*****
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
[log in to unmask]
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:[log in to unmask]] On
>Behalf Of Gert van Cappellen
>Sent: 04 November 2010 20:38
>To: [log in to unmask]
>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<[log in to unmask]> 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.
--
James and Christine Pawley, 21 N. Prospect Ave.
Madison, WI, 53726 Phone: 608-238-3953
|
