DIY FRAP module for widefield

[F Javier Diez Guerra]

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Hi,

For teaching purposes, we would like to perform FRAP experiments in an
inverted widefield fluorescence microscope. Circular ROIs achieved by
closing the epi-illumination field diafragm take too long to bleach and
do not deliver sharp contrast between bleached and non-bleached areas.
We have a Leica DMi8 stage under Metamorph Basic and a basic LED light
source. Sadly, we do not have the resources to purchase Leica's "FRAP
Device for Widefield Microscopy".

I wonder if any lister has successfully managed to build a one-laser
"bleaching" module that could be easily assembled and attached to the
microscope to accomplish ROI bleaching and FRAP experiments, in the
cheap side.

Thanks in advance for the input.


--

Fco. Javier Díez Guerra, PhD
Profesor Titular
Departamento de Biología Molecular
Facultad de Ciencias
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]

[Julio Vazquez-2]

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If your microscope allows, you could try to set the epi-illumination to critical rather than Koehler. This would boost the illumination intensity at the sample several fold.

More information at the link below:

http://www.asiimaging.com/downloads/manuals/Epi-Illumination%20on%20the%20RAMM.pdf

Also, you might be able to find a light source with more power than your standard LED.

Julio.
--

Julio Vazquez
Fred Hutchinson Cancer Research Center
Seattle, WA 98109
fredhutch.org

On Apr 6, 2017, at 9:34 AM, Fco. Javier Díez Guerra wrote:

> *****
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> *****
>
> Hi,
>
> For teaching purposes, we would like to perform FRAP experiments in an inverted widefield fluorescence microscope. Circular ROIs achieved by closing the epi-illumination field diafragm take too long to bleach and do not deliver sharp contrast between bleached and non-bleached areas. We have a Leica DMi8 stage under Metamorph Basic and a basic LED light source. Sadly, we do not have the resources to purchase Leica's "FRAP Device for Widefield Microscopy".
>
> I wonder if any lister has successfully managed to build a one-laser "bleaching" module that could be easily assembled and attached to the microscope to accomplish ROI bleaching and FRAP experiments, in the cheap side.
>
> Thanks in advance for the input.
>
>
> --
>
> Fco. Javier Díez Guerra, PhD
> Profesor Titular
> Departamento de Biología Molecular
> Facultad de Ciencias
> 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]

[Zdenek Svindrych-2]

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Hi Javier,
Haven't managed : -).

Cheap 445 nm diode lasers are strong enough to bleach anything, but galvo
scanning will be difficult to integrate (mainly from the software point of
view).

The cheapest solution would be 445 nm laser combined with the field aperture
trick (just expand the beam to fill the field diaphragm and couple it to the
illumination path together with your current fluorescence illumination;
beware of laser safety, you may even damage the objective lens as the beam
will be focused inside the lens!). The bleaching will be quicker (depends on
fluorophore) and sharper.

If you want something fancier, a DMD from an old projector could serve you
to define the bleaching mask. Expand the beam and put the DMD to a position
conjugate with sample focus (i.e. conjugate with the field diaphragm). You
can drive the projector from auxiliary graphics card. There might be some
issues with angle of the DMD and the beams due to interference and the setup
will be bulky due to the way the driver electronics is connected to the DMD
in typical projectors.

Again, beware of the lasers! The diodes are not single mode, but they still
can blind you in an instant (at 2 Watts of power)...

Good Luck!

zdenek
--
Zdenek Svindrych, Ph.D.
W.M. Keck Center for Cellular Imaging (PLSB 003)
Department of Biology,University of Virginia
409 McCormick Rd, Charlottesville, VA-22904
http://www.kcci.virginia.edu/
tel: [phone]

---------- Původní e-mail ----------
Od: Fco. Javier Díez Guerra <[hidden email]>
Komu: [hidden email]
Datum: 6. 4. 2017 12:44:09
Předmět: DIY FRAP module for widefield
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Hi,

For teaching purposes, we would like to perform FRAP experiments in an
inverted widefield fluorescence microscope. Circular ROIs achieved by
closing the epi-illumination field diafragm take too long to bleach and
do not deliver sharp contrast between bleached and non-bleached areas.
We have a Leica DMi8 stage under Metamorph Basic and a basic LED light
source. Sadly, we do not have the resources to purchase Leica's "FRAP
Device for Widefield Microscopy".

I wonder if any lister has successfully managed to build a one-laser
"bleaching" module that could be easily assembled and attached to the
microscope to accomplish ROI bleaching and FRAP experiments, in the
cheap side.

Thanks in advance for the input.


--

Fco. Javier Díez Guerra, PhD
Profesor Titular
Departamento de Biología Molecular
Facultad de Ciencias
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]
"

[mcammer]

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Excellent suggestion regarding the critical illumination.  Olympus used to sell a T connection part for this for the IX70 so that a lamp aligned for critical illumination with a mask at the field stop position could be used for photoactivation or bleaching while a lamp for Koehler could be used for imaging.  We put shutters on both lamps.  So you could build one, or do it with one lamp changing the lamp focus from one position to the other.
=*===========================================================*=
 Michael Cammer, DART Microscopy Laboratory, NYU Langone Medical Center
    Cell:  914-309-3270 (this is for calling, not texting)    Office: Skirball 2nd Floor main office, back right
      http://ocs.med.nyu.edu/microscopy & http://microscopynotes.com/



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Julio Vazquez
Sent: Thursday, April 06, 2017 1:08 PM
To: [hidden email]
Subject: Re: DIY FRAP module for widefield

If your microscope allows, you could try to set the epi-illumination to critical rather than Koehler. This would boost the illumination intensity at the sample several fold.

More information at the link below:

https://urldefense.proofpoint.com/v2/url?u=http-3A__www.asiimaging.com_downloads_manuals_Epi-2DIllumination-2520on-2520the-2520RAMM.pdf&d=DQIFAw&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=CzWuOZlws16uaSiHCp4uGs1kqG3p1O76dwEcNEVNvqA&s=yHACnqskCpI2cBqurgzhPpcCACgxggqsfg5zN3FbQSM&e= 

Also, you might be able to find a light source with more power than your standard LED.

Julio.
--

Julio Vazquez
Fred Hutchinson Cancer Research Center
Seattle, WA 98109
fredhutch.org

On Apr 6, 2017, at 9:34 AM, Fco. Javier Díez Guerra wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIDaQ&c=eRAMFD45gAfqt84VtBcfhQ&r=ttNYkfRwLB_uJJmzFu8cIdRdnibIA0UXGXDLv4MFuvU&m=FZuV1SXQ4x9smaoOoJOA8uP9Py1f783IagPPB0nllTI&s=nxut6eC4NKCdW8m_G53tujKgQqCCNEF9EHAVvWpZm7Y&e= Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIDaQ&c=eRAMFD45gAfqt84VtBcfhQ&r=ttNYkfRwLB_uJJmzFu8cIdRdnibIA0UXGXDLv4MFuvU&m=FZuV1SXQ4x9smaoOoJOA8uP9Py1f783IagPPB0nllTI&s=-nBY_g519qHI8XIyBk7MYfnTV49DsnDkb7sFxctRrwg&e=  and include the link in your posting.
> *****
>
> Hi,
>
> For teaching purposes, we would like to perform FRAP experiments in an inverted widefield fluorescence microscope. Circular ROIs achieved by closing the epi-illumination field diafragm take too long to bleach and do not deliver sharp contrast between bleached and non-bleached areas. We have a Leica DMi8 stage under Metamorph Basic and a basic LED light source. Sadly, we do not have the resources to purchase Leica's "FRAP Device for Widefield Microscopy".
>
> I wonder if any lister has successfully managed to build a one-laser "bleaching" module that could be easily assembled and attached to the microscope to accomplish ROI bleaching and FRAP experiments, in the cheap side.
>
> Thanks in advance for the input.
>
>
> --
>
> Fco. Javier Díez Guerra, PhD
> Profesor Titular
> Departamento de Biología Molecular
> Facultad de Ciencias
> 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]

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[Ferhan A]

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Hi Javier,
To increase the sharpness of the widefield-bleached region, a masking
filter can be used. There are articles using polycarbonate filters and UV
excitation to induce local UV damage in nuclei of monolayer cultures.
(Volker at al 2001, Mol Cell)
 http://www.sciencedirect.com/science/article/pii/S1097276501002817

*"...To assess whether UV irradiation provokes intranuclear translocation
of repair proteins to the sites of DNA photolesions, we exposed small parts
of the nucleus to UV radiation (“local” irradiation) using a methodology
described in detail by Moné et al. (submitted). For this purpose, monolayer
cultures of human fibroblasts were covered with isopore polycarbonate
filters with pore diameters of either 3 or 8 μm prior to UV irradiation.
These filters block UV light with wavelengths shorter than 300 nm, and,
consequently, only at sites of pores will UV damage be induced..."*

They use low cost, commercially available filters/membranes with very small
holes to allow UV light to selectively enter and damage certain subnuclear
regions. A similar filter with bigger holes (or with a manually drilled
microhole in the middle) can be placed onto your sample (or below your
sample, for inverted microscope) to allow only a very discrete region below
the hole(s) to be excited (hence bleached), then you remove the filter to
observe the recovery. Since the masking filter/membrane will be positioned
very close to your sample (attached to bottom of a coverslip-bottom Petri
dish for the inverted microscope, for example), I expect sharper contrast
between bleached and nonbleached regions. If you use a sample which can be
quickly bleached by UV light, you can try putting your sample (with masking
filter at the bottom) on top of a strong UV lamp or microscope-disconnected
Mercury/Xenon lamphouse (for direct, high power, excitation levels -while
being very careful!) to selectively and quickly bleach multiple regions
(and possibly retinas of your "pupils"), then follow recovery of the
bleached spots on the microscope.
Cheap widefield FRAP? ☑
Ferhan

--

Ferhan Ayaydin, Ph.D.

Cellular Imaging Laboratory

Biological Research Centre

Hungarian Academy of Sciences

Temesvari krt 62.

H6726 Szeged, Hungary

*Applicants wanted for a year long international training course in our
microscopy/imaging lab on CRISPR/Cas9 editing of fluorescent proteins in
plants *

*(7th item at the following website)*:

http://www.brc.hu/international_training_applications.php

Lab website: http://www.brc.hu/core_cellular_imaging.php

[F Javier Diez Guerra]

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Thanks to all that have posted on this issue.

We will try to set up critical illumination to concentrate more light
within the ROI to bleach. Unfortunately our LED source couples directly
to the epi-illumination port of the microscope (no fiber or light guide).

Best regards

Javier