CFP-YFP lines for an inverted confocal microscope

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CFP-YFP lines for an inverted confocal microscope

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Dear All,

We are working on the specs for a new inverted confocal microscope for our core imaging facility. We define this as an high-end confocal with live imaging capability. We would like advice on the following.

1) We are going to have 405, 488, 561 and 640 lines (all solid-state lasers) for this confocal. Is it necessary to have CFP, YFP channels with 448 and 514 laser lines? OR we can use 405 and 488 lines to excite them with dedicated CFP, YFP filter cubes? There is no mention of CFP-YFP FRET after 2013 in the list here. No more CFP-YFP FRET in confocal now a days? Pardon me for my ignorance.

Best regards,
Core Imaging facility Staff,
NTU
Tim Feinstein Tim Feinstein
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Re: CFP-YFP lines for an inverted confocal microscope

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Hi folks,

I agree the rise of solid state lasers has made life harder for FRET specialists (and also better! No more $14,000 power surges...).  One laser, one wavelength, no freebies like that 514 nm line in a Kr/Ar tube.  
       
In my experience 405 nm is really a desperation move for FRET.  Its excitation efficiency is poor, it causes rapid bleaching and it barely excites CFP with more efficiency than any other fluorophore.  In my experience a 442 nm laser is essential for doing quantitative FRET on a confocal microscope*.  As a bonus its cross-excitation of YFP is close to zero.  

You can excite YFP efficiently with a 488 nm laser, and CFP cross-excitation is again near zero.  In fact I am doing FRET right now with 442-488 nm lasers, however, honestly it's not ideal.  The two lasers leave you with a short range to collect CFP emission (~ 450-475 nm), and that's assuming you have a spectrally tuneable emission setup.   Our current Leica scope can move the spectral gates around to collect CFP and YFP more efficiently but it is very time consuming.  If you plan on making FRET a key part of your work then you will really benefit from installing both 442 and 514 nm lasers.  

(*) I'd add that it often pays to reassess whether you may be better off doing FRET with a widefield.  IMO the most effective setup for rigorously quantitative FRET studies, if you don't have a TR/FLIM system available, is a widefield scope with dual emission (either a DualView or a beam splitter with two cameras) and FRET dichroics.  
       
Best,


Tim
       
Timothy Feinstein, Ph.D.
Research Scientist
University of Pittsburgh Department of Developmental Biology

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of SUBSCRIBE CONFOCALMICROSCOPY Anonymous
Sent: Sunday, July 23, 2017 9:35 PM
To: [hidden email]
Subject: CFP-YFP lines for an inverted confocal microscope

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Dear All,

We are working on the specs for a new inverted confocal microscope for our core imaging facility. We define this as an high-end confocal with live imaging capability. We would like advice on the following.

1) We are going to have 405, 488, 561 and 640 lines (all solid-state lasers) for this confocal. Is it necessary to have CFP, YFP channels with 448 and 514 laser lines? OR we can use 405 and 488 lines to excite them with dedicated CFP, YFP filter cubes? There is no mention of CFP-YFP FRET after 2013 in the list here. No more CFP-YFP FRET in confocal now a days? Pardon me for my ignorance.

Best regards,
Core Imaging facility Staff,
NTU
Sylvie Le Guyader Sylvie Le Guyader
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Re: CFP-YFP lines for an inverted confocal microscope

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Hi there

I think that there is no post about cfp-yfp fret on the list because there is no problem about it, not because it is not used. :-)

Most biosensors are available in fluorophores excited at 457 and 514. Exciting with 405 is not an option for live cell imaging (biggest use of biosensors) especially when you need to push the laser to compensate for low excitation efficiency.

Argon lasers are cheaper, can be refurbished when they age and have 3 usable lines. However the 514 and 457 power varies from one laser to the next. Only the power of the 488 line is specified. Make sure you specify in your tender a min power (or % of the 488 line) for the other 2 lines to avoid disappointment upon delivery.

The disadvantage of argon lasers is that they die slowly so their power decreases with time. Another disadvantage is that there is often a long delivery time if you need to replace it. That is not a problem when you order the microscope though.

Another disadvantage is that turning them on /off without cooling shortens their lifetime. Having a good on/off routine can allow your laser to go on for many years. Our users book both the microscope and the laser. This way the previous user knows if they should leave the laser on or turn it off. They turn it off if the laser is not going to be used for 2 hours. If someone books the laser and doesn't show up, it is their responsibility to come and check if the laser has been left on for them. Using this routine we have had very few refurbishing over the past 8 years with an average use of our microscopes at 7h/day over 365 days/year. :-)

I'd go for the argon.

Med vänlig hälsning / Best regards

Sylvie

@@@@@@@@@@@@@@@@@@@@@@@@

Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Hälsovägen 7,
Novum, G lift, floor 6
14157 Huddinge
Sweden
mobile: +46 (0) 73 733 5008
office: +46 (0) 08-524 811 72
LCI website

---- SUBSCRIBE CONFOCALMICROSCOPY Anonymous wrote ----

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Dear All,

We are working on the specs for a new inverted confocal microscope for our core imaging facility. We define this as an high-end confocal with live imaging capability. We would like advice on the following.

1) We are going to have 405, 488, 561 and 640 lines (all solid-state lasers) for this confocal. Is it necessary to have CFP, YFP channels with 448 and 514 laser lines? OR we can use 405 and 488 lines to excite them with dedicated CFP, YFP filter cubes? There is no mention of CFP-YFP FRET after 2013 in the list here. No more CFP-YFP FRET in confocal now a days? Pardon me for my ignorance.

Best regards,
Core Imaging facility Staff,
NTU
S Ram S Ram
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Re: CFP-YFP lines for an inverted confocal microscope

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It really depends on your fluorophore levels. Technically 405 nm will
excite CFP (if I recall CFP excitation spectra has a broad shoulder at
around 405) and YFP can also be excited by 488 nm. Nonetheless, these
wavelengths are not optimal and you might have less than ideal signal if
your fluorophore levels in the sample are not high enough to begin with.

The other issue to consider is the detector. Will you be using a standard
PMT or a high sensitivity variant (like a HyD or GaAsP)?

HTH

Sripad


On Sun, Jul 23, 2017 at 6:34 PM, SUBSCRIBE CONFOCALMICROSCOPY Anonymous <
[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear All,
>
> We are working on the specs for a new inverted confocal microscope for our
> core imaging facility. We define this as an high-end confocal with live
> imaging capability. We would like advice on the following.
>
> 1) We are going to have 405, 488, 561 and 640 lines (all solid-state
> lasers) for this confocal. Is it necessary to have CFP, YFP channels with
> 448 and 514 laser lines? OR we can use 405 and 488 lines to excite them
> with dedicated CFP, YFP filter cubes? There is no mention of CFP-YFP FRET
> after 2013 in the list here. No more CFP-YFP FRET in confocal now a days?
> Pardon me for my ignorance.
>
> Best regards,
> Core Imaging facility Staff,
> NTU
>
Craig Brideau Craig Brideau
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Re: CFP-YFP lines for an inverted confocal microscope

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My lab used a Citrine (YFP)/Cereulean (CFP) Ca2+ FRET indicator with a
405nm and 488nm laser pair with good results. As Sripad mentions, this
mostly depends on how well your fluorophore reaches your sample and how
sensitive your detectors are.

Craig

On Mon, Jul 24, 2017 at 1:57 PM, S Ram <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> It really depends on your fluorophore levels. Technically 405 nm will
> excite CFP (if I recall CFP excitation spectra has a broad shoulder at
> around 405) and YFP can also be excited by 488 nm. Nonetheless, these
> wavelengths are not optimal and you might have less than ideal signal if
> your fluorophore levels in the sample are not high enough to begin with.
>
> The other issue to consider is the detector. Will you be using a standard
> PMT or a high sensitivity variant (like a HyD or GaAsP)?
>
> HTH
>
> Sripad
>
>
> On Sun, Jul 23, 2017 at 6:34 PM, SUBSCRIBE CONFOCALMICROSCOPY Anonymous <
> [hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on http://www.imgur.com and include the link in your
> posting.
> > *****
> >
> > Dear All,
> >
> > We are working on the specs for a new inverted confocal microscope for
> our
> > core imaging facility. We define this as an high-end confocal with live
> > imaging capability. We would like advice on the following.
> >
> > 1) We are going to have 405, 488, 561 and 640 lines (all solid-state
> > lasers) for this confocal. Is it necessary to have CFP, YFP channels with
> > 448 and 514 laser lines? OR we can use 405 and 488 lines to excite them
> > with dedicated CFP, YFP filter cubes? There is no mention of CFP-YFP FRET
> > after 2013 in the list here. No more CFP-YFP FRET in confocal now a days?
> > Pardon me for my ignorance.
> >
> > Best regards,
> > Core Imaging facility Staff,
> > NTU
> >
>
G. Esteban Fernandez G. Esteban Fernandez
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Re: CFP-YFP lines for an inverted confocal microscope

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*****

My users actually report a preference for 405 and 488 on our Zeiss LSM 710
to excite CFP and YFP, even though the system has 458 and 514 Kr-Ar lines,
because the 458 and 514 are weaker. Of course that is not efficient from a
photonics standpoint but for them it is more efficient in terms of scanning
time. I myself use 405 instead of the 458. No one is doing quantitative
work, just mapping where the dyes are. 405 works well enough for CFP in
CFP/GFP/YFP/RFP brainbow/confetti tissues.

-Esteban

On Jul 24, 2017 1:51 PM, "Craig Brideau" <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> My lab used a Citrine (YFP)/Cereulean (CFP) Ca2+ FRET indicator with a
> 405nm and 488nm laser pair with good results. As Sripad mentions, this
> mostly depends on how well your fluorophore reaches your sample and how
> sensitive your detectors are.
>
> Craig
>
> On Mon, Jul 24, 2017 at 1:57 PM, S Ram <[hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on http://www.imgur.com and include the link in your
> posting.
> > *****
> >
> > It really depends on your fluorophore levels. Technically 405 nm will
> > excite CFP (if I recall CFP excitation spectra has a broad shoulder at
> > around 405) and YFP can also be excited by 488 nm. Nonetheless, these
> > wavelengths are not optimal and you might have less than ideal signal if
> > your fluorophore levels in the sample are not high enough to begin with.
> >
> > The other issue to consider is the detector. Will you be using a standard
> > PMT or a high sensitivity variant (like a HyD or GaAsP)?
> >
> > HTH
> >
> > Sripad
> >
> >
> > On Sun, Jul 23, 2017 at 6:34 PM, SUBSCRIBE CONFOCALMICROSCOPY Anonymous <
> > [hidden email]> wrote:
> >
> > > *****
> > > To join, leave or search the confocal microscopy listserv, go to:
> > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > > Post images on http://www.imgur.com and include the link in your
> > posting.
> > > *****
> > >
> > > Dear All,
> > >
> > > We are working on the specs for a new inverted confocal microscope for
> > our
> > > core imaging facility. We define this as an high-end confocal with live
> > > imaging capability. We would like advice on the following.
> > >
> > > 1) We are going to have 405, 488, 561 and 640 lines (all solid-state
> > > lasers) for this confocal. Is it necessary to have CFP, YFP channels
> with
> > > 448 and 514 laser lines? OR we can use 405 and 488 lines to excite them
> > > with dedicated CFP, YFP filter cubes? There is no mention of CFP-YFP
> FRET
> > > after 2013 in the list here. No more CFP-YFP FRET in confocal now a
> days?
> > > Pardon me for my ignorance.
> > >
> > > Best regards,
> > > Core Imaging facility Staff,
> > > NTU
> > >
> >
>
SUBSCRIBE CONFOCALMICROSCOPY Anonymous-3 SUBSCRIBE CONFOCALMICROSCOPY Anonymous-3
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Re: CFP-YFP lines for an inverted confocal microscope

In reply to this post by S Ram
*****
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*****

Dear Sripad, Craig, Esteban, Tim,

Thanks for the insight on CFP YFP imaging in a confocal. It helps me in
deciding.

We are planning to have HyD or GaAsP detector.

I wonder if CFP-YFP FRET is still used in studying protein-protein
interactions OR with the advent of newer imaging technologies, it is slowly
disappearing. In a  recent High-Content Screening Microscopy user group
meeting at our place, one of the speakers showed using bioluminescence
(BRET - a much older concept) as an alternative to FRET. He highlighted
that the former is less tedious, given the fact that FRET requires
background subtractions and assumptions.

Best regards,
Core Imaging Facility Staff
NTU

On 25 July 2017 at 03:57, S Ram <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> It really depends on your fluorophore levels. Technically 405 nm will
> excite CFP (if I recall CFP excitation spectra has a broad shoulder at
> around 405) and YFP can also be excited by 488 nm. Nonetheless, these
> wavelengths are not optimal and you might have less than ideal signal if
> your fluorophore levels in the sample are not high enough to begin with.
>
> The other issue to consider is the detector. Will you be using a standard
> PMT or a high sensitivity variant (like a HyD or GaAsP)?
>
> HTH
>
> Sripad
>
>
> On Sun, Jul 23, 2017 at 6:34 PM, SUBSCRIBE CONFOCALMICROSCOPY Anonymous <
> [hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on http://www.imgur.com and include the link in your
> posting.
> > *****
> >
> > Dear All,
> >
> > We are working on the specs for a new inverted confocal microscope for
> our
> > core imaging facility. We define this as an high-end confocal with live
> > imaging capability. We would like advice on the following.
> >
> > 1) We are going to have 405, 488, 561 and 640 lines (all solid-state
> > lasers) for this confocal. Is it necessary to have CFP, YFP channels with
> > 448 and 514 laser lines? OR we can use 405 and 488 lines to excite them
> > with dedicated CFP, YFP filter cubes? There is no mention of CFP-YFP FRET
> > after 2013 in the list here. No more CFP-YFP FRET in confocal now a days?
> > Pardon me for my ignorance.
> >
> > Best regards,
> > Core Imaging facility Staff,
> > NTU
> >
>
Tim Feinstein Tim Feinstein
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Re: CFP-YFP lines for an inverted confocal microscope

*****
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*****

Hi folks,

BRET and FRET each have their own strengths.  BRET is an ensemble technique like a Western blot, where you measure all of the cells in a well homogenously at the same time.  This will work for many purposes but not so much if you need spatial information (nuclear vs. cytoplasmic, leading edge/trailing edge etc) or you want to filter out inappropriate cells like those with excessive transgene expression.  With either technique need to run careful controls if you want to quantify intermolecular interactions with FRET; intramolecular BRET/FRET, for example biosensors, is much easier and less demanding.  
       
As Craig pointed out a 405 nm line will work fine for biosensors, if bleaching is not a problem.  Cross-talk is not an issue since you're just measuring deviations from baseline.  For intermolecular FRET experiments I'd recommend either a more specific optical setup (442 and 514 lasers), adding dual emission to a widefield scope, or BRET.  

Best,


Tim

Timothy Feinstein, Ph.D.
Research Scientist
University of Pittsburgh Department of Developmental Biology

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Balakrishnan Kannan
Sent: Monday, July 24, 2017 8:58 PM
To: [hidden email]
Subject: Re: CFP-YFP lines for an inverted confocal microscope

*****
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*****

Dear Sripad, Craig, Esteban, Tim,

Thanks for the insight on CFP YFP imaging in a confocal. It helps me in deciding.

We are planning to have HyD or GaAsP detector.

I wonder if CFP-YFP FRET is still used in studying protein-protein interactions OR with the advent of newer imaging technologies, it is slowly disappearing. In a  recent High-Content Screening Microscopy user group meeting at our place, one of the speakers showed using bioluminescence (BRET - a much older concept) as an alternative to FRET. He highlighted that the former is less tedious, given the fact that FRET requires background subtractions and assumptions.

Best regards,
Core Imaging Facility Staff
NTU

On 25 July 2017 at 03:57, S Ram <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.
> umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=01%7C01%7Ctnf8%4
> 0PITT.EDU%7Cf5a3ef189a2e482333e908d4d2f86259%7C9ef9f489e0a04eeb87cc3a5
> 26112fd0d%7C1&sdata=W7Lh6SBbFZ4%2B7hUwYGkeifIP3KmdE3dgei7tnEOvCPk%3D&r
> eserved=0 Post images on
> https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=01%7C01%7Ctnf8%40PITT.EDU%7Cf5a3ef189a2e482333e908d4d2f86259%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1&sdata=S3tvVyqIufLWWSVcksGAHk5rDL1aJ0xiFiKxQjWIX4s%3D&reserved=0 and include the link in your posting.
> *****
>
> It really depends on your fluorophore levels. Technically 405 nm will
> excite CFP (if I recall CFP excitation spectra has a broad shoulder at
> around 405) and YFP can also be excited by 488 nm. Nonetheless, these
> wavelengths are not optimal and you might have less than ideal signal
> if your fluorophore levels in the sample are not high enough to begin with.
>
> The other issue to consider is the detector. Will you be using a
> standard PMT or a high sensitivity variant (like a HyD or GaAsP)?
>
> HTH
>
> Sripad
>
>
> On Sun, Jul 23, 2017 at 6:34 PM, SUBSCRIBE CONFOCALMICROSCOPY
> Anonymous < [hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist
> > s.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=01%7C01%7Ctn
> > f8%40PITT.EDU%7Cf5a3ef189a2e482333e908d4d2f86259%7C9ef9f489e0a04eeb8
> > 7cc3a526112fd0d%7C1&sdata=W7Lh6SBbFZ4%2B7hUwYGkeifIP3KmdE3dgei7tnEOv
> > CPk%3D&reserved=0 Post images on
> > https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.
> > imgur.com&data=01%7C01%7Ctnf8%40PITT.EDU%7Cf5a3ef189a2e482333e908d4d
> > 2f86259%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1&sdata=S3tvVyqIufLWWSV
> > cksGAHk5rDL1aJ0xiFiKxQjWIX4s%3D&reserved=0 and include the link in
> > your
> posting.
> > *****
> >
> > Dear All,
> >
> > We are working on the specs for a new inverted confocal microscope
> > for
> our
> > core imaging facility. We define this as an high-end confocal with
> > live imaging capability. We would like advice on the following.
> >
> > 1) We are going to have 405, 488, 561 and 640 lines (all solid-state
> > lasers) for this confocal. Is it necessary to have CFP, YFP channels
> > with
> > 448 and 514 laser lines? OR we can use 405 and 488 lines to excite
> > them with dedicated CFP, YFP filter cubes? There is no mention of
> > CFP-YFP FRET after 2013 in the list here. No more CFP-YFP FRET in confocal now a days?
> > Pardon me for my ignorance.
> >
> > Best regards,
> > Core Imaging facility Staff,
> > NTU
> >
>
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Re: CFP-YFP lines for an inverted confocal microscope

In reply to this post by Sylvie Le Guyader
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Well Argon Ion lasers produce a lot of heat which needs to be dealt with by expensive air conditioning units and their limited lifetime makes maintenance contracts expensive. It could be much cheaper to run a confocal when we don't need them anymore. I think in a few years they will disappear.
When doing FRET in widefield, watch out for pixel registration.

Best wishes

Andreas

-----Original Message-----
From: "Sylvie Le Guyader" <[hidden email]>
Sent: ‎25/‎07/‎2017 07:38
To: "[hidden email]" <[hidden email]>
Subject: Re: CFP-YFP lines for an inverted confocal microscope

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Hi there

I think that there is no post about cfp-yfp fret on the list because there is no problem about it, not because it is not used. :-)

Most biosensors are available in fluorophores excited at 457 and 514. Exciting with 405 is not an option for live cell imaging (biggest use of biosensors) especially when you need to push the laser to compensate for low excitation efficiency.

Argon lasers are cheaper, can be refurbished when they age and have 3 usable lines. However the 514 and 457 power varies from one laser to the next. Only the power of the 488 line is specified. Make sure you specify in your tender a min power (or % of the 488 line) for the other 2 lines to avoid disappointment upon delivery.

The disadvantage of argon lasers is that they die slowly so their power decreases with time. Another disadvantage is that there is often a long delivery time if you need to replace it. That is not a problem when you order the microscope though.

Another disadvantage is that turning them on /off without cooling shortens their lifetime. Having a good on/off routine can allow your laser to go on for many years. Our users book both the microscope and the laser. This way the previous user knows if they should leave the laser on or turn it off. They turn it off if the laser is not going to be used for 2 hours. If someone books the laser and doesn't show up, it is their responsibility to come and check if the laser has been left on for them. Using this routine we have had very few refurbishing over the past 8 years with an average use of our microscopes at 7h/day over 365 days/year. :-)

I'd go for the argon.

Med vänlig hälsning / Best regards

Sylvie

@@@@@@@@@@@@@@@@@@@@@@@@

Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Hälsovägen 7,
Novum, G lift, floor 6
14157 Huddinge
Sweden
mobile: +46 (0) 73 733 5008
office: +46 (0) 08-524 811 72
LCI website

---- SUBSCRIBE CONFOCALMICROSCOPY Anonymous wrote ----

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Dear All,

We are working on the specs for a new inverted confocal microscope for our core imaging facility. We define this as an high-end confocal with live imaging capability. We would like advice on the following.

1) We are going to have 405, 488, 561 and 640 lines (all solid-state lasers) for this confocal. Is it necessary to have CFP, YFP channels with 448 and 514 laser lines? OR we can use 405 and 488 lines to excite them with dedicated CFP, YFP filter cubes? There is no mention of CFP-YFP FRET after 2013 in the list here. No more CFP-YFP FRET in confocal now a days? Pardon me for my ignorance.

Best regards,
Core Imaging facility Staff,
NTU
Sylvie Le Guyader Sylvie Le Guyader
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Re: CFP-YFP lines for an inverted confocal microscope

In reply to this post by Tim Feinstein
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I totally agree with Tim there.
I also tried FRET with 405 and 488 but got much stronger results with 457/514.
Installing solid state lasers at these wavelengths is a good option but it requires a laser bed that can have more than 4 lines. Ideal but some companies offer this at a much higher price.

Med vänlig hälsning / Best regards
 
Sylvie
 
@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Hälsovägen 7,
Novum, G lift, floor 6
14157 Huddinge
Sweden
mobile: +46 (0) 73 733 5008
office: +46 (0) 08-524 811 72
LCI website


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Feinstein, Timothy N
Sent: den 24 juli 2017 04:21
To: [hidden email]
Subject: Re: CFP-YFP lines for an inverted confocal microscope

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Hi folks,

I agree the rise of solid state lasers has made life harder for FRET specialists (and also better! No more $14,000 power surges...).  One laser, one wavelength, no freebies like that 514 nm line in a Kr/Ar tube.  
       
In my experience 405 nm is really a desperation move for FRET.  Its excitation efficiency is poor, it causes rapid bleaching and it barely excites CFP with more efficiency than any other fluorophore.  In my experience a 442 nm laser is essential for doing quantitative FRET on a confocal microscope*.  As a bonus its cross-excitation of YFP is close to zero.  

You can excite YFP efficiently with a 488 nm laser, and CFP cross-excitation is again near zero.  In fact I am doing FRET right now with 442-488 nm lasers, however, honestly it's not ideal.  The two lasers leave you with a short range to collect CFP emission (~ 450-475 nm), and that's assuming you have a spectrally tuneable emission setup.   Our current Leica scope can move the spectral gates around to collect CFP and YFP more efficiently but it is very time consuming.  If you plan on making FRET a key part of your work then you will really benefit from installing both 442 and 514 nm lasers.  

(*) I'd add that it often pays to reassess whether you may be better off doing FRET with a widefield.  IMO the most effective setup for rigorously quantitative FRET studies, if you don't have a TR/FLIM system available, is a widefield scope with dual emission (either a DualView or a beam splitter with two cameras) and FRET dichroics.  
       
Best,


Tim
       
Timothy Feinstein, Ph.D.
Research Scientist
University of Pittsburgh Department of Developmental Biology

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of SUBSCRIBE CONFOCALMICROSCOPY Anonymous
Sent: Sunday, July 23, 2017 9:35 PM
To: [hidden email]
Subject: CFP-YFP lines for an inverted confocal microscope

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Dear All,

We are working on the specs for a new inverted confocal microscope for our core imaging facility. We define this as an high-end confocal with live imaging capability. We would like advice on the following.

1) We are going to have 405, 488, 561 and 640 lines (all solid-state lasers) for this confocal. Is it necessary to have CFP, YFP channels with 448 and 514 laser lines? OR we can use 405 and 488 lines to excite them with dedicated CFP, YFP filter cubes? There is no mention of CFP-YFP FRET after 2013 in the list here. No more CFP-YFP FRET in confocal now a days? Pardon me for my ignorance.

Best regards,
Core Imaging facility Staff,
NTU
Craig Brideau Craig Brideau
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Re: CFP-YFP lines for an inverted confocal microscope

In reply to this post by Sylvie Le Guyader
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On Mon, Jul 24, 2017 at 1:08 AM, Sylvie Le Guyader <[hidden email]>
wrote:

> Argon lasers are cheaper, can be refurbished when they age and have 3
> usable lines. However the 514 and 457 power varies from one laser to the
> next. Only the power of the 488 line is specified. Make sure you specify in
> your tender a min power (or % of the 488 line) for the other 2 lines to
> avoid disappointment upon delivery.
>

I have a pair of argon lasers that I cycle for refurbishment. When the one
in use starts to show a significant power droop I send the other out for
refurb and swap them. That way I always have one online for the most part.
That said, I am keeping my eyes open for a good diode equivalent system as
my current crop of users mostly only needs 488nm. Unfortunately 488nm tends
to be the most expensive diode line, so the economics don't quite make
sense yet.


> The disadvantage of argon lasers is that they die slowly so their power
> decreases with time. Another disadvantage is that there is often a long
> delivery time if you need to replace it. That is not a problem when you
> order the microscope though.
>

I do recommend monitoring. I have a power meter, and the users have a
calibration with one of those Chroma fluorescent slides that gives us a
hint if the laser is getting weak.


> Another disadvantage is that turning them on /off without cooling shortens
> their lifetime. Having a good on/off routine can allow your laser to go on
> for many years. Our users book both the microscope and the laser. This way
> the previous user knows if they should leave the laser on or turn it off.
> They turn it off if the laser is not going to be used for 2 hours. If
> someone books the laser and doesn't show up, it is their responsibility to
> come and check if the laser has been left on for them. Using this routine
> we have had very few refurbishing over the past 8 years with an average use
> of our microscopes at 7h/day over 365 days/year. :-)
>

A good argon will have a timer wired into its fan. Upon shutdown of the
laser the fan runs for some time afterword, much like a digital projector.
Overall though I find avoiding switching them on and off does make a
significant difference in laser lifespan as you say. Another trick is to
put the laser into low-power 'standby' mode that keeps a minimum arc
current running in the tube. For bright samples sometimes 'standby' power
is enough to image with the 488nm line, although for any of the
secondary/tertiary lines the laser typically must be in 'run' mode. If the
laser must be left on between users, I encourage them to leave it in
standby, and if at all possible, actually work with the laser in standby
mode if the experiment allows.


> I'd go for the argon.
>

They still make the most sense economically at the moment assuming you can
align them yourself when they come back from refurbishment. If you are
relying on hiring an external technician for re-installation then the diode
lasers are more attractive cost-wise.

Craig



>
> Med vänlig hälsning / Best regards
>
> Sylvie
>
> @@@@@@@@@@@@@@@@@@@@@@@@
>
> Sylvie Le Guyader, PhD
> Live Cell Imaging Facility Manager
> Karolinska Institutet- Bionut Dpt
> Hälsovägen 7,
> Novum, G lift, floor 6
> 14157 Huddinge
> Sweden
> mobile: +46 (0) 73 733 5008
> office: +46 (0) 08-524 811 72
> LCI website
>
> ---- SUBSCRIBE CONFOCALMICROSCOPY Anonymous wrote ----
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear All,
>
> We are working on the specs for a new inverted confocal microscope for our
> core imaging facility. We define this as an high-end confocal with live
> imaging capability. We would like advice on the following.
>
> 1) We are going to have 405, 488, 561 and 640 lines (all solid-state
> lasers) for this confocal. Is it necessary to have CFP, YFP channels with
> 448 and 514 laser lines? OR we can use 405 and 488 lines to excite them
> with dedicated CFP, YFP filter cubes? There is no mention of CFP-YFP FRET
> after 2013 in the list here. No more CFP-YFP FRET in confocal now a days?
> Pardon me for my ignorance.
>
> Best regards,
> Core Imaging facility Staff,
> NTU
>
Dr. K N Ganesh Dr. K N Ganesh
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Re: CFP-YFP lines for an inverted confocal microscope

In reply to this post by G. Esteban Fernandez
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Using 405 and 488 gives you more room to collect bigger bandwidth too ..  If you have a dual band notch Primary DM for 405/488 try select manually over the default quad band that will help  you too from collection point of view.  Try using GaAsP over Multi Alkali
Ganesh

Sent from my iPhone

> On 25-Jul-2017, at 3:04 AM, G. Esteban Fernandez <[hidden email]> wrote:
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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> *****
>
> My users actually report a preference for 405 and 488 on our Zeiss LSM 710
> to excite CFP and YFP, even though the system has 458 and 514 Kr-Ar lines,
> because the 458 and 514 are weaker. Of course that is not efficient from a
> photonics standpoint but for them it is more efficient in terms of scanning
> time. I myself use 405 instead of the 458. No one is doing quantitative
> work, just mapping where the dyes are. 405 works well enough for CFP in
> CFP/GFP/YFP/RFP brainbow/confetti tissues.
>
> -Esteban
>
>> On Jul 24, 2017 1:51 PM, "Craig Brideau" <[hidden email]> wrote:
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your posting.
>> *****
>>
>> My lab used a Citrine (YFP)/Cereulean (CFP) Ca2+ FRET indicator with a
>> 405nm and 488nm laser pair with good results. As Sripad mentions, this
>> mostly depends on how well your fluorophore reaches your sample and how
>> sensitive your detectors are.
>>
>> Craig
>>
>>> On Mon, Jul 24, 2017 at 1:57 PM, S Ram <[hidden email]> wrote:
>>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> Post images on http://www.imgur.com and include the link in your
>> posting.
>>> *****
>>>
>>> It really depends on your fluorophore levels. Technically 405 nm will
>>> excite CFP (if I recall CFP excitation spectra has a broad shoulder at
>>> around 405) and YFP can also be excited by 488 nm. Nonetheless, these
>>> wavelengths are not optimal and you might have less than ideal signal if
>>> your fluorophore levels in the sample are not high enough to begin with.
>>>
>>> The other issue to consider is the detector. Will you be using a standard
>>> PMT or a high sensitivity variant (like a HyD or GaAsP)?
>>>
>>> HTH
>>>
>>> Sripad
>>>
>>>
>>> On Sun, Jul 23, 2017 at 6:34 PM, SUBSCRIBE CONFOCALMICROSCOPY Anonymous <
>>> [hidden email]> wrote:
>>>
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> Post images on http://www.imgur.com and include the link in your
>>> posting.
>>>> *****
>>>>
>>>> Dear All,
>>>>
>>>> We are working on the specs for a new inverted confocal microscope for
>>> our
>>>> core imaging facility. We define this as an high-end confocal with live
>>>> imaging capability. We would like advice on the following.
>>>>
>>>> 1) We are going to have 405, 488, 561 and 640 lines (all solid-state
>>>> lasers) for this confocal. Is it necessary to have CFP, YFP channels
>> with
>>>> 448 and 514 laser lines? OR we can use 405 and 488 lines to excite them
>>>> with dedicated CFP, YFP filter cubes? There is no mention of CFP-YFP
>> FRET
>>>> after 2013 in the list here. No more CFP-YFP FRET in confocal now a
>> days?
>>>> Pardon me for my ignorance.
>>>>
>>>> Best regards,
>>>> Core Imaging facility Staff,
>>>> NTU
>>>>
>>>
>>
mcammer mcammer
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Re: CFP-YFP lines for an inverted confocal microscope

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Original questions was best lasers for confocal purchase and has drifted to discussion of best lasers for FRET, but wanted to address original question:   the 405 line is useful for traditional DAPI and for the new Brilliant Violet dyes which are increasingly being used.


Michael Cammer
Research Scientist
DART Microscopy Laboratory

NYU Langone Health
540 First Avenue
SK2 Microscopy Suite
New York, NY  10016

C 914-309-3270
[hidden email]
https://med.nyu.edu/research/research-resources/scientific-cores-shared-resources/microscopy-laboratory
http://microscopynotes.com/ 



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