registration marks on slides

[Kelly Lundsten-3]

*****
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,
Looking for any suggestions for a good product that would allow slides to be removed and replaced on the stage with registration marks embedded in the slide to enable re-alignment of images later via photoshop or another image analysis platform.
 
Any ideas?

Thanks,
Kelly Lundsten

[Craig Brideau]

*****
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.
*****

Slides with embedded cell counting grid, or mark the back of the slide or
top of the coverslip with permanent marker or a scribe?
Craig

On Wed, Aug 7, 2019 at 4:35 PM Kelly Lundsten <
[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 all,
> Looking for any suggestions for a good product that would allow slides to
> be removed and replaced on the stage with registration marks embedded in
> the slide to enable re-alignment of images later via photoshop or another
> image analysis platform.
>
> Any ideas?
>
> Thanks,
> Kelly Lundsten
>

[leavesley]

*****
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 Kelly, Craig,

We have used gridded coverslips from both Electron Microscopy Sciences
and Ibidi.  The etching on the Ibidi coverslips seems more clear and
also shows up in most fluorescence channels, which is helpful, but they
are only made in 28 mm round diameter, which is wider than a standard
slide and may not work with all types of stage inserts (assuming that
you're mounting the coverslip on a slide).

Of note, both of these options are labeled so that you can reproducibly
find locations (e.g., the cells in grid C7, F5, etc.).  We have used
these for comparing single cell imaging results between multiple systems.

Best regards,

Silas


On 8/7/2019 6:07 PM, Craig Brideau 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.
> *****
>
> Slides with embedded cell counting grid, or mark the back of the slide or
> top of the coverslip with permanent marker or a scribe?
> Craig
>
> On Wed, Aug 7, 2019 at 4:35 PM Kelly Lundsten <
> [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 all,
>> Looking for any suggestions for a good product that would allow slides to
>> be removed and replaced on the stage with registration marks embedded in
>> the slide to enable re-alignment of images later via photoshop or another
>> image analysis platform.
>>
>> Any ideas?
>>
>> Thanks,
>> Kelly Lundsten
>>

--
Silas J. Leavesley, Ph.D.
Professor
Department of Chemical and Biomolecular Engineering
Department of Pharmacology
Center for Lung Biology
University of South Alabama
150 Jaguar Drive, SH4129
Mobile, AL 36688
ph: (251)-460-6160
fax: [phone]
web: http://www.southalabama.edu/centers/bioimaging

[Sylvie Le Guyader]

*****
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.
*****

Zell kontakt makes dishes with grids. http://www.zell-kontakt.de/products/imaging-dish/imaging-dish-nano-grid/

Med vänlig hälsning / Best regards

Sylvie

@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Hälsovägen 7C,
Room 7362 (lab)/7840 (office)
14157 Huddinge, Sweden
mobile: [phone]
LCI website
Follow our microscopy blog!

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Silas Leavesley
Sent: 08 August 2019 16:20
To: [hidden email]
Subject: Re: registration marks on slides

*****
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 Kelly, Craig,

We have used gridded coverslips from both Electron Microscopy Sciences and Ibidi.  The etching on the Ibidi coverslips seems more clear and also shows up in most fluorescence channels, which is helpful, but they are only made in 28 mm round diameter, which is wider than a standard slide and may not work with all types of stage inserts (assuming that you're mounting the coverslip on a slide).

Of note, both of these options are labeled so that you can reproducibly find locations (e.g., the cells in grid C7, F5, etc.).  We have used these for comparing single cell imaging results between multiple systems.

Best regards,

Silas


On 8/7/2019 6:07 PM, Craig Brideau 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.
> *****
>
> Slides with embedded cell counting grid, or mark the back of the slide
> or top of the coverslip with permanent marker or a scribe?
> Craig
>
> On Wed, Aug 7, 2019 at 4:35 PM Kelly Lundsten <
> [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 all,
>> Looking for any suggestions for a good product that would allow
>> slides to be removed and replaced on the stage with registration
>> marks embedded in the slide to enable re-alignment of images later
>> via photoshop or another image analysis platform.
>>
>> Any ideas?
>>
>> Thanks,
>> Kelly Lundsten
>>

--
Silas J. Leavesley, Ph.D.
Professor
Department of Chemical and Biomolecular Engineering Department of Pharmacology Center for Lung Biology University of South Alabama
150 Jaguar Drive, SH4129
Mobile, AL 36688
ph: (251)-460-6160
fax: [phone]
web: http://www.southalabama.edu/centers/bioimaging


När du skickar e-post till Karolinska Institutet (KI) innebär detta att KI kommer att behandla dina personuppgifter. Här finns information om hur KI behandlar personuppgifter<https://ki.se/medarbetare/integritetsskyddspolicy>.


Sending email to Karolinska Institutet (KI) will result in KI processing your personal data. You can read more about KI’s processing of personal data here<https://ki.se/en/staff/data-protection-policy>.

[dbstongy]

*****
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 List,
We would like to evaluate our newly installed confocal system with airyscan using Invitrogen 100nm tetra beads.
May I have your kind advice on the tolerance of the chromatic aberration of such a system? And for airyscan confocal, is there a better tool than tetrabeads for the evaluation of system performance, in terms of its super resolution (FWHM measurement) as well as chromatic aberration?
Thank you very much for your advice and sharing in advance.
With best regards,
Iris Zhong (Ms.)
Department of Biological Sciences
National University of Singapore


________________________________

Important: This email is confidential and may be privileged. If you are not the intended recipient, please delete it and notify us immediately; you should not copy or use it for any purpose, nor disclose its contents to any other person. Thank you.

[George McNamara]

*****
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.
*****

Spherotech Ultra Rainbow 8 colors, excite 405 nm - 730 nm (our FV3000RS
has 6 laser lines) emission ~420 - >800 nm (FV3000RS detector #6 has an
780LP, http://confocal.jhu.edu/current-equipment/fv3000 ).

https://www.spherotech.com/AlignmentParticles.htm

URFP-02-2 ... Ultra Rainbow Fluorescent Particles ... 10^10/mL ...
0.1-0.3 µm ... 2 mL ... $245.00

Note: other sizes, volumes available, including product(s) with several
different intensities, I have some data (not shown) for dynamic range of
our confocals [and in future widefield FISHscope) (see product PDFs
below table at web page).

vs mentioned

TetraSpeck ... $276 for 0.5 mL of 0.1 um diameter ...
https://www.thermofisher.com/order/catalog/product/T7279

"TetraSpeck™ microspheres are stained throughout with four different­
fluorescent dyes, yielding beads that each display four well-separated
excitation/emission peaks—365/430 nm (blue), 505/515 nm (green), 560/580
nm (orange), and 660/680 nm (dark red). "

On 8/9/2019 9:27 AM, Tong Yan 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 List,
> We would like to evaluate our newly installed confocal system with airyscan using Invitrogen 100nm tetra beads.
> May I have your kind advice on the tolerance of the chromatic aberration of such a system? And for airyscan confocal, is there a better tool than tetrabeads for the evaluation of system performance, in terms of its super resolution (FWHM measurement) as well as chromatic aberration?
> Thank you very much for your advice and sharing in advance.
> With best regards,
> Iris Zhong (Ms.)
> Department of Biological Sciences
> National University of Singapore
>
>
> ________________________________
>
> Important: This email is confidential and may be privileged. If you are not the intended recipient, please delete it and notify us immediately; you should not copy or use it for any purpose, nor disclose its contents to any other person. Thank you.

[dbstongy]

*****
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 George,

Thank you very much for the resourceful information.

One more thing about the confocal system evaluation is the chromatic aberration. Normally how much is the lateral and axial shift among all the four colours of a confocal/airyscan system?

Best regards

Iris

> On 11 Aug 2019, at 4:30 AM, George McNamara <[hidden email]> wrote:
>
> Spherotech Ultra Rainbow 8 colors, excite 405 nm - 730 nm (our FV3000RS has 6 laser lines) emission ~420 - >800 nm (FV3000RS detector #6 has an 780LP, http://confocal.jhu.edu/current-equipment/fv3000 ).
>
> https://www.spherotech.com/AlignmentParticles.htm
>
> URFP-02-2 ... Ultra Rainbow Fluorescent Particles ... 10^10/mL ... 0.1-0.3 µm ... 2 mL ... $245.00
>
> Note: other sizes, volumes available, including product(s) with several different intensities, I have some data (not shown) for dynamic range of our confocals [and in future widefield FISHscope) (see product PDFs below table at web page).
>
> vs mentioned
>
> TetraSpeck ... $276 for 0.5 mL of 0.1 um diameter ... https://www.thermofisher.com/order/catalog/product/T7279
>
> "TetraSpeck™ microspheres are stained throughout with four different­ fluorescent dyes, yielding beads that each display four well-separated excitation/emission peaks—365/430 nm (blue), 505/515 nm (green), 560/580 nm (orange), and 660/680 nm (dark red). "
>
>> On 8/9/2019 9:27 AM, Tong Yan 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 List,
>> We would like to evaluate our newly installed confocal system with airyscan using Invitrogen 100nm tetra beads.
>> May I have your kind advice on the tolerance of the chromatic aberration of such a system? And for airyscan confocal, is there a better tool than tetrabeads for the evaluation of system performance, in terms of its super resolution (FWHM measurement) as well as chromatic aberration?
>> Thank you very much for your advice and sharing in advance.
>> With best regards,
>> Iris Zhong (Ms.)
>> Department of Biological Sciences
>> National University of Singapore
>>
>>
>> ________________________________
>>
>> Important: This email is confidential and may be privileged. If you are not the intended recipient, please delete it and notify us immediately; you should not copy or use it for any purpose, nor disclose its contents to any other person. Thank you.

________________________________

Important: This email is confidential and may be privileged. If you are not the intended recipient, please delete it and notify us immediately; you should not copy or use it for any purpose, nor disclose its contents to any other person. Thank you.

[George McNamara]

*****
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 Iris,

Chromatic aberration: Yes.

Some web links are:

new Olympus X-Line objective lenses, for example the teaser page,

https://www.olympus-lifescience.com/en/landing/x-line/

See online and/or local representatives, for more info on X-Line, most
are corrected (see O' content for details) for 400-1000 nm, a couple to
~450 - ~650 nm.

You can also discuss microscope aberrations with the local
representatives of Zeiss with respect to AiryScan (1) and AiryScan 2,
and whatever other confocal and super duper resolution microscopes you have.

See Brown & Cole led papers, especially PubMed hits #3 and #4,

https://www.ncbi.nlm.nih.gov/pubmed/?term=brown+c+cole+r

//

Not just the objective lenses are in play, for good and/or bad. I had
never paid much attention to this one:

Zeiss does some correction(s) in the tube lens, whereas the other major
microscope companies do all their corrections in the objective lens.

So: how good is the Zeiss tube lens?

Has Zeiss changed (improved? cheapened?) the tube lens in the 'couple of
decades' since it would have been introduced with their 'infinity'
corrected objective lenses?

With respect to some or all of the vendors, any glass in the
epi-illumination path could also impact the excitation side quality of
widefield fluorescence (and reflection) microscopes.


enjoy,

George

On 8/10/2019 6:40 PM, Tong Yan wrote:

> Dear George,
>
> Thank you very much for the resourceful information.
>
> One more thing about the confocal system evaluation is the chromatic aberration. Normally how much is the lateral and axial shift among all the four colours of a confocal/airyscan system?
>
> Best regards
>
> Iris
>
>> On 11 Aug 2019, at 4:30 AM, George McNamara <[hidden email]> wrote:
>>
>> Spherotech Ultra Rainbow 8 colors, excite 405 nm - 730 nm (our FV3000RS has 6 laser lines) emission ~420 - >800 nm (FV3000RS detector #6 has an 780LP, http://confocal.jhu.edu/current-equipment/fv3000 ).
>>
>> https://www.spherotech.com/AlignmentParticles.htm
>>
>> URFP-02-2 ... Ultra Rainbow Fluorescent Particles ... 10^10/mL ... 0.1-0.3 µm ... 2 mL ... $245.00
>>
>> Note: other sizes, volumes available, including product(s) with several different intensities, I have some data (not shown) for dynamic range of our confocals [and in future widefield FISHscope) (see product PDFs below table at web page).
>>
>> vs mentioned
>>
>> TetraSpeck ... $276 for 0.5 mL of 0.1 um diameter ... https://www.thermofisher.com/order/catalog/product/T7279
>>
>> "TetraSpeck™ microspheres are stained throughout with four different­ fluorescent dyes, yielding beads that each display four well-separated excitation/emission peaks—365/430 nm (blue), 505/515 nm (green), 560/580 nm (orange), and 660/680 nm (dark red)."
>>
>>> On 8/9/2019 9:27 AM, Tong Yan 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 List,
>>> We would like to evaluate our newly installed confocal system with airyscan using Invitrogen 100nm tetra beads.
>>> May I have your kind advice on the tolerance of the chromatic aberration of such a system? And for airyscan confocal, is there a better tool than tetrabeads for the evaluation of system performance, in terms of its super resolution (FWHM measurement) as well as chromatic aberration?
>>> Thank you very much for your advice and sharing in advance.
>>> With best regards,
>>> Iris Zhong (Ms.)
>>> Department of Biological Sciences
>>> National University of Singapore
>>>
>>>
>>> ________________________________
>>>
>>> Important: This email is confidential and may be privileged. If you are not the intended recipient, please delete it and notify us immediately; you should not copy or use it for any purpose, nor disclose its contents to any other person. Thank you.
> ________________________________
>
> Important: This email is confidential and may be privileged. If you are not the intended recipient, please delete it and notify us immediately; you should not copy or use it for any purpose, nor disclose its contents to any other person. Thank you.

[Steffen Dietzel]

*****
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,

when I tried 0.1 µm TetraSpec beads many years back, I found them not
bright enough and bleaching very fast. They may have improved since then.

For PSF measurements, we found that 80 nm gold beads are much more
convenient than fluorescent beads: no bleaching and much brighter
signals to start with (DOI:10.1017/S1431927619000060
<https://doi.org/10.1017/S1431927619000060>). Your instrument must allow
reflection confocal microscopy. With simple confocals using glass
filters as dichroics this is usually not a problem since the dichroic
will let pass enough of the 'excitation' light. You have to be able to
take out the detection filter or set you spectral scanning unit to the
'excitation' wavelength.

Obviously, if you do the reflection PSF for several wavelengths, you
also can check for chromatic aberration. Strictly speaking your
fluorescent channels will be a little bit different, since the detection
wavelength is a few dozens nm longer than the reflection.

As for expected results, Leica made a huge point that their 100x STED
white objective has a chromatic aberration of less than 50 nm in z. In
conclusion, 'normal' objectives will have considerably more. I would
expect up to a few 100 nm in z for NA 1.4 objectives and more for
smaller NAs. In that context let me add that we found that the immersion
oil also may have a considerable impact on the chromatic aberration. I'd
suggest to only use the oil recommended by the manufacturer of the
system. Or else test for chromatic aberration (and resolution).

Best

Steffen

Am 11.08.2019 um 01:55 schrieb George McNamara:

> *****
> 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 Iris,
>
> Chromatic aberration: Yes.
>
> Some web links are:
>
> new Olympus X-Line objective lenses, for example the teaser page,
>
> https://www.olympus-lifescience.com/en/landing/x-line/
>
> See online and/or local representatives, for more info on X-Line, most
> are corrected (see O' content for details) for 400-1000 nm, a couple
> to ~450 - ~650 nm.
>
> You can also discuss microscope aberrations with the local
> representatives of Zeiss with respect to AiryScan (1) and AiryScan 2,
> and whatever other confocal and super duper resolution microscopes you
> have.
>
> See Brown & Cole led papers, especially PubMed hits #3 and #4,
>
> https://www.ncbi.nlm.nih.gov/pubmed/?term=brown+c+cole+r
>
> //
>
> Not just the objective lenses are in play, for good and/or bad. I had
> never paid much attention to this one:
>
> Zeiss does some correction(s) in the tube lens, whereas the other
> major microscope companies do all their corrections in the objective
> lens.
>
> So: how good is the Zeiss tube lens?
>
> Has Zeiss changed (improved? cheapened?) the tube lens in the 'couple
> of decades' since it would have been introduced with their 'infinity'
> corrected objective lenses?
>
> With respect to some or all of the vendors, any glass in the
> epi-illumination path could also impact the excitation side quality of
> widefield fluorescence (and reflection) microscopes.
>
>
> enjoy,
>
> George
>
> On 8/10/2019 6:40 PM, Tong Yan wrote:
>> Dear George,
>>
>> Thank you very much for the resourceful information.
>>
>> One more thing about the confocal system evaluation is the chromatic
>> aberration. Normally how much is the lateral and axial shift among
>> all the four colours of a confocal/airyscan system?
>>
>> Best regards
>>
>> Iris
>>
>>> On 11 Aug 2019, at 4:30 AM, George McNamara
>>> <[hidden email]> wrote:
>>>
>>> Spherotech Ultra Rainbow 8 colors, excite 405 nm - 730 nm (our
>>> FV3000RS has 6 laser lines) emission ~420 - >800 nm (FV3000RS
>>> detector #6 has an 780LP,
>>> http://confocal.jhu.edu/current-equipment/fv3000 ).
>>>
>>> https://www.spherotech.com/AlignmentParticles.htm
>>>
>>> URFP-02-2 ... Ultra Rainbow Fluorescent Particles ... 10^10/mL ...
>>> 0.1-0.3 µm ... 2 mL ... $245.00
>>>
>>> Note: other sizes, volumes available, including product(s) with
>>> several different intensities, I have some data (not shown) for
>>> dynamic range of our confocals [and in future widefield FISHscope)
>>> (see product PDFs below table at web page).
>>>
>>> vs mentioned
>>>
>>> TetraSpeck ... $276 for 0.5 mL of 0.1 um diameter ...
>>> https://www.thermofisher.com/order/catalog/product/T7279
>>>
>>> "TetraSpeck™ microspheres are stained throughout with four
>>> different­ fluorescent dyes, yielding beads that each display four
>>> well-separated excitation/emission peaks—365/430 nm (blue), 505/515
>>> nm (green), 560/580 nm (orange), and 660/680 nm (dark red)."
>>>
>>>> On 8/9/2019 9:27 AM, Tong Yan 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 List,
>>>> We would like to evaluate our newly installed confocal system with
>>>> airyscan using Invitrogen 100nm tetra beads.
>>>> May I have your kind advice on the tolerance of the chromatic
>>>> aberration of such a system? And for airyscan confocal, is there a
>>>> better tool than tetrabeads for the evaluation of system
>>>> performance, in terms of its super resolution (FWHM measurement) as
>>>> well as chromatic aberration?
>>>> Thank you very much for your advice and sharing in advance.
>>>> With best regards,
>>>> Iris Zhong (Ms.)
>>>> Department of Biological Sciences
>>>> National University of Singapore
>>>>
>>>>
>>>> ________________________________
>>>>
>>>> Important: This email is confidential and may be privileged. If you
>>>> are not the intended recipient, please delete it and notify us
>>>> immediately; you should not copy or use it for any purpose, nor
>>>> disclose its contents to any other person. Thank you.
>> ________________________________
>>
>> Important: This email is confidential and may be privileged. If you
>> are not the intended recipient, please delete it and notify us
>> immediately; you should not copy or use it for any purpose, nor
>> disclose its contents to any other person. Thank you.
>

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Biomedical Center (BMC)
Head of the Core Facility Bioimaging

Großhaderner Straße 9
D-82152 Planegg-Martinsried
Germany

http://www.bioimaging.bmc.med.uni-muenchen.de