5 color imaging (6 color would be even better!)

[Aromis Storm]

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Dear Everyone:

I have been trying to visualize a macromolecular structure with 4 color
imaging. I have been doing this with spinning disc microscopy as I need
complete sections of cells also to know the distribution of the structure
inside the cells in 3D. So far things are working properly BUT ...

We now would like to upgrade to 5 color imaging as we want to visualize
additional components of the structure. I checked the spinning disc setting
myself and I asked several senior users of spinning disc and got the opinion
that "5 color imaging with spinning disc without bleed-through is hardly
possible, if possible at all". The main reason being that it is not very
likely to set up emission filter combinations for 5 color imaging without
bleed-through. Spectral mixing is technically possible but not very
promising.

I also learned that 5 color imaging is possible with laser scanning
microscopy with which one can specifically define the emission band pass
range. But with laser scanning microscope it will cost me much more time to
collect enough data for statistical analysis.

So my questions would be: can anyone of you share your
experience/opinion/literatures on 5 color imaging? I also tried to look for
literatures describing multi-color (> 4 color) imaging (I think there must
be decent papers about it) but so far I have not found any. Maybe some of
you have some nice papers about this?

Thank you very much for your input.

Nice weekend!

Aromis  

[Kevin Ryan]

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You might find Garini et al 1998 "Signal to Noise Analysis of Multiple Color Fluorescence Imaging Microscopy" (http://itmhrt.ca/students/files/April2008/pdfs_for_journal_clubs/pdf4_Signal_to_noise_analysis-Cytometry_1999-Y_Garini.pdf), a useful reference.


Kevin Ryan
Media Cybernetics, Inc.


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Aromis Storm
Sent: Friday, February 08, 2013 9:13 AM
To: [hidden email]
Subject: 5 color imaging (6 color would be even better!)

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Dear Everyone:

I have been trying to visualize a macromolecular structure with 4 color imaging. I have been doing this with spinning disc microscopy as I need complete sections of cells also to know the distribution of the structure inside the cells in 3D. So far things are working properly BUT ...

We now would like to upgrade to 5 color imaging as we want to visualize additional components of the structure. I checked the spinning disc setting myself and I asked several senior users of spinning disc and got the opinion that "5 color imaging with spinning disc without bleed-through is hardly possible, if possible at all". The main reason being that it is not very likely to set up emission filter combinations for 5 color imaging without bleed-through. Spectral mixing is technically possible but not very promising.

I also learned that 5 color imaging is possible with laser scanning microscopy with which one can specifically define the emission band pass range. But with laser scanning microscope it will cost me much more time to collect enough data for statistical analysis.

So my questions would be: can anyone of you share your experience/opinion/literatures on 5 color imaging? I also tried to look for literatures describing multi-color (> 4 color) imaging (I think there must be decent papers about it) but so far I have not found any. Maybe some of you have some nice papers about this?

Thank you very much for your input.

Nice weekend!

Aromis  

[George McNamara]

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) Hi Kevin,

Thanks for referencing my former colleagues at ASI. I also recommend
Yuval and my 2006 review at
DOI: 10.1002/cyto.a.20311
and perusal of other articles (including Carl Boswell and I) in the rest
of the 2006 spectral imaging issue
http://onlinelibrary.wiley.com/doi/10.1002/cyto.a.v69a:8/issuetoc
(back issues of Cytometry are free online).

As for 5 colors (actually 6 with a separate filter cube for DAPI), the
ASI SKY system has about 800 references:

http://www.ncbi.nlm.nih.gov/pubmed?term=spectral%20karyotyp*

using (essentially) Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 594
(or Texas Red), Cy5 (or Alexa fluor 647), Cy5.5

With the same instrument Tsurui et al 2007 (
PMID:
10769049) acquired seven colors (but with 3 filter cubes):
Alexa488 and Alexa532, for Alexa546, Alexa568, and Alexa594, and for Cy5
and Cy5.5.

Not that I would buy a SKY instrument for one experiment (though my
former colleagues at CHLA might be willing to sell theirs).

If I wanted to buy a new instrument, I would contact Jeremy Lerner at
LightForm Inc, http://www.lightforminc.com/ to order a PARISS with solid
state lighting (I would go for a Lumencor SPECTRA X) and excitation slit
(ideally some light output from the X to closely match the slit). This
would result in a pretty sweet linescan confocal (ditch the disc) -
Jeremy routinely puts on a motorized stage for "pushbroom" acquisition.

If you want to go fast, Keith Lidke (UNM), updated from an earlier
Sandia National Labs clone of Jeremy's PARISS, have developed a fast
scanning module - see the "Hyperspectral Microscope Layout" page at
http://www.systemscenters.org/wordpress/wp-content/uploads/2011/01/nm-ncbs-aug-8-11-lidke.pdf
Not clear from that presentation, but Keith is tracking 8 colors quantum
dots at 30 fps with his instrument (single excitation, 30 full field
spectral images per sec).

***

A little chemistry can also go a long way ... if certain colors are NOT
being colocalized, you could buy or make a FRET construct. Alan Waggoner
had a presentation circa 1996 (a CSHL immunocytochemistry course) with
nearly 100% FRET from Cy3 to Cy5. Likewise in the FP world, Miyawaki
published CY11.5 with nearly 100% FRET, and CyPet-YPet was almost as
high due to non-covalent heterodimerization.

Of course most core facility confocal microscopes (the Leica SP5's and
Zeiss LSM710's I manage for examples) have 405, 458, 488, 561, 594, and
633 nm lasers - can excite different dyes (or FPs) with each.

Getting better resolution out of existing (point scanning) confocals ...
See  PMID 23361088 ("3B" ImageJ plugin, only just obtained the plugin,
have not had time to get it running on my PC - some file location issue
I have not figured out yet) and 22357945 (Munck PiMP ImageJ plugin ...
but you'll need the plugin file locally, not the silly VIB Citrix
server, to do PiMP usefully). I've done two color PiMP - works great
(SP5 or LSM710, plan apo 63x/1.4NA, 30 nm pixel size, filter size = 1.6,
16-bit output). Ther is a non-linear step (Rainer Heintzmann came up
with that critical idea), so don't bother with intensity quantitation
(thresholding and counting pixels should be fine).

George
p.s. best wishes with the controls. Unlikely I would be a reviewer (and
don't suggest me!), but I would expect to see proof that both your
"single marker' and your FMOs (fluorescence minus one) controls worked well.

On 2/8/2013 1:29 PM, Kevin Ryan wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> You might find Garini et al 1998 "Signal to Noise Analysis of Multiple Color Fluorescence Imaging Microscopy" (http://itmhrt.ca/students/files/April2008/pdfs_for_journal_clubs/pdf4_Signal_to_noise_analysis-Cytometry_1999-Y_Garini.pdf), a useful reference.
>
>
> Kevin Ryan
> Media Cybernetics, Inc.
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Aromis Storm
> Sent: Friday, February 08, 2013 9:13 AM
> To: [hidden email]
> Subject: 5 color imaging (6 color would be even better!)
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Everyone:
>
> I have been trying to visualize a macromolecular structure with 4 color imaging. I have been doing this with spinning disc microscopy as I need complete sections of cells also to know the distribution of the structure inside the cells in 3D. So far things are working properly BUT ...
>
> We now would like to upgrade to 5 color imaging as we want to visualize additional components of the structure. I checked the spinning disc setting myself and I asked several senior users of spinning disc and got the opinion that "5 color imaging with spinning disc without bleed-through is hardly possible, if possible at all". The main reason being that it is not very likely to set up emission filter combinations for 5 color imaging without bleed-through. Spectral mixing is technically possible but not very promising.
>
> I also learned that 5 color imaging is possible with laser scanning microscopy with which one can specifically define the emission band pass range. But with laser scanning microscope it will cost me much more time to collect enough data for statistical analysis.
>
> So my questions would be: can anyone of you share your experience/opinion/literatures on 5 color imaging? I also tried to look for literatures describing multi-color (>  4 color) imaging (I think there must be decent papers about it) but so far I have not found any. Maybe some of you have some nice papers about this?
>
> Thank you very much for your input.
>
> Nice weekend!
>
> Aromis
>
>    

[Rosemary.White]

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Dear Aromis,

5-colour imaging should be possible without bleed-through by sequential scanning on a regular confocal. Not sure how this is done with spinning disc instruments, but if possible, this is an option.

cheers,
Rosemary

Dr Rosemary White
CSIRO Plant Industry
GPO Box 1600
Canberra, ACT 2601
Australia

T 61 2 6246 5475
F 61 2 6246 5334
________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of Aromis Storm [[hidden email]]
Sent: Saturday, 9 February 2013 1:13 a.m.
To: [hidden email]
Subject: 5 color imaging (6 color would be even better!)

*****
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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Dear Everyone:

I have been trying to visualize a macromolecular structure with 4 color
imaging. I have been doing this with spinning disc microscopy as I need
complete sections of cells also to know the distribution of the structure
inside the cells in 3D. So far things are working properly BUT ...

We now would like to upgrade to 5 color imaging as we want to visualize
additional components of the structure. I checked the spinning disc setting
myself and I asked several senior users of spinning disc and got the opinion
that "5 color imaging with spinning disc without bleed-through is hardly
possible, if possible at all". The main reason being that it is not very
likely to set up emission filter combinations for 5 color imaging without
bleed-through. Spectral mixing is technically possible but not very
promising.

I also learned that 5 color imaging is possible with laser scanning
microscopy with which one can specifically define the emission band pass
range. But with laser scanning microscope it will cost me much more time to
collect enough data for statistical analysis.

So my questions would be: can anyone of you share your
experience/opinion/literatures on 5 color imaging? I also tried to look for
literatures describing multi-color (> 4 color) imaging (I think there must
be decent papers about it) but so far I have not found any. Maybe some of
you have some nice papers about this?

Thank you very much for your input.

Nice weekend!

Aromis

[Johannes Helm]

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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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Good evening,

Kevin and George have already provided good answers. What I write in the
following lines is more of a concept than a practical solution in case of
camera detection based systems.

Two very interesting - to my mind - approaches have been described and
successfully used by Åslund, Carlsson, Liljeborg and others in the 1990s
on a conventional CLSM. The approaches, both called "IMS Technique", are
based on lock-in detection principles:

In brief:

Provided, one has n e |N >1 dye substances, which can be excited at
available laser wavelengths without any cross excitation, the resp. laser
beams each exciting one, and one only, out of the n dyes can be modulated
using, e.g., n electro optic modulators at n individual frequencies and
phases.

First approach:
If the periods of the n modulation frequencies are long compared to the
decay times of the dye substances, the resulting n fluorescences will then
also be modulated at the same frequencies and phases (at which, in this
case, phase shifts related to the different decay times of the dyes can be
neglected). Irrespectively of any spectral overlap of the fluorscence
spectra of the dyes, the resulting fluorescence signals as detected by one
photomultiplier can be read out via n lock-in amplifiers and be recorded
at negligibly large cross talk to n channels.

Second approach (basically a life time method):
If the periods of the modulation frequencies are short scompared to the
decay times of the lasers, the resulting fluorescences will then also be
modulated at
the same frequencies but different phases (in this case, other then in the
first approach, phase differences as induced by the different decay times
of the different dyes cannot be neglected but are the detection
magnitude). If the n dyes have n different decay times, at which any Delta
T between any two dyes must be large enough to cause a specific phase
shift of the fluorescences, n lock-in amplifiers can be used to read out
the resp. signals from one photomultiplier - with sufficiently small
transition time spread - at negligible cross talk into n channels.

A task as far as I know not done on a regular basis and possibly un-done -
besides the financial challenge, of course - for this method to be applied
to spinning disk systems including camera detection would be to read out
the signals as detected by the camera via lock-in amplifiers.
Alternatively, one might try to use PMT arrays.

References:

E.g.

K. Carlsson, N. Åslund, K. Mossberg & J. Philip, "Simultaneous confocal
recording of multiple fluorescent labels with improved channel separation"
J. Microsc. 176, 287-299 (1994).

K. Carlsson & B. Ulfhake, "Improved fluorophore separation with IMS
confocal microscopy" NeuroReport, 6(8), 1169-1173 (1995).

K. Carlsson, "Signal-to-noise ratio for confocal microscopy when using the
IMS technique" Micron 26, 317-322 (1995)

P.J. Helm, O. Franksson & K. Carlsson, "A confocal scanning laser
microscope for quantitative ratiometric 3D measurements of [Ca2+] and Ca2+
diffusion in living cells stained with Fura-2" Pfluegers Arch - Eur J
Physiol, 429, 672-681 (1995).

K. Carlsson, A. Liljeborg, "Confocal fluorescence microscopy using
spectral and lifetime information to simultaneuosly record four
fluorophores with high channel separation", J. Microsc. 185, 37-46 (1997).

PJ Helm, A Patwardhan, and EMM Manders (1997),
A study of the precision of confocal, ratiometric, Fura-2 based measurements,
Cell Calcium 22(4):287-298

K. Carlsson & A. Liljeborg, "Simultaneous confocal lifetime imaging of
multiple fluorophores using the Intensity-modulated Multiple-wavelength
Scanning (IMS) technique," J. Microsc. 191, 119-127 (1998).

as well as

Åslund, N.; Carlsson, K.S. Apparatus for Quantitative Imaging of Multiple
Fluorophores.
U.S. Patent 5,294,799, 15 March 1994.

Åslund, N.; Carlsson, K.S. Apparatus for Quantitative Imaging of Multiple
Fluorophores Using Dual Detectors. U.S. Patent 5,418,371, 23 May 1995.



Best wishes,

Johannes


--
P. Johannes Helm, M.Sc. PhD
Seniorengineer
CMBN
University of Oslo
Institute of Basic Medical Science
Department of Anatomy
Postboks 1105 - Blindern
NO-0317 Oslo

Voice: [phone]
Fax: [phone]

WWW: folk.uio.no/jhelm

> *****
> To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Everyone:
>
> I have been trying to visualize a macromolecular structure with 4 color
imaging. I have been doing this with spinning disc microscopy as I need
complete sections of cells also to know the distribution of the
structure
> inside the cells in 3D. So far things are working properly BUT ...
>
> We now would like to upgrade to 5 color imaging as we want to visualize
additional components of the structure. I checked the spinning disc
setting
> myself and I asked several senior users of spinning disc and got the
opinion
> that "5 color imaging with spinning disc without bleed-through is hardly
possible, if possible at all". The main reason being that it is not very
likely to set up emission filter combinations for 5 color imaging
without
> bleed-through. Spectral mixing is technically possible but not very
promising.
>
> I also learned that 5 color imaging is possible with laser scanning
microscopy with which one can specifically define the emission band pass
range. But with laser scanning microscope it will cost me much more time
to
> collect enough data for statistical analysis.
>
> So my questions would be: can anyone of you share your
> experience/opinion/literatures on 5 color imaging? I also tried to look for
> literatures describing multi-color (> 4 color) imaging (I think there
must
> be decent papers about it) but so far I have not found any. Maybe some
of
> you have some nice papers about this?
>
> Thank you very much for your input.
>
> Nice weekend!
>
> Aromis
>

[Aromis Storm]

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Dear Everyone:

Thank you all for your input. Just back from my weekend so I need some time
to digest these info especially with the suggested papers. Also I'll have an
appointment with the head of our imaging center to discuss about it so I
guess I might get a feasible plan.

What confuses me a bit is that it seems that 5 color imaging is really
doable (even routine) but the people around me ,including those who started
to use confocal microscopy from the 1st generation, are not very positive
about this. As a person coming from conventional molecular biology
background I'll try to understand the discrepancy.

After collecting/understanding enough info I'll try to compose a 'protocol'
of 5/6 color imaging as I presume more people might be interested in it.

More questions from me can be expected.

Thanks again.

Best wishes,

Aro


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Good evening,

Kevin and George have already provided good answers. What I write in the
following lines is more of a concept than a practical solution in case of
camera detection based systems.

Two very interesting - to my mind - approaches have been described and
successfully used by Åslund, Carlsson, Liljeborg and others in the 1990s on
a conventional CLSM. The approaches, both called "IMS Technique", are based
on lock-in detection principles:


In brief:

Provided, one has n e |N >1 dye substances, which can be excited at
available laser wavelengths without any cross excitation, the resp. laser
beams each exciting one, and one only, out of the n dyes can be modulated
using, e.g., n electro optic modulators at n individual frequencies and
phases.

First approach:
If the periods of the n modulation frequencies are long compared to the
decay times of the dye substances, the resulting n fluorescences will then
also be modulated at the same frequencies and phases (at which, in this
case, phase shifts related to the different decay times of the dyes can be
neglected). Irrespectively of any spectral overlap of the fluorscence
spectra of the dyes, the resulting fluorescence signals as detected by one
photomultiplier can be read out via n lock-in amplifiers and be recorded at
negligibly large cross talk to n channels.

Second approach (basically a life time method):
If the periods of the modulation frequencies are short scompared to the
decay times of the lasers, the resulting fluorescences will then also be
modulated at the same frequencies but different phases (in this case, other
then in the first approach, phase differences as induced by the different
decay times of the different dyes cannot be neglected but are the detection
magnitude). If the n dyes have n different decay times, at which any Delta T
between any two dyes must be large enough to cause a specific phase shift of
the fluorescences, n lock-in amplifiers can be used to read out the resp.
signals from one photomultiplier - with sufficiently small transition time
spread - at negligible cross talk into n channels.

A task as far as I know not done on a regular basis and possibly un-done -
besides the financial challenge, of course - for this method to be applied
to spinning disk systems including camera detection would be to read out the
signals as detected by the camera via lock-in amplifiers.
Alternatively, one might try to use PMT arrays.

References:

E.g.

K. Carlsson, N. Åslund, K. Mossberg & J. Philip, "Simultaneous confocal
recording of multiple fluorescent labels with improved channel separation"
J. Microsc. 176, 287-299 (1994).

K. Carlsson & B. Ulfhake, "Improved fluorophore separation with IMS confocal
microscopy" NeuroReport, 6(8), 1169-1173 (1995).

K. Carlsson, "Signal-to-noise ratio for confocal microscopy when using the
IMS technique" Micron 26, 317-322 (1995)

P.J. Helm, O. Franksson & K. Carlsson, "A confocal scanning laser microscope
for quantitative ratiometric 3D measurements of [Ca2+] and Ca2+ diffusion in
living cells stained with Fura-2" Pfluegers Arch - Eur J Physiol, 429,
672-681 (1995).

K. Carlsson, A. Liljeborg, "Confocal fluorescence microscopy using spectral
and lifetime information to simultaneuosly record four fluorophores with
high channel separation", J. Microsc. 185, 37-46 (1997).

PJ Helm, A Patwardhan, and EMM Manders (1997), A study of the precision of
confocal, ratiometric, Fura-2 based measurements, Cell Calcium 22(4):287-298

K. Carlsson & A. Liljeborg, "Simultaneous confocal lifetime imaging of
multiple fluorophores using the Intensity-modulated Multiple-wavelength
Scanning (IMS) technique," J. Microsc. 191, 119-127 (1998).

as well as

Åslund, N.; Carlsson, K.S. Apparatus for Quantitative Imaging of Multiple
Fluorophores.
U.S. Patent 5,294,799, 15 March 1994.

Åslund, N.; Carlsson, K.S. Apparatus for Quantitative Imaging of Multiple
Fluorophores Using Dual Detectors. U.S. Patent 5,418,371, 23 May 1995.



Best wishes,

Johannes


--
P. Johannes Helm, M.Sc. PhD
Seniorengineer
CMBN
University of Oslo
Institute of Basic Medical Science
Department of Anatomy
Postboks 1105 - Blindern
NO-0317 Oslo

Voice: [phone]
Fax: [phone]

WWW: folk.uio.no/jhelm

> *****
> To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Everyone:
>
> I have been trying to visualize a macromolecular structure with 4
> color
imaging. I have been doing this with spinning disc microscopy as I need
complete sections of cells also to know the distribution of the structure
> inside the cells in 3D. So far things are working properly BUT ...
>
> We now would like to upgrade to 5 color imaging as we want to
> visualize
additional components of the structure. I checked the spinning disc setting
> myself and I asked several senior users of spinning disc and got the
opinion
> that "5 color imaging with spinning disc without bleed-through is
> hardly
possible, if possible at all". The main reason being that it is not very
likely to set up emission filter combinations for 5 color imaging without
> bleed-through. Spectral mixing is technically possible but not very
promising.
>
> I also learned that 5 color imaging is possible with laser scanning
microscopy with which one can specifically define the emission band pass
range. But with laser scanning microscope it will cost me much more time to
> collect enough data for statistical analysis.
>
> So my questions would be: can anyone of you share your
> experience/opinion/literatures on 5 color imaging? I also tried to
> look for literatures describing multi-color (> 4 color) imaging (I
> think there
must
> be decent papers about it) but so far I have not found any. Maybe some
of
> you have some nice papers about this?
>
> Thank you very much for your input.
>
> Nice weekend!
>
> Aromis
>

[Steffen Dietzel]

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Dear Aro,

doing 5 colors routinely on a clsm usually is a problem of having the
right lasers in the system. Not many are equiped with five lasers. If
you can excite them, you can separate Dapi, Fitc/Alexa488, Cy3,
C3.5/Texas Red and Cy5. We did this with 405, 488, 561, 594 and 633. You
have to do it sequentially, not paralel since eg the green dyes show up
pretty strongly in the orange channel when excited simultaneously. See
the following paper for more on this

Towards many colors in FISH on 3D-preserved interphase nuclei.
Walter J, Joffe B, Bolzer A, Albiez H, Benedetti PA, Müller S, Speicher
MR, Cremer T, Cremer M, Solovei I.
Cytogenet Genome Res. 2006;114(3-4):367-78. Review.
http://www.karger.com/Article/FullText/94227#SA4


On a widefield system, in addition to those, DEAC, Cy5.5 and Cy7 have
been used to get 8-color labeling. The key to success was to use very
narrow bandpass filters, so you end up with rather dim images but good
signal to noise.

On top of that, these colors were used for combinatorial labeling to
identify all 24 human chromosomes by a unique combination of colors. See
for example another paper from my former collegues:

PLoS Biol. 2005 May;3(5):e157. Epub 2005 Apr 26.
Three-dimensional maps of all chromosomes in human male fibroblast
nuclei and prometaphase rosettes.
Bolzer A, Kreth G, Solovei I, Koehler D, Saracoglu K, Fauth C, Müller S,
Eils R, Cremer C, Speicher MR, Cremer T.
http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.0030157
doi:10.1371/journal.pbio.0030157

The first paper in that series was
Karyotyping human chromosomes by combinatorial multi-fluor FISH.
Speicher MR, Gwyn Ballard S, Ward DC.
Nat Genet. 1996 Apr;12(4):368-75.


Best regards

Steffen


On 10.02.2013 23:16, Aromis wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Everyone:
>
> Thank you all for your input. Just back from my weekend so I need some time
> to digest these info especially with the suggested papers. Also I'll have an
> appointment with the head of our imaging center to discuss about it so I
> guess I might get a feasible plan.
>
> What confuses me a bit is that it seems that 5 color imaging is really
> doable (even routine) but the people around me ,including those who started
> to use confocal microscopy from the 1st generation, are not very positive
> about this. As a person coming from conventional molecular biology
> background I'll try to understand the discrepancy.
>
> After collecting/understanding enough info I'll try to compose a 'protocol'
> of 5/6 color imaging as I presume more people might be interested in it.
>
> More questions from me can be expected.
>
> Thanks again.
>
> Best wishes,
>
> Aro
>
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Good evening,
>
> Kevin and George have already provided good answers. What I write in the
> following lines is more of a concept than a practical solution in case of
> camera detection based systems.
>
> Two very interesting - to my mind - approaches have been described and
> successfully used by Åslund, Carlsson, Liljeborg and others in the 1990s on
> a conventional CLSM. The approaches, both called "IMS Technique", are based
> on lock-in detection principles:
>
>
> In brief:
>
> Provided, one has n e |N>1 dye substances, which can be excited at
> available laser wavelengths without any cross excitation, the resp. laser
> beams each exciting one, and one only, out of the n dyes can be modulated
> using, e.g., n electro optic modulators at n individual frequencies and
> phases.
>
> First approach:
> If the periods of the n modulation frequencies are long compared to the
> decay times of the dye substances, the resulting n fluorescences will then
> also be modulated at the same frequencies and phases (at which, in this
> case, phase shifts related to the different decay times of the dyes can be
> neglected). Irrespectively of any spectral overlap of the fluorscence
> spectra of the dyes, the resulting fluorescence signals as detected by one
> photomultiplier can be read out via n lock-in amplifiers and be recorded at
> negligibly large cross talk to n channels.
>
> Second approach (basically a life time method):
> If the periods of the modulation frequencies are short scompared to the
> decay times of the lasers, the resulting fluorescences will then also be
> modulated at the same frequencies but different phases (in this case, other
> then in the first approach, phase differences as induced by the different
> decay times of the different dyes cannot be neglected but are the detection
> magnitude). If the n dyes have n different decay times, at which any Delta T
> between any two dyes must be large enough to cause a specific phase shift of
> the fluorescences, n lock-in amplifiers can be used to read out the resp.
> signals from one photomultiplier - with sufficiently small transition time
> spread - at negligible cross talk into n channels.
>
> A task as far as I know not done on a regular basis and possibly un-done -
> besides the financial challenge, of course - for this method to be applied
> to spinning disk systems including camera detection would be to read out the
> signals as detected by the camera via lock-in amplifiers.
> Alternatively, one might try to use PMT arrays.
>
> References:
>
> E.g.
>
> K. Carlsson, N. Åslund, K. Mossberg&  J. Philip, "Simultaneous confocal
> recording of multiple fluorescent labels with improved channel separation"
> J. Microsc. 176, 287-299 (1994).
>
> K. Carlsson&  B. Ulfhake, "Improved fluorophore separation with IMS confocal
> microscopy" NeuroReport, 6(8), 1169-1173 (1995).
>
> K. Carlsson, "Signal-to-noise ratio for confocal microscopy when using the
> IMS technique" Micron 26, 317-322 (1995)
>
> P.J. Helm, O. Franksson&  K. Carlsson, "A confocal scanning laser microscope
> for quantitative ratiometric 3D measurements of [Ca2+] and Ca2+ diffusion in
> living cells stained with Fura-2" Pfluegers Arch - Eur J Physiol, 429,
> 672-681 (1995).
>
> K. Carlsson, A. Liljeborg, "Confocal fluorescence microscopy using spectral
> and lifetime information to simultaneuosly record four fluorophores with
> high channel separation", J. Microsc. 185, 37-46 (1997).
>
> PJ Helm, A Patwardhan, and EMM Manders (1997), A study of the precision of
> confocal, ratiometric, Fura-2 based measurements, Cell Calcium 22(4):287-298
>
> K. Carlsson&  A. Liljeborg, "Simultaneous confocal lifetime imaging of
> multiple fluorophores using the Intensity-modulated Multiple-wavelength
> Scanning (IMS) technique," J. Microsc. 191, 119-127 (1998).
>
> as well as
>
> Åslund, N.; Carlsson, K.S. Apparatus for Quantitative Imaging of Multiple
> Fluorophores.
> U.S. Patent 5,294,799, 15 March 1994.
>
> Åslund, N.; Carlsson, K.S. Apparatus for Quantitative Imaging of Multiple
> Fluorophores Using Dual Detectors. U.S. Patent 5,418,371, 23 May 1995.
>
>
>
> Best wishes,
>
> Johannes
>
>
> --
> P. Johannes Helm, M.Sc. PhD
> Seniorengineer
> CMBN
> University of Oslo
> Institute of Basic Medical Science
> Department of Anatomy
> Postboks 1105 - Blindern
> NO-0317 Oslo
>
> Voice: [phone]
> Fax: [phone]
>
> WWW: folk.uio.no/jhelm
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear Everyone:
>>
>> I have been trying to visualize a macromolecular structure with 4
>> color
> imaging. I have been doing this with spinning disc microscopy as I need
> complete sections of cells also to know the distribution of the structure
>> inside the cells in 3D. So far things are working properly BUT ...
>>
>> We now would like to upgrade to 5 color imaging as we want to
>> visualize
> additional components of the structure. I checked the spinning disc setting
>> myself and I asked several senior users of spinning disc and got the
> opinion
>> that "5 color imaging with spinning disc without bleed-through is
>> hardly
> possible, if possible at all". The main reason being that it is not very
> likely to set up emission filter combinations for 5 color imaging without
>> bleed-through. Spectral mixing is technically possible but not very
> promising.
>>
>> I also learned that 5 color imaging is possible with laser scanning
> microscopy with which one can specifically define the emission band pass
> range. But with laser scanning microscope it will cost me much more time to
>> collect enough data for statistical analysis.
>>
>> So my questions would be: can anyone of you share your
>> experience/opinion/literatures on 5 color imaging? I also tried to
>> look for literatures describing multi-color (>  4 color) imaging (I
>> think there
> must
>> be decent papers about it) but so far I have not found any. Maybe some
> of
>> you have some nice papers about this?
>>
>> Thank you very much for your input.
>>
>> Nice weekend!
>>
>> Aromis
>>
>

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

Mail room:
Marchioninistr. 15, D-81377 München

Building location:
Marchioninistr. 27,  München-Großhadern

[Andreas Bruckbauer]

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Dear all,

related to this, there was an interesting presentation at the last FOM meeting by Prashant Prabhat from Semrock: "Nine-color widefield fluorescent microscopy using standard optical filters". While not spinning disk, it was quite remarkable, I don't remember the details but i am sure Semrock will be able to provide some information (no commercial interest).

best wishes

Andreas

 

 

 

-----Original Message-----
From: Aromis <[hidden email]>
To: CONFOCALMICROSCOPY <[hidden email]>
Sent: Sun, 10 Feb 2013 22:36
Subject: Re: 5 color imaging (6 color would be even better!)


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*****

Dear Everyone:

Thank you all for your input. Just back from my weekend so I need some time
to digest these info especially with the suggested papers. Also I'll have an
appointment with the head of our imaging center to discuss about it so I
guess I might get a feasible plan.

What confuses me a bit is that it seems that 5 color imaging is really
doable (even routine) but the people around me ,including those who started
to use confocal microscopy from the 1st generation, are not very positive
about this. As a person coming from conventional molecular biology
background I'll try to understand the discrepancy.

After collecting/understanding enough info I'll try to compose a 'protocol'
of 5/6 color imaging as I presume more people might be interested in it.

More questions from me can be expected.

Thanks again.

Best wishes,

Aro


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*****

Good evening,

Kevin and George have already provided good answers. What I write in the
following lines is more of a concept than a practical solution in case of
camera detection based systems.

Two very interesting - to my mind - approaches have been described and
successfully used by Åslund, Carlsson, Liljeborg and others in the 1990s on
a conventional CLSM. The approaches, both called "IMS Technique", are based
on lock-in detection principles:


In brief:

Provided, one has n e |N >1 dye substances, which can be excited at
available laser wavelengths without any cross excitation, the resp. laser
beams each exciting one, and one only, out of the n dyes can be modulated
using, e.g., n electro optic modulators at n individual frequencies and
phases.

First approach:
If the periods of the n modulation frequencies are long compared to the
decay times of the dye substances, the resulting n fluorescences will then
also be modulated at the same frequencies and phases (at which, in this
case, phase shifts related to the different decay times of the dyes can be
neglected). Irrespectively of any spectral overlap of the fluorscence
spectra of the dyes, the resulting fluorescence signals as detected by one
photomultiplier can be read out via n lock-in amplifiers and be recorded at
negligibly large cross talk to n channels.

Second approach (basically a life time method):
If the periods of the modulation frequencies are short scompared to the
decay times of the lasers, the resulting fluorescences will then also be
modulated at the same frequencies but different phases (in this case, other
then in the first approach, phase differences as induced by the different
decay times of the different dyes cannot be neglected but are the detection
magnitude). If the n dyes have n different decay times, at which any Delta T
between any two dyes must be large enough to cause a specific phase shift of
the fluorescences, n lock-in amplifiers can be used to read out the resp.
signals from one photomultiplier - with sufficiently small transition time
spread - at negligible cross talk into n channels.

A task as far as I know not done on a regular basis and possibly un-done -
besides the financial challenge, of course - for this method to be applied
to spinning disk systems including camera detection would be to read out the
signals as detected by the camera via lock-in amplifiers.
Alternatively, one might try to use PMT arrays.

References:

E.g.

K. Carlsson, N. Åslund, K. Mossberg & J. Philip, "Simultaneous confocal
recording of multiple fluorescent labels with improved channel separation"
J. Microsc. 176, 287-299 (1994).

K. Carlsson & B. Ulfhake, "Improved fluorophore separation with IMS confocal
microscopy" NeuroReport, 6(8), 1169-1173 (1995).

K. Carlsson, "Signal-to-noise ratio for confocal microscopy when using the
IMS technique" Micron 26, 317-322 (1995)

P.J. Helm, O. Franksson & K. Carlsson, "A confocal scanning laser microscope
for quantitative ratiometric 3D measurements of [Ca2+] and Ca2+ diffusion in
living cells stained with Fura-2" Pfluegers Arch - Eur J Physiol, 429,
672-681 (1995).

K. Carlsson, A. Liljeborg, "Confocal fluorescence microscopy using spectral
and lifetime information to simultaneuosly record four fluorophores with
high channel separation", J. Microsc. 185, 37-46 (1997).

PJ Helm, A Patwardhan, and EMM Manders (1997), A study of the precision of
confocal, ratiometric, Fura-2 based measurements, Cell Calcium 22(4):287-298

K. Carlsson & A. Liljeborg, "Simultaneous confocal lifetime imaging of
multiple fluorophores using the Intensity-modulated Multiple-wavelength
Scanning (IMS) technique," J. Microsc. 191, 119-127 (1998).

as well as

Åslund, N.; Carlsson, K.S. Apparatus for Quantitative Imaging of Multiple
Fluorophores.
U.S. Patent 5,294,799, 15 March 1994.

Åslund, N.; Carlsson, K.S. Apparatus for Quantitative Imaging of Multiple
Fluorophores Using Dual Detectors. U.S. Patent 5,418,371, 23 May 1995.



Best wishes,

Johannes


--
P. Johannes Helm, M.Sc. PhD
Seniorengineer
CMBN
University of Oslo
Institute of Basic Medical Science
Department of Anatomy
Postboks 1105 - Blindern
NO-0317 Oslo

Voice: [phone]
Fax: [phone]

WWW: folk.uio.no/jhelm

> *****
> To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Everyone:
>
> I have been trying to visualize a macromolecular structure with 4
> color
imaging. I have been doing this with spinning disc microscopy as I need
complete sections of cells also to know the distribution of the structure
> inside the cells in 3D. So far things are working properly BUT ...
>
> We now would like to upgrade to 5 color imaging as we want to
> visualize
additional components of the structure. I checked the spinning disc setting
> myself and I asked several senior users of spinning disc and got the
opinion
> that "5 color imaging with spinning disc without bleed-through is
> hardly
possible, if possible at all". The main reason being that it is not very
likely to set up emission filter combinations for 5 color imaging without
> bleed-through. Spectral mixing is technically possible but not very
promising.
>
> I also learned that 5 color imaging is possible with laser scanning
microscopy with which one can specifically define the emission band pass
range. But with laser scanning microscope it will cost me much more time to
> collect enough data for statistical analysis.
>
> So my questions would be: can anyone of you share your
> experience/opinion/literatures on 5 color imaging? I also tried to
> look for literatures describing multi-color (> 4 color) imaging (I
> think there
must
> be decent papers about it) but so far I have not found any. Maybe some
of
> you have some nice papers about this?
>
> Thank you very much for your input.
>
> Nice weekend!
>
> Aromis
>

 

[Daniela Malide]

*****
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*****

We have a recent publication in Blood -imaging 5 fluorescent proteins on a
commercial Leica SP5 confocal system:

Dynamic clonal analysis of murine hematopoietic stem and progenitor cells
marked by 5 fluorescent proteins using confocal and multiphoton microscopy

Daniela Malide, Jean-Yves Métais and Cynthia E. Dunbar
Blood December 20, 2012 vol. 120 no. 26 e105-e116
 
http://bloodjournal.hematologylibrary.org/content/120/26/e105.long

I can email a pdf of the manuscript if interested -as require subscription to
Blood.

Daniela Malide
Staff Scientist
NHLBI Light microscopy core facility

[George McNamara]

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*****

Hi Daniela,

First paragraph of your discussion states, " ... mCherry (based on our
novel observation of mCherry photoconversion to a red-shifted species)"

where is data on this?

Is this referring to your observation,

"reference spectra for each FP (Figure 1A). Unexpectedly, we observed
that the mCherry spectrum was dependent on the excitation wavelength,
and that 594 nm excitation resulted in ~12-nm-peak red-shift compared
with 561-nm excitation."

which is more likely to be instrument than protein related.

Are you going to add a blue FP?
Are you adding fusion proteins, ex. cyan-yellow (re: CY11.5, CyPet-YPet)
with moderate to high FRET, enabling more colors (if using sequential
scanning ... may also better take advantage of MP/NDDs)?
How about using plasma membrane targeting to outline the cell surfaces?
This would leave a cleaner signal for taking advantage of my Tattletales
concept to multiplex fluorescent reporters and live cells
(http://works.bepress.com/gmcnamara/26/ - best so start with the Feb 5
pdf and ppt).

Sincerely,

George


On 2/15/2013 1:22 PM, Daniela Malide wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> We have a recent publication in Blood -imaging 5 fluorescent proteins on a
> commercial Leica SP5 confocal system:
>
> Dynamic clonal analysis of murine hematopoietic stem and progenitor cells
> marked by 5 fluorescent proteins using confocal and multiphoton microscopy
>
> Daniela Malide, Jean-Yves Métais and Cynthia E. Dunbar
> Blood December 20, 2012 vol. 120 no. 26 e105-e116
>
> http://bloodjournal.hematologylibrary.org/content/120/26/e105.long
>
> I can email a pdf of the manuscript if interested -as require subscription to
> Blood.
>
> Daniela Malide
> Staff Scientist
> NHLBI Light microscopy core facility
>
>    

[Dmitry Sokolov]

*****
To join, leave or search the confocal microscopy listserv, go to:
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*****

Hi George,

could you please comment more on:
> "reference spectra for each FP (Figure 1A). Unexpectedly, we observed
> that the mCherry spectrum was dependent on the excitation wavelength,
> and that 594 nm excitation resulted in ~12-nm-peak red-shift compared
> with 561-nm excitation."
>
> which is more likely to be instrument than protein related.
What experiment would diagnose the reason for the peak shift?
Would you suggest the reference on the continuous ("3D") fluorescence
landscape of mCherry please?

Thank you,
Dmitry

*Advanced Knowledge Management*
for *MICROSCOPY *and *Image Analysis *
------------------------------------------------------------------------
*Dmitry Sokolov*, Ph.D.
Mob: *[phone]***
[hidden email] <mailto:[hidden email]>



17.02.2013 7:01, George McNamara ?????:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Daniela,
>
> First paragraph of your discussion states, " ... mCherry (based on our
> novel observation of mCherry photoconversion to a red-shifted species)"
>
> where is data on this?
>
> Is this referring to your observation,
>
> "reference spectra for each FP (Figure 1A). Unexpectedly, we observed
> that the mCherry spectrum was dependent on the excitation wavelength,
> and that 594 nm excitation resulted in ~12-nm-peak red-shift compared
> with 561-nm excitation."
>
> which is more likely to be instrument than protein related.
>
> Are you going to add a blue FP?
> Are you adding fusion proteins, ex. cyan-yellow (re: CY11.5,
> CyPet-YPet) with moderate to high FRET, enabling more colors (if using
> sequential scanning ... may also better take advantage of MP/NDDs)?
> How about using plasma membrane targeting to outline the cell
> surfaces? This would leave a cleaner signal for taking advantage of my
> Tattletales concept to multiplex fluorescent reporters and live cells
> (http://works.bepress.com/gmcnamara/26/ - best so start with the Feb 5
> pdf and ppt).
>
> Sincerely,
>
> George
>
>
> On 2/15/2013 1:22 PM, Daniela Malide wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> We have a recent publication in Blood -imaging 5 fluorescent proteins
>> on a
>> commercial Leica SP5 confocal system:
>>
>> Dynamic clonal analysis of murine hematopoietic stem and progenitor
>> cells
>> marked by 5 fluorescent proteins using confocal and multiphoton
>> microscopy
>>
>> Daniela Malide, Jean-Yves Métais and Cynthia E. Dunbar
>> Blood December 20, 2012 vol. 120 no. 26 e105-e116
>>
>> http://bloodjournal.hematologylibrary.org/content/120/26/e105.long
>>
>> I can email a pdf of the manuscript if interested -as require
>> subscription to
>> Blood.
>>
>> Daniela Malide
>> Staff Scientist
>> NHLBI Light microscopy core facility
>>

[George McNamara]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Dmitry,

I am unaware of any publications on excitation-emission matrix
spectroscopy ("3D landscape") of mCherry. Vitaly and others may want to
comment on any odd behaviors of mCherry. It has cousins, rsCherry and
rsCherryRev for example, that do interesting things. There is also the
issue that measurements in cells and tissues can differ from pure protein.

Diagnosing peak shift using 561 vs 594 excitation ... spectral scans of:
1. test with solution of pure mCherry, rather than cells/tissue.
2. excite (pure solution) with 514 nm - should be the same as 561 nm
excitation, whether using identical spectral range as with 561, or
starting at shorter wavelength (i.e. at 525 nm).
3. They have an MP laser - in fact, MP-OPO, so Daniela could use
multiple MP(OPO) wavelengths and spectral emission scanning.
4. Buy a PARISS (www.lightforminc.com) with excitation confocal slit and
appropriate excitation options (including excitation slit), or go visit
Keith Lidke to use his clone
http://www.systemscenters.org/wordpress/wp-content/uploads/2011/01/nm-ncbs-aug-8-11-lidke.pdf 

and test on this imaging spectrometer.

Also: never rely on just the peak. The area under the curve of a
fluorescence spectrum carries a lot more information.

Bob Zucker & Robert Lief published a couple of papers showing various
artifacts with early spectral confocal microscopes. When they used the
latter's MIDL calibrated lamp, most (all?) Leica SP1's had an
interesting behavior where the spectra differed depending on start
wavelength (i.e. 500 vs 501, vs 502 vs 503 vs 504 nm).

George
p.s. bonus: some confocal microscopes adjust the pinhole size based on
(excitation) wavelength. For example, LSM710 if the user clicks "1 Airy"
button after switching the laser line selected. The pinhole is part of
the emission path, so this affects the spectral resolution (probably not
enough for most LSM users to notice if going from 561 to 594 nm). More
generally, any manuscript that uses a spectral confocal microscope and
fails to state pinhole size is a waste of effort. Easy test anyone with
a spectral confocal microscope can perform: compare spectra at pinhole 1
vs wide open.

On 2/16/2013 10:48 PM, Dmitry Sokolov wrote:

> Hi George,
>
> could you please comment more on:
>> "reference spectra for each FP (Figure 1A). Unexpectedly, we observed
>> that the mCherry spectrum was dependent on the excitation wavelength,
>> and that 594 nm excitation resulted in ~12-nm-peak red-shift compared
>> with 561-nm excitation."
>>
>> which is more likely to be instrument than protein related.
> What experiment would diagnose the reason for the peak shift?
> Would you suggest the reference on the continuous ("3D") fluorescence
> landscape of mCherry please?
>
> Thank you,
> Dmitry
>
> * Advanced Knowledge Management*
> for *MICROSCOPY *and *Image Analysis *
> ------------------------------------------------------------------------
> * Dmitry Sokolov*, Ph.D.
> Mob: *[phone]** *
> [hidden email] <mailto:[hidden email]>
>
>
>
> 17.02.2013 7:01, George McNamara ?????:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi Daniela,
>>
>> First paragraph of your discussion states, " ... mCherry (based on
>> our novel observation of mCherry photoconversion to a red-shifted
>> species)"
>>
>> where is data on this?
>>
>> Is this referring to your observation,
>>
>> "reference spectra for each FP (Figure 1A). Unexpectedly, we observed
>> that the mCherry spectrum was dependent on the excitation wavelength,
>> and that 594 nm excitation resulted in ~12-nm-peak red-shift compared
>> with 561-nm excitation."
>>
>> which is more likely to be instrument than protein related.
>>
>> Are you going to add a blue FP?
>> Are you adding fusion proteins, ex. cyan-yellow (re: CY11.5,
>> CyPet-YPet) with moderate to high FRET, enabling more colors (if
>> using sequential scanning ... may also better take advantage of
>> MP/NDDs)?
>> How about using plasma membrane targeting to outline the cell
>> surfaces? This would leave a cleaner signal for taking advantage of
>> my Tattletales concept to multiplex fluorescent reporters and live
>> cells (http://works.bepress.com/gmcnamara/26/ - best so start with
>> the Feb 5 pdf and ppt).
>>
>> Sincerely,
>>
>> George
>>
>>
>> On 2/15/2013 1:22 PM, Daniela Malide wrote:
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> We have a recent publication in Blood -imaging 5 fluorescent
>>> proteins on a
>>> commercial Leica SP5 confocal system:
>>>
>>> Dynamic clonal analysis of murine hematopoietic stem and progenitor
>>> cells
>>> marked by 5 fluorescent proteins using confocal and multiphoton
>>> microscopy
>>>
>>> Daniela Malide, Jean-Yves Métais and Cynthia E. Dunbar
>>> Blood December 20, 2012 vol. 120 no. 26 e105-e116
>>>
>>> http://bloodjournal.hematologylibrary.org/content/120/26/e105.long
>>>
>>> I can email a pdf of the manuscript if interested -as require
>>> subscription to
>>> Blood.
>>>
>>> Daniela Malide
>>> Staff Scientist
>>> NHLBI Light microscopy core facility
>>>
>

[Dmitry Sokolov]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Geourge,

your comments are summarised in the knowledge network format.
Would you agree with what is published here?
http://confocal-manawatu.pbworks.com/w/page/63713266/Fluorescence%20Peak%20Shift%20Factors

Dear All, your comments and suggestions would be highly appreciated.

Cheers,
Dmitry

*Advanced Knowledge Management*
for *MICROSCOPY *and *Image Analysis *
------------------------------------------------------------------------
*Dmitry Sokolov*, Ph.D.
Mob: *[phone]***
[hidden email] <mailto:[hidden email]>




18.02.2013 5:30, George McNamara ?????:

> Hi Dmitry,
>
> I am unaware of any publications on excitation-emission matrix
> spectroscopy ("3D landscape") of mCherry. Vitaly and others may want
> to comment on any odd behaviors of mCherry. It has cousins, rsCherry
> and rsCherryRev for example, that do interesting things. There is also
> the issue that measurements in cells and tissues can differ from pure
> protein.
>
> Diagnosing peak shift using 561 vs 594 excitation ... spectral scans of:
> 1. test with solution of pure mCherry, rather than cells/tissue.
> 2. excite (pure solution) with 514 nm - should be the same as 561 nm
> excitation, whether using identical spectral range as with 561, or
> starting at shorter wavelength (i.e. at 525 nm).
> 3. They have an MP laser - in fact, MP-OPO, so Daniela could use
> multiple MP(OPO) wavelengths and spectral emission scanning.
> 4. Buy a PARISS (www.lightforminc.com) with excitation confocal slit
> and appropriate excitation options (including excitation slit), or go
> visit Keith Lidke to use his clone
> http://www.systemscenters.org/wordpress/wp-content/uploads/2011/01/nm-ncbs-aug-8-11-lidke.pdf 
>
> and test on this imaging spectrometer.
>
> Also: never rely on just the peak. The area under the curve of a
> fluorescence spectrum carries a lot more information.
>
> Bob Zucker & Robert Lief published a couple of papers showing various
> artifacts with early spectral confocal microscopes. When they used the
> latter's MIDL calibrated lamp, most (all?) Leica SP1's had an
> interesting behavior where the spectra differed depending on start
> wavelength (i.e. 500 vs 501, vs 502 vs 503 vs 504 nm).
>
> George
> p.s. bonus: some confocal microscopes adjust the pinhole size based on
> (excitation) wavelength. For example, LSM710 if the user clicks "1
> Airy" button after switching the laser line selected. The pinhole is
> part of the emission path, so this affects the spectral resolution
> (probably not enough for most LSM users to notice if going from 561 to
> 594 nm). More generally, any manuscript that uses a spectral confocal
> microscope and fails to state pinhole size is a waste of effort. Easy
> test anyone with a spectral confocal microscope can perform: compare
> spectra at pinhole 1 vs wide open.
>
> On 2/16/2013 10:48 PM, Dmitry Sokolov wrote:
>> Hi George,
>>
>> could you please comment more on:
>>> "reference spectra for each FP (Figure 1A). Unexpectedly, we
>>> observed that the mCherry spectrum was dependent on the excitation
>>> wavelength, and that 594 nm excitation resulted in ~12-nm-peak
>>> red-shift compared with 561-nm excitation."
>>>
>>> which is more likely to be instrument than protein related.
>> What experiment would diagnose the reason for the peak shift?
>> Would you suggest the reference on the continuous ("3D") fluorescence
>> landscape of mCherry please?
>>
>> Thank you,
>> Dmitry
>>
>> *Advanced Knowledge Management*
>> for *MICROSCOPY *and *Image Analysis *
>> ------------------------------------------------------------------------
>> *Dmitry Sokolov*, Ph.D.
>> Mob: *[phone]***
>> [hidden email] <mailto:[hidden email]>
>>
>>
>>
>> 17.02.2013 7:01, George McNamara ?????:
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Hi Daniela,
>>>
>>> First paragraph of your discussion states, " ... mCherry (based on
>>> our novel observation of mCherry photoconversion to a red-shifted
>>> species)"
>>>
>>> where is data on this?
>>>
>>> Is this referring to your observation,
>>>
>>> "reference spectra for each FP (Figure 1A). Unexpectedly, we
>>> observed that the mCherry spectrum was dependent on the excitation
>>> wavelength, and that 594 nm excitation resulted in ~12-nm-peak
>>> red-shift compared with 561-nm excitation."
>>>
>>> which is more likely to be instrument than protein related.
>>>
>>> Are you going to add a blue FP?
>>> Are you adding fusion proteins, ex. cyan-yellow (re: CY11.5,
>>> CyPet-YPet) with moderate to high FRET, enabling more colors (if
>>> using sequential scanning ... may also better take advantage of
>>> MP/NDDs)?
>>> How about using plasma membrane targeting to outline the cell
>>> surfaces? This would leave a cleaner signal for taking advantage of
>>> my Tattletales concept to multiplex fluorescent reporters and live
>>> cells (http://works.bepress.com/gmcnamara/26/ - best so start with
>>> the Feb 5 pdf and ppt).
>>>
>>> Sincerely,
>>>
>>> George
>>>
>>>
>>> On 2/15/2013 1:22 PM, Daniela Malide wrote:
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> We have a recent publication in Blood -imaging 5 fluorescent
>>>> proteins on a
>>>> commercial Leica SP5 confocal system:
>>>>
>>>> Dynamic clonal analysis of murine hematopoietic stem and progenitor
>>>> cells
>>>> marked by 5 fluorescent proteins using confocal and multiphoton
>>>> microscopy
>>>>
>>>> Daniela Malide, Jean-Yves Métais and Cynthia E. Dunbar
>>>> Blood December 20, 2012 vol. 120 no. 26 e105-e116
>>>>
>>>> http://bloodjournal.hematologylibrary.org/content/120/26/e105.long
>>>>
>>>> I can email a pdf of the manuscript if interested -as require
>>>> subscription to
>>>> Blood.
>>>>
>>>> Daniela Malide
>>>> Staff Scientist
>>>> NHLBI Light microscopy core facility
>>>>
>>
>