA
>
>office:  AHSC 4212         email: [hidden email]
>voice:  520-626-2824       fax:  520-626-2097
>
>http://swehsc.pharmacy.arizona.edu/exppath/
>Home of: "Microscopy and Imaging Resources on the WWW"
>
>
>-----Original Message-----
>From: Confocal Microscopy List
>[mailto:[hidden email]] On
>Behalf Of Steffen Dietzel
>Sent: Thursday, October 04, 2012 7:30 AM
>To: [hidden email]
>Subject: Re: Oil vs water objectives
>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>I second Michael's comment.
>
>Apart from that, when you have an aqueous(!)
>preparation, spherical aberration decreases
>image quality faster for the oil objective. So,
>directly behind the coverslip the oil objective
>(1.4) will give you the better image due to the
>higher NA, at some depth both objectives will
>give you similar quality and beyond that, the
>water immersion will be better.
>
>I seem to remember that for oil 1.4 and water 1.2 the crossing point is
>10-20 µm into the sample.
>
>If you have the Handbook laying around, check
>the chapter on Lens aberrations. Chapter 20 by
>Hell and Stelzer in the second edition, chapter
>20 by Egner and Hell in the 3rd edition.
>
>Needles to say that all above considerations
>assume that both objectives have the same
>transparency for excitation and emission
>wavelengths - which they probably don't. So we
>are back at Michael's comment: You've got to
>test it. Beads attached to the coverslip and the
>slide (various preparations with various
>distances between the two) give good test
>objects.
>
>Steffen
>
>On 04.10.2012 15:15, Gabriel Lapointe wrote:
>>  *****
>>  To join, leave or search the confocal microscopy listserv, go to:
>>  http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>  *****
>>
>>  Hi,
>>
>>  I have a user who insist that using a 1,27NA water immersion objective
>>  is brighter and would give better images than using a 1,4NA oil
>>  immersion. I understand that deeper into the media that would be true.
>>  But, in that particular case, we are talking about imaging GFP at less
>>  than 100 micron away with a spinning disk.
>>
>>  So, I was wondering at which distance from the coverslips do we start
>>  seeing benefits of using a water immersion objective over an oil
>>  objective in aqueous media.
>>
>>  Thanks for your help.
>>
>>  Sincerely
>>  *Gabriel Lapointe, M.Sc.*
>  > Lab Manager / Microscopy Specialist
>>  Concordia University, Biology Department
>>  7141 Sherbrooke St. West SP 534
>>  Montréal QC H4B 1R6 Canada
>>  [hidden email]
>>  cmac.concordia.ca
>>  http://gabriellapointe.ca
>>
>
>
>--
>------------------------------------------------------------
>Steffen Dietzel, PD Dr. rer. nat
>Ludwig-Maximilians-Universität München
>Walter-Brendel-Zentrum für experimentelle
>Medizin (WBex) Head of light microscopy
>
>Mail room:
>Marchioninistr. 15, D-81377 München
>
>Building location:
>Marchioninistr. 27,  München-Großhadern


--
James and Christine Pawley, PO Box 2348, 5446
Burley Place (PO Box 2348), Sechelt, BC, Canada,
V0N3A0, 604-885-0840 NEW! Cell (when I remember
to turn it on!) 1-765-637-1917,
<[hidden email]>
Guy Cox-2 Guy Cox-2
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Re: Oil vs water objectives

In reply to this post by Scot C Kuo
*****
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*****

Nobody seems to have mentioned so far that the NA of an oil objective will NOT be 1.4 if it is imaging a sample in water.  The maximum it can be is 1.33 - the refractive index of water.  Anything over this will be beyond the critical angle and rays will not reach the specimen (in excitation) or the objective (in emission).  So the oil objective has little or no advantage in NA and as Scot pointed out, the spherical aberration becomes horrendous very rapidly.  So Gabriel's user is quite right.  Actually I'd be surprised if you could see anything 100µm (0.1mm) into water with the oil lens.

So why do some people say they do better with an oil lens when imaging very close to the coverslip?  The suggestion has been made in this list that they are seeing evanescent wave enhancement of fluorescence, and it seems highly believable to me.  Those rays between NA 1.33 and 1.4 cannot reach the sample in the far field, but their evanescent wave can give a TIRF image, and we see this superimposed on the regular far-field image.    

There is one further caveat, which Mark hinted at.  If this is a Yokogawa spinning disk system it is designed for a 100x objective, and if used with 60x both pinhole size and pupil filling will not be optimal.  But 100x water immersion objectives are rare beasts.  (Apparently there are design constraints which prevent a Yokogawa head being optimised for a 60x objective).

                                                                    Guy

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Scot C Kuo
Sent: Friday, 5 October 2012 2:35 AM
To: [hidden email]
Subject: Re: Oil vs water objectives

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

I've measured it quantitatively and the performance difference flips surprisingly close to the coverslip (see supplemental info, Fisher & Kuo 2009 PNAS 106, 133-138).  For an Olympus 60x U-PlanApoS lenses, comparing 1.2 and 1.4 NA, the flip happens ~8 microns into an aqueous sample.  For fluorescence closer than ~8um, oil is brighter, whereas for objects further, water immersion is brighter.  If lenses aren't matched, then the cross-over can happen elsewhere, but the relative shapes of the curves are the same.  Oil lenses (1.4NA) will have half the brightness by ~50um.

For the information you've provided (higher NA on water lens), I'd expect the cross-over to be closer to the coverslip surface.

-- Scot

============================================================================
...............Scot C. Kuo (410) 955-4536; email:[hidden email]...............
...Director, Microscope Facility, JHU-SOM, www.hopkinsmedicine.org/micfac...
..Assoc Professor, Biomedical Engineering & Cell Biology, www.jhu.edu/cmml..


----- Original Message -----
From: Gabriel Lapointe <[hidden email]>
Date: Thursday, October 4, 2012 9:16 am
Subject: [CONFOCALMICROSCOPY] Oil vs water objectives
To: [hidden email]


> *****
>  To join, leave or search the confocal microscopy listserv, go to:
>  
>  *****
>  
>  Hi,
>  
>  I have a user who insist that using a 1,27NA water immersion
> objective is
>  brighter and would give better images than using a 1,4NA oil
> immersion. I
>  understand that deeper into the media that would be true. But, in that
>  particular case, we are talking about imaging GFP at less than 100 micron
>  away with a spinning disk.
>  
>  So, I was wondering at which distance from the coverslips do we start
>  seeing benefits of using a water immersion objective over an oil objective
>  in aqueous media.
>  
>  Thanks for your help.
>  
>  Sincerely
>  *Gabriel Lapointe, M.Sc.*
>  Lab Manager / Microscopy Specialist
>  Concordia University, Biology Department
>  7141 Sherbrooke St. West SP 534
>  Montréal QC H4B 1R6 Canada
>  [hidden email]
>  cmac.concordia.ca
>  
John Oreopoulos John Oreopoulos
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Re: Oil vs water objectives

*****
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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Just to add to Guy's last point, I recently read an interesting paper by Thomas Burghardt which thoroughly investigated the effect he just described (about evanescent wave enhancement near the coverslip):

Burghardt, T.P., Evanescent field shapes excitation profile under axial epi-illumination. Journal of Biomedical Optics, 2012. 17(6).

Here is a section in the discussion that I think fits your description, Guy:

"The highest NA objectives available are TIRF objectives for through-the-objective total internal reflection because they achieve excitation incidence angles beyond critical angle for the glass/ aqueous interface. Generally, TIRF or epi-illumination excitations pertain to evanescent or propagating field microscopies that are appropriate for different applications. I show here that the TIRF objective under common axial epi-illumination conditions produces an evanescent field that favorably remodels the excitation volume for samples near the coverslip.

Point source fluorescent spheres were imaged from a region where the excitation evanescent field contributes to excitation and from a region where the evanescent field is necessarily absent. To do so, I constructed a microfluidic PDMS spacer that separates two glass coverslips (Fig. 1). The lower coverslip optically contacts the oil immersion objective whereas the upper coverslip has an intervening 20um-thick slab of water. The 100um objective working distance ensures that either object can be brought into focus by vertical movement of the objective. Objects at the lower coverslip are subjected to both evanescent and propagating exciting fields whereas objects at the upper coverslip feel only the propagating field. Figure 7 shows a one-beam intensity profile measured by axial translation of the objective over 1500 nm, indicating the narrowing effect of the evanescent field. Profile computation agrees with observation. Figure 5 indicates the expected half-width remodeling of the axial dependence for exciting light as a function of probe position relative to the lower coverslip interface. I also observed a 2- to 4-fold intensity enhancement for the fluorescent sphere at the lower coverslip that is attributable to the discontinuous enhancement of the exciting normal electric field on the aqueous side at the lower coverslip, the selective collection of near-field emission from a sphere at the lower coverslip, and the effect of light scattering in the intervening water layer on both exciting and emission light for the sphere at the upper coverslip. Other effects may be significant, including the presence of the aqueous/glass interface at the upper coverslip...

... evanescent excitation contributes to observed fluorescence whenever a TIRF objective is used and suggests that the sample material nearest the coverslip disproportionally contributes to the observed fluorescence signal."

To this point I would add that even a 1.4 NA oil immersion objective is technically a TIRF objective since even this NA subtends (just barely) an angle greater than the glass-water interface critical angle.

Cheers,

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


On 2012-10-04, at 8:40 PM, Guy Cox wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Nobody seems to have mentioned so far that the NA of an oil objective will NOT be 1.4 if it is imaging a sample in water.  The maximum it can be is 1.33 - the refractive index of water.  Anything over this will be beyond the critical angle and rays will not reach the specimen (in excitation) or the objective (in emission).  So the oil objective has little or no advantage in NA and as Scot pointed out, the spherical aberration becomes horrendous very rapidly.  So Gabriel's user is quite right.  Actually I'd be surprised if you could see anything 100µm (0.1mm) into water with the oil lens.
>
> So why do some people say they do better with an oil lens when imaging very close to the coverslip?  The suggestion has been made in this list that they are seeing evanescent wave enhancement of fluorescence, and it seems highly believable to me.  Those rays between NA 1.33 and 1.4 cannot reach the sample in the far field, but their evanescent wave can give a TIRF image, and we see this superimposed on the regular far-field image.    
>
> There is one further caveat, which Mark hinted at.  If this is a Yokogawa spinning disk system it is designed for a 100x objective, and if used with 60x both pinhole size and pupil filling will not be optimal.  But 100x water immersion objectives are rare beasts.  (Apparently there are design constraints which prevent a Yokogawa head being optimised for a 60x objective).
>
>                                                                    Guy
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Scot C Kuo
> Sent: Friday, 5 October 2012 2:35 AM
> To: [hidden email]
> Subject: Re: Oil vs water objectives
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I've measured it quantitatively and the performance difference flips surprisingly close to the coverslip (see supplemental info, Fisher & Kuo 2009 PNAS 106, 133-138).  For an Olympus 60x U-PlanApoS lenses, comparing 1.2 and 1.4 NA, the flip happens ~8 microns into an aqueous sample.  For fluorescence closer than ~8um, oil is brighter, whereas for objects further, water immersion is brighter.  If lenses aren't matched, then the cross-over can happen elsewhere, but the relative shapes of the curves are the same.  Oil lenses (1.4NA) will have half the brightness by ~50um.
>
> For the information you've provided (higher NA on water lens), I'd expect the cross-over to be closer to the coverslip surface.
>
> -- Scot
>
> ============================================================================
> ...............Scot C. Kuo (410) 955-4536; email:[hidden email]...............
> ...Director, Microscope Facility, JHU-SOM, www.hopkinsmedicine.org/micfac...
> ..Assoc Professor, Biomedical Engineering & Cell Biology, www.jhu.edu/cmml..
>
>
> ----- Original Message -----
> From: Gabriel Lapointe <[hidden email]>
> Date: Thursday, October 4, 2012 9:16 am
> Subject: [CONFOCALMICROSCOPY] Oil vs water objectives
> To: [hidden email]
>
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>>
>> *****
>>
>> Hi,
>>
>> I have a user who insist that using a 1,27NA water immersion
>> objective is
>> brighter and would give better images than using a 1,4NA oil
>> immersion. I
>> understand that deeper into the media that would be true. But, in that
>> particular case, we are talking about imaging GFP at less than 100 micron
>> away with a spinning disk.
>>
>> So, I was wondering at which distance from the coverslips do we start
>> seeing benefits of using a water immersion objective over an oil objective
>> in aqueous media.
>>
>> Thanks for your help.
>>
>> Sincerely
>> *Gabriel Lapointe, M.Sc.*
>> Lab Manager / Microscopy Specialist
>> Concordia University, Biology Department
>> 7141 Sherbrooke St. West SP 534
>> Montréal QC H4B 1R6 Canada
>> [hidden email]
>> cmac.concordia.ca
>>
Martin Offterdinger Martin Offterdinger
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Re: Oil vs water objectives

In reply to this post by Gabriel Lapointe-4
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Hi Gabriel
An excellent discussion about oil vs water can be found here (no affiliation)
http://www.microscopyu.com/articles/optics/waterimmersionobjectives.html

It is not only about brightness, more important is the loss in resolution imho.
Moreover in the 'oil case' you can also get color shifts between the
channels (..different wavelengths will result in different refraction at the
interface...).
All these problems do not only arise at sample thicknesses in the range of
50 to 100 microns but much earlier. 5-10 microns may give you a noticeable
effect already.

SO for me water lenses are the lenses of choice when it comes to
high-resolution live cell imaging (except for TIRF of course)


best

Martin
***********************
Martin Offterdinger, PhD
Medical University Innsbruck
CCB
Division of Neurobiochemistry
Biooptics
Innrain 80-82, 1st floor, room 01.370
A-6020 Innsbruck
Austria
***********************
James Pawley James Pawley
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Re: Oil vs water objectives

In reply to this post by John Oreopoulos
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Very good points!

I am sure that Guy and John (and T.P. Burkhardt!)
are describing a very important effect.

Perhaps it is also worth pointing out that this
effect will only occur if the epi-illumination
system really fills the full NA of the specific
objective in use. (i.e., On a given set-up, it
might work with 100x NA 1.4 but not so well with
a 40x 1.4, where the diameter of the entrance
pupil is 2.5x larger).

Also, deconvolution systems assume that the total
excitation illumination  power does not vary with
z (although its power density in mw/µm*2 will, of
course, vary with the distance from the focus
plane). Therefore, what we might call
TIRF-enhancement will tend to  exaggerate
quantitative results from features near the glass
surface. It will also add more out-of-focus light
to every plane than is justified by the
fluorescent markers present in the specimen and
it very seems likely to distort the z-axis of any
the PSF standard obtained from a sub-resolution
bead located at the glass surface.

I agree that a straight PSF obtained in this way
would not, in any case, have the proper SA
correction for planes inside the specimen,
however some workers have reduced this problem by
using an NA 1.4 objective with an immersion oil
having an RI that is higher than that specified.
This can work pretty well for a specific, narrow
range of distances into the cell (exactly where
these planes are depends on RI of the oil and the
working distance of the objective, which defines
how thick the layer of the "wrong" oil will be).
However, it only works well if one determines the
PSF using small objects located within this focus
range.

Cheers,

Jim Pawley

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Just to add to Guy's last point, I recently read
>an interesting paper by Thomas Burghardt which
>thoroughly investigated the effect he just
>described (about evanescent wave enhancement
>near the coverslip):
>
>Burghardt, T.P., Evanescent field shapes
>excitation profile under axial epi-illumination.
>Journal of Biomedical Optics, 2012. 17(6).
>
>Here is a section in the discussion that I think fits your description, Guy:
>
>"The highest NA objectives available are TIRF
>objectives for through-the-objective total
>internal reflection because they achieve
>excitation incidence angles beyond critical
>angle for the glass/ aqueous interface.
>Generally, TIRF or epi-illumination excitations
>pertain to evanescent or propagating field
>microscopies that are appropriate for different
>applications. I show here that the TIRF
>objective under common axial epi-illumination
>conditions produces an evanescent field that
>favorably remodels the excitation volume for
>samples near the coverslip.
>
>Point source fluorescent spheres were imaged
>from a region where the excitation evanescent
>field contributes to excitation and from a
>region where the evanescent field is necessarily
>absent. To do so, I constructed a microfluidic
>PDMS spacer that separates two glass coverslips
>(Fig. 1). The lower coverslip optically contacts
>the oil immersion objective whereas the upper
>coverslip has an intervening 20um-thick slab of
>water. The 100um objective working distance
>ensures that either object can be brought into
>focus by vertical movement of the objective.
>Objects at the lower coverslip are subjected to
>both evanescent and propagating exciting fields
>whereas objects at the upper coverslip feel only
>the propagating field. Figure 7 shows a one-beam
>intensity profile measured by axial translation
>of the objective over 1500 nm, indicating the
>narrowing effect of the evanescent field.
>Profile computation agrees with observation.
>Figure 5 indicates the expected half-width
>remodeling of the axial dependence for exciting
>light as a function of probe position relative
>to the lower coverslip interface. I also
>observed a 2- to 4-fold intensity enhancement
>for the fluorescent sphere at the lower
>coverslip that is attributable to the
>discontinuous enhancement of the exciting normal
>electric field on the aqueous side at the lower
>coverslip, the selective collection of
>near-field emission from a sphere at the lower
>coverslip, and the effect of light scattering in
>the intervening water layer on both exciting and
>emission light for the sphere at the upper
>coverslip. Other effects may be significant,
>including the presence of the aqueous/glass
>interface at the upper coverslip...
>
>... evanescent excitation contributes to
>observed fluorescence whenever a TIRF objective
>is used and suggests that the sample material
>nearest the coverslip disproportionally
>contributes to the observed fluorescence signal."
>
>To this point I would add that even a 1.4 NA oil
>immersion objective is technically a TIRF
>objective since even this NA subtends (just
>barely) an angle greater than the glass-water
>interface critical angle.
>
>Cheers,
>
>John Oreopoulos
>Research Assistant
>Spectral Applied Research
>Richmond Hill, Ontario
>Canada
>www.spectral.ca
>
>
>On 2012-10-04, at 8:40 PM, Guy Cox wrote:
>
>>  *****
>>  To join, leave or search the confocal microscopy listserv, go to:
>>  http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>  *****
>>
>>  Nobody seems to have mentioned so far that the
>>NA of an oil objective will NOT be 1.4 if it is
>>imaging a sample in water.  The maximum it can
>>be is 1.33 - the refractive index of water.
>>Anything over this will be beyond the critical
>>angle and rays will not reach the specimen (in
>>excitation) or the objective (in emission).  So
>>the oil objective has little or no advantage in
>>NA and as Scot pointed out, the spherical
>>aberration becomes horrendous very rapidly.  So
>>Gabriel's user is quite right.  Actually I'd be
>>surprised if you could see anything 100µm
>>(0.1mm) into water with the oil lens.
>>
>>  So why do some people say they do better with
>>an oil lens when imaging very close to the
>>coverslip?  The suggestion has been made in
>>this list that they are seeing evanescent wave
>>enhancement of fluorescence, and it seems
>>highly believable to me.  Those rays between NA
>>1.33 and 1.4 cannot reach the sample in the far
>>field, but their evanescent wave can give a
>>TIRF image, and we see this superimposed on the
>>regular far-field image.
>>
>>  There is one further caveat, which Mark hinted
>>at.  If this is a Yokogawa spinning disk system
>>it is designed for a 100x objective, and if
>>used with 60x both pinhole size and pupil
>>filling will not be optimal.  But 100x water
>>immersion objectives are rare beasts.
>>(Apparently there are design constraints which
>>prevent a Yokogawa head being optimised for a
>>60x objective).
>>
>>                                                                     Guy
>>
>>  -----Original Message-----
>>  From: Confocal Microscopy List
>>[mailto:[hidden email]] On
>>Behalf Of Scot C Kuo
>>  Sent: Friday, 5 October 2012 2:35 AM
>>  To: [hidden email]
>>  Subject: Re: Oil vs water objectives
>>
>>  *****
>>  To join, leave or search the confocal microscopy listserv, go to:
>>  http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>  *****
>>
>>  I've measured it quantitatively and the
>>performance difference flips surprisingly close
>>to the coverslip (see supplemental info, Fisher
>>& Kuo 2009 PNAS 106, 133-138).  For an Olympus
>>60x U-PlanApoS lenses, comparing 1.2 and 1.4
>>NA, the flip happens ~8 microns into an aqueous
>>sample.  For fluorescence closer than ~8um, oil
>>is brighter, whereas for objects further, water
>>immersion is brighter.  If lenses aren't
>>matched, then the cross-over can happen
>>elsewhere, but the relative shapes of the
>>curves are the same.  Oil lenses (1.4NA) will
>>have half the brightness by ~50um.
>>
>>  For the information you've provided (higher NA
>>on water lens), I'd expect the cross-over to be
>>closer to the coverslip surface.
>>
>>  -- Scot
>>
>>  ============================================================================
>>  ...............Scot C. Kuo (410) 955-4536; email:[hidden email]...............
>>  ...Director, Microscope Facility, JHU-SOM, www.hopkinsmedicine.org/micfac...
>>  ..Assoc Professor, Biomedical Engineering & Cell Biology, www.jhu.edu/cmml..
>>
>>
>>  ----- Original Message -----
>>  From: Gabriel Lapointe <[hidden email]>
>>  Date: Thursday, October 4, 2012 9:16 am
>>  Subject: [CONFOCALMICROSCOPY] Oil vs water objectives
>>  To: [hidden email]
>>
>>
>>>  *****
>>>  To join, leave or search the confocal microscopy listserv, go to:
>>>
>>>  *****
>>>
>>>  Hi,
>>>
>>>  I have a user who insist that using a 1,27NA water immersion
>>>  objective is
>>>  brighter and would give better images than using a 1,4NA oil
>  >> immersion. I
>>>  understand that deeper into the media that would be true. But, in that
>>>  particular case, we are talking about imaging GFP at less than 100 micron
>  >> away with a spinning disk.
>>>
>>>  So, I was wondering at which distance from the coverslips do we start
>>>  seeing benefits of using a water immersion objective over an oil objective
>>>  in aqueous media.
>>>
>>>  Thanks for your help.
>>>
>>>  Sincerely
>>>  *Gabriel Lapointe, M.Sc.*
>>>  Lab Manager / Microscopy Specialist
>>>  Concordia University, Biology Department
>>>  7141 Sherbrooke St. West SP 534
>>>  Montréal QC H4B 1R6 Canada
>>>  [hidden email]
>>>  cmac.concordia.ca
>>>


--
James and Christine Pawley, PO Box 2348, 5446
Burley Place (PO Box 2348), Sechelt, BC, Canada,
V0N3A0, 604-885-0840 NEW! Cell (when I remember
to turn it on!) 1-765-637-1917,
<[hidden email]>
Guy Cox-2 Guy Cox-2
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Re: Oil vs water objectives

In reply to this post by Cammer, Michael
That's not really true.  If you cannot validate the method, how can you trust your image of your sample?  

                                                                                Guy

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Cammer, Michael
Sent: Friday, 5 October 2012 2:20 AM
To: [hidden email]
Subject: Re: Oil vs water objectives

Also, while people have generated various test samples of collagen and latex and acrylic and fat globules and glass beads and carrageen gum and dissolved Scotch tape glue etc., the only test sample that really answers the question is the sample itself.  
________________________________________________________
Michael Cammer, Assistant Research Scientist Skirball Institute of Biomolecular Medicine
Lab: (212) 263-3208  Cell: (914) 309-3270


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Steffen Dietzel
Sent: Thursday, October 04, 2012 10:30 AM
To: [hidden email]
Subject: Re: Oil vs water objectives

So we are back at Michael's comment: You've got to test it. Beads attached to the coverslip and the slide (various preparations with various distances between the two) give good test objects.

Steffen


George McNamara George McNamara
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Re: Oil vs water objectives

In reply to this post by James Pawley
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Hi Jim and listserv,

Jim wrote, "I am convinced that it was because they just didn't want to
take the time to adjust the collar properly. "

An additional explanation is that the microscope vendors have done a
poor job making critical microscope features - such as the correction
collar - easy to reach and use. Of course the customer buying an
anti-ergonomic microscope are also contributing to the problem.

One solution of course is to "automate everything" - which simply
changes the issue from being physically out of reach physically to one
of budget.

The vendors should be able to design microscopes - and nanoscopes - that
have excellent ergonomics and optics, and customers should take the time
to buy such microscopes.

Another option with respect to S.A. is to move the correction outside
the microscope, as with the mSAC

https://www.intelligent-imaging.com/msac.php

though this makes me wonder what happens if the mSAC is used with an
objective lens whose "correction" collar is (badly) misadjusted.

George


On 10/4/2012 7:26 PM, James Pawley wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi all,
>
> Looks like you have received a lot of good advice, but I would like to
> add one thing.
>
> Yes, do a test BUT don't forget to be very careful in adjusting the SA
> correction collar on the water lens. )Find a point object: focus up
> and down and adjust for symmetry (i.e., for the same image a little
> above focus as a little below focus. DON'T just try to "make it
> sharper!" The collar changes the focus plane.)
>
> The collars usually have markings in terms of the coverslip thickness
> that they are designed to correct for. One can easily see the
> difference created by mis-adjusting a 1.2 NA objective by 2
> µm-of-glass-replaced-by-water (my favorite unit for "measuring" SA).
>
> When modern water objectives first emerged in the early 1990s, many
> folk bought them and then went back to their oil objectives and I am
> convinced that it was because they just didn't want to take the time
> to adjust the collar properly.
>
> Because, if you do adjust it properly, you will get better (brighter?
> higher resolution?) images of aqeous specimens beyond about 3 µm
>
> Best,
>
> Jim Pawley,
>
> Near Harbin, China.
>
>
>
>> Make sure the coverslip is orthogonal to the image axis with your
>> water objective.
>>
>> A common aberration with water-immersion objective lenses.
>> J Microsc. 2004 Oct;216(Pt 1):49-51.
>> http://www.ncbi.nlm.nih.gov/pubmed/15369482
>>
>> Doug
>>
>> ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
>> Douglas W. Cromey, M.S. - Associate Scientific Investigator
>> Dept. of Cellular & Molecular Medicine, University of Arizona
>> 1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA
>>
>> office:  AHSC 4212         email: [hidden email]
>> voice:  520-626-2824       fax:  520-626-2097
>>
>> http://swehsc.pharmacy.arizona.edu/exppath/
>> Home of: "Microscopy and Imaging Resources on the WWW"
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List
>> [mailto:[hidden email]] On Behalf Of Steffen Dietzel
>> Sent: Thursday, October 04, 2012 7:30 AM
>> To: [hidden email]
>> Subject: Re: Oil vs water objectives
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> I second Michael's comment.
>>
>> Apart from that, when you have an aqueous(!) preparation, spherical
>> aberration decreases image quality faster for the oil objective. So,
>> directly behind the coverslip the oil objective (1.4) will give you
>> the better image due to the higher NA, at some depth both objectives
>> will give you similar quality and beyond that, the water immersion
>> will be better.
>>
>> I seem to remember that for oil 1.4 and water 1.2 the crossing point is
>> 10-20 µm into the sample.
>>
>> If you have the Handbook laying around, check the chapter on Lens
>> aberrations. Chapter 20 by Hell and Stelzer in the second edition,
>> chapter 20 by Egner and Hell in the 3rd edition.
>>
>> Needles to say that all above considerations assume that both
>> objectives have the same transparency for excitation and emission
>> wavelengths - which they probably don't. So we are back at Michael's
>> comment: You've got to test it. Beads attached to the coverslip and
>> the slide (various preparations with various distances between the
>> two) give good test objects.
>>
>> Steffen
>>
>> On 04.10.2012 15:15, Gabriel Lapointe wrote:
>>>  *****
>>>  To join, leave or search the confocal microscopy listserv, go to:
>>>  http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>  *****
>>>
>>>  Hi,
>>>
>>>  I have a user who insist that using a 1,27NA water immersion objective
>>>  is brighter and would give better images than using a 1,4NA oil
>>>  immersion. I understand that deeper into the media that would be true.
>>>  But, in that particular case, we are talking about imaging GFP at less
>>>  than 100 micron away with a spinning disk.
>>>
>>>  So, I was wondering at which distance from the coverslips do we start
>>>  seeing benefits of using a water immersion objective over an oil
>>>  objective in aqueous media.
>>>
>>>  Thanks for your help.
>>>
>>>  Sincerely
>>>  *Gabriel Lapointe, M.Sc.*
>> > Lab Manager / Microscopy Specialist
>>>  Concordia University, Biology Department
>>>  7141 Sherbrooke St. West SP 534
>>>  Montréal QC H4B 1R6 Canada
>>>  [hidden email]
>>>  cmac.concordia.ca
>>>  http://gabriellapointe.ca
>>>
>>
>>
>> --
>> ------------------------------------------------------------
>> Steffen Dietzel, PD Dr. rer. nat
>> Ludwig-Maximilians-Universität München
>> Walter-Brendel-Zentrum für experimentelle Medizin (WBex) Head of
>> light microscopy
>>
>> Mail room:
>> Marchioninistr. 15, D-81377 München
>>
>> Building location:
>> Marchioninistr. 27,  München-Großhadern
>
>
Steffen Dietzel Steffen Dietzel
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Re: Oil vs water objectives

In reply to this post by Cammer, Michael
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On 04.10.2012 18:20, Cammer, Michael wrote:
> Also, while people have generated various test samples of
collagen and latex and acrylic and fat globules and glass
beads and carrageen gum and dissolved Scotch tape glue etc.,
the only test sample that really answers the question is the
sample itself.

Again, I agree, in particular if you look at tissues with more or less
unpredictable optical properties. Still, bead preparations allow to
demonstrate the general effect very clearly and thus convincingly. They
also allow quantification and sometimes hard numbers help to convince
people. Some person might argue that they don't care about resolution
since s/he is planning to use a 1 µm pixel size anyway. But you still
can convince these people if you can show them that they will also loose
a lot of intensity.
I use such a demonstration to argue for the usage of 170 µm coverslips
and a good embedding medium.

On 05.10.2012 02:40, Guy Cox wrote:
> Nobody seems to have mentioned so far that the NA of an oil
objective will NOT be 1.4 if it is imaging a sample in water.
The maximum it can be is 1.33 - the refractive index of water.
...
> So the oil objective has little or no advantage in NA

Is that really true? If there is oil between the coverslip and the oil
objective, but water immersion for the water objective, this should make
a noticeable difference in effective acceptance angle, should it not?
(Assuming the object is rather close to the coverslip).

Steffen


--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
Head of light microscopy

Mail room:
Marchioninistr. 15, D-81377 München

Building location:
Marchioninistr. 27,  München-Großhadern
Guy Cox-2 Guy Cox-2
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Re: Oil vs water objectives

OK, so let's get into this in (minimal) depth.

The reason an oil, or water, objective has a higher resolution than a water one is that the wavelength of light in oil, or water, is shorter than in air so resolution is automatically better.  However the homogeneous medium has to go all the way to the objective, otherwise the acceptance angle is reduced by the refractive index change - and since both effects depend directly on the refractive index the loss exactly cancels out the gain.  (See Chapter 1 of Optical Imaging Techniques in Cell Biology).  

Now if we have an oil immersion objective of (say) NA 1.0, it will still have that NA even imaging into water because the change in refractive index means that the lens can collect a wider angle and that will compensate exactly for the longer wavelength (as Steffen says).  However this cosy relationship breaks down once we reach the critical angle, since rays leaving the sample in a downward direction are obviously never going to reach the objective.  Likewise, in confocal illumination, rays beyond the critical angle will experience total internal reflection and will never reach the sample.  Therefore an oil immersion lens cannot have an NA of more than 1.33 when imaging into water, and every oil immersion lens of NA greater than 1.33 will have an  effective NA of  1.33 when imaging into water.  

I don't want to write pages on this, but I think if you just draw it out with a pencil and paper it will be quite clear.  There is neither magic nor deep physics involved!

                                                               Guy

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Steffen Dietzel
Sent: Friday, 5 October 2012 10:29 PM
To: [hidden email]
Subject: Re: Oil vs water objectives

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On 04.10.2012 18:20, Cammer, Michael wrote:
> Also, while people have generated various test samples of
collagen and latex and acrylic and fat globules and glass beads and carrageen gum and dissolved Scotch tape glue etc., the only test sample that really answers the question is the sample itself.

Again, I agree, in particular if you look at tissues with more or less unpredictable optical properties. Still, bead preparations allow to demonstrate the general effect very clearly and thus convincingly. They also allow quantification and sometimes hard numbers help to convince people. Some person might argue that they don't care about resolution since s/he is planning to use a 1 µm pixel size anyway. But you still can convince these people if you can show them that they will also loose a lot of intensity.
I use such a demonstration to argue for the usage of 170 µm coverslips and a good embedding medium.

On 05.10.2012 02:40, Guy Cox wrote:
> Nobody seems to have mentioned so far that the NA of an oil
objective will NOT be 1.4 if it is imaging a sample in water.
The maximum it can be is 1.33 - the refractive index of water.
...
> So the oil objective has little or no advantage in NA

Is that really true? If there is oil between the coverslip and the oil objective, but water immersion for the water objective, this should make a noticeable difference in effective acceptance angle, should it not?
(Assuming the object is rather close to the coverslip).

Steffen


--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex) Head of light microscopy

Mail room:
Marchioninistr. 15, D-81377 München

Building location:
Marchioninistr. 27,  München-Großhadern
Guy Cox-2 Guy Cox-2
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Re: Oil vs water objectives

In reply to this post by George McNamara
*****
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Maybe the users are partly to blame by demanding inverted microscopes.  I am convinced that at least 50% of the time an upright would do the job better, and be much easier to use.  But how is it that people will happily spend hours each week learning to correct their golf swing, yet they will not spend minutes learning how to adjust a microscope properly?

                                                              Guy

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of George McNamara
Sent: Friday, 5 October 2012 8:56 PM
To: [hidden email]
Subject: Re: Oil vs water objectives

*****
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Hi Jim and listserv,

Jim wrote, "I am convinced that it was because they just didn't want to take the time to adjust the collar properly. "

An additional explanation is that the microscope vendors have done a poor job making critical microscope features - such as the correction collar - easy to reach and use. Of course the customer buying an anti-ergonomic microscope are also contributing to the problem.

One solution of course is to "automate everything" - which simply changes the issue from being physically out of reach physically to one of budget.

The vendors should be able to design microscopes - and nanoscopes - that have excellent ergonomics and optics, and customers should take the time to buy such microscopes.

Another option with respect to S.A. is to move the correction outside the microscope, as with the mSAC

https://www.intelligent-imaging.com/msac.php

though this makes me wonder what happens if the mSAC is used with an objective lens whose "correction" collar is (badly) misadjusted.

George


On 10/4/2012 7:26 PM, James Pawley wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi all,
>
> Looks like you have received a lot of good advice, but I would like to
> add one thing.
>
> Yes, do a test BUT don't forget to be very careful in adjusting the SA
> correction collar on the water lens. )Find a point object: focus up
> and down and adjust for symmetry (i.e., for the same image a little
> above focus as a little below focus. DON'T just try to "make it
> sharper!" The collar changes the focus plane.)
>
> The collars usually have markings in terms of the coverslip thickness
> that they are designed to correct for. One can easily see the
> difference created by mis-adjusting a 1.2 NA objective by 2
> µm-of-glass-replaced-by-water (my favorite unit for "measuring" SA).
>
> When modern water objectives first emerged in the early 1990s, many
> folk bought them and then went back to their oil objectives and I am
> convinced that it was because they just didn't want to take the time
> to adjust the collar properly.
>
> Because, if you do adjust it properly, you will get better (brighter?
> higher resolution?) images of aqeous specimens beyond about 3 µm
>
> Best,
>
> Jim Pawley,
>
> Near Harbin, China.
>
>
>
>> Make sure the coverslip is orthogonal to the image axis with your
>> water objective.
>>
>> A common aberration with water-immersion objective lenses.
>> J Microsc. 2004 Oct;216(Pt 1):49-51.
>> http://www.ncbi.nlm.nih.gov/pubmed/15369482
>>
>> Doug
>>
>> ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
>> Douglas W. Cromey, M.S. - Associate Scientific Investigator Dept. of
>> Cellular & Molecular Medicine, University of Arizona
>> 1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA
>>
>> office:  AHSC 4212         email: [hidden email]
>> voice:  520-626-2824       fax:  520-626-2097
>>
>> http://swehsc.pharmacy.arizona.edu/exppath/
>> Home of: "Microscopy and Imaging Resources on the WWW"
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List
>> [mailto:[hidden email]] On Behalf Of Steffen
>> Dietzel
>> Sent: Thursday, October 04, 2012 7:30 AM
>> To: [hidden email]
>> Subject: Re: Oil vs water objectives
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> I second Michael's comment.
>>
>> Apart from that, when you have an aqueous(!) preparation, spherical
>> aberration decreases image quality faster for the oil objective. So,
>> directly behind the coverslip the oil objective (1.4) will give you
>> the better image due to the higher NA, at some depth both objectives
>> will give you similar quality and beyond that, the water immersion
>> will be better.
>>
>> I seem to remember that for oil 1.4 and water 1.2 the crossing point
>> is
>> 10-20 µm into the sample.
>>
>> If you have the Handbook laying around, check the chapter on Lens
>> aberrations. Chapter 20 by Hell and Stelzer in the second edition,
>> chapter 20 by Egner and Hell in the 3rd edition.
>>
>> Needles to say that all above considerations assume that both
>> objectives have the same transparency for excitation and emission
>> wavelengths - which they probably don't. So we are back at Michael's
>> comment: You've got to test it. Beads attached to the coverslip and
>> the slide (various preparations with various distances between the
>> two) give good test objects.
>>
>> Steffen
>>
>> On 04.10.2012 15:15, Gabriel Lapointe wrote:
>>>  *****
>>>  To join, leave or search the confocal microscopy listserv, go to:
>>>  http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>  *****
>>>
>>>  Hi,
>>>
>>>  I have a user who insist that using a 1,27NA water immersion
>>> objective  is brighter and would give better images than using a
>>> 1,4NA oil  immersion. I understand that deeper into the media that would be true.
>>>  But, in that particular case, we are talking about imaging GFP at
>>> less  than 100 micron away with a spinning disk.
>>>
>>>  So, I was wondering at which distance from the coverslips do we
>>> start  seeing benefits of using a water immersion objective over an
>>> oil  objective in aqueous media.
>>>
>>>  Thanks for your help.
>>>
>>>  Sincerely
>>>  *Gabriel Lapointe, M.Sc.*
>> > Lab Manager / Microscopy Specialist
>>>  Concordia University, Biology Department
>>>  7141 Sherbrooke St. West SP 534
>>>  Montréal QC H4B 1R6 Canada
>>>  [hidden email]
>>>  cmac.concordia.ca
>>>  http://gabriellapointe.ca
>>>
>>
>>
>> --
>> ------------------------------------------------------------
>> Steffen Dietzel, PD Dr. rer. nat
>> Ludwig-Maximilians-Universität München Walter-Brendel-Zentrum für
>> experimentelle Medizin (WBex) Head of light microscopy
>>
>> Mail room:
>> Marchioninistr. 15, D-81377 München
>>
>> Building location:
>> Marchioninistr. 27,  München-Großhadern
>
>
Phillips, Thomas E. Phillips, Thomas E.
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Re: Oil vs water objectives

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

I fully agree that an upright is almost always easier to use but how is it better in 50% of the cases? I would argue that an inverted is essential for at least 30% of the cases and there is no work around for using an upright when you have to deal with things like plates or chambers.  

Thomas E. Phillips, Ph.D
Professor of Biological Sciences
Director, Molecular Cytology Core
2 Tucker Hall
University of Missouri
Columbia, MO 65211-7400
573-882-4712 (office)
573-882-0123 (fax)
[hidden email]

http://www.biology.missouri.edu/faculty/phillips.html
http://www.biotech.missouri.edu/mcc/


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Guy Cox
Sent: Friday, October 05, 2012 8:16 AM
To: [hidden email]
Subject: Re: Oil vs water objectives

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

Maybe the users are partly to blame by demanding inverted microscopes.  I am convinced that at least 50% of the time an upright would do the job better, and be much easier to use.  But how is it that people will happily spend hours each week learning to correct their golf swing, yet they will not spend minutes learning how to adjust a microscope properly?

                                                              Guy

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of George McNamara
Sent: Friday, 5 October 2012 8:56 PM
To: [hidden email]
Subject: Re: Oil vs water objectives

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

Hi Jim and listserv,

Jim wrote, "I am convinced that it was because they just didn't want to take the time to adjust the collar properly. "

An additional explanation is that the microscope vendors have done a poor job making critical microscope features - such as the correction collar - easy to reach and use. Of course the customer buying an anti-ergonomic microscope are also contributing to the problem.

One solution of course is to "automate everything" - which simply changes the issue from being physically out of reach physically to one of budget.

The vendors should be able to design microscopes - and nanoscopes - that have excellent ergonomics and optics, and customers should take the time to buy such microscopes.

Another option with respect to S.A. is to move the correction outside the microscope, as with the mSAC

https://www.intelligent-imaging.com/msac.php

though this makes me wonder what happens if the mSAC is used with an objective lens whose "correction" collar is (badly) misadjusted.

George


On 10/4/2012 7:26 PM, James Pawley wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi all,
>
> Looks like you have received a lot of good advice, but I would like to
> add one thing.
>
> Yes, do a test BUT don't forget to be very careful in adjusting the SA
> correction collar on the water lens. )Find a point object: focus up
> and down and adjust for symmetry (i.e., for the same image a little
> above focus as a little below focus. DON'T just try to "make it
> sharper!" The collar changes the focus plane.)
>
> The collars usually have markings in terms of the coverslip thickness
> that they are designed to correct for. One can easily see the
> difference created by mis-adjusting a 1.2 NA objective by 2
> µm-of-glass-replaced-by-water (my favorite unit for "measuring" SA).
>
> When modern water objectives first emerged in the early 1990s, many
> folk bought them and then went back to their oil objectives and I am
> convinced that it was because they just didn't want to take the time
> to adjust the collar properly.
>
> Because, if you do adjust it properly, you will get better (brighter?
> higher resolution?) images of aqeous specimens beyond about 3 µm
>
> Best,
>
> Jim Pawley,
>
> Near Harbin, China.
>
>
>
>> Make sure the coverslip is orthogonal to the image axis with your
>> water objective.
>>
>> A common aberration with water-immersion objective lenses.
>> J Microsc. 2004 Oct;216(Pt 1):49-51.
>> http://www.ncbi.nlm.nih.gov/pubmed/15369482
>>
>> Doug
>>
>> ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
>> Douglas W. Cromey, M.S. - Associate Scientific Investigator Dept. of
>> Cellular & Molecular Medicine, University of Arizona
>> 1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA
>>
>> office:  AHSC 4212         email: [hidden email]
>> voice:  520-626-2824       fax:  520-626-2097
>>
>> http://swehsc.pharmacy.arizona.edu/exppath/
>> Home of: "Microscopy and Imaging Resources on the WWW"
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List
>> [mailto:[hidden email]] On Behalf Of Steffen
>> Dietzel
>> Sent: Thursday, October 04, 2012 7:30 AM
>> To: [hidden email]
>> Subject: Re: Oil vs water objectives
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> I second Michael's comment.
>>
>> Apart from that, when you have an aqueous(!) preparation, spherical
>> aberration decreases image quality faster for the oil objective. So,
>> directly behind the coverslip the oil objective (1.4) will give you
>> the better image due to the higher NA, at some depth both objectives
>> will give you similar quality and beyond that, the water immersion
>> will be better.
>>
>> I seem to remember that for oil 1.4 and water 1.2 the crossing point
>> is
>> 10-20 µm into the sample.
>>
>> If you have the Handbook laying around, check the chapter on Lens
>> aberrations. Chapter 20 by Hell and Stelzer in the second edition,
>> chapter 20 by Egner and Hell in the 3rd edition.
>>
>> Needles to say that all above considerations assume that both
>> objectives have the same transparency for excitation and emission
>> wavelengths - which they probably don't. So we are back at Michael's
>> comment: You've got to test it. Beads attached to the coverslip and
>> the slide (various preparations with various distances between the
>> two) give good test objects.
>>
>> Steffen
>>
>> On 04.10.2012 15:15, Gabriel Lapointe wrote:
>>>  *****
>>>  To join, leave or search the confocal microscopy listserv, go to:
>>>  http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>  *****
>>>
>>>  Hi,
>>>
>>>  I have a user who insist that using a 1,27NA water immersion
>>> objective  is brighter and would give better images than using a
>>> 1,4NA oil  immersion. I understand that deeper into the media that would be true.
>>>  But, in that particular case, we are talking about imaging GFP at
>>> less  than 100 micron away with a spinning disk.
>>>
>>>  So, I was wondering at which distance from the coverslips do we
>>> start  seeing benefits of using a water immersion objective over an
>>> oil  objective in aqueous media.
>>>
>>>  Thanks for your help.
>>>
>>>  Sincerely
>>>  *Gabriel Lapointe, M.Sc.*
>> > Lab Manager / Microscopy Specialist
>>>  Concordia University, Biology Department
>>>  7141 Sherbrooke St. West SP 534
>>>  Montréal QC H4B 1R6 Canada
>>>  [hidden email]
>>>  cmac.concordia.ca
>>>  http://gabriellapointe.ca
>>>
>>
>>
>> --
>> ------------------------------------------------------------
>> Steffen Dietzel, PD Dr. rer. nat
>> Ludwig-Maximilians-Universität München Walter-Brendel-Zentrum für
>> experimentelle Medizin (WBex) Head of light microscopy
>>
>> Mail room:
>> Marchioninistr. 15, D-81377 München
>>
>> Building location:
>> Marchioninistr. 27,  München-Großhadern
>
>
James Pawley James Pawley
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Re: Oil vs water objectives

In reply to this post by Guy Cox-2
*****
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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

>OK, so let's get into this in (minimal) depth.
>
>The reason an oil, or water, objective has a
>higher resolution than a water one is that the
>wavelength of light in oil, or water, is shorter
>than in air so resolution is automatically
>better.  However the homogeneous medium has to
>go all the way to the objective, otherwise the
>acceptance angle is reduced by the refractive
>index change - and since both effects depend
>directly on the refractive index the loss
>exactly cancels out the gain.  (See Chapter 1 of
>Optical Imaging Techniques in Cell Biology).
>
>Now if we have an oil immersion objective of
>(say) NA 1.0, it will still have that NA even
>imaging into water because the change in
>refractive index means that the lens can collect
>a wider angle and that will compensate exactly
>for the longer wavelength (as Steffen says).
>However this cosy relationship breaks down once
>we reach the critical angle, since rays leaving
>the sample in a downward direction are obviously
>never going to reach the objective.  Likewise,
>in confocal illumination, rays beyond the
>critical angle will experience total internal
>reflection and will never reach the sample.
>Therefore an oil immersion lens cannot have an
>NA of more than 1.33 when imaging into water,
>and every oil immersion lens of NA greater than
>1.33 will have an  effective NA of  1.33 when
>imaging into water.
>
>I don't want to write pages on this, but I think
>if you just draw it out with a pencil and paper
>it will be quite clear.  There is neither magic
>nor deep physics involved!
>
>                                                                Guy


Very clear.

But what I would like to add is that, even before
you reach the critical angle for TOTAL internal
reflection, the interface will still reflect some
fraction of the incident light. This fraction
increases as you get closer to the critical
angle. As a result, it doesn't make much sense to
make an NA 1.33 water lens. Not only would the
correction collar be very finicky, but the rays
between 1.20 and 1.33 would be increasingly
attenuated by "non-total" reflections taking
place at all (3?) of the flat, horizontal
glass-water surfaces.

Cheers,

Jim P.


>-----Original Message-----
>From: Confocal Microscopy List
>[mailto:[hidden email]] On
>Behalf Of Steffen Dietzel
>Sent: Friday, 5 October 2012 10:29 PM
>To: [hidden email]
>Subject: Re: Oil vs water objectives
>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>On 04.10.2012 18:20, Cammer, Michael wrote:
>>  Also, while people have generated various test samples of
>collagen and latex and acrylic and fat globules
>and glass beads and carrageen gum and dissolved
>Scotch tape glue etc., the only test sample that
>really answers the question is the sample itself.
>
>Again, I agree, in particular if you look at
>tissues with more or less unpredictable optical
>properties. Still, bead preparations allow to
>demonstrate the general effect very clearly and
>thus convincingly. They also allow
>quantification and sometimes hard numbers help
>to convince people. Some person might argue that
>they don't care about resolution since s/he is
>planning to use a 1 µm pixel size anyway. But
>you still can convince these people if you can
>show them that they will also loose a lot of
>intensity.
>I use such a demonstration to argue for the
>usage of 170 µm coverslips and a good embedding
>medium.
>
>On 05.10.2012 02:40, Guy Cox wrote:
>>  Nobody seems to have mentioned so far that the NA of an oil
>objective will NOT be 1.4 if it is imaging a sample in water.
>The maximum it can be is 1.33 - the refractive index of water.
>...
>>  So the oil objective has little or no advantage in NA
>
>Is that really true? If there is oil between the
>coverslip and the oil objective, but water
>immersion for the water objective, this should
>make a noticeable difference in effective
>acceptance angle, should it not?
>(Assuming the object is rather close to the coverslip).
>
>Steffen
>
>
>--
>------------------------------------------------------------
>Steffen Dietzel, PD Dr. rer. nat
>Ludwig-Maximilians-Universität München
>Walter-Brendel-Zentrum für experimentelle
>Medizin (WBex) Head of light microscopy
>
>Mail room:
>Marchioninistr. 15, D-81377 München
>
>Building location:
>Marchioninistr. 27,  München-Großhadern


--
James and Christine Pawley, PO Box 2348, 5446
Burley Place (PO Box 2348), Sechelt, BC, Canada,
V0N3A0, 604-885-0840 NEW! Cell (when I remember
to turn it on!) 1-765-637-1917,
<[hidden email]>
Alison North Alison North
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Re: Oil vs water objectives

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

Hi George, Guy and all,

I agree that the manufacturers have generally made it very difficult to
adjust the correction collars on the lenses when using an inverted
microscope.   It cracks me up that the Olympus guys designed their
unbelievably lovely silicone oil objective, which has a collar that
should be adjusted for use at different temperatures, but then placed
the marks to line up in a position which is invisible to the user when
the objectives are mounted on an inverted microscope.  Needless to say,
I have pointed this out to them with some force :-).  Likewise, I have
had to remove the aquastop from my Zeiss 780 confocal system altogether,
because I found it so impossible to reach beneath the motorized stage
AND the aquastop to try to get to the correction collar.  BUT THERE IS
HOPE FOR US ALL!!!   I am greatly enjoying the new 40x water objective
on my Leica confocal system with a MOTORIZED correction collar that is
controllable through the software.  Finally, even the most nervous of my
users can adjust the collar optimally without worrying about tweaking
the wrong thing with their fingers stuck up under the stage.

So now I am making myself a pain in the neck, telling all of the other
vendors how much I love this objective lens - pretty much every time I
see them.  I am hoping that if enough of us do this the other
manufacturers may follow suit with motorized collars.... Please join me
in this endeavour?

All the best,
Alison


On 10/5/2012 6:55 AM, George McNamara wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Jim and listserv,
>
> Jim wrote, "I am convinced that it was because they just didn't want
> to take the time to adjust the collar properly. "
>
> An additional explanation is that the microscope vendors have done a
> poor job making critical microscope features - such as the correction
> collar - easy to reach and use. Of course the customer buying an
> anti-ergonomic microscope are also contributing to the problem.
>
> One solution of course is to "automate everything" - which simply
> changes the issue from being physically out of reach physically to one
> of budget.
>
> The vendors should be able to design microscopes - and nanoscopes -
> that have excellent ergonomics and optics, and customers should take
> the time to buy such microscopes.
>
> Another option with respect to S.A. is to move the correction outside
> the microscope, as with the mSAC
>
> https://www.intelligent-imaging.com/msac.php
>
> though this makes me wonder what happens if the mSAC is used with an
> objective lens whose "correction" collar is (badly) misadjusted.
>
> George
>

--
Alison J. North, Ph.D.,
Research Associate Professor and
Senior Director of the Bio-Imaging Resource Center,
The Rockefeller University,
1230 York Avenue,
New York,
NY 10065.
Tel: office ++ 212 327 7488
Tel: lab     ++ 212 327 7486
Fax:         ++ 212 327 7489
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