Demonstrating the field and aperture diaphragm as well as darkfield illumination.

[Smith, Benjamin E.]

*****
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.
*****

Hey Microscopists,
  For those of you teaching microscopy courses, it can be challenging to explain the field and aperture diaphragm and how they effect the cone of light emerging from the condenser.  On a whim, I filled a cuvette with fluorescein in order to visualize the cone of light.  The demonstration proved quite successful, so I made a movie of the demonstration, showing how the field diaphragm affects the width of the cone without impacting its angle, and the aperture diaphragm impacts the angle of the cone without affecting its width.  Having the students see the cone also went a long way in helping them understand why axial resolution goes down with a lower NA.

Here is the link to the movie: https://youtu.be/06CQ6IIaDWs

The cuvette also allowed me to show the hollow cone generated in phase contrast and darkfield:
http://imgur.com/hswMRjH
http://imgur.com/Zl87dcr

The sectored ray in oblique illumination:
http://imgur.com/oerMkk0

As well as the solid cone in brightfield:
http://imgur.com/KZP3Sv0
http://imgur.com/akeiI0T

Small disclaimer, the oblique was a "poor man's" oblique done my misaligning the darkfield annulus and partially closing the aperture diaphragm, but I also like to show "poor man's" oblique to students to show how thinking a little outside the box can allow you to get the most out of your microscope.

Hope this helps,
  Ben Smith

Benjamin E. Smith, Ph.D.
Samuel Roberts Noble Microscopy Laboratory
Research Scientist, Confocal Facility Manager
University of Oklahoma
Norman, OK 73019
E-mail: [hidden email]
Voice   405-325-4391
FAX  [phone]
http://www.microscopy.ou.edu/


________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Gelman, Laurent [[hidden email]]
Sent: Friday, September 04, 2015 8:53 AM
To: [hidden email]
Subject: Re: FRAP review

Dear Alby,

I would be very interested as well...

Thanks a lot,

Laurent.



-----Ursprüngliche Nachricht-----
Von: Confocal Microscopy List [mailto:[hidden email]] Im Auftrag von Alberto Diaspro
Gesendet: Montag, 31. August 2015 08:31
An: [hidden email]
Betreff: FRAP review

*****
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.
*****

Friends
after a hard job, hopefully you can be intersted in this FRAP review.
Ciao
Alby

 Niklas Lorén, Joel Hagman, Jenny K. Jonasson, Hendrik Deschout, Diana Bernin, Francesca Cella-Zanacchi, Alberto Diaspro, James G. McNally, Marcel Ameloot, Nick Smisdom, Magnus Nydén, Anne-Marie Hermansson, Mats Rudemo and Kevin Braeckmans (2015). Fluorescence recovery after photobleaching in material and life sciences: putting theory into practice. Quarterly Reviews of Biophysics, 48, pp 323-387.

doi:10.1017/S0033583515000013.

[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.
*****

We taught a class on precisely this at the Canadian Light Microscopy
workshop a few months back. Instead of fluorescein we used fluorescent
plastic rods. Much less cleanup!

http://www.delviesplastics.com/mm5/merchant.mvc?Screen=CTGY&Store_Code=DPI&Category_Code=Cast_Acrylic_Rod
The fluorescent plastic colors further down the page work well.

Craig

On Wed, Sep 23, 2015 at 3:26 PM, Smith, Benjamin E. <[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.
> *****
>
> Hey Microscopists,
>   For those of you teaching microscopy courses, it can be challenging to
> explain the field and aperture diaphragm and how they effect the cone of
> light emerging from the condenser.  On a whim, I filled a cuvette with
> fluorescein in order to visualize the cone of light.  The demonstration
> proved quite successful, so I made a movie of the demonstration, showing
> how the field diaphragm affects the width of the cone without impacting its
> angle, and the aperture diaphragm impacts the angle of the cone without
> affecting its width.  Having the students see the cone also went a long way
> in helping them understand why axial resolution goes down with a lower NA.
>
> Here is the link to the movie: https://youtu.be/06CQ6IIaDWs
>
> The cuvette also allowed me to show the hollow cone generated in phase
> contrast and darkfield:
> http://imgur.com/hswMRjH
> http://imgur.com/Zl87dcr
>
> The sectored ray in oblique illumination:
> http://imgur.com/oerMkk0
>
> As well as the solid cone in brightfield:
> http://imgur.com/KZP3Sv0
> http://imgur.com/akeiI0T
>
> Small disclaimer, the oblique was a "poor man's" oblique done my
> misaligning the darkfield annulus and partially closing the aperture
> diaphragm, but I also like to show "poor man's" oblique to students to show
> how thinking a little outside the box can allow you to get the most out of
> your microscope.
>
> Hope this helps,
>   Ben Smith
>
> Benjamin E. Smith, Ph.D.
> Samuel Roberts Noble Microscopy Laboratory
> Research Scientist, Confocal Facility Manager
> University of Oklahoma
> Norman, OK 73019
> E-mail: [hidden email]
> Voice   405-325-4391
> FAX  [phone]
> http://www.microscopy.ou.edu/
>
>
> ________________________________________
> From: Confocal Microscopy List [[hidden email]] on
> behalf of Gelman, Laurent [[hidden email]]
> Sent: Friday, September 04, 2015 8:53 AM
> To: [hidden email]
> Subject: Re: FRAP review
>
> Dear Alby,
>
> I would be very interested as well...
>
> Thanks a lot,
>
> Laurent.
>
>
>
> -----Ursprüngliche Nachricht-----
> Von: Confocal Microscopy List [mailto:[hidden email]]
> Im Auftrag von Alberto Diaspro
> Gesendet: Montag, 31. August 2015 08:31
> An: [hidden email]
> Betreff: FRAP review
>
> *****
> 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.
> *****
>
> Friends
> after a hard job, hopefully you can be intersted in this FRAP review.
> Ciao
> Alby
>
>  Niklas Lorén, Joel Hagman, Jenny K. Jonasson, Hendrik Deschout, Diana
> Bernin, Francesca Cella-Zanacchi, Alberto Diaspro, James G. McNally, Marcel
> Ameloot, Nick Smisdom, Magnus Nydén, Anne-Marie Hermansson, Mats Rudemo and
> Kevin Braeckmans (2015). Fluorescence recovery after photobleaching in
> material and life sciences: putting theory into practice. Quarterly Reviews
> of Biophysics, 48, pp 323-387.
>
> doi:10.1017/S0033583515000013.
>

[Martin Wessendorf-2]

*****
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 Dr. Smith--

Excellent--thanks.  Nice video!

Martin Wessendorf



On 9/23/2015 4:26 PM, Smith, Benjamin E. wrote:

> Hey Microscopists,
>    For those of you teaching microscopy courses, it can be challenging to explain the field and aperture diaphragm and how they effect the cone of light emerging from the condenser.  On a whim, I filled a cuvette with fluorescein in order to visualize the cone of light.  The demonstration proved quite successful, so I made a movie of the demonstration, showing how the field diaphragm affects the width of the cone without impacting its angle, and the aperture diaphragm impacts the angle of the cone without affecting its width.  Having the students see the cone also went a long way in helping them understand why axial resolution goes down with a lower NA.
>
> Here is the link to the movie: https://youtu.be/06CQ6IIaDWs
>
> The cuvette also allowed me to show the hollow cone generated in phase contrast and darkfield:
> http://imgur.com/hswMRjH
> http://imgur.com/Zl87dcr
>
> The sectored ray in oblique illumination:
> http://imgur.com/oerMkk0
>
> As well as the solid cone in brightfield:
> http://imgur.com/KZP3Sv0
> http://imgur.com/akeiI0T
>
> Small disclaimer, the oblique was a "poor man's" oblique done my misaligning the darkfield annulus and partially closing the aperture diaphragm, but I also like to show "poor man's" oblique to students to show how thinking a little outside the box can allow you to get the most out of your microscope.
>
> Hope this helps,
>    Ben Smith
>
> Benjamin E. Smith, Ph.D.
> Samuel Roberts Noble Microscopy Laboratory
> Research Scientist, Confocal Facility Manager
> University of Oklahoma
> Norman, OK 73019
> E-mail: [hidden email]
> Voice   405-325-4391
> FAX  [phone]
> http://www.microscopy.ou.edu/

--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455                    e-mail: [hidden email]

[Mark Clymer]

*****
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.
*****

Great demonstrations!  For an even cheaper approach, try the original lemon-lime Gatorade in a cuvette or media bottle.  Field and aperture diaphragm operation is important to demonstrate for fluorescence/reflected light techniques, too!  Such a demonstration works well to illustrate the importance of matching the NA above and below the specimen plane.

Traditionally, we would use uranium glass cubes, but they're nearly impossible to find now.

Mark Clymer
Director of Marketing, Datacolor Scientific
Mobile: [phone]
[hidden email]
[hidden email]




-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Smith, Benjamin E.
Sent: Wednesday, September 23, 2015 5:26 PM
To: [hidden email]
Subject: Demonstrating the field and aperture diaphragm as well as darkfield illumination.

*****
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.
*****

Hey Microscopists,
  For those of you teaching microscopy courses, it can be challenging to explain the field and aperture diaphragm and how they effect the cone of light emerging from the condenser.  On a whim, I filled a cuvette with fluorescein in order to visualize the cone of light.  The demonstration proved quite successful, so I made a movie of the demonstration, showing how the field diaphragm affects the width of the cone without impacting its angle, and the aperture diaphragm impacts the angle of the cone without affecting its width.  Having the students see the cone also went a long way in helping them understand why axial resolution goes down with a lower NA.

Here is the link to the movie: https://youtu.be/06CQ6IIaDWs

The cuvette also allowed me to show the hollow cone generated in phase contrast and darkfield:
http://imgur.com/hswMRjH
http://imgur.com/Zl87dcr

The sectored ray in oblique illumination:
http://imgur.com/oerMkk0

As well as the solid cone in brightfield:
http://imgur.com/KZP3Sv0
http://imgur.com/akeiI0T

Small disclaimer, the oblique was a "poor man's" oblique done my misaligning the darkfield annulus and partially closing the aperture diaphragm, but I also like to show "poor man's" oblique to students to show how thinking a little outside the box can allow you to get the most out of your microscope.

Hope this helps,
  Ben Smith

Benjamin E. Smith, Ph.D.
Samuel Roberts Noble Microscopy Laboratory Research Scientist, Confocal Facility Manager University of Oklahoma Norman, OK 73019
E-mail: [hidden email]
Voice   405-325-4391
FAX  [phone]
http://www.microscopy.ou.edu/


________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Gelman, Laurent [[hidden email]]
Sent: Friday, September 04, 2015 8:53 AM
To: [hidden email]
Subject: Re: FRAP review

Dear Alby,

I would be very interested as well...

Thanks a lot,

Laurent.



-----Ursprüngliche Nachricht-----
Von: Confocal Microscopy List [mailto:[hidden email]] Im Auftrag von Alberto Diaspro
Gesendet: Montag, 31. August 2015 08:31
An: [hidden email]
Betreff: FRAP review

*****
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.
*****

Friends
after a hard job, hopefully you can be intersted in this FRAP review.
Ciao
Alby

 Niklas Lorén, Joel Hagman, Jenny K. Jonasson, Hendrik Deschout, Diana Bernin, Francesca Cella-Zanacchi, Alberto Diaspro, James G. McNally, Marcel Ameloot, Nick Smisdom, Magnus Nydén, Anne-Marie Hermansson, Mats Rudemo and Kevin Braeckmans (2015). Fluorescence recovery after photobleaching in material and life sciences: putting theory into practice. Quarterly Reviews of Biophysics, 48, pp 323-387.

doi:10.1017/S0033583515000013.

[Patrick Van Oostveldt]

*****
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,

Instead of fluoresceine you can also use diluted milk. It scatters and does not need high intensity light or a dark class room ans even explains why milk is white ( because it scatters white light).

Patrick Van Oostveldt
Sint-Denijslaan 199
9000 GENT

Mobile [phone]
Sent from my iPad

> On 23 Sep 2015, at 23:46, Martin Wessendorf <[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 Dr. Smith--
>
> Excellent--thanks.  Nice video!
>
> Martin Wessendorf
>
>
>
>> On 9/23/2015 4:26 PM, Smith, Benjamin E. wrote:
>> Hey Microscopists,
>>   For those of you teaching microscopy courses, it can be challenging to explain the field and aperture diaphragm and how they effect the cone of light emerging from the condenser.  On a whim, I filled a cuvette with fluorescein in order to visualize the cone of light.  The demonstration proved quite successful, so I made a movie of the demonstration, showing how the field diaphragm affects the width of the cone without impacting its angle, and the aperture diaphragm impacts the angle of the cone without affecting its width.  Having the students see the cone also went a long way in helping them understand why axial resolution goes down with a lower NA.
>>
>> Here is the link to the movie: https://youtu.be/06CQ6IIaDWs
>>
>> The cuvette also allowed me to show the hollow cone generated in phase contrast and darkfield:
>> http://imgur.com/hswMRjH
>> http://imgur.com/Zl87dcr
>>
>> The sectored ray in oblique illumination:
>> http://imgur.com/oerMkk0
>>
>> As well as the solid cone in brightfield:
>> http://imgur.com/KZP3Sv0
>> http://imgur.com/akeiI0T
>>
>> Small disclaimer, the oblique was a "poor man's" oblique done my misaligning the darkfield annulus and partially closing the aperture diaphragm, but I also like to show "poor man's" oblique to students to show how thinking a little outside the box can allow you to get the most out of your microscope.
>>
>> Hope this helps,
>>   Ben Smith
>>
>> Benjamin E. Smith, Ph.D.
>> Samuel Roberts Noble Microscopy Laboratory
>> Research Scientist, Confocal Facility Manager
>> University of Oklahoma
>> Norman, OK 73019
>> E-mail: [hidden email]
>> Voice   405-325-4391
>> FAX  [phone]
>> http://www.microscopy.ou.edu/
>
> --
> Martin Wessendorf, Ph.D.                   office: [phone]
> Assoc Prof, Dept Neuroscience                 lab: [phone]
> University of Minnesota             Preferred FAX: [phone]
> 6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
> Minneapolis, MN  55455                    e-mail: [hidden email]

[JOEL B. SHEFFIELD]

*****
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.
*****

On Thu, Sep 24, 2015 at 10:33 AM, JOEL B. SHEFFIELD <[hidden email]> wrote:

> I have been following this discussion with great interest.  We have been
> able to show the shape of the cone of light from the phase ring by folding
> a piece of paper at a right angle, and placing it on the microscope stage.
> It becomes a useful way to demonstrate dark field as well.
>

See this image:  http://imgur.com/gallery/zpQHAy1/new

>
>
>
> Joel B. Sheffield, Ph.D
> Department of Biology
> Temple University
> Philadelphia, PA 19122
> Voice: 215 204 8839
> e-mail: [hidden email]
> URL:  *http://tinyurl.com/khbouft <http://tinyurl.com/khbouft>*
>
> On Thu, Sep 24, 2015 at 9:55 AM, patrick van oostveldt <
> [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,
>>
>> Instead of fluoresceine you can also use diluted milk. It scatters and
>> does not need high intensity light or a dark class room ans even explains
>> why milk is white ( because it scatters white light).
>>
>> Patrick Van Oostveldt
>> Sint-Denijslaan 199
>> 9000 GENT
>>
>> Mobile [phone]
>> Sent from my iPad
>>
>> > On 23 Sep 2015, at 23:46, Martin Wessendorf <[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 Dr. Smith--
>> >
>> > Excellent--thanks.  Nice video!
>> >
>> > Martin Wessendorf
>> >
>> >
>> >
>> >> On 9/23/2015 4:26 PM, Smith, Benjamin E. wrote:
>> >> Hey Microscopists,
>> >>   For those of you teaching microscopy courses, it can be challenging
>> to explain the field and aperture diaphragm and how they effect the cone of
>> light emerging from the condenser.  On a whim, I filled a cuvette with
>> fluorescein in order to visualize the cone of light.  The demonstration
>> proved quite successful, so I made a movie of the demonstration, showing
>> how the field diaphragm affects the width of the cone without impacting its
>> angle, and the aperture diaphragm impacts the angle of the cone without
>> affecting its width.  Having the students see the cone also went a long way
>> in helping them understand why axial resolution goes down with a lower NA.
>> >>
>> >> Here is the link to the movie: https://youtu.be/06CQ6IIaDWs
>> >>
>> >> The cuvette also allowed me to show the hollow cone generated in phase
>> contrast and darkfield:
>> >> http://imgur.com/hswMRjH
>> >> http://imgur.com/Zl87dcr
>> >>
>> >> The sectored ray in oblique illumination:
>> >> http://imgur.com/oerMkk0
>> >>
>> >> As well as the solid cone in brightfield:
>> >> http://imgur.com/KZP3Sv0
>> >> http://imgur.com/akeiI0T
>> >>
>> >> Small disclaimer, the oblique was a "poor man's" oblique done my
>> misaligning the darkfield annulus and partially closing the aperture
>> diaphragm, but I also like to show "poor man's" oblique to students to show
>> how thinking a little outside the box can allow you to get the most out of
>> your microscope.
>> >>
>> >> Hope this helps,
>> >>   Ben Smith
>> >>
>> >> Benjamin E. Smith, Ph.D.
>> >> Samuel Roberts Noble Microscopy Laboratory
>> >> Research Scientist, Confocal Facility Manager
>> >> University of Oklahoma
>> >> Norman, OK 73019
>> >> E-mail: [hidden email]
>> >> Voice   405-325-4391
>> >> FAX  [phone]
>> >> http://www.microscopy.ou.edu/
>> >
>> > --
>> > Martin Wessendorf, Ph.D.                   office: [phone]
>> > Assoc Prof, Dept Neuroscience                 lab: [phone]
>> > University of Minnesota             Preferred FAX: [phone]
>> > 6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
>> > Minneapolis, MN  55455                    e-mail: [hidden email]
>>
>
>

[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.
*****

Thank you all for sharing your nice images and movies.

May I suggest that you put such material on the web with a creative
commons license tag. That way it will be easier for others to reuse such
images in their own presentations for teaching they wish to share on the
web, with their students or for general reuse. Or in other projects such
as Wikipedia.

An example would be the "Creative Commons Attribution
Sharealike"-License described at
https://creativecommons.org/licenses/by-sa/4.0/
Or one of the other licenses presented there that fit your personal
preferences.

Steffen

> --
> ------------------------------------------------------------
> Steffen Dietzel, PD Dr. rer. nat
> Ludwig-Maximilians-Universität München
> Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
> Head of light microscopy
>
> Marchioninistr. 27
> D-81377 München
> Germany