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GFP/YFP colocalization?

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Howard Berg Howard Berg
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GFP/YFP colocalization?

Hi all:

A paper for ''The Plant Cell'' (Raffaele et al.) just came online and  
several figures are presented showing colocalization of GFP and YFP-
tagged proteins.  I am not surprised that they colocalized because I  
expect there would be a severe bleedthrough for these two.  Am I  
missing something here or have they presented an artifact?  Their  
methods state that they used sequential acquisition (on a Leica TCS  
SP2).

Howard



R. Howard Berg, Ph.D.
Director, Integrated Microscopy Facility
Danforth Plant Science Center
975 N. Warson Rd.
St. Louis, MO 63132

ph 314-587-1261    fx 314-587-1361   cell 314-378-2409
[hidden email]    www.danforthcenter.org
visit this educational resource: http://www.danforthcenter.org/Cells/
JOEL B. SHEFFIELD JOEL B. SHEFFIELD
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Re: GFP/YFP colocalization?

The key is that the Leica allows you to set precise spectral settings
for emission detection.  Sequential acquisition on the Leica SP2
allows you to set these to limit bleed-through by looking at one
channel at a time.  For each z slice, the microscope switches between
the settings.  It's as if you were switching filter sets and
superimposing the images.  On the other hand, it would have to be
carefully controlled.  I would like to see that the "GFP" channel
showed no signal from a YFP source, and vice-versa.

 

Joel


Date sent:       Wed, 27 May 2009 08:15:29 -0500
Send reply to:   Confocal Microscopy List
<[hidden email]>
From:           Howard Berg <[hidden email]>
Subject:         GFP/YFP colocalization?
To:             [hidden email]

> Hi all:
>
> A paper for ''The Plant Cell'' (Raffaele et al.) just came online and  
> several figures are presented showing colocalization of GFP and YFP-
> tagged proteins.  I am not surprised that they colocalized because I  
> expect there would be a severe bleedthrough for these two.  Am I  
> missing something here or have they presented an artifact?  Their  
> methods state that they used sequential acquisition (on a Leica TCS  
> SP2).
>
> Howard
>
>
>
> R. Howard Berg, Ph.D.
> Director, Integrated Microscopy Facility
> Danforth Plant Science Center
> 975 N. Warson Rd.
> St. Louis, MO 63132
>
> ph 314-587-1261    fx 314-587-1361   cell 314-378-2409
> [hidden email]    www.danforthcenter.org
> visit this educational resource: http://www.danforthcenter.org/Cells/



Joel B. Sheffield, Ph.D
Department of Biology
Temple University
Philadelphia, PA 19122
Voice: 215 204 8839
e-mail: [hidden email]
URL:  http://astro.temple.edu/~jbs
John Oreopoulos John Oreopoulos
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Re: GFP/YFP colocalization?

The authors cite a paper by Brandizzi et al (2002) in the same journal  
within their methods section at the end of the paper. Brandizzi's  
methods section details the filter settings and sequential laser  
acquisition that was used. Even with these settings there still may be  
a bit of bleedthrough, but it will be minimalized. To be absolutely  
sure, I agree with Joel that a set of controls with just GFP or just  
YFP should be imaged and observed in both channels (and presented  
somewhere in the publication).

John Oreopoulos

On 27-May-09, at 9:39 AM, Joel Sheffield <[hidden email]> wrote:

> The key is that the Leica allows you to set precise spectral settings
> for emission detection.  Sequential acquisition on the Leica SP2
> allows you to set these to limit bleed-through by looking at one
> channel at a time.  For each z slice, the microscope switches between
> the settings.  It's as if you were switching filter sets and
> superimposing the images.  On the other hand, it would have to be
> carefully controlled.  I would like to see that the "GFP" channel
> showed no signal from a YFP source, and vice-versa.
>
>
>
> Joel
>
>
> Date sent:          Wed, 27 May 2009 08:15:29 -0500
> Send reply to:      Confocal Microscopy List
> <[hidden email]>
> From:               Howard Berg <[hidden email]>
> Subject:            GFP/YFP colocalization?
> To:                 [hidden email]
>
>> Hi all:
>>
>> A paper for ''The Plant Cell'' (Raffaele et al.) just came online and
>> several figures are presented showing colocalization of GFP and YFP-
>> tagged proteins.  I am not surprised that they colocalized because I
>> expect there would be a severe bleedthrough for these two.  Am I
>> missing something here or have they presented an artifact?  Their
>> methods state that they used sequential acquisition (on a Leica TCS
>> SP2).
>>
>> Howard
>>
>>
>>
>> R. Howard Berg, Ph.D.
>> Director, Integrated Microscopy Facility
>> Danforth Plant Science Center
>> 975 N. Warson Rd.
>> St. Louis, MO 63132
>>
>> ph 314-587-1261    fx 314-587-1361   cell 314-378-2409
>> [hidden email]    www.danforthcenter.org
>> visit this educational resource: http://www.danforthcenter.org/Cells/
>
>
>
> Joel B. Sheffield, Ph.D
> Department of Biology
> Temple University
> Philadelphia, PA 19122
> Voice: 215 204 8839
> e-mail: [hidden email]
> URL:  http://astro.temple.edu/~jbs
Gregg Sobocinski Gregg Sobocinski
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Re: GFP/YFP colocalization?

In reply to this post by Howard Berg
Does the paper mention any spectral unmixing? I know that some software can mathematically separate GFP and YFP, but proving that you've done it correctly would be tough.  A lot of variables on both the technical and algorithmic techniques.

I've seen spectral unmixing performed to separate GFP-YFP signals, but only when the labels were distinct. I also am interested how one proves GFP-YFP colocalization, since there are people here who would like to do that as well.

~Gregg

Gregg Sobocinski
Imaging Specialist/Microscopist
University of Michigan, MCDB Dept.
Ann Arbor, Michigan
USA

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Howard Berg
Sent: Wednesday, May 27, 2009 9:15 AM
To: [hidden email]
Subject: GFP/YFP colocalization?

Hi all:

A paper for ''The Plant Cell'' (Raffaele et al.) just came online and  
several figures are presented showing colocalization of GFP and YFP-
tagged proteins.  I am not surprised that they colocalized because I  
expect there would be a severe bleedthrough for these two.  Am I  
missing something here or have they presented an artifact?  Their  
methods state that they used sequential acquisition (on a Leica TCS  
SP2).

Howard



R. Howard Berg, Ph.D.
Director, Integrated Microscopy Facility
Danforth Plant Science Center
975 N. Warson Rd.
St. Louis, MO 63132

ph 314-587-1261    fx 314-587-1361   cell 314-378-2409
[hidden email]    www.danforthcenter.org
visit this educational resource: http://www.danforthcenter.org/Cells/
Howard Berg Howard Berg
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Re: GFP/YFP colocalization?

No spectral unmixing was done.  Nor was there any presentation of  
control images showing lack of bleedthrough in GFP- or YFP-only cells...

I agree that unmixing works well for these two fluorophores, provided  
an appropriate level of expression for them both is achieved.

Howard



On May 27, 2009, at 9:25 AM, Sobocinski, Gregg wrote:

> Does the paper mention any spectral unmixing? I know that some  
> software can mathematically separate GFP and YFP, but proving that  
> you've done it correctly would be tough.  A lot of variables on both  
> the technical and algorithmic techniques.
>
> I've seen spectral unmixing performed to separate GFP-YFP signals,  
> but only when the labels were distinct. I also am interested how one  
> proves GFP-YFP colocalization, since there are people here who would  
> like to do that as well.
>
> ~Gregg
>
> Gregg Sobocinski
> Imaging Specialist/Microscopist
> University of Michigan, MCDB Dept.
> Ann Arbor, Michigan
> USA
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]
> ] On Behalf Of Howard Berg
> Sent: Wednesday, May 27, 2009 9:15 AM
> To: [hidden email]
> Subject: GFP/YFP colocalization?
>
> Hi all:
>
> A paper for ''The Plant Cell'' (Raffaele et al.) just came online and
> several figures are presented showing colocalization of GFP and YFP-
> tagged proteins.  I am not surprised that they colocalized because I
> expect there would be a severe bleedthrough for these two.  Am I
> missing something here or have they presented an artifact?  Their
> methods state that they used sequential acquisition (on a Leica TCS
> SP2).
>
> Howard
>
>
>
> R. Howard Berg, Ph.D.
> Director, Integrated Microscopy Facility
> Danforth Plant Science Center
> 975 N. Warson Rd.
> St. Louis, MO 63132
>
> ph 314-587-1261    fx 314-587-1361   cell 314-378-2409
> [hidden email]    www.danforthcenter.org
> visit this educational resource: http://www.danforthcenter.org/Cells/
Boswell, Carl A - (cboswell) Boswell, Carl A - (cboswell)
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Re: GFP/YFP colocalization?

In reply to this post by John Oreopoulos
An issue to remember when considering bleed-through is the overlap of the
excitation spectra.  If GFP can be "partially" excited by the wavelength(s)
used for YFP, and the emission filters have enough overlap, then imagng
exclusively for YFP will not guarantee seeing only YFP.   In that case one
would be exciting GFP also, and possibly seeing it in the YFP channel.
Sequential imaging does not solve this problem.

It behoves the conciencious microscopist to maintain awareness of the
excitation and emission bandwidth of fluorescent reagents and assess their
results accordingly; simply knowing ex max and em max is not enough.  There
are several websites that do a good job of displaying these spectra.

Controls should have been presented, and the reviewers should have caught
this.

C

Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX 520-621-3709
----- Original Message -----
From: "John Oreopoulos" <[hidden email]>
To: <[hidden email]>
Sent: Wednesday, May 27, 2009 6:59 AM
Subject: Re: GFP/YFP colocalization?


> The authors cite a paper by Brandizzi et al (2002) in the same journal
> within their methods section at the end of the paper. Brandizzi's  methods
> section details the filter settings and sequential laser  acquisition that
> was used. Even with these settings there still may be  a bit of
> bleedthrough, but it will be minimalized. To be absolutely  sure, I agree
> with Joel that a set of controls with just GFP or just  YFP should be
> imaged and observed in both channels (and presented  somewhere in the
> publication).
>
> John Oreopoulos
>
> On 27-May-09, at 9:39 AM, Joel Sheffield <[hidden email]> wrote:
>
>> The key is that the Leica allows you to set precise spectral settings
>> for emission detection.  Sequential acquisition on the Leica SP2
>> allows you to set these to limit bleed-through by looking at one
>> channel at a time.  For each z slice, the microscope switches between
>> the settings.  It's as if you were switching filter sets and
>> superimposing the images.  On the other hand, it would have to be
>> carefully controlled.  I would like to see that the "GFP" channel
>> showed no signal from a YFP source, and vice-versa.
>>
>>
>>
>> Joel
>>
>>
>> Date sent:          Wed, 27 May 2009 08:15:29 -0500
>> Send reply to:      Confocal Microscopy List
>> <[hidden email]>
>> From:               Howard Berg <[hidden email]>
>> Subject:            GFP/YFP colocalization?
>> To:                 [hidden email]
>>
>>> Hi all:
>>>
>>> A paper for ''The Plant Cell'' (Raffaele et al.) just came online and
>>> several figures are presented showing colocalization of GFP and YFP-
>>> tagged proteins.  I am not surprised that they colocalized because I
>>> expect there would be a severe bleedthrough for these two.  Am I
>>> missing something here or have they presented an artifact?  Their
>>> methods state that they used sequential acquisition (on a Leica TCS
>>> SP2).
>>>
>>> Howard
>>>
>>>
>>>
>>> R. Howard Berg, Ph.D.
>>> Director, Integrated Microscopy Facility
>>> Danforth Plant Science Center
>>> 975 N. Warson Rd.
>>> St. Louis, MO 63132
>>>
>>> ph 314-587-1261    fx 314-587-1361   cell 314-378-2409
>>> [hidden email]    www.danforthcenter.org
>>> visit this educational resource: http://www.danforthcenter.org/Cells/
>>
>>
>>
>> Joel B. Sheffield, Ph.D
>> Department of Biology
>> Temple University
>> Philadelphia, PA 19122
>> Voice: 215 204 8839
>> e-mail: [hidden email]
>> URL:  http://astro.temple.edu/~jbs
>
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