1. Fluorescence Applications in Molecular Neurobiology
Justin W. Taraska, William N. Zagotta 
Neuron 
Volume 66, Issue 2, Pages (April 2010)
DOI: /j.neuron
Copyright © 2010 Elsevier Inc. Terms and Conditions

2. Figure 1 Fluorescence Colocalization Analysis
(A) A fibroblast expressing clathrin-dsRED stained with an antibody against the AP2 complex (B). Almost perfect colocation of both proteins is seen in the merged image (C). (D) TIRF images from a live cell where GFP-labeled dynamin-1 was recruited to an individual dsRED-labeled clathrin-coated pit during endocytosis. Modified and reprinted from Merrifield et al. (2002). The scale bar is 10 μm in (B) and 1 μm in (D).
Neuron  , DOI: ( /j.neuron )
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3. Figure 2
Three-Cube and Spectral FRET for Analysis of Protein-Protein Interactions
(A) Cartoon of FRET between CFP and YFP. (B) Excitation (dotted lines) and emission spectra (solid shapes) of CFP and YFP. For three-cube FRET, three filter cubes are used to collect portions of the spectrum of CFP and YFP. First, the intensity of the donor emission (green arrow) with excitation light at the donor's peak excitation wavelength (blue arrow) (IDD). Second, the intensity of the acceptor emission (red arrow) upon excitation near the acceptor's peak excitation wavelength (orange arrow) (IAA). Third, the intensity of the acceptor (red arrow) upon excitation near the donor's peak excitation wavelength (blue arrow) (IDA). Equations utilizing these three values (IDD, IAA, and IDA) can then be used to calculate the amount of FRET in the sample. Spectra based on Patterson et al. (2001). (C) For spectral FRET, the entire spectrum of the donor and acceptor are used to calculate the amount of acceptor sensitization due to FRET. This is done by determining the amount of YFP emission due to FRET after correction for CFP fluorescence and the direct excitation of the acceptor by the donor wavelength. From these values, the amount of FRET expressed by ratio A can be determined (D). The amount of direct excitation of YFP is determined in a separate experiment with samples containing only YFP and is expressed by ratio A0.
Neuron  , DOI: ( /j.neuron )
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4. Figure 3 Bimolecular Complementation Analysis
To test for the binding of two proteins (a and b), each protein is fused to a fragment of yellow fluorescent protein. On their own, these two fragments of YFP are not fluorescent. If the two protein fusion partners bind together, however, the two fragments of YFP combine to form a fluorescent molecule.
Neuron  , DOI: ( /j.neuron )
Copyright © 2010 Elsevier Inc. Terms and Conditions

5. Figure 4
Fluorescence Intensity Ratio Measurement to Determine Subunit Stoichiometry
(A) Scatter plot of CNGA1 ion channel subunits labeled with CFP and CNGB1 subunits labeled with YFP.
(B) Scatter plot of CNGA1 subunits labeled with YFP and CNGB1 subunits labeled with CFP. From the slope of the two graphs, the relative ratio of subunits (3:1) can be determined.
Reprinted from Zheng and Zagotta (2004).
Neuron  , DOI: ( /j.neuron )
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6. Figure 5 Fluorescence Bleaching to Determine Subunit Stoichiometry
(A) An image of an oocyte membrane sparsely labeled with GFP-containing membrane proteins.
(B) Traces showing four bleaching steps in a tetrameric CNGA1 channel, two bleaching steps in a complex containing two GFP-labeled NMDA subunits, and one bleaching step in a monomeric calcium channel construct. The number of steps in a diffraction-limited spot tracks the number of subunits in the complex.
Reprinted and modified from Ulbrich and Isacoff (2007).
Neuron  , DOI: ( /j.neuron )
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7. Figure 6 Fluorescence Intensity Measurements of Conformational Changes
(A) Cartoon of a single subunit of the tetrameric voltage-gated Shaker potassium channel. Individual residues in an extracellular loop (red box) were labeled with a cysteine-reactive dye.
(B) Cartoon showing the experimental design. The membrane voltage was depolarized in a step (black trace) opening channels and producing ionic current (blue trace). Changes in the intensity of the fluorescence (red trace) were measured and tracked the opening of the channels.
(C) Fluorescence traces from individual fluorophore-labeled residues in the S3–S4 extracellular loop of the channel during voltage steps.
Reprinted and modified from Pathak et al. (2007).
Neuron  , DOI: ( /j.neuron )
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8. Figure 7 Intramolecular FRET to Map Distances in Protein Structures
(A) GFP can be attached to the channel and used as a FRET donor to single residues labeled with the red fluorophore Alexa-568.
(B) By measuring the amount of donor quenched (blue arrow) after the addition of the acceptor (gray arrow), the quantity of FRET can be determined.
Reprinted and modified from Taraska and Zagotta (2007).
Neuron  , DOI: ( /j.neuron )
Copyright © 2010 Elsevier Inc. Terms and Conditions

9. Figure 8 LRET Measurements in Ion Channels
(A) Cartoon showing LRET between a chelated lanthanide ion and bodipy-maleimide.
(B) In channels, a fluorescently labeled pore-bound toxin was used as an acceptor for lanthanide attached to the channel.
Figure based on Posson and Selvin (2008).
Neuron  , DOI: ( /j.neuron )
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10. Figure 9 Transition Metal Ion FRET
(A) A nickel ion bound to di-histidine motifs in an α helix can be used as a distance-dependent energy acceptor for small fluorophores.
(B) In peptides, the amount of energy transfer decreases the farther the metal is positioned from the fluorophore. Error bars are standard error of the mean.
(C) In HCN2, di-histidine motifs were used to position a metal ion (blue) near a cysteine-modified fluorophore (green). FRET was used to map the structure and conformational movements of the C helix during ligand binding.
(D) More FRET was seen in the ligand-bound state, demonstrating that the C helix moved closer to the cysteine-attached fluorophore. Error bars are standard error of the mean.
Modified and reprinted from Taraska et al. (2009a).
Neuron  , DOI: ( /j.neuron )
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11. Figure 10 Quenching of Bimane by Photo-Induced Electron Transfer
(A) In T4 lysozyme, a tryptophan (red) was engineered into an α helix near a bimane-modified cysteine (blue).
(B) Tryptophan-induced quenching of the bimane's fluorescence was seen in steady-state fluorescence measurements.
(C) Electron-transfer-induced quenching could also be seen in the shortening of bimane's lifetime.
Modified and reprinted from Mansoor et al. (2002).
Neuron  , DOI: ( /j.neuron )
Copyright © 2010 Elsevier Inc. Terms and Conditions