Build spectral database

[NkrAb]

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

I have acquired fluorescent emission spectra and other data for a number of
fluorescent probes. Does anyone have suggestions to how I can easily build a
spectral database where people can get access to the belonging spectra/data
when they choose a probe? E.g. any suitable software for doing this?

[Russell Spear]

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

Hi

Check the following

http://www.sylvester.org/AICF/pubspectra.zip
.
Don't want to have to reinvent the wheel, if it has already been done.

Cheers
Russ



On 1/18/2011 9:28 AM, NkrAb wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I have acquired fluorescent emission spectra and other data for a number of
> fluorescent probes. Does anyone have suggestions to how I can easily build a
> spectral database where people can get access to the belonging spectra/data
> when they choose a probe? E.g. any suitable software for doing this?

[Boswell, Carl A - (cboswell)]

If you are looking for something that will let you insert data and immediately produce an interactive graphics page for the web, then no, there is not suitable software.  However there are several useful database packages that will let you extract the data appropriately if you can write the scripts.  SQL is a standard for databases, and Access is useful, though maybe not as powerful.  

As Russ mentioned, there are sites that have done this already, some of which are specific to the company that makes the labels, and some that are more "generic", such as Jonathan Lindsey's PhotoChemCAD: http://www.photochemcad.com/ , the Cornell DRBIO 2P spectra:  http://www.drbio.cornell.edu/personnel.html , and ours: http://www.mcb.arizona.edu/ipc/fret/.  A new version still being finalized is: http://www.spx.arizona.edu/spectra/ which  should soon have the ability to allow downloading of any data displayed.  We will also be adding spectral properties of objectives as we get them.  Either of the last two sites welcome spectral data from anyone, not only for fluorescent labels but for light sources, filters, mirrors and objectives.

C

Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
Univ. of Arizona
520-954-7053
FAX [phone]


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of NkrAb
Sent: Tuesday, January 18, 2011 8:28 AM
To: [hidden email]
Subject: Build spectral database

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

I have acquired fluorescent emission spectra and other data for a number of fluorescent probes. Does anyone have suggestions to how I can easily build a spectral database where people can get access to the belonging spectra/data when they choose a probe? E.g. any suitable software for doing this?

[Renato A. Mortara]

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

Hello,

Having being in the first of Jim Pawley's course I feel embarassed to ask
this, but the doubt is haunting me. Here it goes:

The system and approach: nucleoli of eukaryotic cells double immunolabeled
for pairs of antigens. Z-sectioning through the structures (some quite
spherical, others very disperse and granular) the fluorescence intensity
profile of some of these pairs of antigens appear distinct, ie. the axial of
distribution of different antigens varies among samples - that were NOT
necessarily imaged or PREPARED on the same day.

The question: is it valid to compare these distribution patterns
(intensities along z axis) of antigen pairs between these different samples
(nucleoli of cells A x cells B)?

Many thanks !!

Best

Renato

Renato A. Mortara
Parasitology Division
UNIFESP - Escola Paulista de Medicina
Rua Botucatu, 862, 6th floor
São Paulo, SP
04023-062
Brazil
Phone: [phone]
Fax:     [phone]
email: [hidden email]
home page: www.ecb.epm.br/~ramortara

[George McNamara]

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

Hi Renato,

Don't waste clean image analysis on bad samples!

It sounds like your sample preparation, immunostaining and/or imaging
conditions are highly variable. How about locking down these conditions.
and satisfying yourself that you have consistent imaging conditions. If
you have two very good monoclonal antibodies that bind far apart on the
same protein (specifically, farther than efficient FRET distance, or use
fluorophores with large spectral separation to minimize spectral
overlap, such as Alexa Fluor 488 and 647 ... hopefully without
introducing chromatic aberrations!), you should be able to find
conditions where the distribution patterns are consistent. If any of
your favorite proteins have a fluorescent protein tag, you can also take
advantage of it.

Effective permeabilization of nuclei is more difficult than
permeabilizing the plasma membrane. I believe nucleoli are especially
high density structures - may be difficult to get full size antibodies
to penetrate efficiently into them (assuming you have good nuclear
permeabilization).

Since you are fixing and permeabilizing the cells, you have your choice
of mounting media. I suggest you consider 2,2'-thiodiethanol for your
final mounting medium and use your best oil immersion objective lens.
See Staudt ... Hell 2007 (Staudt's PhD dissertation is available online
- might have more tips), along with Stan Vitha's listserv post on
gradual infiltration.

2,2'-thiodiethanol: a new water soluble mounting medium for high
resolution optical microscopy. </pubmed/17131355>Staudt T, Lang MC,
Medda R, Engelhardt J, Hell SW. Microsc Res Tech. 2007
Jan;70(1):1-9.PMID: 17131355


There are plenty of literature on fluorescence colocalization. A few I
recommend include:

Multi-image colocalization and its statistical significance.
</pubmed/20858446> Fletcher PA, Scriven DR, Schulson MN, Moore ED.
Biophys J. 2010 Sep 22;99(6):1996-2005.PMID: 20858446

Quantifying colocalization by correlation: the Pearson correlation
coefficient is superior to the Mander's overlap coefficient.
</pubmed/20653013> Adler J, Parmryd I. Cytometry A. 2010
Aug;77(8):733-42.PMID: 20653013

Replicate-based noise corrected correlation for accurate measurements of
colocalization. </pubmed/18387047>Adler J, Pagakis SN, Parmryd I. J
Microsc. 2008 Apr;230(Pt 1):121-33.PMID: 18387047


Sincerely,

George
p.s. not specific for a single nucleolar protein, but I want to draw
your attention to this fascinating paper:

In vitro and intracellular production of peptide-encapsulated
fluorescent silver nanoclusters. </pubmed/17285671> Yu J, Patel SA,
Dickson RM. Angew Chem Int Ed Engl. 2007;46(12):2028-30. No abstract
available. PMID: 17285671




On 1/20/2011 6:03 AM, Renato Mortara wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello,
>
> Having being in the first of Jim Pawley's course I feel embarassed to ask
> this, but the doubt is haunting me. Here it goes:
>
> The system and approach: nucleoli of eukaryotic cells double immunolabeled
> for pairs of antigens. Z-sectioning through the structures (some quite
> spherical, others very disperse and granular) the fluorescence intensity
> profile of some of these pairs of antigens appear distinct, ie. the axial of
> distribution of different antigens varies among samples - that were NOT
> necessarily imaged or PREPARED on the same day.
>
> The question: is it valid to compare these distribution patterns
> (intensities along z axis) of antigen pairs between these different samples
> (nucleoli of cells A x cells B)?
>
> Many thanks !!
>
> Best
>
> Renato
>
> Renato A. Mortara
> Parasitology Division
> UNIFESP - Escola Paulista de Medicina
> Rua Botucatu, 862, 6th floor
> São Paulo, SP
> 04023-062
> Brazil
> Phone: [phone]
> Fax:     [phone]
> email: [hidden email]
> home page: www.ecb.epm.br/~ramortara
>
>    

[Adams,Henry P]

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

Renato,
Another suggestion, if you haven't already done this, is to post fix in 2-4% formaldehyde after you have finished staining so your antibodies remain bound and maintain their positions between prep times. This may help.
hank adams

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Renato Mortara
Sent: Thursday, January 20, 2011 5:04 AM
To: [hidden email]
Subject: Is this approach valid ?

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

Hello,

Having being in the first of Jim Pawley's course I feel embarassed to ask
this, but the doubt is haunting me. Here it goes:

The system and approach: nucleoli of eukaryotic cells double immunolabeled
for pairs of antigens. Z-sectioning through the structures (some quite
spherical, others very disperse and granular) the fluorescence intensity
profile of some of these pairs of antigens appear distinct, ie. the axial of
distribution of different antigens varies among samples - that were NOT
necessarily imaged or PREPARED on the same day.

The question: is it valid to compare these distribution patterns
(intensities along z axis) of antigen pairs between these different samples
(nucleoli of cells A x cells B)?

Many thanks !!

Best

Renato

Renato A. Mortara
Parasitology Division
UNIFESP - Escola Paulista de Medicina
Rua Botucatu, 862, 6th floor
São Paulo, SP
04023-062
Brazil
Phone: [phone]
Fax:     [phone]
email: [hidden email]
home page: www.ecb.epm.br/~ramortara