Basic live cell imaging question...

[Guy Cox-2]

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This isn't really right.  It's true that it would be possible to design
an oil immersion lens which would be corrected for imaging in water (but
you would have to adjust the correction collar for each focal position).
Such a lens is not manufactured (though you might be able to fake it
with a TIRF lens with a correction collar).  What's more it would be a
totally pointless exercise.  You have to bear in mind that the reason RI
comes into the equation is because it shortens the wavelength of light.
So if the sample is in water the NA is sin alpha x the refractive index
of water NOT the refractive index of oil.  Now it's true that the oil
lens could therefore in principle offer an effective sin alpha higher
that the 0.95 offered by the water lens but it will be clear that this
improvement cannot be greater than 5% (and will actually be much less).
This would be way more than offset by the SA problems.  A corrected,
water-immersion coverslip lens will ALWAYS be the best option.  

 

 
Guy

 

Optical Imaging Techniques in Cell Biology

by Guy Cox    CRC Press / Taylor & Francis

     http://www.guycox.com/optical.htm
<http://www.guycox.com/optical.htm>

______________________________________________

Associate Professor Guy Cox, MA, DPhil(Oxon)

Australian Centre for Microscopy & Microanalysis,

Madsen Building F09, University of Sydney, NSW 2006

 

Phone [phone]     Fax [phone]

             Mobile [phone]

______________________________________________

      http://www.guycox.net <http://www.guycox.net>

 

 

From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Barbara Foster
Sent: Saturday, 6 November 2010 3:36 AM
To: [hidden email]
Subject: Re: Basic live cell imaging question...

 

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HI, Kees

This is a little difficult to discuss without benefit of diagrams, but
let's give it a try.
Several things to keep in mind:
a. If the RI of an object exactly matches the RI of the surroundings
there will be NO contrast and therefore you will not be able to see the
object.
b. The cells are mounted in, essentially water.
c. If you think of a cell as a point, light emerging from that point
will travel in all directions.  The more you capture, the better, not
only because the signal is stronger, but that light carries information
about orientation, spacing, and edges (See diffraction theory)
d. As the imaging information leaves the cells, it is that information
which is subject to the RI of the surrounding medium.  As that light
leaves the cell it will approach a variety of interfaces at the
water/coverslip and the coverslip/air or oil.  
e. Here are the RI's we need:
Water (in which the cells are mounted): 1.33
Glass coverslip: ~ 1.51
Air: 1.00
Oil: 1.5121
According to Snell's Law, light will refract (bend) as it crosses an
interface at an angle.  The amount of bend is directly related to the
ratio of the RI's on either side of the interface.  Further, if light
passes from higher refractive index into lower refractive index, it will
bend AWAY from the normal (in the simplest case, essentially, the
optical axis of the microscope).  If moving from lower into higher, it
will bend TOWARD the optical axis.  So, if light moves from water (1.33)
into a glass coverslip (1.51), it will move TOWARD the optical axis.
This is good because it allows the objective to capture more of that
information-carrying light.
f.  However, when the light emerges from the coverslip, one of three
things happens.
<If it moves into air (from RI 1.51 to RI = 1.00), it is strongly
refracted, often out of the collecting angle of the objective, resulting
in a loss of both resolution and edge information.
<If it moves into water (water immersion objective... RI=1.51 to RI =
1.33), less information is lost
<If it moves into oil (oil immersion objective... RI=1.51 to RI=1.51),
the maximum amount of information is retained.

So... the long answer to your question is: oil immersion is really the
best for observing fine detail in living cells.  Dipping objectives are
also helpful because they significantly reduce the number of interfaces.
One caveat: you must carefully attend to their cleanliness.  In actual
fact, to keep them healthy, live cells are mounted in saline solutions.
If you don't clean off the objective after every use with a cloth
dampened (not sopping wet) with distilled water, the salt will dry on
the objective, corroding both the casing and marring the glass.  I've
visited some very prestigious labs where, unfortunately, that simple
step was not practiced.  The results were very sad, indeed.

Hope this is helpful.

Good hunting,
Barbara Foster, President and Sr. Consultant
Microscopy/Microscopy Education
W: www.MicroscopyEducation.com

Working in confocal or fluorescence? Take part in our latest survey.
Visit www.MicroscopyEducation.com for details.  Survey has been extended
to November 5th.

At 09:00 PM 11/4/2010, you wrote:
>I always wonder if a water immersion lens is better than an oil
immersion lens for live cell imaging. Both have the wrong RI for cells.
Water too low, oil too high. However, the NA of an oil lens is higher
than from a water lens, so the oil objective should be more light
efficient and your resolution should be a little better. Or am I
wrong....?
>
>kees
>
>-----Original Message-----
>From: Confocal Microscopy List
[mailto:[hidden email]] On Behalf Of Gert van
Cappellen

>Sent: 04 November 2010 20:38
>To: [hidden email]
>Subject: Re: Basic live cell imaging question...
>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>  I'm quite sure the cells will also survive an oil immersion lens and
>normally this gives enough information for single cells. However a

water

>immersion lens is better but certainly not necessary.
>
>Best regards,
>Gert
>
>Op 4-11-2010 2:03, Axel Kurt Preuss schreef:
>> You need a water immersion object or have to build one
>>
>>
>>     Cheers
>>
>> Axel
>> -----
>> Axel
>> Axel K Preuss, PhD,
>> Central Imaging, IMCB, A*Star, 61 Biopolis Dr, 6-19B, Singapore

138673,  sent from 9271.5622
>>
>>
>> On Nov 4, 2010, at 4:06 AM, Gert van
Cappellen<[hidden email]>  wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>>   Culture your cells on a round coverslip. Take an object glas glue
a
>>> square piece of non-toxic rubber with a round hole on it. Fill this
with
>>> CO2 satured medium somewaht more as the volume of the hole. Put your
>>> coverslip on it, with the cells to the medium off course. Press it
>>> gently down and the glass will seal itself to the rubber ring. Now
your
>>> cells will survive for a couple of hours, so you can do the first
>>> imaging. For real experiments you have to find a way to heat the
object

>>> glass to 37C.
>>>
>>> Good luck, Gert
>>>
>>> Op 29-10-2010 21:00, Dolphin, Colin schreef:
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> We would like to do live cell imaging - mammalian cell lines - but

only have direct access to an upright Olympus BX61. We don't really need
complicated perfusion chambers, etc just something simple. We're real
neophytes so all suggestions gratefully received.
>>>>
>>>> Colin
>>>>
>> Note: This message may contain confidential information. If this
Email/Fax has been sent to you by mistake, please notify the sender and
delete it immediately. Thank you.

________________________________

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[Guy Cox-2]

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Seriously off-topic.

 

I am trying to trace Rayleigh's original resolution publication.   The
reference I have is:

 

Investigations in optics with special reference to the spectroscope.  1.
Resolution or separating power of optical instruments.  Phil Mag 8.
261-274, 1880

 

The problem is that when I go to the Phil Mag archives it isn't there!
Can any historically-minded list members help?

 

 
Guy

 

Optical Imaging Techniques in Cell Biology

by Guy Cox    CRC Press / Taylor & Francis

     http://www.guycox.com/optical.htm
<http://www.guycox.com/optical.htm>

______________________________________________

Associate Professor Guy Cox, MA, DPhil(Oxon)

Australian Centre for Microscopy & Microanalysis,

Madsen Building F09, University of Sydney, NSW 2006

 

Phone [phone]     Fax [phone]

             Mobile [phone]

______________________________________________

      http://www.guycox.net <http://www.guycox.net>

 

 

 

[Martin Wessendorf-2]

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Dear Guy--

On 11/7/2010 6:33 AM, Guy Cox wrote:
> I am trying to trace Rayleigh's original resolution publication.   The
> reference I have is:
>
> Investigations in optics with special reference to the spectroscope.  1.
> Resolution or separating power of optical instruments.  Phil Mag 8.
> 261-274, 1880
>
> The problem is that when I go to the Phil Mag archives it isn't there!
> Can any historically-minded list members help?

To help avoid false leads, where did you find that citation?

Thanks--

Martin Wessendorf
--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455                    e-mail: [hidden email]

[Guy Cox-2]

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OK, Wikipedia had links to a Cambridge University Press bibliography,
published in 1899.

 

http://www.archive.org/details/scientificpaper01raylgoog 

 

This led me to that reference.  But the  Phil Mag archives for 1880
showed no trace of Rayleigh.

 

                                                      Guy

 

Optical Imaging Techniques in Cell Biology

by Guy Cox    CRC Press / Taylor & Francis

     http://www.guycox.com/optical.htm
<http://www.guycox.com/optical.htm>

______________________________________________

Associate Professor Guy Cox, MA, DPhil(Oxon)

Australian Centre for Microscopy & Microanalysis,

Madsen Building F09, University of Sydney, NSW 2006

 

Phone [phone]     Fax [phone]

             Mobile [phone]

______________________________________________

      http://www.guycox.net <http://www.guycox.net>

 

 

From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Martin Wessendorf
Sent: Sunday, 7 November 2010 11:45 PM
To: [hidden email]
Subject: Re: Historical question

 

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Dear Guy--

On 11/7/2010 6:33 AM, Guy Cox wrote:
> I am trying to trace Rayleigh's original resolution publication.   The
> reference I have is:
>
> Investigations in optics with special reference to the spectroscope.
1.
> Resolution or separating power of optical instruments.  Phil Mag 8.
> 261-274, 1880
>
> The problem is that when I go to the Phil Mag archives it isn't there!
> Can any historically-minded list members help?

To help avoid false leads, where did you find that citation?

Thanks--

Martin Wessendorf
--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455                    e-mail: [hidden email]

________________________________

No virus found in this message.
Checked by AVG - www.avg.com
Version: 10.0.1153 / Virus Database: 424/3241 - Release Date: 11/06/10

________________________________

No virus found in this message.
Checked by AVG - www.avg.com
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[gradice]

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Guy,

Try this:  Google books has a scanned copy of his collected works, and it is there.

http://tinyurl.com/254u4w8



Gary Radice






On Nov 7, 2010, at 7:50 AM, Guy Cox wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> OK, Wikipedia had links to a Cambridge University Press bibliography,
> published in 1899.
>
>
>
> http://www.archive.org/details/scientificpaper01raylgoog 
>
>
>
> This led me to that reference.  But the  Phil Mag archives for 1880
> showed no trace of Rayleigh.
>
>
>
>                                                      Guy
>
>
>
> Optical Imaging Techniques in Cell Biology
>
> by Guy Cox    CRC Press / Taylor & Francis
>
>     http://www.guycox.com/optical.htm
> <http://www.guycox.com/optical.htm>
>
> ______________________________________________
>
> Associate Professor Guy Cox, MA, DPhil(Oxon)
>
> Australian Centre for Microscopy & Microanalysis,
>
> Madsen Building F09, University of Sydney, NSW 2006
>
>
>
> Phone [phone]     Fax [phone]
>
>             Mobile [phone]
>
> ______________________________________________
>
>      http://www.guycox.net <http://www.guycox.net>
>
>
>
>
>
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Martin Wessendorf
> Sent: Sunday, 7 November 2010 11:45 PM
> To: [hidden email]
> Subject: Re: Historical question
>
>
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Guy--
>
> On 11/7/2010 6:33 AM, Guy Cox wrote:
>> I am trying to trace Rayleigh's original resolution publication.   The
>> reference I have is:
>>
>> Investigations in optics with special reference to the spectroscope.
> 1.
>> Resolution or separating power of optical instruments.  Phil Mag 8.
>> 261-274, 1880
>>
>> The problem is that when I go to the Phil Mag archives it isn't there!
>> Can any historically-minded list members help?
>
> To help avoid false leads, where did you find that citation?
>
> Thanks--
>
> Martin Wessendorf
> --
> Martin Wessendorf, Ph.D.                   office: [phone]
> Assoc Prof, Dept Neuroscience                 lab: [phone]
> University of Minnesota             Preferred FAX: [phone]
> 6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
> Minneapolis, MN  55455                    e-mail: [hidden email]
>
> ________________________________
>
> No virus found in this message.
> Checked by AVG - www.avg.com
> Version: 10.0.1153 / Virus Database: 424/3241 - Release Date: 11/06/10
>
> ________________________________
>
> No virus found in this message.
> Checked by AVG - www.avg.com
> Version: 10.0.1153 / Virus Database: 424/3241 - Release Date: 11/06/10

[Gary G. Li]

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A Google search shows that the year of publication may be 1879 or 1876,
instead of 1880.

Lord Rayleigh, Investigations in optics with special reference to the
spectroscope, Phil. Mag. 8, 261-274 (1879)

Lord Rayleigh, Philosophical Magazine 8 (1876) 261-274.

Gary

On Sun, Nov 7, 2010 at 9:01 AM, gradice <[hidden email]> wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Guy,
>
> Try this:  Google books has a scanned copy of his collected works, and it
is there.

>
> http://tinyurl.com/254u4w8
>
>
>
> Gary Radice
>
>
>
>
>
>
> On Nov 7, 2010, at 7:50 AM, Guy Cox wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> OK, Wikipedia had links to a Cambridge University Press bibliography,
>> published in 1899.
>>
>>
>>
>> http://www.archive.org/details/scientificpaper01raylgoog
>>
>>
>>
>> This led me to that reference.  But the  Phil Mag archives for 1880
>> showed no trace of Rayleigh.
>>
>>
>>
>>                                                      Guy
>>
>>
>>
>> Optical Imaging Techniques in Cell Biology
>>
>> by Guy Cox    CRC Press / Taylor & Francis
>>
>>     http://www.guycox.com/optical.htm
>> <http://www.guycox.com/optical.htm>
>>
>> ______________________________________________
>>
>> Associate Professor Guy Cox, MA, DPhil(Oxon)
>>
>> Australian Centre for Microscopy & Microanalysis,
>>
>> Madsen Building F09, University of Sydney, NSW 2006
>>
>>
>>
>> Phone [phone]     Fax [phone]
>>
>>             Mobile [phone]
>>
>> ______________________________________________
>>
>>      http://www.guycox.net <http://www.guycox.net>
>>
>>
>>
>>
>>
>> From: Confocal Microscopy List [mailto:[hidden email]]
>> On Behalf Of Martin Wessendorf
>> Sent: Sunday, 7 November 2010 11:45 PM
>> To: [hidden email]
>> Subject: Re: Historical question
>>
>>
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear Guy--
>>
>> On 11/7/2010 6:33 AM, Guy Cox wrote:
>>> I am trying to trace Rayleigh's original resolution publication.   The
>>> reference I have is:
>>>
>>> Investigations in optics with special reference to the spectroscope.
>> 1.
>>> Resolution or separating power of optical instruments.  Phil Mag 8.
>>> 261-274, 1880
>>>
>>> The problem is that when I go to the Phil Mag archives it isn't there!
>>> Can any historically-minded list members help?
>>
>> To help avoid false leads, where did you find that citation?
>>
>> Thanks--
>>
>> Martin Wessendorf
>> --
>> Martin Wessendorf, Ph.D.                   office: [phone]
>> Assoc Prof, Dept Neuroscience                 lab: [phone]
>> University of Minnesota             Preferred FAX: [phone]
>> 6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
>> Minneapolis, MN  55455                    e-mail: [hidden email]
>>

[Alice Rodriguez Diaz]

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  Also see :
Abbe,E., 1884, Note on the proper definition of the amplifying power of
a lens or lens-system. J. Royal Micosc. Soc. 4:348-351

Alice Rodriguez Diaz

On 117/2010 8:33 AM, Guy Cox wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Seriously off-topic.
>
>
>
> I am trying to trace Rayleigh's original resolution publication.   The
> reference I have is:
>
>
>
> Investigations in optics with special reference to the spectroscope.  1.
> Resolution or separating power of optical instruments.  Phil Mag 8.
> 261-274, 1880
>
>
>
> The problem is that when I go to the Phil Mag archives it isn't there!
> Can any historically-minded list members help?
>
>
>
>
> Guy
>
>
>
> Optical Imaging Techniques in Cell Biology
>
> by Guy Cox    CRC Press / Taylor&  Francis
>
>       http://www.guycox.com/optical.htm
> <http://www.guycox.com/optical.htm>
>
> ______________________________________________
>
> Associate Professor Guy Cox, MA, DPhil(Oxon)
>
> Australian Centre for Microscopy&  Microanalysis,
>
> Madsen Building F09, University of Sydney, NSW 2006
>
>
>
> Phone [phone]     Fax [phone]
>
>               Mobile [phone]
>
> ______________________________________________
>
>        http://www.guycox.net<http://www.guycox.net>
>
>
>
>
>
>

[James Pawley]

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Dear John,

I take your points. Thank you for finding the references.

I was thinking that my last email was too long
already. So I just assumed that the TIRF was
using illumination through the objective. Were
this so, some provision (besides the barrier
filter) must be made to prevent the totally
reflected light from reaching the camera and this
is usually a beam stop somewhere in NA-space that
must reduce the NA (although, unless conical
illumination is used, not in all directions). I
agree that this would not be necessary if TIRF
were done in the original manner with
trans-illumination using a prism on the back side
of the slide. One caution I might mention: in
epi-illuminated fluorescence, a lot of the light
that seems to be passing through the BFP from the
specimen may in fact be excitation light
reflected by the many optical surfaces between
the BFP and the specimen. Although most of this
light should be removed by the barrier filters
(Returning excited light can also reflect but
here is less of it.), when the signal levels are
low (as in TIRF), simply imaging this light could
give you the impression that more of the NA is
being used for imaging than is the case.

The hairy quantum mechanics does not have to do
with the math (although that would be even
hairier!) but with simply defining where "the
optical surface" is. Is it the glass? Or is it
the edge of the often-fairly-high RI cell cortex
wherever the cell is "touching" the glass
surface? Or is it a little of both (As the cell
cortex probably has a lower RI than that of
glass, maybe there are two interfaces but then
the change in RI at each interface is lower and
the critical angle becomes much more oblique,
probably more oblique than you can get with
epi-illumination.)? How far apart do these
surfaces have to be to be considered separate?
One wavelength? half? a quarter? Is it a sliding
scale?

To make matters worse, if these glass-contacting
"objects" (i.e., the patches of cortex) are small
in xy, a considerable fraction of the
water-object interface is not parallel to the
glass surface and may even be perpendicular to
it, meaning that total internal reflection is not
going to occur on the "sides" and excitation
light will "leak" out. And perhaps emitted light
will leak in. Is this important? It will probably
depend on the fraction of the glass surface
covered with small RI-objects. And what their RI
is. And how "thick" they are.).

There are more questions: How close do the
surfaces of the cell and the glass have to be to
be counted as touching? 1nM? 100nm? If you do
backscattered light confocal of the glass surface
with a hi-NA lens, the focal contacts appear
darker, so some light isn't being reflected.
Where did it go?

So, when I was saying that the light used to form
the image in TIRF was that coming from about NA
1.25, I was referring to light excited within the
evanescent layer (say within 100 nm of the glass)
but from dye molecules that are "in the water"
rather than those optically connected to the
glass. When I was suggesting that you might do
better using an oil lens when imaging the fibers
on the glass surface in DIC, I was thinking of
the "fibrous features" as being close enough to
the glass to be considered as part of it.
However, I agree that this is a bit of a fudge as
contrast in DIC depends on orientation as well as
the geometry of the RI changes and it is VERY
hard measure the DIC-PSF in the shear direction
as each point object is imaged as a light blob
next to a dark one (or vice versa). Is the PSF
the diameter of one of these blobs (easier to
measure), or of both of them together (Clearly,
if there were two objects located near to each
other along the shear direction, the contrast
between them would overlap and cancel out if the
light blob of one coincided with the dark blob of
the second.)? And the light pattern in the BFP
really can't tell you what angles of light are
contributing to the DIC image because this
pattern of light depends on the position of the
slider and the interference that produces the
contrast uses only some of the light that passes
through the BFP. This doesn't even mention the
orientation-dependent reflection effects that
partially depolarize the light passing through
refractive optics at high NA.)

So I think that Barbara is right about an oil
lens being good for this type of "thin" DIC
specimen, but would be wrong to suggest that an
oil lens would be better to record any data from
focus planes more than a few 100 nms into the
aqueous solution.

And Guy is quite right to direct our attention to
the fact that the only reason that RI even comes
into the discussion is because it reduces the
wavelength of the light at the location where the
interaction takes place, and that it is this
wavelength that sets the diffraction limit.

After that it is just a matter of arranging some
optics that will permit you to create an
aberration-free image of this interaction. As
optics are made of glass, and living specimens
are made of water (plus some other stuff), there
must be an interface between these two materials
somewhere and, if the optics are to be aberration
free, refraction (and reflection!) at this
interface must be accounted for. For most cells,
this is best done by using water objectives but
ONLY with the condition that you carefully adjust
the correction collar separately for every
specimen (because coverslips are not all the same
thickness (+/- 1µm), and the cell RI may differ
from that of water by a variable amount. In fact,
if the medium between the lens and the coverslip
has an RI different from that of the "cell",
obtaining aberration-free images, will require
that you readjust the collar for different depths
into the specimen.). Even this care will not
eliminate the effects of the sometimes pronounced
variation of RI within each cell.

So, now you see what a can of worms we have to
analyze, aren't you glad I left this out the
first time?

Keep you lenses clean,

Jim Pawley

*********************************************************************************
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 11-23, 2011, UBC, Vancouver Canada
Info: http://www.3dcourse.ubc.ca/
               "If it ain't diffraction, it must be statistics." Anon.

>
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Hi Jim,
>
>I'm wondering if you can clear up a few things
>related to this topic. You said in your last
>email that the higher NA rays in a water-glass
>situation do not get into the objective, even in
>the case of TIRF. There is a very nice paper by
>Jorg Enderlein's group that uses a very simple
>technique to measure the actual NA of an
>objective which involves viewing the
>distribution of light rays in the back focal
>plane of an objective with the use of a Bertrand
>lens:
>
>http://www.opticsinfobase.org/oe/abstract.cfm?uri=oe-13-23-9409
>
>And another similar paper by Mattheyses and Axelrod:
>
>http://spiedl.aip.org/getabs/servlet/GetabsServlet?prog=normal&id=JBOPFO000010000005054007000001&idtype=cvips&gifs=yes&ref=no
>
>Both of these papers clearly show that a large
>percentage of the light actually does propagate
>into the objective lens beyond the critical
>angle. However, you did also allude to "thin
>aqueous biological" in vitro specimens, the ones
>that are typical of single-molecule experiments
>where the material under investigation has been
>separated from the cell, and both of these
>papers made their measurements under similar
>conditions, so maybe this is only a special case.
>
>My question is, does this situation only occur
>for fluorescent objects that are very near the
>glass-water interface? That is to say, if I were
>to view a fluorescent cellular sample the same
>way (with a Bertrand lens, widefield
>illumination, focused slightly into the cell),
>would I see that most of the light propagates
>into the oil immersion objective BELOW the
>critical angle (and hence the effective lower
>NA)?
>
>As for a "quantum mechanical explanation" of why
>the other situation works, it seems that Hellen
>and Axelrod came up with an explanation some
>time ago that does not involve quantum mechanics
>at all, and it can be explained using classical
>electrodynamics... but I've had trouble
>following this paper because it gets rather
>complicated with the math:
>
>Hellen, E.H. and D. Axelrod, Fluorescence
>emission at dielectric and metal-film
>interfaces. Journal of the Optical Society of
>America B-Optical Physics, 1987. 4(3): p.
>337-350.
>
>Axelrod, D., Emission of fluorescence at an
>interface, in Methods in cell biology, T.
>Langsing and Y. Wang, Editors. 1989, Academic
>Press: San Diego. p. 399-416.
>
>There is a very simple diagram in the second
>book chapter that basically shows very oblique
>light rays emitted by a fluorescent molecule
>near an interface (the near-field) can turn into
>propagating evanescent waves that get coupled
>back into the higher index medium. This website
>talks a bit more about this (see Figure 6):
>
>http://micro.magnet.fsu.edu/primer/techniques/fluorescence/tirf/tirfintro.html
>
>John Oreopoulos
>
>
>
>On 2010-11-05, at 11:33 AM, James Pawley wrote:
>
>>  *****
>>  To join, leave or search the confocal microscopy listserv, go to:
>>  http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>  *****
>  >
>>>  I always wonder if a water immersion lens is
>>>better than an oil immersion lens for live
>>>cell imaging. Both have the wrong RI for
>>>cells. Water too low, oil too high. However,
>>>the NA of an oil lens is higher than from a
>>>water lens, so the oil objective should be
>>>more light efficient and your resolution
>>>should be a little better. Or am I wrong....?
>>>
>>>  kees
>>
>>
>>  Hi Kees,
>>
>>  Yes, I am afraid that you are wrong. The
>>higher NA rays, that an NA 1.4 lens might
>>accept if it were used with an oiled specimen,
>>are totally reflected at the water-glass
>>interface and do not get into the objective.
>>Oil lenses work well for TIRF because the
>>"specimen" is just the thin layer near the
>>interface in which fluorescence is excited by
>>the evanescent wave. But the cone of light that
>>actually forms the image (i.e., not counting
>>the part that is probably used for the
>>high-angle excitation) will still be about NA
>>1.25.
>>
>>  The other "thin aqueous biological specimens"
>>that were productively viewed with an oil
>>objective were the various fiber systems
>>(isolated microtubules, actin filaments etc.
>>lying on a slide, in media) that were
>>visualized using video-enhanced contrast DIC by
>>Shinya Inoue and Robert Allen back in the early
>>1980s. Although the exact quantum-mechanical
>>explanation of the interactions near this
>>specimen is beyond me, it seems likely that
>>these structures were so close to the glass
>>that they were essentially part of it in terms
>>of maximum angle at which scattered rays could
>>enter the objective. In any case, the raw
>>resolution of the recorded images was about
>>that of a normal  NA 1.4 objective with the
>>green light that they used. The ability to
>>visualize much smaller structures was more a
>>"detection of their presence on a clear
>>background" than a matter of resolving them.
>>
>>  The rationale for using oil in those days was
>>that there really weren't any good, high-NA
>>water objectives available.  (Here is a good
>>place to reemphasize Guy's point about the
>>absolute necessity of carefully adjusting the
>>coverslip correction collar. At NA 1.2, aim for
>>an accuracy of <+/- 2µm of water-replaced-by
>>glass. This is less important with oil
>>objectives because oil and coverslip have about
>>the same RI.)
>>
>>  As the RI of water is about 1.33, one might
>>assume that one could use an objective with an
>>NA of up to 1.33. The reason this doesn't help
>>much is that, although the rays between 1.2 and
>>1.33 may not be totally reflected at the
>>water-glass interface, they are still highly
>>reflected. (Just remember how bright the image
>>of the sun is when it reflects off a pane of
>>glass at glancing incidence. Although some
>>sunlight is still being refracted through the
>>glass into the building, it will be dim because
>>most of the light was reflected.)
>>
>>  And finally, as mentioned by others, there is
>>the matter of spherical aberration (apparently
>>my favorite topic!). How is this important? If
>>you are looking at large fluorescent objects
>>(say >1µm), then it isn't quite so important.
>>Assuming it is not lost to reflection at the
>>glass-water interface, a larger NA lens will
>>accept more light and if it is not absorbed or
>>reflected while passing through the optics,
>>this light will end up in the image somewhere
>>close to its proper location, making the blob
>>appear brighter than it would be otherwise.
>>However, if you are hoping that the higher NA
>>of the oil lens will yield  better resolution
>>on a water specimen, forget it. As Rimas
>>Juskaitis makes clear in Chapter 11 of The
>>Handbook, even under optimal conditions (i.e.,
>>the right oil, temp etc) the best 1.4
>>objectives then available were only free from
>>phase error up to NA 1.35 (i.e., the rays
>>between 1.35 and 1.4 were passing the objective
>>but not being focused properly and hence not
>>contributing the a reduction in the imaged size
>>of a point object.) I don't know how the newer
>>1.45, 1.49 and 1.65 objectives would perform
>>under his very stringent test conditions but I
>>would like to point out that he only got the
>>old ones to work at 1.35 by controlling the oil
>>temperature to +/- 1deg C.
>  >
>>  Once SA is present, the image of a point
>>object gets bigger in a complicated way (it is
>>hard to define the PSF of an aberrated image
>>with a single number.). This means not only
>>that the resolution is reduced, but that that
>>the brightest part of this image will be dimmer
>>than it would have been without the aberration.
>>i.e. The high-NA oil image of a point object
>>will be dimmer than the aberration-free image
>>from a water lens with slightly lower
>>"faceplate" NA. The best plan is to always
>>include small (<.3µm) fluorescent beads in your
>>preparations and before you start imaging "for
>>real," focus up and down on the beads "by eye"
>>to make sure that the slightly-out-of-focus
>>image seen above focus, looks very much like
>>that seen the same amount below focus. If this
>>is true, then SA should not be a problem.
>  >
>>  What I am trying to say is that resolution
>>isn't always proportional to the number on the
>>side of the objective. Everything else has to
>>be "perfect" and it seldom is. As you point
>>out, cells are neither water or oil. It is
>>worse than that. Their internal RI is extremely
>>variable, which is why DIC and phase show
>>strong contrast of subcellular details.
>>
>>  But that is another story for another hour...
>>
>>  Regards,
>>
>>  Jim Pawley
>>
>>
>>*********************************************************************************
>>  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
>>11-23, 2011, UBC, Vancouver Canada
>>  Info: http://www.3dcourse.ubc.ca/ Applications still being accepted
>>       "If it ain't diffraction, it must be statistics." Anon.
>>
>>
>>
>>
>>>  -----Original Message-----
>>>  From: Confocal Microscopy List
>>>[mailto:[hidden email]] On
>>>Behalf Of Gert van Cappellen
>>>  Sent: 04 November 2010 20:38
>>>  To: [hidden email]
>>>  Subject: Re: Basic live cell imaging question...
>>>
>>>  *****
>>>  To join, leave or search the confocal microscopy listserv, go to:
>>>  http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>  *****
>>>
>>>   I'm quite sure the cells will also survive an oil immersion lens and
>>>  normally this gives enough information for single cells. However a water
>>>  immersion lens is better but certainly not necessary.
>>>
>>>  Best regards,
>>>  Gert
>>>
>>>  Op 4-11-2010 2:03, Axel Kurt Preuss schreef:
>>>>  You need a water immersion object or have to build one
>>>>
>>>>
>>>>      Cheers
>>>>
>>>>  Axel
>>>>  -----
>>>>  Axel K Preuss, PhD,
>>>>  Central Imaging, IMCB, A*Star, 61 Biopolis
>>>>Dr, 6-19B, Singapore 138673,  sent from
>>>>9271.5622
>>>>
>>>>
>>>>  On Nov 4, 2010, at 4:06 AM, Gert van
>>>>Cappellen<[hidden email]>
>>>>wrote:
>>>>
>>>>>  *****
>>>>>  To join, leave or search the confocal microscopy listserv, go to:
>>>>>  http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>>  *****
>>>>>
>>>>>    Culture your cells on a round coverslip. Take an object glas glue a
>>>>>  square piece of non-toxic rubber with a round hole on it. Fill this with
>>>>>  CO2 satured medium somewaht more as the volume of the hole. Put your
>>>>>  coverslip on it, with the cells to the medium off course. Press it
>>>>>  gently down and the glass will seal itself to the rubber ring. Now your
>>>>>  cells will survive for a couple of hours, so you can do the first
>>>>>  imaging. For real experiments you have to find a way to heat the object
>>>>>  glass to 37C.
>>>>>
>>>>>  Good luck, Gert
>>>>>
>>>>>  Op 29-10-2010 21:00, Dolphin, Colin schreef:
>>>>>>  *****
>>>>>>  To join, leave or search the confocal microscopy listserv, go to:
>>>>>>  http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>>>  *****
>>>>>>
>>>>>>  We would like to do live cell imaging -
>>>>>>mammalian cell lines - but only have direct
>>>>>>access to an upright Olympus BX61. We don't
>>>>>>really need complicated perfusion chambers,
>>>>>>etc just something simple. We're real
>>>>>>neophytes so all suggestions gratefully
>>>>>>received.
>>>>>>
>>>>>>  Colin
>>>>>>
>>>>  Note: This message may contain confidential
>>>>information. If this Email/Fax has been sent
>>>>to you by mistake, please notify the sender
>>>>and delete it immediately. Thank you.
>  >
>>
>

--

[Mark Cannell]

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*****

The correct reference according to the publisher is:

Rayleigh(1879) 'XXXI. Investigations in optics, with special reference  
to the spectroscope', Philosophical
Magazine Series 5, 8: 49, 261 — 274

Cheers

On 8/11/2010, at 3:44 AM, Gary G. Li wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> A Google search shows that the year of publication may be 1879 or  
> 1876,
> instead of 1880.
>
> Lord Rayleigh, Investigations in optics with special reference to the
> spectroscope, Phil. Mag. 8, 261-274 (1879)
>
> Lord Rayleigh, Philosophical Magazine 8 (1876) 261-274.
>
> Gary
>
> On Sun, Nov 7, 2010 at 9:01 AM, gradice <[hidden email]> wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Guy,
>>
>> Try this:  Google books has a scanned copy of his collected works,  
>> and it
> is there.
>>
>> http://tinyurl.com/254u4w8
>>
>>
>>
>> Gary Radice
>>
>>
>>
>>
>>
>>
>> On Nov 7, 2010, at 7:50 AM, Guy Cox wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> OK, Wikipedia had links to a Cambridge University Press  
>>> bibliography,
>>> published in 1899.
>>>
>>>
>>>
>>> http://www.archive.org/details/scientificpaper01raylgoog
>>>
>>>
>>>
>>> This led me to that reference.  But the  Phil Mag archives for 1880
>>> showed no trace of Rayleigh.
>>>
>>>
>>>
>>>                                                     Guy
>>>
>>>
>>>
>>> Optical Imaging Techniques in Cell Biology
>>>
>>> by Guy Cox    CRC Press / Taylor & Francis
>>>
>>>    http://www.guycox.com/optical.htm
>>> <http://www.guycox.com/optical.htm>
>>>
>>> ______________________________________________
>>>
>>> Associate Professor Guy Cox, MA, DPhil(Oxon)
>>>
>>> Australian Centre for Microscopy & Microanalysis,
>>>
>>> Madsen Building F09, University of Sydney, NSW 2006
>>>
>>>
>>>
>>> Phone [phone]     Fax [phone]
>>>
>>>            Mobile [phone]
>>>
>>> ______________________________________________
>>>
>>>     http://www.guycox.net <http://www.guycox.net>
>>>
>>>
>>>
>>>
>>>
>>> From: Confocal Microscopy List [mailto:[hidden email]
>>> ]
>>> On Behalf Of Martin Wessendorf
>>> Sent: Sunday, 7 November 2010 11:45 PM
>>> To: [hidden email]
>>> Subject: Re: Historical question
>>>
>>>
>>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Dear Guy--
>>>
>>> On 11/7/2010 6:33 AM, Guy Cox wrote:
>>>> I am trying to trace Rayleigh's original resolution  
>>>> publication.   The
>>>> reference I have is:
>>>>
>>>> Investigations in optics with special reference to the  
>>>> spectroscope.
>>> 1.
>>>> Resolution or separating power of optical instruments.  Phil Mag 8.
>>>> 261-274, 1880
>>>>
>>>> The problem is that when I go to the Phil Mag archives it isn't  
>>>> there!
>>>> Can any historically-minded list members help?
>>>
>>> To help avoid false leads, where did you find that citation?
>>>
>>> Thanks--
>>>
>>> Martin Wessendorf
>>> --
>>> Martin Wessendorf, Ph.D.                   office: [phone]
>>> Assoc Prof, Dept Neuroscience                 lab: [phone]
>>> University of Minnesota             Preferred FAX: [phone]
>>> 6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
>>> Minneapolis, MN  55455                    e-mail: [hidden email]
>>>

[Jeremy Adler-3]

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*****

re explanation from James Pawley, shown below.

If the limitation on the useful NA of a water objective is reflection  
of light from the coverslip at shallow glancing angles, would an anti  
reflection coating on the coverslip reduce the reflection and thereby  
increase the useful NA ?

quoting James Pawley
As the RI of water is about 1.33, one might assume that one could use  
an objective with
an NA of up to 1.33. The reason this doesn't help much is that,  
although the rays between
1.2 and 1.33 may not be totally reflected at the water-glass  
interface, they are still
highly reflected. (Just remember how bright the image of the sun is  
when it reflects off
a pane of glass at glancing incidence. Although some sunlight is still  
being refracted
through the glass into the building, it will be dim because most of  
the light was
reflected.)

[Guy Cox-2]

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

Gary,

 

        I cannot access this - all I get is a couple of paragraphs.
Google seems to regard anyone outside the USA as a second-class citizen
even though the copyright issues are exactly the same!  If you are able
to access the entire paper I'd be eternally grateful if you can download
it and forward it to me.   My problem is that I can access the Phil Mag
archives but the paper doesn't seem to be there!

 

 
Guy

 

Optical Imaging Techniques in Cell Biology

by Guy Cox    CRC Press / Taylor & Francis

     http://www.guycox.com/optical.htm
<http://www.guycox.com/optical.htm>

______________________________________________

Associate Professor Guy Cox, MA, DPhil(Oxon)

Australian Centre for Microscopy & Microanalysis,

Madsen Building F09, University of Sydney, NSW 2006

 

Phone [phone]     Fax [phone]

             Mobile [phone]

______________________________________________

      http://www.guycox.net <http://www.guycox.net>

 

 

From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of gradice
Sent: Monday, 8 November 2010 1:02 AM
To: [hidden email]
Subject: Re: Historical question

 

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

Guy,

Try this:  Google books has a scanned copy of his collected works, and
it is there.

http://tinyurl.com/254u4w8



Gary Radice






On Nov 7, 2010, at 7:50 AM, Guy Cox wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> OK, Wikipedia had links to a Cambridge University Press bibliography,
> published in 1899.
>
>
>
> http://www.archive.org/details/scientificpaper01raylgoog
>
>
>
> This led me to that reference.  But the  Phil Mag archives for 1880
> showed no trace of Rayleigh.
>
>
>
>                                                      Guy
>
>
>
> Optical Imaging Techniques in Cell Biology
>
> by Guy Cox    CRC Press / Taylor & Francis
>
>     http://www.guycox.com/optical.htm
> <http://www.guycox.com/optical.htm>
>
> ______________________________________________
>
> Associate Professor Guy Cox, MA, DPhil(Oxon)
>
> Australian Centre for Microscopy & Microanalysis,
>
> Madsen Building F09, University of Sydney, NSW 2006
>
>
>
> Phone [phone]     Fax [phone]
>
>             Mobile [phone]
>
> ______________________________________________
>
>      http://www.guycox.net <http://www.guycox.net>
>
>
>
>
>
> From: Confocal Microscopy List

[mailto:[hidden email]]

> On Behalf Of Martin Wessendorf
> Sent: Sunday, 7 November 2010 11:45 PM
> To: [hidden email]
> Subject: Re: Historical question
>
>
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Guy--
>
> On 11/7/2010 6:33 AM, Guy Cox wrote:
>> I am trying to trace Rayleigh's original resolution publication.

The
>> reference I have is:
>>
>> Investigations in optics with special reference to the spectroscope.
> 1.
>> Resolution or separating power of optical instruments.  Phil Mag 8.
>> 261-274, 1880
>>
>> The problem is that when I go to the Phil Mag archives it isn't
there!

>> Can any historically-minded list members help?
>
> To help avoid false leads, where did you find that citation?
>
> Thanks--
>
> Martin Wessendorf
> --
> Martin Wessendorf, Ph.D.                   office: [phone]
> Assoc Prof, Dept Neuroscience                 lab: [phone]
> University of Minnesota             Preferred FAX: [phone]
> 6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
> Minneapolis, MN  55455                    e-mail: [hidden email]
>
> ________________________________
>
> No virus found in this message.
> Checked by AVG - www.avg.com
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>
> ________________________________
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________________________________

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[Patrick Van Oostveldt]

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Dear James,

Some other question;
It seems to me that wavelength also has an impact on this. Is a color  
shift visible in these circumstances, or can a simple demo be set -up  
for this?

Patrick



  Quoting "Jeremy Adler" <[hidden email]>:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> re explanation from James Pawley, shown below.
>
> If the limitation on the useful NA of a water objective is  
> reflection of light from the coverslip at shallow glancing angles,  
> would an anti reflection coating on the coverslip reduce the  
> reflection and thereby increase the useful NA ?
>
> quoting James Pawley
> As the RI of water is about 1.33, one might assume that one could  
> use an objective with
> an NA of up to 1.33. The reason this doesn't help much is that,  
> although the rays between
> 1.2 and 1.33 may not be totally reflected at the water-glass  
> interface, they are still
> highly reflected. (Just remember how bright the image of the sun is  
> when it reflects off
> a pane of glass at glancing incidence. Although some sunlight is  
> still being refracted
> through the glass into the building, it will be dim because most of  
> the light was
> reflected.)
>

--
Dep. Moleculaire Biotechnologie
Coupure links 653
B 9000 GENT

tel [phone]
fax [phone]

[Marrison, J.L.]

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CONFOCAL MICROSCOPY

4-Day Intensive Hands-On Course

Website: www.york.ac.uk/depts/biol/tf/imaging_course.htm

Basics, Live-cell imaging, FRET, FRAP and Spectral Unmixing

12 - 15 April 2011
Technology Facility, Dept. Biology
University of York, UK

Instructors will include

Peter O'Toole, University of York
Jo Marrison, University of York
Les Borland, Carl Zeiss
and special guest lecture and practical session with Daniel Zicha (CRUK).

Tutorials and practical sessions will cover both basic and advanced
confocal microscopy. The course will enable participants to realise the
potential behind many developing confocal techniques and realise the
simplicity of applying the techniques to a broad range of sample types
and fluorescent labels. Both novice and more experienced users welcome.

Preliminary Programme
DAY AIMS
Day 1: Basics, Familiarisation, x,y,z and multicolour imaging.
Day 2: Live-cell imaging with spinning disk confocal microscopy, FRAP on
laser scanning confocal.
Day 3: Advanced Techniques covering spectral unmixing and FRET with
fluorescent proteins.
Day 4: Further advances. More samples to explore confocal limits,
introduction to MP and time for participants own samples.

Thursday evening will include a special guest speaker and course meal.

The course will utilise many different sample types ranging from
cultured monolayers to plant cells. A diverse range of fluorescent
labels will also be used, which will include various fluorescent
proteins (CFP, GFP and YFP) and classic antibody labels. Over the four
days, at least four confocal microscopes (2x Zeiss LSM 710 and 2x LSM
510 META on both an invert and upright microscopes, Spinning Disk
Confocal Microscope) will be utilised.

Accommodation (en-suite), breakfast and lunches will be provided for the
length of the course.  Evening meals will be provided on both Tuesday
and Thursday evenings.

Places are limited to 12 participants to permit full hands-on practice,
so please book early to avoid disappointment.
For further information, please visit the course website at:
www.york.ac.uk/depts/biol/tf/imaging-cytometry/imaging_course.htm

or contact Margaret Newton 01904 328821, [hidden email]

--
Dr Peter O'Toole
Head of Imaging and Cytometry
Technology Facility
Department of Biology (Area 15)
University of York
YORK
YO10 5DD

Tel : [phone]
Fax : [phone]
email : [hidden email]
www.york.ac.uk/biology/tf

EMAIL DISCLAIMER http://www.york.ac.uk/docs/disclaimer/email.htm

[Guy Cox-2]

*****
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*****

Sorry, this wasn't intended to go to the list.  But thank you all who
helped, I've now nailed it down and have the article both from the
Cambridge collected papers published in 1899 and from the journal site.
Curiously, though Google won't let me look at the scan of the 1899
volume I can access it from another site - and it's actually the Google
scan!  

 

Investigations in optics, with special reference to the spectroscope.

 

[Phil Mag VIII. pp. 261-274, 403-411, 477-486. 1879; IX. pp. 40-55, 1880

 

#1, Resolving, or Separating, Power of Optical Instruments.

 

This means that #1 is indeed in 1879, not 1880 - I didn't see this
clearly when all I accessed was the contents.  And that is the one we
are interested in.

 

So the formal reference for this is:

 

Investigations in optics, with special reference to the spectroscope.
1879.  #1, Resolving, or Separating, Power of Optical Instruments.  Phil
Mag (ser 5) VIII. pp. 261-274

 

Thanks again to all of you.  

 

 
Guy

 

 

Optical Imaging Techniques in Cell Biology

by Guy Cox    CRC Press / Taylor & Francis

     http://www.guycox.com/optical.htm
<http://www.guycox.com/optical.htm>

______________________________________________

Associate Professor Guy Cox, MA, DPhil(Oxon)

Australian Centre for Microscopy & Microanalysis,

Madsen Building F09, University of Sydney, NSW 2006

 

Phone [phone]     Fax [phone]

             Mobile [phone]

______________________________________________

      http://www.guycox.net <http://www.guycox.net>

 

 

From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Guy Cox
Sent: Monday, 8 November 2010 7:04 PM
To: [hidden email]
Subject: Re: Historical question

 

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

Gary,



        I cannot access this - all I get is a couple of paragraphs.
Google seems to regard anyone outside the USA as a second-class citizen
even though the copyright issues are exactly the same!  If you are able
to access the entire paper I'd be eternally grateful if you can download
it and forward it to me.   My problem is that I can access the Phil Mag
archives but the paper doesn't seem to be there!




Guy



Optical Imaging Techniques in Cell Biology

by Guy Cox    CRC Press / Taylor & Francis

     http://www.guycox.com/optical.htm
<http://www.guycox.com/optical.htm>

______________________________________________

Associate Professor Guy Cox, MA, DPhil(Oxon)

Australian Centre for Microscopy & Microanalysis,

Madsen Building F09, University of Sydney, NSW 2006



Phone [phone]     Fax [phone]

             Mobile [phone]

______________________________________________

      http://www.guycox.net <http://www.guycox.net>





From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of gradice
Sent: Monday, 8 November 2010 1:02 AM
To: [hidden email]
Subject: Re: Historical question



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*****

Guy,

Try this:  Google books has a scanned copy of his collected works, and
it is there.

http://tinyurl.com/254u4w8



Gary Radice






On Nov 7, 2010, at 7:50 AM, Guy Cox wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> OK, Wikipedia had links to a Cambridge University Press bibliography,
> published in 1899.
>
>
>
> http://www.archive.org/details/scientificpaper01raylgoog
>
>
>
> This led me to that reference.  But the  Phil Mag archives for 1880
> showed no trace of Rayleigh.
>
>
>
>                                                      Guy
>
>
>
> Optical Imaging Techniques in Cell Biology
>
> by Guy Cox    CRC Press / Taylor & Francis
>
>     http://www.guycox.com/optical.htm
> <http://www.guycox.com/optical.htm>
>
> ______________________________________________
>
> Associate Professor Guy Cox, MA, DPhil(Oxon)
>
> Australian Centre for Microscopy & Microanalysis,
>
> Madsen Building F09, University of Sydney, NSW 2006
>
>
>
> Phone [phone]     Fax [phone]
>
>             Mobile [phone]
>
> ______________________________________________
>
>      http://www.guycox.net <http://www.guycox.net>
>
>
>
>
>
> From: Confocal Microscopy List

[mailto:[hidden email]]

> On Behalf Of Martin Wessendorf
> Sent: Sunday, 7 November 2010 11:45 PM
> To: [hidden email]
> Subject: Re: Historical question
>
>
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Guy--
>
> On 11/7/2010 6:33 AM, Guy Cox wrote:
>> I am trying to trace Rayleigh's original resolution publication.

The
>> reference I have is:
>>
>> Investigations in optics with special reference to the spectroscope.
> 1.
>> Resolution or separating power of optical instruments.  Phil Mag 8.
>> 261-274, 1880
>>
>> The problem is that when I go to the Phil Mag archives it isn't
there!

>> Can any historically-minded list members help?
>
> To help avoid false leads, where did you find that citation?
>
> Thanks--
>
> Martin Wessendorf
> --
> Martin Wessendorf, Ph.D.                   office: [phone]
> Assoc Prof, Dept Neuroscience                 lab: [phone]
> University of Minnesota             Preferred FAX: [phone]
> 6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
> Minneapolis, MN  55455                    e-mail: [hidden email]
>
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[Guy Cox-2]

Yes, there is a very simple solution – use an NA 1.2 water lens!  

 

                                                                               Guy

 

Optical Imaging Techniques in Cell Biology

by Guy Cox    CRC Press / Taylor & Francis

     http://www.guycox.com/optical.htm <http://www.guycox.com/optical.htm>

______________________________________________

Associate Professor Guy Cox, MA, DPhil(Oxon)

Australian Centre for Microscopy & Microanalysis,

Madsen Building F09, University of Sydney, NSW 2006

 

Phone [phone]     Fax [phone]

             Mobile [phone]

______________________________________________

      http://www.guycox.net <http://www.guycox.net>

 

 

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Vitaly Boyko
Sent: Saturday, 6 November 2010 12:46 AM
To: [hidden email]
Subject: Re: Basic live cell imaging question...

 

Effective NA 1.2 of a buffer sample is good news - better
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Guy,

Effective NA 1.2 of a buffer sample is good news - better sampling
(reduced/negligeable undersampling) when 100x NA 1.4 is used either with the
16um/pixel EMCCD or 2x2 binned 6.4um/pixel CCD. Is there a (simple) solution to
spherical aberrations which are pronounced especially in the image corners?

Vitaly
301-515-7833




________________________________
From: Guy Cox <[hidden email]>
To: [hidden email]
Sent: Fri, November 5, 2010 7:22:05 AM
Subject: Re: Basic live cell imaging question...

High NA water lenses have correction collars to adjust for the refractive
index.  So long as you know how to adjust it – and it’s simple, but it is
essential – it will give you a far better result than an oil lens.  The supposed
higher NA of an oil lens is imaginary – if the sample is in ‘medium’ the
effective NA is RI of ‘medium’ x sin alpha.  The figure written on the lens
implies that the sample is in a mountant of RI 1.515.  If the sample is in water
the real NA of your NA 1.4 lens is 1.2.  But the extreme uncorrected spherical
aberration involved means that you won’t get anything like the resolution you’d
expect from a lens of NA 1.2.




The whole concept of “Numerical Aperture” is an unfortunate accident of
history.  If we stuck to the actual parameter – RI x sin alpha – all these
misconceptions would not arise.




                                                                               
      Guy



Optical Imaging Techniques in Cell Biology

by Guy Cox    CRC Press / Taylor & Francis

    http://www.guycox.com/optical.htm <http://www.guycox.com/optical.htm>

______________________________________________

Associate Professor Guy Cox, MA, DPhil(Oxon)

Australian Centre for Microscopy & Microanalysis,

Madsen Building F09, University of Sydney, NSW 2006



Phone [phone]    Fax [phone]

            Mobile [phone]

______________________________________________

      http://www.guycox.net <http://www.guycox.net>





From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Straatman, Kees R. (Dr.)
Sent: Friday, 5 November 2010 7:51 PM
To: [hidden email]
Subject: Re: Basic live cell imaging question...



I always wonder if a water immersion lens is better than an oil immersion lens
for live cell imaging. Both have the wrong RI for cells. Water too low, oil too
high. However, the NA of an oil lens is higher than from a water lens, so the
oil objective should be more light efficient and your resolution should be a
little better. Or am I wrong....?

kees

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Gert van Cappellen
Sent: 04 November 2010 20:38
To: [hidden email]
Subject: Re: Basic live cell imaging question...

*****
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*****

  I'm quite sure the cells will also survive an oil immersion lens and
normally this gives enough information for single cells. However a water
immersion lens is better but certainly not necessary.

Best regards,
Gert

Op 4-11-2010 2:03, Axel Kurt Preuss schreef:

> You need a water immersion object or have to build one
>
>
>    Cheers
>
> Axel
> —————
> Axel K Preuss, PhD,
> Central Imaging, IMCB, A*Star, 61 Biopolis Dr, 6-19B, Singapore 138673,  sent
>from 9271.5622
>
>
> On Nov 4, 2010, at 4:06 AM, Gert van Cappellen<[hidden email]>
>wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>>  Culture your cells on a round coverslip. Take an object glas glue a
>> square piece of non-toxic rubber with a round hole on it. Fill this with
>> CO2 satured medium somewaht more as the volume of the hole. Put your
>> coverslip on it, with the cells to the medium off course. Press it
>> gently down and the glass will seal itself to the rubber ring. Now your
>> cells will survive for a couple of hours, so you can do the first
>> imaging. For real experiments you have to find a way to heat the object
>> glass to 37C.
>>
>> Good luck, Gert
>>
>> Op 29-10-2010 21:00, Dolphin, Colin schreef:
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> We would like to do live cell imaging - mammalian cell lines - but only have
>>>direct access to an upright Olympus BX61. We don't really need complicated
>>>perfusion chambers, etc just something simple. We're real neophytes so all
>>>suggestions gratefully received.
>>>
>>> Colin
>>>
> Note: This message may contain confidential information. If this Email/Fax has
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>Thank you.

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