Whole animal fluorescence imaging

[kspencer007]

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Hello all;
        Are you using whole animal infrared fluorescence imaging? What equipment are you using? Any suggestions? We don't need bioluminescence, but possibly doing more than one fluor would be nice in the future. Right now, the needs are for Katushka (Ex at 55nm, EM at 635nm), imaging brain subregions for screening assays. We are looking at the IVIS Spectrum, Fluor Vivo, Li-Cor Pearl, and iBox Scientia. Other thoughts and suggestions?
        Thanks.
        Kathy Spencer
        The Scripps Research Institute

[George McNamara]

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Hi Kathy,

Prof. Nagai gave a talk at the Cold Spring Harbor Lab "Single cell
analysis" workshop that included a video of a SuperStar tumor cells
implanted mouse (injected with coelenterazine) imaged while walking
around in its cage. Basically, RLuc8-m1 has ~5% quantum yield
(photons/coelenterzine reaction) whereas the fusion protein resonance
energy transfer is efficient and Venus has good quantum yield (next
~70%). I suggested to Prof. Nagai that he work with Brian Rabinovich
(MDACC, Houston) who previously published on enhanced firefly luciferase
(PNAS paper) and had also enhanced RLuc and GLuc. Of course Venus can
also be used post-vivo for fluorescence widefield, confocal, multiphoton
(i.e. thick mount), and nanoscopy.

        High performance genetically-encoded auto-luminescent Ca2+
        indicators, SuperBRACs

        Takeharu Nagai , Yu-Fen Chang, Kenta Saito, Kazuki Horikawa,
        Tomoki Matsuda
        Research Institute for Electronic Science
        Hokkaido University
        Kita20, Nishi10, Kita-ku, Sapporo 001-0020, Japan
        E-mail : [hidden email]
        KEY WORDS: Bioluminescence, luciferase, fluorescent protein,
        BRET, Ca2+, auto-luminescent, indicator, imaging, living cells.
        Bioluminescent proteins such as luciferase are a powerful tool
        for monitoring biological processes including gene expression in
        living organisms since bioluminescent signals can be acquired
        without an external light source; bioluminescence imaging is
        thus completely free from phototoxicity and auto-fluorescence
        from the specimen, enabling signal detection with high
        signal-to-noise ratio. These properties make bioluminescent
        proteins potentially superior to fluorescent proteins as a
        bioimaging tool [1]. However, bioluminescence signals are too
        dim to be measured in real time, requiring longer exposure than
        fluorescence imaging that takes less than 1 second. To overcome
        this drawback, Renilla reniformis luciferase was conducted on
        random mutagenesis to improve the intensity, yielding
        Rluc8-m1.Then, the luminescence intensity was further increased
        by fusion of the Rluc8-m1 to a yellow fluorescent protein, Venus
        with high BRET efficiency. The chimeric protein, *SuperStar*,
        showed 1,000-fold brighter luminescence than the commercially
        available Rluc, enabling observation of subcellular structure at
        the single-cell level in real time at more than 1 Hz. We then
        applied the SuperStar to design Ca2+ indicators based on
        reconstitution of split- SuperStar fused with the Ca2+ binding
        domain, calmodulin (CaM) and M13, yielding SperBRAC. The dim
        signal of SperBRAC in the absence of Ca2+ increased owing to
        Ca2+ dependent reconstitution of the split-SuperStar moiety. By
        optimizing the split position and linker length between CaM-M13
        [2], we achieved expansion of the dynamic range up to 500%, and
        obtained a series of Ca2+ indicators with different Ca2+
        affinity by which we succeeded long-term video rate luminescent
        Ca2+ imaging in several types of cells.
        [1] K. Saito, N. Hatsugai, K.Horikawa, K. Kobayashi, T.
        Matsu-ura, K. Mikoshiba, and T. Nagai, "Auto-luminescent
        genttically-encoded ratiometric indicator for real-time Ca2+
        imaging at the single cell level, " PLoS ONE, 5, e9935 (2010)
        [2] K. Horikawa, Y. Yamada, T. Matsuda, K. Kobayashi, M.
        Hashimoto, T. Matsu-ura, A. Miyawaki, T. Michikawa, K.
        Mikoshiba, and T. Nagai, "Spontaneous network activity
        visualized by ultra-sensitive Ca2+ indicators, yellow
        cameloen-Nano, " Nature Methods 7, 729-732 (2010)


See also pdf pages 13, 24 and 25 of  
http://www.qimaging.jp/pdf/photomic%20Bioimaging.pdf

Another core at the U has a Xenogen IVIS Spectrum, that is well used.
Glad to see you did not mention the Kodak/Carestream boxes so I do not
have to write anything disrespectful of those products.

Enjoy,

George

On 8/12/2011 12:19 PM, Kathryn Spencer wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello all;
> Are you using whole animal infrared fluorescence imaging? What equipment are you using? Any suggestions? We don't need bioluminescence, but possibly doing more than one fluor would be nice in the future. Right now, the needs are for Katushka (Ex at 55nm, EM at 635nm), imaging brain subregions for screening assays. We are looking at the IVIS Spectrum, Fluor Vivo, Li-Cor Pearl, and iBox Scientia. Other thoughts and suggestions?
> Thanks.
> Kathy Spencer
> The Scripps Research Institute
>
>    

--


George McNamara, PhD
Analytical Imaging Core Facility
University of Miami

[Jeremy Adler-4]

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Perkin Elmer offer Fluorescence Molecular Tomography, 3D imaging of  
live mice using long wagelengths.



Quoting Kathryn Spencer <[hidden email]>:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello all;
> Are you using whole animal infrared fluorescence imaging? What  
> equipment are you using? Any suggestions? We don't need  
> bioluminescence, but possibly doing more than one fluor would be  
> nice in the future. Right now, the needs are for Katushka (Ex at  
> 55nm, EM at 635nm), imaging brain subregions for screening assays.  
> We are looking at the IVIS Spectrum, Fluor Vivo, Li-Cor Pearl, and  
> iBox Scientia. Other thoughts and suggestions?
> Thanks.
> Kathy Spencer
> The Scripps Research Institute
>

Jeremy Adler
IGP
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Sweden

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