http://confocal-microscopy-list.275.s1.nabble.com/Protective-window-for-a-confocal-microscope-tp7591445p7591487.html
I purchased the item #47-838 which is a 30-mm VIS-coated silica window with the parameters:
thickness 4mm, parallelism <5 arcsec, surface quality 20-10, and transmitted wavefront lambda/10.
1.) the opening in the filter holder is tilted, so the protective window after the installation is also tilted in the optic path,
2.) the protective filter is 30mm in diameter, which is a bit small for filter holder (32 mm), and to make it sit tight I will have to order an appropriate O-ring. Only then the protective window should provide full protection against dripping oil. But even without the O-ring, the protective window is larger than the aperture of the filter slider, so it sits nicely in place.
3.) I checked the PSFs of 200nm beads, and I cannot tell the difference in images with and without protective window. I could not measure resolutions, because I slightly overexposed the PSFs, but PSF shapes in xy, xz, and yz planes looked the same to me.
4.) I noticed a slight increase in lateral shift between the green and far red channels: from ca. 30 nm to ca. 50 nm. This is a bit surprising to me, as there should be no bending of rays, and besides the Abbe number is not so bad (67.7). I did not check green vs red channel, because I do not have a proper prep at the moment.
In summary, I think the protective window is suitable for confocal imaging. For critical applications, it can be easily removed from the path. I feel much safer when this protective window is in the path, especially these days, when I am not so often at the institute because of COVID-19 situation.
If I discover any more disadvantages of this protective window, I will let you know.
Following advice of Zdenek (thank you!) on the importance of TWE I decided to order this:
Its transmitted wavefront is lambda/10, and it shows very good parallelism, surface finish and transmission for VIS light, which is sufficient for us.
I guess the opening for filters in the filter slider is angled, but I will check this. And the slider can be removed anytime from the optic path in no time, so it will be very easy to check the imaging with and without the protective window.
PS. In my previous mail I meant 40x/1.3 EC Plan-Neofluar lens rather than "Fluar", which is a different lens (and less suitable for confocal). So, I meant one thing but somehow wrote the other. Sorry for that.
filter in the infinity space. And, indeed, most flat surfaces (windows,
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> Post images on
http://www.imgur.com and include the link in your posting.
> *****
>
> Hi All,
>
> Staying on the question, in regards to putting a piece of glass in that
> slider (which would make our lives easier), should it be placed on an
> angle, to minimize reflections? But, will that increase aberration?
>
> Good question Tomasz.
>
> On Tue, Oct 27, 2020 at 7:46 PM Zdenek Svindrych <
[hidden email]>
> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> >
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> > Post images on
http://www.imgur.com and include the link in your
> posting.
> > *****
> >
> > Hi Tomasz,
> >
> > back to your question. The filter holder is there to hold a filter. Any
> > decent glass or fused silica AR window should do the trick.
> > As far as I remember, the hole is quite big, around 30 mm diameter
> (bigger
> > than the standard 25 mm filters), which may limit your options.
> >
> > Most filters and windows have specified surface flatness (reflected wave
> > error), but it's actually transmitted wave error (TWE) that matters here.
> > Usually, windows with single digit flatness (in lambda or fringe units)
> > will have TWE << 1/5 lambda, and should be OK.
> >
> > Thicker substrate (2 - 3 mm) usually means precision optics, and is a
> safe
> > bet. Hard coating is a must, if you want to wipe oil off of it regularly.
> > All reputable brands use hard coatings these days, 1% loss (per pass) is
> > OK.
> >
> > Sapphire is hard to work with and comes with a price premium. Fused
> silica,
> > e.g.
> >
> >
>
https://www.edmundoptics.com/p/30mm-dia-vis-nir-coated-1lambda-fused-silica-window/10158/> > (no commercial interest) looks like a good option, but BK-7 will work
> well,
> > too.
> >
> > If you happen to have a piece of AR-coated glass at hand, just put it
> there
> > and look at some beads. If you're not able to tell the difference, you're
> > good to go. You can even try a glass coverslip, the 4% loss (the
> excitation
> > loss is not an issue, you can always crank up the laser) will be hardly
> > noticeable. Coverslips are not polished, so the TWE is generally bad, but
> > with some luck you can find a good one in the stack...
> >
> > Best, zdenek
> >
> >
> > On Tue, Oct 27, 2020 at 1:18 PM Tomek Węgierski <
[hidden email]>
> > wrote:
> >
> > > *****
> > > To join, leave or search the confocal microscopy listserv, go to:
> > >
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> > > Post images on
http://www.imgur.com and include the link in your
> > posting.
> > > *****
> > >
> > > Dear All,
> > >
> > > Thank you very much for so many responses, although the discussion
> went a
> > > bit into a direction that I did not anticipate ??
> > > But yes, it is all about preventing oil from getting into the
> microscope.
> > > Our 40x/1.3 Fluar lens takes a lot of oil and I frequently have to
> clean
> > > its bottom side from oil. In contrast, I never saw any oil on the back
> of
> > > 63x/1.4 even though it is used as frequently as the 40x.
> > >
> > > I have to admit I have been skeptical about hair elastics, because when
> > > they saturate with oil they will just let it go. IMHO, it is replacing
> > "oil
> > > cleaning" approach with "oil buffering" approach. The "cleaning"
> approach
> > > has worked for us for many years. It failed when an unexperienced user
> > (who
> > > apparently did not take seriously what I was talking during the
> training)
> > > made two mistakes: did not clean the lens and let it stay in the optic
> > path
> > > after the work instead of changing for 10x. In addition, it was a
> holiday
> > > time, the system was not used for the next few days, giving the oil
> > plenty
> > > of time. So, it was a coincidence of several factors, but it can happen
> > > again.
> > >
> > > I think, hearing so many positive opinions about hair elastics, I will
> > > give them the 2nd chance in our facility. Aqua-Stop in my opinion is
> > > overpriced, and we do not currently do any experiments under perfusion
> to
> > > justify such purchase (this may change in the future). However, I would
> > not
> > > like to give up on the protective window strategy. I think placing a
> > glass
> > > element in the infinity space is not unusual. It would function as the
> > last
> > > stronghold against the oil. And it would be considerably cheaper than
> > > Aqua-Stop. I would of course test PSF. If PSF is not affected, I guess
> > the
> > > protective window is OK with imaging. So, if you still have some advice
> > > which one would be most suitable, I would appreciate it.
> > >
> > > Best,
> > > Tomasz
> > >
> > > Tomasz Wegierski, PhD
> > > International Institute of Molecular and Cell Biology
> > > Trojdena 4, 02-109 Warsaw, POLAND
> > > tel: +48-22 597 0763
> > > fax: +48 22 597 0715
> > >
http://www.iimcb.gov.pl/> > > ________________________________
> > > From: Confocal Microscopy List <
[hidden email]> on
> > > behalf of Frohlich, Victoria <
[hidden email]>
> > > Sent: Tuesday, October 27, 2020 4:38 PM
> > > To:
[hidden email] <
[hidden email]
> >
> > > Subject: Re: Protective window for a confocal microscope?
> > >
> > > *****
> > > To join, leave or search the confocal microscopy listserv, go to:
> > >
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> > > Post images on
http://www.imgur.com and include the link in your
> > posting.
> > > *****
> > >
> > > Just my 2 cents.
> > > Scrunchies work well but are not fool-proof. Cotton not polyester
> > > material are more absorbent.
> > > In addition to scrunchies I suggest wrapping the lens barrel near the
> > nose
> > > of the objective with plumbers tape to prevent oil from seeping into
> the
> > > barrel. It can be done so that the lens retraction still functions.
> > Then
> > > once the lens is on the nosepiece of the scope the plumbers tape can be
> > > wrapped there as well.
> > >
> > > I've also used thin rubber sheets or parafilm sheets, as well as filter
> > > paper (thanks Mike S.!), to draw oil away from the lens and nosepiece.
> > >
> > > Best method, however, is to teach users proper oil application and
> ensure
> > > they clean up after themselves.
> > >
> > > Regards,
> > > Vickie
> > >
> > > Victoria Centonze Frohlich, PhD
> > > Director, Light Microscopy Shared Resource
> > > Cell & Tissue Imaging Center
> > > St. Jude Children’s Research Hospital
> > > 262 Danny Thomas Place
> > > MS 312
> > > Memphis, TN 38105
> > >
> > > Office: D1052D
> > > Phone and Digital Fax: 1-901-595-2536
> > > Cell: 1-901-907-2596
> > > Paper Fax: 1-901-595-2909
> > > Email:
[hidden email]
> > >
> > > CTIC-LM Location: D1055 and D1056
> > > Facility Phone: 1-901-595-3439
> > >
> > >
http://home.stjude.org/cell-tissue-imaging/Pages/default.aspx> > >
> > > Facility Acknowledgement for publications:
> > > “Images were acquired at the Cell & Tissue Imaging Center which is
> > > supported by SJCRH and NCI P30 CA021765.”
> > >
> > > -----Original Message-----
> > > From: Confocal Microscopy List <
[hidden email]> On
> > > Behalf Of Michael Stanley
> > > Sent: Tuesday, October 27, 2020 8:19 AM
> > > To:
[hidden email]
> > > Subject: Re: Protective window for a confocal microscope?
> > >
> > > Caution: External Sender
> > >
> > >
> > > *****
> > > To join, leave or search the confocal microscopy listserv, go to:
> > >
> > >
> >
>
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> > >
> >
>
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> > > *****
> > >
> > > in the 'old days' in the core we used to cut filter paper and then
> bore a
> > > hole with a cork-bore slightly smaller than the lens. slide 2-3 (or as
> > > many as will fit) layers of the filter paper over the objective. this
> > will
> > > not stop a "flood" of oil (which sounds very possible for some) but it
> > will
> > > hold much more than you would think. and, the most important feature
> of
> > > this little trick is that the oil will show very quickly on the paper
> and
> > > hopefully will trigger a response.!
> > >
> > > some of the darker filter papers will show the oil better, but this
> will
> > > vary by manufacturer.
> > >
> > > no commercial interest here, just trying to protect all the optics in
> the
> > > system.
> > >
> > > michael
> > >
> > > c. michael stanley, phd
> > > senior applications scientist
> > > chroma technology corp.
> > > 10 imtec lane
> > > bellows falls, vt 05101
> > >
[hidden email]
> > >
[hidden email]
> > >
> > >
> > > -----Original Message-----
> > > From: Confocal Microscopy List <
[hidden email]> On
> > > Behalf Of Gary Laevsky
> > > Sent: Tuesday, October 27, 2020 10:19 AM
> > > To:
[hidden email]
> > > Subject: Re: Protective window for a confocal microscope?
> > >
> > > *****
> > > To join, leave or search the confocal microscopy listserv, go to:
> > >
> > >
> >
>
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> > >
> >
>
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> > > *****
> > >
> > > The hair ties work great with objectives that have enough of a neck for
> > > the tie to sit on, next to the barrel.
> > >
> > > Our 40X objective (most frequently used) has no/very small neck, so the
> > > hair ties don't work.
> > >
> > > I fairly regularly (bi-weekly) have to take the objective off, store it
> > in
> > > it's case upside down, and put it in a 37C oven. Ridiculous, in a bad
> > way,
> > > how much oil comes out. The 40X also doesn't have "lip" that acts as a
> > bit
> > > of a reservoir (unlike the 60 and 100).
> > >
> > > On one of my heavily used systems, I have to go so far as to dismantle
> > the
> > > top (stage, objective turret, and fluorescent turret) to get to the
> piece
> > > of glass that prevents leakage into the body (amazingly awesome feature
> > of
> > > the stand). Takes me 15 minutes. Although I shouldn't have to do that
> > ...
> > >
> > > On Tue, Oct 27, 2020 at 9:13 AM Cammer, Michael <
> > >
[hidden email]> wrote:
> > >
> > > > *****
> > > > To join, leave or search the confocal microscopy listserv, go to:
> > > >
>
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> > > > ictoria.frohlich%40STJUDE.ORG
> %7C6b89a1a0f4cc42f5937e08d87a8d1047%7C223
> > > >
> 40fa892264871b677d3b3e377af72%7C0%7C0%7C637394093573459000%7CUnknown%7
> > > >
> CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXV
> > > >
> CI6Mn0%3D%7C1000&sdata=q2oFBgmsbEfra%2BIBWB6i8xwEJ%2B8SF%2F59WSo3Y
> > > > %2BKvFKg%3D&reserved=0 Post images on
> > > >
> > >
> >
>
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> > > > *****
> > > >
> > > > Aquastop works well for big spills when installed correctly and has
> > > > proven effective on some of our Zeiss scopes. But we did have a user
> > > > who spilled over or around it on our Zeiss 710, so it is not
> foolproof.
> > > >
> > > >
> > > > Hair elastics are great for the oil.
> > > >
> > > >
> > > > Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory
> > > >
> > > > NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York,
> > > > NY
> > > > 10016
> > > >
> > > >
[hidden email]<mailto:
[hidden email]>
> > > >
>
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> > > >
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> > > >
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> > > >
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> > > >
> > > > Voice direct only, no text or messages: 1-914-309-3270 and
> > > > 1-646-501-0567
> > > >
> > > >
> > > >
> > > > ________________________________
> > > > From: Confocal Microscopy List <
[hidden email]> on
> > > > behalf of Doug Richardson <
[hidden email]>
> > > > Sent: Tuesday, October 27, 2020 7:41:45 AM
> > > > To:
[hidden email]
> > > > Subject: Re: Protective window for a confocal microscope?
> > > >
> > > > [EXTERNAL]
> > > >
> > > > *****
> > > > To join, leave or search the confocal microscopy listserv, go to:
> > > >
> > > >
>
https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-> > > >
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> > > >
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> > > > Pgwni6bBeSRyqYRAE&e=
> > > > Post images on
> > > >
>
https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=Dw> > > >
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> > > > and include the link in your posting.
> > > > *****
> > > >
> > > > I would highly recommend the aquastop system from Zeiss. It is
> > > > overpriced for some pieces of plastic, but it forms a very tight seal
> > > > and pays for itself many times over. It is far superior to hair
> > > > elastics/glove fingers/condoms which we used in the past.
> > > >
> > > > Doug
> > > >
> > > > Get Outlook for Android
> > > >
> > > > ________________________________
> > > > From: Confocal Microscopy List <
[hidden email]> on
> > > > behalf of Tomek Węgierski <
[hidden email]>
> > > > Sent: Tuesday, October 27, 2020 7:12:10 AM
> > > > To:
[hidden email]
> > > > <
[hidden email]>
> > > > Subject: Protective window for a confocal microscope?
> > > >
> > > > *****
> > > > To join, leave or search the confocal microscopy listserv, go to:
> > > >
> > > >
>
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> > > > Post images on
> > > >
>
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> > > > and include the link in your posting.
> > > > *****
> > > >
> > > > Dear All,
> > > > I am considering a purchase of a protective window for our Zeiss
> > > > LSM800 and I would like to hear your advice whether this is a good
> > > > idea, and if yes, which one to buy.
> > > > The reason is our LSM800 is on an inverted stand and we already got a
> > > > leakage of oil once which went down onto Optovar. And this was
> despite
> > > > my regular checks of objectives (both sides) for leaking oil and
> > > > extensive education of users on how important it is not to use too
> > > > much oil and to clean lenses, especially on inverted stands.
> > > > Zeiss Observer has a dummy slider with an opening for 32 mm filters.
> I
> > > > hope, a protective window there should stop the oil, if the leakage
> > > > happens again.
> > > > 1) do you think placing a protective window in a confocal microscope
> > > > is a good idea or not really, because the imaging will be affected?
> > > > This would be in infinity space, so I guess it should not be
> > > > detrimental....
> > > > 2) which characteristics of protective window I should take into
> > > > consideration (substrate, thickness, coating, flatness, surface
> > > > quality, parallelism)? Currently I am inclined into protective
> glasses
> > > > by Edmund Optics such as 1-mm thick MgF2-coated Sapphire window,
> which
> > > > has high transmission, or 2-mm thick MgF2-coated B270 window, which
> > > > has a bit worse transmission but better surface parameters (and is
> > > > much cheaper).
> > > > best regards,
> > > > Tomasz
> > > >
> > > >
> > > > Tomasz Wegierski, PhD
> > > > International Institute of Molecular and Cell Biology Trojdena 4,
> > > > 02-109 Warsaw, POLAND
> > > > tel: +48-22 597 0763
> > > > fax: +48 22 597 0715
> > > >
> > > >
>
https://urldefense.proofpoint.com/v2/url?u=http-3A__www.iimcb.gov.pl_&> > > >
> d=DwIFBA&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDi
> > > >
> PlC50tp7rW2nBkvV7fujQf0RknE5bU&m=keg1PHlpRuZ6hHabv7CvVJ2rsWSRa_2pkARNB
> > > > tS-dFQ&s=u7qHptX43byvVfMe-Zn04ev5KF4-ToW1ss01pq0eVtM&e=
> > > >
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> > >
> > > --
> > > Best,
> > >
> > > Gary Laevsky, Ph.D.
> > > Director, Confocal Imaging Facility
> > > Nikon Center of Excellence
> > > Co-Founder, North Atlantic Microscopy Society (NAMS)
> > >
> > >
> >
>
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> > --
> > --
> > Zdenek Svindrych, Ph.D.
> > Research Scientist - Microscopy Imaging Specialist
> > Department of Biochemistry and Cell Biology
> > Geisel School of Medicine at Dartmouth
> >
>
>
> --
> Best,
>
> Gary Laevsky, Ph.D.
> Director, Confocal Imaging Facility
> Nikon Center of Excellence
> Co-Founder, North Atlantic Microscopy Society (NAMS)
>
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> Princeton University
> Princeton, New Jersey, 08544-1014
> (O) 609 258 5432
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>