photobleaching/photoactivation on widefields

[F Javier Diez Guerra]

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Dear confocalists,

I would like to know your experiences in implementing spatially
restricted illumination to wide field microscopes. We are thinking to
combine FRAP, photoactivation/photoconversion, optogenetics, uncaging,
etc with the speed of widefield acquisition, and have a limited budget.

Thanks in advance for your input (offline, if you wish).

Javier

--

Fco. Javier Díez Guerra, PhD
Profesor Titular
Centro de Biología Molecular Severo Ochoa
C/ Nicolás Cabrera, 1
Universidad Autónoma
Ctra Colmenar Viejo Km 15
Cantoblanco, 28049 Madrid
SPAIN

phone:  [phone]
e-mail: [hidden email]

[Craig Brideau]

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The standard method is to use a digital mirror or spatial light modulator
for something like this for fast switching. If I had to do it on a budget I
would try using an iris or manually cut a pattern into some black foil in a
conjugate plane. An inverse reticle might also be useful.

Craig


On Wed, Apr 11, 2018 at 10:58 AM, Fco. Javier Díez Guerra <
[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 confocalists,
>
> I would like to know your experiences in implementing spatially restricted
> illumination to wide field microscopes. We are thinking to combine FRAP,
> photoactivation/photoconversion, optogenetics, uncaging, etc with the
> speed of widefield acquisition, and have a limited budget.
>
> Thanks in advance for your input (offline, if you wish).
>
> Javier
>
> --
>
> Fco. Javier Díez Guerra, PhD
> Profesor Titular
> Centro de Biología Molecular Severo Ochoa
> C/ Nicolás Cabrera, 1
> <https://maps.google.com/?q=C/+Nicol%C3%A1s+Cabrera,+1&entry=gmail&source=g>
> Universidad Autónoma
> Ctra Colmenar Viejo Km 15
> Cantoblanco, 28049 Madrid
> SPAIN
>
> phone:  [phone]
> e-mail: [hidden email]
>

[Benjamin Smith]

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To expand on what Craig said, a DLP (digital micromirror) is the best way
to rapidly switch between different illumination patterns.  Basically,
couple the needed LED wavelengths into a single liquid light guide, and
then use this as an illumination source for the DLP.  DLinnovations (no
commercial interest) have a DLP already setup to accept a liquid light
guide.  Then, use an Arduino to coordinate the LED and DMD switching.  Even
with a standard LED driver, you would easily get sub-millisecond switch
times.

If a fixed pattern is okay, then another way we've done patterned
illumination is to use an LED and pinholes (or any other desired mask) on a
turret coming in though the trinoc mount.  Basically, use a pair of
condenser lenses to focus the LED onto the mask, then collimate the image
with one more condenser lens.  If you need white light or multiple
wavelengths, Edmund Optics (no commercial interest) makes aspheric
achromats which would minimize the chromatic aberration. An added bonus is
that this setup is very compact.

The DLP approach would cost about $6,000 to $7,000, while the mask approach
would cost about $400 to $500.  If you are flexible with wavelengths and
don't need high resolution (or speeds faster than 60 Hz) then a PicoDLP
with an achromat to collimate the beam will give you structured patterns
for about $500 to $700.  We have this on one of our rigs, and it is
surprisingly bright.

Hope this helps,
   Ben Smith



On Wed, Apr 11, 2018 at 11:24 AM, Craig Brideau <[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.
> *****
>
> The standard method is to use a digital mirror or spatial light modulator
> for something like this for fast switching. If I had to do it on a budget I
> would try using an iris or manually cut a pattern into some black foil in a
> conjugate plane. An inverse reticle might also be useful.
>
> Craig
>
>
> On Wed, Apr 11, 2018 at 10:58 AM, Fco. Javier Díez Guerra <
> [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 confocalists,
> >
> > I would like to know your experiences in implementing spatially
> restricted
> > illumination to wide field microscopes. We are thinking to combine FRAP,
> > photoactivation/photoconversion, optogenetics, uncaging, etc with the
> > speed of widefield acquisition, and have a limited budget.
> >
> > Thanks in advance for your input (offline, if you wish).
> >
> > Javier
> >
> > --
> >
> > Fco. Javier Díez Guerra, PhD
> > Profesor Titular
> > Centro de Biología Molecular Severo Ochoa
> > C/ Nicolás Cabrera, 1
> > <https://maps.google.com/?q=C/+Nicol%C3%A1s+Cabrera,+1&
> entry=gmail&source=g>
> > Universidad Autónoma
> > Ctra Colmenar Viejo Km 15
> > Cantoblanco, 28049 Madrid
> > SPAIN
> >
> > phone:  [phone]
> > e-mail: [hidden email]
> >
>

--
Benjamin E. Smith, Ph. D.
Imaging Specialist, Vision Science
University of California, Berkeley
195 Life Sciences Addition
Berkeley, CA  94720-3200
Tel  [phone]
Fax [phone]
e-mail: [hidden email]
http://vision.berkeley.edu/?page_id=5635 <http://vision.berkeley.edu/>