GFP/YFP colocalization?
Locked
|
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 |
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 |
|
|
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 |
|
|
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/ |
|
|
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) |
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 > |
«
Return to Confocal Microscopy List
|
1 view|%1 views
| Free forum by Nabble | Edit this page |