Re: fluorescent reference slides/beads for spinning disc - Vendor response

[Barbara Foster]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hi,

MME provides these slides, as well as several of our dealers,
including SPI and Ted Pella.

I see that the link to the spectra has disappeared from the website
(www.MicroscopyEducation.com) but can re-install over the weekend for
those of you who are interested.  In the meantime, if you need the
spectra, I can email to those who contact me (off-line).

These slides were designed originally by Dr. P. C. Cheng  of
SUNY/Buffalo and were tested extensively in his classes for over a
year.  Both confocal and regular fluorescence users have found them
valuable for testing intensity, laser settings, and field flatness.
Again, answers to FAQs are available on the website.

Hope this was helpful.

Best regards,
Barbara Foster, President

Microscopy/Microscopy Education
7101 Royal Glen Trail, Suite A
McKinney TX 75070
P: [phone]
Skype: fostermme
W: www.MicroscopyEducation.com


MME is now scheduling customized, on-site courses through July
2008.  Call us today for details.

We are sorry to report that Optimizing Light Microscopy for the
Biological and Clinical Laboratory is no longer available.




At 12:21 PM 2/1/2008, you wrote:

>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>Whenever I want to test even signal intensity distribution across a
>field of view, I use translucent fluorescent plastic slides. At a
>given setting, ideally, all pixels should have the same intensity
>value. I once picked up a complimentary set of 4 different coloured
>ones from the Chroma booth at a meeting. They should also be useful
>to compare intensities between filters having similar ex/em specs.
>Judy
>
>Judy Trogadis
>Bio-Imaging Coordinator
>St. Michael's Hospital, 7Queen
>30 Bond St.
>Toronto, ON M5B 1W8, Canada
>ph:  416-864-6060  x6337
>pager: 416-685-9219
>fax: [phone]
>[hidden email]
>
>
> >>> Hanspeter Niederstrasser <[hidden email]> 02/01/08 1:26 PM >>>
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>We're testing new probes and filters for our microscope and amongst the
>things we wanted to check was signal loss btw different filters as well
>as intensity distribution across the view field (this is separate from
>the filters of course).  I found Invitrogen's FluoSpheres as well as
>Fluor-Ref fluorescent slides (Ted Pella being one vendor here:
><http://www.tedpella.com/histo_html/fluor.htm>).
>
>The reference slides are obviously much more economical, but they don't
>list their emission spectra, and since the filters we're testing have
>fairly different OD spectra, we would like the references to be as
>similar to our probes.  What are people's experiences and
>recommendations with these references or others you have used?
>
>Hanspeter
>
>--
>Hanspeter Niederstrasser, Ph.D.      Dept. of Microbiology
>hn2157 at columbia dot edu           701 W. 168th St.
>Chang Lab                            New York, NY 10032
>Columbia University

[Pedro Camello]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

I´m trying to set a Biorad 1024 (Nikon 60x, oil) to get optical slices of
labelled cells. Using a sample of 200 nm fluorescent beads I got Z
resolution of 327 nm (average of 6 beads, range 110 to 520 nm) with the
minimum iris (0.7).

My first question is: if I want to make 1 µ optical slices, should I get
the resolution for higher iris aperture untill I get 1 µ axial resolution?
(I´ve recorded vertical sections for the same beads from 0.7 to 8 iris
apertute)

The other question is about the axial (vertical) images generated by the
confocal. I expected to get an elliptical image for each bead, stretching
from top to bottom of the image. However, the lowest part of each bead is
shifted to the right, so that the fluorescent ellipses are slightly
tilted. It is supposed that the microscope is correctly aligned (the laser
has been recently replaced and the engineer from Zeiss checked
everything). If alignment is fine, could be that internal settings
regarding magnification and objetive are wrong? I made the measurements
using square pixels, but this feature depends on numbers entered in the
settings by the engineer.

I would appreciate some help for this artifact before starting real
experiments.

Thanks in advance.

Pedro


--
Dr Pedro J Camello
Dpt Physiology
Faculty of Veterinary Sciences
University of Extremadura
10071 Caceres
Spain
Ph: 927257100 Extension 1321
Fax:[phone]

[Julio Vazquez]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal -

Hello Pedro, 

When you talk about the Iris, I assume you are talking about the Pinhole (in Airy units), right?

I don't know specifically about the Biorad microscope, but the way I understand it, "resolution" is given by the objective (NA and wavelength). The pinhole determines the thickness of the sample region from which light is able to reach the detector (although defocused). If you opened the pinhole completely, you would no longer have a confocal, but a widefield microscope. The resolution. as generally defined, should only change minimally, and mainly because the contrast of your sections in confocal mode is much better than that of widefield sections.  One formula I have for axial resolution on a confocal is r = 1.4X lambda x n /NA^2 (from the Olympus microscopy web site: http://www.olympusfluoview.com/theory/resolutionintro.html ), which involves the wavelength, the refractive index, and the numerical aperture. 

Bu opening the pinhole, you increase the thickness of the sections you collect, and the idea is that if you set the pinhole to get a section "thickness" of let's say 2 microns, you should collect sections at 1 micron interval, so that you don't loose information.  However, if your objective can resolve let's say 0.5 microns, you should collect sections at 0.2-0.25 microns (that is with roughly Nyquist sampling), so that you will effectively achieve the nominal resolution.  It's easy to see that if you collect your sections at 5 microns interval, even with the best objective in the world you will not get 0.5 micron resolution.  To get optimal resolution, you need to sample properly, both in x/y and in z. Maybe, this is why you get so much variability between beads (110-550 nm)

If you are setting the pinhole to 0.7 Airy units, the system will be very sensitive to any slight misalignment, and you may get severe image degradation (loss of brightness, contrast, and probably resolution too). In that situation (or even at 1 Airy units), check pinhole alignment before you do any measurements.

As for the asymmetric PSF, I don't know what the reason could be. I suspect a slightly misaligned pinhole, or most likely spherical aberration. When we measure PSFs, we get variations depending on the objective used (not all of them are perfect). Also, some old bead slides we have are mounted in media that are far from optimal.Deltavision provides kits of immersion oils with slightly different refractive index, specifically to pick the one that matches a specific sample. Achieving a perfectly symmetrical PSF is not easy...

--

Julio Vazquez

Fred Hutchinson Cancer Research Center

Seattle, WA 98109-1024

http://www.fhcrc.org/

 

==

On Feb 3, 2008, at 7:14 AM, Pedro J Camello wrote:

Search the CONFOCAL archive at

http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

I´m trying to set a Biorad 1024 (Nikon 60x, oil) to get optical slices of

labelled cells. Using a sample of 200 nm fluorescent beads I got Z

resolution of 327 nm (average of 6 beads, range 110 to 520 nm) with the

minimum iris (0.7).

My first question is: if I want to make 1 µ optical slices, should I get

the resolution for higher iris aperture untill I get 1 µ axial resolution?

(I´ve recorded vertical sections for the same beads from 0.7 to 8 iris

apertute)

The other question is about the axial (vertical) images generated by the

confocal. I expected to get an elliptical image for each bead, stretching

from top to bottom of the image. However, the lowest part of each bead is

shifted to the right, so that the fluorescent ellipses are slightly

tilted. It is supposed that the microscope is correctly aligned (the laser

has been recently replaced and the engineer from Zeiss checked

everything). If alignment is fine, could be that internal settings

regarding magnification and objetive are wrong? I made the measurements

using square pixels, but this feature depends on numbers entered in the

settings by the engineer.

I would appreciate some help for this artifact before starting real

experiments.

Thanks in advance.

Pedro

-- 

Dr Pedro J Camello

Dpt Physiology

Faculty of Veterinary Sciences

University of Extremadura

10071 Caceres

Spain

Ph: 927257100 Extension 1321

Fax:[phone]

[mmodel]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

I think tilted axis means coma and could be caused by coverglass placed at an angle or dirt on the optics.
Also, I assume that 327 nm is half the peak width - is that right? I don't think though that the number 110 nm for even half-width is very believable. Laser fluctuations may cause errors in profile measurements. If bleaching is not a problem, I would try to collect PSF at a large zoom and, and if possible, using vibration isolation.
Best wishes -

Mike

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Pedro J Camello
Sent: Sunday, February 03, 2008 10:15 AM
To: [hidden email]
Subject: Help with Z slices

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

I´m trying to set a Biorad 1024 (Nikon 60x, oil) to get optical slices of
labelled cells. Using a sample of 200 nm fluorescent beads I got Z
resolution of 327 nm (average of 6 beads, range 110 to 520 nm) with the
minimum iris (0.7).

My first question is: if I want to make 1 µ optical slices, should I get
the resolution for higher iris aperture untill I get 1 µ axial resolution?
(I´ve recorded vertical sections for the same beads from 0.7 to 8 iris
apertute)

The other question is about the axial (vertical) images generated by the
confocal. I expected to get an elliptical image for each bead, stretching
from top to bottom of the image. However, the lowest part of each bead is
shifted to the right, so that the fluorescent ellipses are slightly
tilted. It is supposed that the microscope is correctly aligned (the laser
has been recently replaced and the engineer from Zeiss checked
everything). If alignment is fine, could be that internal settings
regarding magnification and objetive are wrong? I made the measurements
using square pixels, but this feature depends on numbers entered in the
settings by the engineer.

I would appreciate some help for this artifact before starting real
experiments.

Thanks in advance.

Pedro


--
Dr Pedro J Camello
Dpt Physiology
Faculty of Veterinary Sciences
University of Extremadura
10071 Caceres
Spain
Ph: 927257100 Extension 1321
Fax:[phone]

[Monique Vasseur]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

What kind of microscope are you using?  Usually Nikon objectives are not compatible whit other brands of microscope.

Monique Vasseur
Microscopie et imagerie
Département de biochimie
Université de Montréal
C.P. 6128, succursale Centre-ville
Montréal QC    H3C 3J7   Canada
tél. [phone] poste 5148
-----Message d'origine-----
De : Confocal Microscopy List [mailto:[hidden email]] De la part de Pedro J Camello
Envoyé : 3 février 2008 10:15
À : [hidden email]
Objet : Help with Z slices

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

I´m trying to set a Biorad 1024 (Nikon 60x, oil) to get optical slices of
labelled cells. Using a sample of 200 nm fluorescent beads I got Z
resolution of 327 nm (average of 6 beads, range 110 to 520 nm) with the
minimum iris (0.7).

My first question is: if I want to make 1 µ optical slices, should I get
the resolution for higher iris aperture untill I get 1 µ axial resolution?
(I´ve recorded vertical sections for the same beads from 0.7 to 8 iris
apertute)

The other question is about the axial (vertical) images generated by the
confocal. I expected to get an elliptical image for each bead, stretching
from top to bottom of the image. However, the lowest part of each bead is
shifted to the right, so that the fluorescent ellipses are slightly
tilted. It is supposed that the microscope is correctly aligned (the laser
has been recently replaced and the engineer from Zeiss checked
everything). If alignment is fine, could be that internal settings
regarding magnification and objetive are wrong? I made the measurements
using square pixels, but this feature depends on numbers entered in the
settings by the engineer.

I would appreciate some help for this artifact before starting real
experiments.

Thanks in advance.

Pedro


--
Dr Pedro J Camello
Dpt Physiology
Faculty of Veterinary Sciences
University of Extremadura
10071 Caceres
Spain
Ph: 927257100 Extension 1321
Fax:[phone]

[Kevin Braeckmans]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear Pedro,

I have seen a tilted PSF on our Biorad MRC1024 as well in the past. It
turned out to be a misalignment of the laser -  or at least, it could be
corrected by adjusting the entrance angle of the laser beam into the
scanhead.

Best regards,

Kevin


Kevin Braeckmans, Ph.D.
Lab. General Biochemistry and Physical Pharmacy
Ghent University
Harelbekestraat 72
9000 Ghent
Belgium
Tel: [phone]
Fax: [phone]

> -----Oorspronkelijk bericht-----
> Van: Confocal Microscopy List [mailto:[hidden email]]
> Namens Pedro J Camello
> Verzonden: zondag 3 februari 2008 16:15
> Aan: [hidden email]
> Onderwerp: Help with Z slices
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> I´m trying to set a Biorad 1024 (Nikon 60x, oil) to get optical slices
> of
> labelled cells. Using a sample of 200 nm fluorescent beads I got Z
> resolution of 327 nm (average of 6 beads, range 110 to 520 nm) with the
> minimum iris (0.7).
>
> My first question is: if I want to make 1 µ optical slices, should I
> get
> the resolution for higher iris aperture untill I get 1 µ axial
> resolution?
> (I´ve recorded vertical sections for the same beads from 0.7 to 8 iris
> apertute)
>
> The other question is about the axial (vertical) images generated by
> the
> confocal. I expected to get an elliptical image for each bead,
> stretching
> from top to bottom of the image. However, the lowest part of each bead
> is
> shifted to the right, so that the fluorescent ellipses are slightly
> tilted. It is supposed that the microscope is correctly aligned (the
> laser
> has been recently replaced and the engineer from Zeiss checked
> everything). If alignment is fine, could be that internal settings
> regarding magnification and objetive are wrong? I made the measurements
> using square pixels, but this feature depends on numbers entered in the
> settings by the engineer.
>
> I would appreciate some help for this artifact before starting real
> experiments.
>
> Thanks in advance.
>
> Pedro
>
>
> --
> Dr Pedro J Camello
> Dpt Physiology
> Faculty of Veterinary Sciences
> University of Extremadura
> 10071 Caceres
> Spain
> Ph: 927257100 Extension 1321
> Fax:[phone]