Possibility to visualize far-red signal by eye?

[SUBSCRIBE CONFOCALMICROSCOPY Sara Smith]

Dear lister,

 

Just wondering if there is such a configuration that would allow me to see the infrared signal directly by eye?

 

I always believed that human eye is not able to detect the far-red signal till one of my colleague argued with me that there might be a way to make this signal visible.

So just want to ask around to see if it is possible.

 

Thanks!

 

Yee

[Eric Scarfone]

Hi Yee,
It all depends on the strength of your signal and on the configuration
of your filters. if you allow the whole emission spectra of Cy5 for
example to pass through your emission filters and if your signal is
strong enough, your eyes will be able to see the lower half of the spectra!
Eric

Eric Scarfone, PhD, CNRS,
Center for Hearing and communication Research
Department of Clinical Neuroscience
Karolinska Institutet

Postal Address:
CFH, M1:02
Karolinska Hospital,
SE-171 76 Stockholm, Sweden

Work:  [phone],
Cell:  [phone]
Fax:   [phone]

email:  [hidden email]
http://www.ki.se/cfh/


----- Original Message -----
From: yee sarah <[hidden email]>
Date: Wednesday, February 3, 2010 10:04 pm
Subject: Possibility to visualize far-red signal by eye?
To: [hidden email]

> Dear lister,
>
>
>
> Just wondering if there is such a configuration that would allow me
> to see
> the infrared signal directly by eye?
>
>
>
> I always believed that human eye is not able to detect the far-red
> signaltill one of my colleague argued with me that there might be a
> way to make
> this signal visible.
>
> So just want to ask around to see if it is possible.
>
>
>
> Thanks!
>
>
>
> Yee
>

[leoncio vergara]

TOPRO3 is visible with a Texas Red filter set.

________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of Eric Scarfone [[hidden email]]
Sent: Wednesday, February 03, 2010 3:23 PM
To: [hidden email]
Subject: Re: Possibility to visualize far-red signal by eye?

Hi Yee,
It all depends on the strength of your signal and on the configuration
of your filters. if you allow the whole emission spectra of Cy5 for
example to pass through your emission filters and if your signal is
strong enough, your eyes will be able to see the lower half of the spectra!
Eric

Eric Scarfone, PhD, CNRS,
Center for Hearing and communication Research
Department of Clinical Neuroscience
Karolinska Institutet

Postal Address:
CFH, M1:02
Karolinska Hospital,
SE-171 76 Stockholm, Sweden

Work:  [phone],
Cell:  [phone]
Fax:   [phone]

email:  [hidden email]
http://www.ki.se/cfh/


----- Original Message -----
From: yee sarah <[hidden email]>
Date: Wednesday, February 3, 2010 10:04 pm
Subject: Possibility to visualize far-red signal by eye?
To: [hidden email]

> Dear lister,
>
>
>
> Just wondering if there is such a configuration that would allow me
> to see
> the infrared signal directly by eye?
>
>
>
> I always believed that human eye is not able to detect the far-red
> signaltill one of my colleague argued with me that there might be a
> way to make
> this signal visible.
>
> So just want to ask around to see if it is possible.
>
>
>
> Thanks!
>
>
>
> Yee
>

[Jacqueline Ross]

Hi Sarah,

We have one fluorescence microscope with a Cy filter from Semrock. I
have seen Alexa 647 with this filter cube but it was extremely bright
signal on that occasion (too bright!) such that it was also bleeding
through into the Texas Red filter! The graduate student involved had to
dilute down her fluorophore accordingly in order to do 4 colour imaging.

Kind regards,

Jacqui

Jacqueline Ross

Biomedical Imaging Microscopist
Biomedical Imaging Research Unit
School of Medical Sciences
Faculty of Medical & Health Sciences
The University of Auckland
Private Bag 92019
Auckland, NEW ZEALAND

Tel: [phone] Ext 87438
Fax: [phone]

http://www.fmhs.auckland.ac.nz/sms/biru/


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Eric Scarfone
Sent: Thursday, 4 February 2010 10:24 a.m.
To: [hidden email]
Subject: Re: Possibility to visualize far-red signal by eye?

Hi Yee,
It all depends on the strength of your signal and on the configuration
of your filters. if you allow the whole emission spectra of Cy5 for
example to pass through your emission filters and if your signal is
strong enough, your eyes will be able to see the lower half of the
spectra!
Eric

Eric Scarfone, PhD, CNRS,
Center for Hearing and communication Research
Department of Clinical Neuroscience
Karolinska Institutet

Postal Address:
CFH, M1:02
Karolinska Hospital,
SE-171 76 Stockholm, Sweden

Work:  [phone],
Cell:  [phone]
Fax:   [phone]

email:  [hidden email]
http://www.ki.se/cfh/


----- Original Message -----
From: yee sarah <[hidden email]>
Date: Wednesday, February 3, 2010 10:04 pm
Subject: Possibility to visualize far-red signal by eye?
To: [hidden email]

> Dear lister,
>
>
>
> Just wondering if there is such a configuration that would allow me
> to see
> the infrared signal directly by eye?
>
>
>
> I always believed that human eye is not able to detect the far-red
> signaltill one of my colleague argued with me that there might be a
> way to make
> this signal visible.
>
> So just want to ask around to see if it is possible.
>
>
>
> Thanks!
>
>
>
> Yee
>

[Julio Vazquez]

The ability to see near infra-red light depends on the specific wavelength, the intensity, and the individual, so I guess it all depends on what you want to see and under what conditions. With the right filter, I can see some far red dyes, assuming the signal is relatively decent, the background is low, and my eyes got used to the dark for a short time. Otherwise, night vision goggles come to mind, but I doubt this would be a practical solution (at least for microscopy)...

--

Julio Vazquez

Fred Hutchinson Cancer Research Center

Seattle, WA 98109-1024

http://www.fhcrc.org/

==

On Feb 3, 2010, at 1:04 PM, yee sarah wrote:

Dear lister,

 

Just wondering if there is such a configuration that would allow me to see the infrared signal directly by eye?

 

I always believed that human eye is not able to detect the far-red signal till one of my colleague argued with me that there might be a way to make this signal visible.

So just want to ask around to see if it is possible.

 

Thanks!

 

Yee

[Martin Wessendorf-2]

Dear Dr. Yee--

yee sarah wrote:

> Just wondering if there is such a configuration that would allow me to
> see the infrared signal directly by eye?

My recollection is that the human eye can detect photons out past 1000
nm wavelength--it's just not very efficient at doing so.  Cy5 is
certainly visible by eye if the staining is bright enough (e.g., I've
seen intracellularly filled neurons stained with Cy5)--you need a
630-650 nm exciter and a 665-700 nm barrier filter (or similar filters)
to see it with a mercury lamp.  A high NA objective will help.

Good luck!

Martin Wessendorf

--
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]

[George Peeters-2]

Scientific ICCD cameras, (Stanford Photonics Inc)  can image out to 850 to 900 nm with gains of 35,000 to 40,000. Beyond that our Governments start to get restrictive about who has access to these.

Best regards,

George A. Peeters MD, MS

President,  Solamere Technology Group Inc

1427 Perry Ave

Salt Lake City, UT 84103

www.solameretech.com

801 322-2645 office          801 322-2645 fax

On Feb 3, 2010, at 3:55 PM, Martin Wessendorf wrote:

Dear Dr. Yee--

yee sarah wrote:

Just wondering if there is such a configuration that would allow me to see the infrared signal directly by eye?

My recollection is that the human eye can detect photons out past 1000 nm wavelength--it's just not very efficient at doing so.  Cy5 is certainly visible by eye if the staining is bright enough (e.g., I've seen intracellularly filled neurons stained with Cy5)--you need a 630-650 nm exciter and a 665-700 nm barrier filter (or similar filters) to see it with a mercury lamp.  A high NA objective will help.

Good luck!

Martin Wessendorf

--
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]

[Peter Gabriel Pitrone]

I bought a cheap 5mW 532nm green laser pointer from a Chinese dealer (http://www.dealextreme.com/products.dx/category.911) that has no IR filter. last week I left it in my car for a few hours (in sub-freezing temperatures) and when I checked the beam spot on the floor, it was a deep red that was hard to see until the crystal in the laser warmed up enough to generate a second harmonic. I assume to get 5mW of 532 nm light that 10's if not 100's of mWs 1064nm are used due to the suboptimal crystals used in 10.00 euro laser pointers.

So that being said, if you have enough power it is possible to see "invisible" light.

Pete

On Feb 3, 2010, at 23:55 PM, Martin Wessendorf wrote:

> Dear Dr. Yee--
>
> yee sarah wrote:
>
>> Just wondering if there is such a configuration that would allow me to see the infrared signal directly by eye?
>
> My recollection is that the human eye can detect photons out past 1000 nm wavelength--it's just not very efficient at doing so.  Cy5 is certainly visible by eye if the staining is bright enough (e.g., I've seen intracellularly filled neurons stained with Cy5)--you need a 630-650 nm exciter and a 665-700 nm barrier filter (or similar filters) to see it with a mercury lamp.  A high NA objective will help.
>
> Good luck!
>
> Martin Wessendorf
>
> --
> 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]

[George McNamara]

Hi Yee,

By definition, infrared photons are invisible to the human eye. So, any visible emission seen from Cy5, Alexa Fluor 647, CF647 (www.biotium.com) is not infrared.

On a more practical note, the longpass mKate filter set I have from Chroma, excitation around the Hg arc lamp 578 nm line (still excites Cy3 and Alexa Fluor 568, for good or bad depending on the experiment) and 600-700 nm emission, or our Zeiss longpass Cy3 filter set (Hg arc lamp 546 nm line), lets me see Cy5 and Alexa Fluor 647 (or at least bright immunofluorescence, ex. insulin in adult pancreatic beta cells). I usually have the room lights off in the microscope room.

Below are two references for Ferri et al papers on filter design.


Direct eye visualization of Cy5 fluorescence for immunocytochemistry and in situ hybridization.
Ferri GL, Isola J, Berger P, Giro G.
J Histochem Cytochem. 2000 Mar;48(3):437-44.PMID: 10681398


Quadruple immunofluorescence: a direct visualization method.
Ferri GL, Gaudio RM, Castello IF, Berger P, Giro G.
J Histochem Cytochem. 1997 Feb;45(2):155-8.PMID: 9016305 [PubMed - indexed for MEDLINE] Related articles



http://www.thefreedictionary.com/infrared

infrared

Relating to the invisible part of the electromagnetic spectrum with wavelengths longer than those of visible red light but shorter than those of microwaves. See more at electromagnetic spectrum.

A Closer Look In 1800 the astronomer Sir William Herschel discovered infrared light while exploring the relationship between heat and light. Herschel used a prism to split a beam of sunlight into a spectrum and then placed a thermometer in each of the bands of light. When he placed the thermometer just outside the red band, where there was no visible color, the temperature rose, as if light were shining on the thermometer. Further experiment showed that this invisible radiation behaved like visible light in many ways; for example, it could be reflected by a mirror. Infrared radiation is simply electromagnetic radiation with a lower frequency than visible light, having longer wavelengths of 0.7 micrometer to 1 millimeter. Ultraviolet radiation, like infrared radiation, lies just outside the visible part of the spectrum, but with higher frequencies; some animals, such as bees, are capable of seeing such radiation. Both infrared and ultraviolet radiation are often referred to as forms of light, though they cannot be seen by human beings. Heat energy is often transferred in the form of infrared radiation, which is given off from an object as a result of molecular collisions within it. Molecules typically have a characteristic infrared absorption spectrum, and infrared spectroscopy is a common technique for identifying the molecular structure of substances. Astronomers similarly analyze the infrared radiation emitted by celestial bodies to determine their temperature and composition.

The American Heritage® Science Dictionary Copyright © 2005 by Houghton Mifflin Company. Published by Houghton Mifflin Company. All rights reserved.

Enjoy,

George



At 04:04 PM 2/3/2010, you wrote:

Dear lister,

 

Just wondering if there is such a configuration that would allow me to see the infrared signal directly by eye?

 

I always believed that human eye is not able to detect the far-red signal till one of my colleague argued with me that there might be a way to make this signal visible.

So just want to ask around to see if it is possible.

 

Thanks!

 

Yee

George McNamara, Ph.D.
Image Core Manager
Analytical Imaging Core Facility
University of Miami, Miller School of Medicine
Miami, FL 33136
[hidden email]
[hidden email]
305-243-8436 office
http://www.sylvester.org/AICF (Analytical Imaging Core Facility)
http://www.sylvester.org/AICF/pubspectra.zip (the entire 2000+ spectra .xlsx file is in the zip file)
http://home.earthlink.net/~geomcnamara

[SUBSCRIBE CONFOCALMICROSCOPY Sara Smith]

Thank you all for taking time to answer my question on/off line!

I learned a great deal from all the answers!

It is good to know that the some far red dyes could be visible if I have the right filter set and good eyes!

Thanks again!

 

Yee

On Thu, Feb 4, 2010 at 11:11 PM, George McNamara <[hidden email]> wrote:

Hi Yee,

By definition, infrared photons are invisible to the human eye. So, any visible emission seen from Cy5, Alexa Fluor 647, CF647 (www.biotium.com) is not infrared.

On a more practical note, the longpass mKate filter set I have from Chroma, excitation around the Hg arc lamp 578 nm line (still excites Cy3 and Alexa Fluor 568, for good or bad depending on the experiment) and 600-700 nm emission, or our Zeiss longpass Cy3 filter set (Hg arc lamp 546 nm line), lets me see Cy5 and Alexa Fluor 647 (or at least bright immunofluorescence, ex. insulin in adult pancreatic beta cells). I usually have the room lights off in the microscope room.

Below are two references for Ferri et al papers on filter design.


Direct eye visualization of Cy5 fluorescence for immunocytochemistry and in situ hybridization.
Ferri GL, Isola J, Berger P, Giro G.
J Histochem Cytochem. 2000 Mar;48(3):437-44.PMID: 10681398


Quadruple immunofluorescence: a direct visualization method.
Ferri GL, Gaudio RM, Castello IF, Berger P, Giro G.
J Histochem Cytochem. 1997 Feb;45(2):155-8.PMID: 9016305 [PubMed - indexed for MEDLINE] Related articles



http://www.thefreedictionary.com/infrared

infrared

Relating to the invisible part of the electromagnetic spectrum with wavelengths longer than those of visible red light but shorter than those of microwaves. See more at electromagnetic spectrum.

A Closer Look In 1800 the astronomer Sir William Herschel discovered infrared light while exploring the relationship between heat and light. Herschel used a prism to split a beam of sunlight into a spectrum and then placed a thermometer in each of the bands of light. When he placed the thermometer just outside the red band, where there was no visible color, the temperature rose, as if light were shining on the thermometer. Further experiment showed that this invisible radiation behaved like visible light in many ways; for example, it could be reflected by a mirror. Infrared radiation is simply electromagnetic radiation with a lower frequency than visible light, having longer wavelengths of 0.7 micrometer to 1 millimeter. Ultraviolet radiation, like infrared radiation, lies just outside the visible part of the spectrum, but with higher frequencies; some animals, such as bees, are capable of seeing such radiation. Both infrared and ultraviolet radiation are often referred to as forms of light, though they cannot be seen by human beings. Heat energy is often transferred in the form of infrared radiation, which is given off from an object as a result of molecular collisions within it. Molecules typically have a characteristic infrared absorption spectrum, and infrared spectroscopy is a common technique for identifying the molecular structure of substances. Astronomers similarly analyze the infrared radiation emitted by celestial bodies to determine their temperature and composition.

The American Heritage® Science Dictionary Copyright © 2005 by Houghton Mifflin Company. Published by Houghton Mifflin Company. All rights reserved.

Enjoy,

George

At 04:04 PM 2/3/2010, you wrote:

Dear lister,

 

Just wondering if there is such a configuration that would allow me to see the infrared signal directly by eye?

 

I always believed that human eye is not able to detect the far-red signal till one of my colleague argued with me that there might be a way to make this signal visible.

So just want to ask around to see if it is possible.

 

Thanks!

 

Yee

George McNamara, Ph.D.
Image Core Manager
Analytical Imaging Core Facility
University of Miami, Miller School of Medicine
Miami, FL 33136
[hidden email]
[hidden email]
305-243-8436 office
http://www.sylvester.org/AICF (Analytical Imaging Core Facility)
http://www.sylvester.org/AICF/pubspectra.zip (the entire 2000+ spectra .xlsx file is in the zip file)
http://home.earthlink.net/~geomcnamara

[Saar Oz]

Hi all,

We have no success in labeling actin in fixed cells with Coumarin Phalloidin (Sigma).

Does anyone have a previous experience with this compound, or experience with other Phalloidin compounds which can be applied?

(PFA vs. methanol fixation, concentration,  methanol as the dissolving reagent etc.)

 

Thanks!

 

Saar

[Michal Jarnik]

Saar,

We regularly use Phalloidin-Alexa (mostly 568) from invitrogen, works pretty well for us after % PF fixation.

Michal
-----Original Message-----
From: Confocal Microscopy List on behalf of Saar Oz
Sent: Fri 2/5/2010 4:35 AM
To: [hidden email]
Subject: Coumarin Phalloidin

Hi all,

We have no success in labeling actin in fixed cells with Coumarin Phalloidin
(Sigma).

Does anyone have a previous experience with this compound, or experience
with other Phalloidin compounds which can be applied?

(PFA vs. methanol fixation, concentration,  methanol as the dissolving
reagent etc.)



Thanks!



Saar

[Guenter Giese]

Hi Saar,

 

check this article. It contains a lot of useful information:

 

Vielkind, U. and Swierenga, S. H. (1989). A simple fixation procedure for immunofluorescent detection of different cytoskeletal components within the same cell. Histochemistry 91, 81-8.

 

 

Best,

 

Guenter

 

---

Von: Confocal Microscopy List [mailto:[hidden email]] Im Auftrag von Saar Oz
Gesendet: Freitag, 5. Februar 2010 10:35
An: [hidden email]
Betreff: Coumarin Phalloidin

Hi all,

We have no success in labeling actin in fixed cells with Coumarin Phalloidin (Sigma).

Does anyone have a previous experience with this compound, or experience with other Phalloidin compounds which can be applied?

(PFA vs. methanol fixation, concentration,  methanol as the dissolving reagent etc.)

 

Thanks!

 

Saar

[phil laissue]

Hi Saar,

what kind of cell? In any case, success will strongly depend on the protocol. Rhodamine Phalloidin & PFA worked for us in one filamentous fungus (doesn't work in all), although the staining protocol had to be tweaked (e.g. on ice, no or little spinning) for best results. You may also consider live staining (if possible) - Lifeact (1) being a nice option (first 17aa of Abp140, plus GFP) working in many cells (2,3, and our fil. fungus (although we used full-length Abp140)), but not on all isoforms (4).
Cheers

Philippe

___________________________________________
philippe laissue, phd - bioimaging manager
department of biological sciences
university of essex, colchester CO4 3SQ, UK


1.
Nat Methods. 2008 Jul;5(7):605-7. Epub 2008 Jun 8.
Lifeact: a versatile marker to visualize F-actin.
Riedl J, Crevenna AH, Kessenbrock K, Yu JH, Neukirchen D, Bista M, Bradke F, Jenne D, Holak TA, Werb Z, Sixt M, Wedlich-Soldner R.
2.
Lifeact-mEGFP reveals a dynamic apical F-actin network in tip growing plant cells.
Vidali L, Rounds CM, Hepler PK, Bezanilla M.
PLoS One. 2009 May 29;4(5):e5744.
3.
J Cell Sci. 2009 Oct 1;122(Pt 19):3492-501. Epub 2009 Sep 1.
Moesin orchestrates cortical polarity of melanoma tumour cells to initiate 3D invasion.
Estecha A, Sánchez-Martín L, Puig-Kröger A, Bartolomé RA, Teixidó J, Samaniego R, Sánchez-Mateos P.
4.
Nat Methods. 2009 May;6(5):317.
Lifeact cannot visualize some forms of stress-induced twisted F-actin.
Munsie LN, Caron N, Desmond CR, Truant R.

[McKnight, David]

I would be very surprised to get ay detectable signal out at a micron.
There are two accepted regimes for the human visual response, the
photopic response, which is the normal 'bright adjusted' vision, and
scotopic vision, which is dark adjusted.

The photopic curve is quite weak in the 700-800 nm range (about 0.5%
peak response at 700, and about 0.01% peak response at 750nm)

The scotopic response is somewhat blue shifted from the photopic one (It
peaks at 507nm, rather than 555nm) and you get around two orders of
magnitude less perception once you are above 700nm.

David


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Martin Wessendorf
Sent: Wednesday, February 03, 2010 10:55 PM
To: [hidden email]
Subject: Re: Possibility to visualize far-red signal by eye?

Dear Dr. Yee--

yee sarah wrote:

> Just wondering if there is such a configuration that would allow me to

> see the infrared signal directly by eye?

My recollection is that the human eye can detect photons out past 1000
nm wavelength--it's just not very efficient at doing so.  Cy5 is
certainly visible by eye if the staining is bright enough (e.g., I've
seen intracellularly filled neurons stained with Cy5)--you need a
630-650 nm exciter and a 665-700 nm barrier filter (or similar filters)
to see it with a mercury lamp.  A high NA objective will help.

Good luck!

Martin Wessendorf

--
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]

[Guy Cox-2]

George,

          That's all just a matter of definition.  Down to 800nm is
quite visible but very dim.  It all looks the same colour from about 650
onwards.  Human vision doesn't cut off, it just gradually fades out.
But if we are talking about practical labelling with sensible dye
loadings, I have to say that I could not see, by eye, the image from
bacterial cells strongly labelled with Atto 647 which gave excellent
images in confocal and STED.  

                                       Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net
 


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of George McNamara
Sent: Friday, 5 February 2010 3:11 PM
To: [hidden email]
Subject: Re: Possibility to visualize far-red signal by eye?

Hi Yee,

By definition, infrared photons are invisible to
the human eye. So, any visible emission seen from
Cy5, Alexa Fluor 647, CF647 (www.biotium.com) is not infrared.

On a more practical note, the longpass mKate
filter set I have from Chroma, excitation around
the Hg arc lamp 578 nm line (still excites Cy3
and Alexa Fluor 568, for good or bad depending on
the experiment) and 600-700 nm emission, or our
Zeiss longpass Cy3 filter set (Hg arc lamp 546 nm
line), lets me see Cy5 and Alexa Fluor 647 (or at
least bright immunofluorescence, ex. insulin in
adult pancreatic beta cells). I usually have the
room lights off in the microscope room.

Below are two references for Ferri et al papers on filter design.


<http://www.ncbi.nlm.nih.gov/sites//pubmed/10681398?itool=EntrezSystem2.
PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=1>Direct
eye visualization of Cy5 fluorescence for
immunocytochemistry and in situ hybridization.
Ferri GL, Isola J, Berger P, Giro G.
J Histochem Cytochem. 2000 Mar;48(3):437-44.PMID: 10681398


<http://www.ncbi.nlm.nih.gov/sites//pubmed/9016305?itool=EntrezSystem2.P
Entrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=2>Quadruple

immunofluorescence: a direct visualization method.
Ferri GL, Gaudio RM, Castello IF, Berger P, Giro G.
J Histochem Cytochem. 1997 Feb;45(2):155-8.PMID:
9016305 [PubMed - indexed for
MEDLINE]<http://www.ncbi.nlm.nih.gov/sites//sites/entrez?db=pubmed&cmd=l
ink&linkname=pubmed_pubmed&uid=9016305&ordinalpos=2>Related
articles



http://www.thefreedictionary.com/infrared
infrared
Relating to the invisible part of the
electromagnetic spectrum with wavelengths longer
than those of visible red light but shorter than
those of microwaves. See more at
<http://www.thefreedictionary.com/electromagnetic+spectrum>electromagnet
ic
spectrum.
A Closer Look In 1800 the astronomer Sir William
Herschel discovered infrared light while
exploring the relationship between heat and
light. Herschel used a prism to split a beam of
sunlight into a spectrum and then placed a
thermometer in each of the bands of light. When
he placed the thermometer just outside the red
band, where there was no visible color, the
temperature rose, as if light were shining on the
thermometer. Further experiment showed that this
invisible radiation behaved like visible light in
many ways; for example, it could be reflected by
a mirror. Infrared radiation is simply
electromagnetic radiation with a lower frequency
than visible light, having longer wavelengths of
0.7 micrometer to 1 millimeter. Ultraviolet
radiation, like infrared radiation, lies just
outside the visible part of the spectrum, but
with higher frequencies; some animals, such as
bees, are capable of seeing such radiation. Both
infrared and ultraviolet radiation are often
referred to as forms of light, though they cannot
be seen by human beings. Heat energy is often
transferred in the form of infrared radiation,
which is given off from an object as a result of
molecular collisions within it. Molecules
typically have a characteristic infrared
absorption spectrum, and infrared spectroscopy is
a common technique for identifying the molecular
structure of substances. Astronomers similarly
analyze the infrared radiation emitted by
celestial bodies to determine their temperature and composition.
The American Heritage(r) Science Dictionary
Copyright (c) 2005 by Houghton Mifflin Company.
Published by
<http://www.eref-trade.hmco.com/>Houghton Mifflin Company. All rights
reserved.


Enjoy,

George



At 04:04 PM 2/3/2010, you wrote:

>Dear lister,
>
>
>
>Just wondering if there is such a configuration
>that would allow me to see the infrared signal directly by eye?
>
>
>
>I always believed that human eye is not able to
>detect the far-red signal till one of my
>colleague argued with me that there might be a way to make this signal

visible.

>
>So just want to ask around to see if it is possible.
>
>
>
>Thanks!
>
>
>
>Yee

George McNamara, Ph.D.
Image Core Manager
Analytical Imaging Core Facility
University of Miami, Miller School of Medicine
Miami, FL 33136
[hidden email]
[hidden email]
305-243-8436 office
http://www.sylvester.org/AICF (Analytical Imaging Core Facility)
http://www.sylvester.org/AICF/pubspectra.zip (the
entire 2000+ spectra .xlsx file is in the zip file)
http://home.earthlink.net/~geomcnamara

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18:35:00

[Martin Wessendorf-2]

McKnight, David wrote:
> I would be very surprised to get ay detectable signal out at a micron.
> There are two accepted regimes for the human visual response, the
> photopic response, which is the normal 'bright adjusted' vision, and
> scotopic vision, which is dark adjusted.

See: Griffin DR, Hubbard R and Wald G: "The sensitivity of the human eye
to infra-red radiation" J. Opt Soc Am 37: 546, 1947.

It's available at:
http://www.opticsinfobase.org/josa/issue.cfm?volume=37&issue=7

The sensitivity at 1000 nm was over 12 orders of magnitude lower than
that at 550 nm, but was still measurable.  (Sensitivity at 650 nm was a
mere 3-4 orders of magnitude lower!)

Martin
--
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]

[Julio Vazquez]

We typically don't bother putting far-red filter sets on the eye piece side of our microscopes, but one of our users needed to take confocal images of virus-infected cells labeled with Qdot 655 in human skin biopsies. The foci were very rare, and therefore unlikely to be hit upon just by random luck. We purchased a Q655 filter set, and we had no trouble finding the foci visually after dimming the lights and letting our eyes adapt. The emission filter was a 20 or 40 nm bandpass around 655. The foci were moderately bright, but we could also see enough glow in the background to see the overall tissue. 

Julio Vazquez

Fred Hutchinson Cancer Research Center

Seattle, WA 98109-1024

http://www.fhcrc.org

==

On Feb 5, 2010, at 8:16 AM, Martin Wessendorf wrote:

McKnight, David wrote:

I would be very surprised to get ay detectable signal out at a micron.

There are two accepted regimes for the human visual response, the

photopic response, which is the normal 'bright adjusted' vision, and

scotopic vision, which is dark adjusted. 

See: Griffin DR, Hubbard R and Wald G: "The sensitivity of the human eye

to infra-red radiation" J. Opt Soc Am 37: 546, 1947.

It's available at:

http://www.opticsinfobase.org/josa/issue.cfm?volume=37&issue=7

The sensitivity at 1000 nm was over 12 orders of magnitude lower than

that at 550 nm, but was still measurable.  (Sensitivity at 650 nm was a

mere 3-4 orders of magnitude lower!)

Martin

-- 

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]

[Mario-2]

Saar,

For me, using PFA fixation with and without 0.2-0.5% Triton followed
by phalloidin-FITC, phalloidin-rhodamine,
phalloidin-biotin+streptavidin-fluorophoreofchoice has always proven
successful except in the rare instance when the phalloidin itself was
defective.

If you need to use a UV excitable dye with blue emission, which are
characteristic of the coumarins I am familiar with, I strongly
suggest that you try a vendor other than Sigma. Unless some newer
synthetic method is now available, phalloidin cannot be derivatized
using a free amino group (doesn't have one- phallicidin does) and
requires relatively fancy chemistry to build a fluorophore phalloidin
conjugate by assembling it from "scratch." I once considered doing
this myself but decided it was too much trouble. Using
phalloidin-biotin as a primary with streptavidin and any number of
fluorophores always worked very well (Molecular Probes/Invitrogen).

You don't mention the kinds of cells you are using, but I assume your
complaint indicates that you get no staining, not that you don't care
for the morphology. Depending on the cell type and the cell growth
conditions, you can get very long actin filaments typically in
fibroblasts (5 um or longer) or very short spiky (~1 um) ones.

Anyway, if you are not getting any staining and you are using UV
ext./blue emission, your phalloidin is probably inactive. Make Sigma
give you a credit and try a different vendor.

Mario

>Hi all,
>We have no success in labeling actin in fixed cells with Coumarin
>Phalloidin (Sigma).
>Does anyone have a previous experience with this compound, or
>experience with other Phalloidin compounds which can be applied?
>(PFA vs. methanol fixation, concentration,  methanol as the
>dissolving reagent etc.)
>
>Thanks!
>
>Saar

--
________________________________________________________________________________
Mario M. Moronne, Ph.D.

[hidden email]
[hidden email]