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Dear Alby,
please may I ak you to be so kind as to send me a copy or a
pdf of your paper quoted below?
Thanks in advance,
Jurek
On 12 May 2002 at 7:58, Alberto Diaspro wrote:
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1Ānfocal
>
> Dear Angela,
> I hope the following paper can contribute to one of the
> issues raised by Jim. Reprints are available. 1: Diaspro A.
> et al. Influence of refractive-index mismatch in
> high-resolution three-dimensional confocal microscopy. Appl
> Opt. 2002 Feb 1;41(4):685-90.
>
> Bye
> ALby
>
> ............................................................
> ............... DON'T MISS FOCUS ON MICROSCOPY!
> http://www.focusonmicroscopy.org
> ------------------------------------------------------------
> ---------------- Alberto "Alby" Diaspro -
> [log in to unmask] INFM & Department of Physics, Genoa
> Univ Via Dodecaneso 33, 16146 Genoa, Italy fax
> +39-010314218; tel+39-0103536426
> -------------------------
> ---------------- Confocal and Two-Photon Microscopy:
> Foundations, Applications and Advances Alberto Diaspro
> ISBN: 0-471-40920-0 Wiley, 2002
> http://www.wiley.com/cda/product/0,,0471409200,00.html
> ------------------------------------------------------------
> ----------------
>
> > Da: James Pawley <[log in to unmask]>
> > Risposta: Confocal Microscopy List
> > <[log in to unmask]> Data: Fri, 10 May
> > 2002 20:47:35 -0500 A: [log in to unmask]
> > Oggetto: Re: Axial distortion
> >
> > Search the CONFOCAL archive at
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> > Dear Angela,
> >
> > Just wanted to add a few cautions to Fred's and helpful
> > long reply.
> >
> > Given the absence of good instructions from Zeiss about
> > the use of the very-expensive c-Apo Water lenses, the URL
> > Fred suggests, provides a lot of useful information.
> >
> > However, it also makes some suggestions I find hard to
> > agree with.
> >
> > 1. The standard 0.17 mm coverglass that most objectives
> > are designed for use with is actually closest to a #1,5
> > not a #1, (but every box is different!)
> >
> > 2. Making a vertical scan through the coverslip to measure
> > its thickness is problematic as SA effects the apparent
> > position of the reflections.
> >
> > This is further complicated by the fact that the amount of
> > light is reflected at a water/glass interface depends on
> > the angle that the light approaches the interface. Light
> > that comes in normal (i.e., low-NA light) is only slightly
> > reflected: light that approaches near the angle
> > corresponding to 1.2 NA is almost totally reflected.
> > Consequently the reflected light signal is one that
> > consists predominantly of high NA rays, which in the
> > presence of SA, focus at a very different height than low
> > NA rays. This throws off the calculation.
> >
> > While it should be possible to adjust the collar for no-SA
> > on the far side of the coverslip, this cannot be done for
> > the near side (where only a dipping-water lens would
> > produce aberration-free images.).
> >
> > Bear in mind that the collar is calibrated in steps of 2
> > microns-of-glass- replaced-by-water. Focusing on the top
> > surface of the coverslip, involves the SA produced by 170
> > microns-of-glass-replaced-by-water. It is possible that
> > the suggestion that one set the software to assume an RI
> > of 1.146 instead of 1.51 for the coverglass, is an attempt
> > to trick the software into compensating for this problem
> > but I am a bit doubtful about this.
> >
> > A better plan is to buy, for about $150, and then use, a
> > Mitutoyo digital micrometer that reads out directly in
> > microns.
> >
> > Unfortunately, this is not enough because it seems that
> > the coverslip correction collars are often not to be
> > properly calibrated. We had one that gave the
> > aberration-free images when adjusted 30
> > microns-of-glass-replaced-by-water away from the nominal
> > setting.
> >
> > What to do?
> >
> > Pray for dust! or some other small object that reasonably
> > approximates a point object ( (especially when viewed in
> > dark field! Molecular Probes fluorescent beads also do
> > fine too!).
> >
> > Focus up and down through the bead while viewing through
> > the occular (may need higher power than usual) and adjust
> > the collar until the "rings-above" look as similar as
> > possible to the "rings below". Once you have done this
> > once with a coverslip of measured thickness, note the
> > setting of your collar and use the offset (if any) to
> > guide future settings with coverslips of other known
> > thicknesses.
> >
> > 3. Any air lens is designed to provide SA-free imaging at
> > only one depth beneath the coverslip. If you use a dry
> > lens that has a correction collar, you have some control
> > over how far below the air-glass surface this SA-free
> > plane is located, but, unless the sample has the RI of
> > Air, the happy state will only exist a few microns above
> > and below the ideal plane (How far above or below
> > decreases with increasing NA.).
> >
> > 4. Simple, arithmetic, z-distance corrections only work at
> > low NA (<0.3?). At higher NA, all rays contribute to the
> > image near a plane for which SA has been corrected. Above
> > or below, this is not true and the farther you get into
> > the "wrong" medium the less you know, as it depends on,
> > amongst other things, how well your objective BFP is
> > filled (If it isn't filled, the lens is actually operating
> > at low NA, at least on the excitation side and, if you use
> > a large pinhole, on the collection side too.) and on the
> > the optical inhomogeneity of the specimen between the
> > bottom of the coverslip and the focus plane.
> >
> > Best plan is to include some spheres at different levels
> > and see how they fare at different heights.
> >
> > 5. The main reason for PSF broadening is usually SA. Which
> > is where we started. (I will soon put a PDF of a simple
> > chapter on SA on my WWW site at
> > http://www.wisc.edu/zoology/faculty/fac/Paw/pawpapers.html
> > )
> >
> > That's it for now. TGIF!
> >
> > Jim P.
> >
> >
> >>
> >>> ----------
> >>> From: Angela Klaus
> >>> Reply To: Confocal Microscopy List
> >>> Sent: Thursday, May 9, 2002 10:39 PM
> >>> To: [log in to unmask]
> >>> Subject: Axial distortion
> >>>
> >>> Search the CONFOCAL archive at
> >>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >>>
> >>> Hi all...
> >>>
> >>> I've been trying to determine the best way to correct
> >>> for (and understand) axial distortion in a thick
> >>> specimen (greater than 50 um) composed of a material of
> >>> unknown refractive index. I am using a 20x objective
> >>> (NA=0.75) on a Zeiss 510 LSM. If someone could comment
> >>> on the following, I would be most appreciative:
> >>>
> >>> Axial distortion can be attributed to:
> >>>
> >>> 1. Refractive index mismatch between immersion (air in
> >>> this case) and mounting medium (a glycerin-based
> >>> gelatin, RI = 1.57)
> >>>
> >>> The purpose of the "mounting" medium has always been to
> >>> eliminate
> >> refractive mismatch between specimen and ITS immediate
> >> environment. So, one should consider the 'specimen' to
> >> be that which lies between coverglass and slide in a
> >> traditional preparation. The plan-apochromatic 'air'
> >> objective is DESIGNED to overcome both chromatic and
> >> spherical aberrations both within the objective and
> >> between it and the surface of the specimen (normally
> >> glass). It should not be expected to overcome mismatches
> >> between specimen and mountant.
> >>
> >>> 2. Point spread function broadening
> >>>
> >>> This means that changes in axial scaling with increasing
> >>> depth are linear,
> >> as a correlate that as the depth increases the diameter
> >> of the PSF increases.
> >>
> >>> 3. Refractive index mismatch between the sample itself
> >>> and the mounting medium
> >>>
> >>> Back to (1). In the absence of knowledge of the R.I. of
> >>> the real specimen,
> >> your scaling factor is likely NOT usable, because you
> >> still don't know the REAL mismatch.
> >>
> >>> There is an empiric method that I have used in the past,
> >>> but it is
> >> initially NOT inexpensive. I have a set of R.I.
> >> standards from Cargille that I have used to determine the
> >> R.I. 'as close as my eye can determine'. I have a match
> >> when the specimen is most invisible in brightfield.
> >>
> >>> There is a lot of good information at:
> >> http://www.olympusmicro.com/primer/techniques/index.html
> >>
> >> Also, here:
> >> http://www.olympusmicro.com/primer/techniques/technojava.
> >> html where there is much on the interaction of light with
> >> specimen object.
> >>
> >>> I have imaged 3.4 um fluorescent beads under the same
> >>> mounting and imaging conditions as the sample and find
> >>> that I need to use a correction factor of ~0.5 to obtain
> >>> a near spherical reconstruction of the beads. I have a
> >>> paper (Visser and Oud, 1994, Scanning 16:198-200) which
> >>> states that axial distortion can be corrected for by
> >>> simply determining the ratio of the lateral and axial
> >>> sizes of the sphere and dividing all vertical dimensions
> >>> by this elongation factor. I've read several other
> >>> papers on axial distortion, but most deal with oil
> >>> immersion rather than air lenses, and I am not sure if
> >>> they apply to my particular conditions.
> >>>
> >>> Here you might want to refer to a recent offering on
> >>> this list: James
> >> Pawley,
> >> Subj: Re: Lo-Mag/Hi-NA lenses?
> >>
> >> And, here's a site with instructions for a c-APO water
> >> lens of the type that he mentioned:
> >> http://physiology.umaryland.edu/core/confocal/c-apo.htm
> >>
> >>> But also look at Total Internal Reflection Fluorescence
> >>> Microscopy at:
> >> http://www.olympusmicro.com/primer/techniques/fluorescenc
> >> e/tirf/tirfhome.htm l
> >>
> >>> My general question is: am I approaching this problem
> >>> in a reasonable way without knowing the refractive index
> >>> of the material composing the specimen itself? My
> >>> background is primarily in electron optics, so I
> >>> apologize in advance if my understanding of light optics
> >>> is a bit sketchy.
> >>>
> >>> In SEM the question is moot. In TEM, most of us don't
> >>> have much experience
> >> with using the instrument in a phase mode, but the
> >> explanations would be the same. In getting at the
> >> resolution along the z-axis, R.I. matching is a critical
> >> matter. Thus, if you can determine the R.I. of the
> >> unknown, you will be in the game, though, that knowledge
> >> may NOT provide an inexpensive path to success. It is
> >> clear that you have a material that is at least
> >> semi-transparent with embedded parts that are fluorescent
> >> or at least reflective - you can tell the difference. I
> >> have methacrylate corrosion casts of microvasculatures
> >> that happen to be laced with a dye that is fluorescent.
> >> If I immerse the thing in water, the R.I. mismatches are
> >> unreasonable. If, however, I immerse it in one of my
> >> Cargille oils at the R.I. of methacrylate, then I get
> >> great results. In that way, knowing the R.I. of the
> >> specimen is VERY helpful, and even at low powers
> >> z-distortions appear to be minimal.
> >>
> >>> I think of the problem this way. If I was given a
> >>> specimen with five
> >> sandwiched layers of material each of identical thickness
> >> and each with a different R.I. How could I correctly
> >> achieve an essentially accurate reconstruction if my view
> >> was through the layers rather than from the 'side'. A
> >> vertical section would likely provide a precise answer in
> >> TEM, even when using phase mode (I think!), but a
> >> through-focus series of a thick section, or of the whole
> >> sandwich, might not provide the same precision. This
> >> problem points out the value of using confocal imaging
> >> for examination of specimens with fluorescent parts that
> >> are otherwise transparent - i.e. matched with the R.I. of
> >> the mounting medium. When the 'specimen' is effectively
> >> transparent, the fluorescent object becomes a volume of
> >> point sources of light.
> >>
> >> Final comment. It is curious that in discussions of this
> >> type, including the one referenced above, any mismatch
> >> between the real specimen and its immediate environment
> >> is ignored. The "immersion" fluid (that between
> >> objective and coverglass) is considered primarily
> >> responsible for any mismatch. Cells and aqueous media
> >> are considered to have similar R.I.'s, and dehydrated
> >> tissues are said to have R.I.'s closer to that of glass,
> >> but plastics can be below that of glass. A water
> >> immersion objective, corrected for use with a coverglass,
> >> is considered to make the coverglass transparent (or
> >> negligible?) in a preparation with a specimen in water
> >> covered with a coverglass. The water permits the lens to
> >> 'gather'/accept a larger cone of light and thus, provide
> >> greater lateral resolution. In questions about the
> >> z-axis, one is always trying to decide if the focus for
> >> the exciting light is in the same position as that for
> >> the emitted light, since the objective lens is performing
> >> both functions. The concern for this is greater for
> >> multi-photon microscopy, because the wavelength of the
> >> exciting light is often much longer than that of the
> >> emitted light. R.I. mismatching only exacerbates the
> >> misalignment of focus points on the z-axis for different
> >> wavelengths of light. R.I. matching for specimen and
> >> surround is similar to correcting that space for
> >> chromatic aberration.
> >>
> >>> Any comments would be greatly appreciated.
> >>>
> >>> All best,
> >>>
> >>> Angela
> >>> -----------------------------------------
> >>> Angela V. Klaus, Ph.D.
> >>> Director, Microscopy and Imaging Facility
> >>> American Museum of Natural History
> >>> Central Park West at 79th Street
> >>> New York, NY 10024 USA
> >>> Email: [log in to unmask]
> >>> Tel: 212-769-5977
> >>> Fax: 212-496-3480
> >>> -----------------------------------------
> >>>
> >>>
> >
> > --
> > ****************************************
> > 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] "A scientist is not
> > one who can answer questions but one who can question
> > answers." Theodore Schick Jr., Skeptical Enquirer,
> > 21-2:39
> >
Jurek Dobrucki, PhD
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