FRET analyses

Moss et al., 2009 Moss F.J.

Imoukhuede P.I.

Scott K.

Hu J.

Jankowsky J.L.

Quick M.W.

Lester H.A. GABA transporter function, oligomerization state, and anchoring: correlates with subcellularly resolved FRET. Richler et al., 2008 Richler E.

Chaumont S.

Shigetomi E.

Sagasti A.

Khakh B.S. Tracking transmitter-gated P2X cation channel activation in vitro and in vivo. Son et al., 2009 Son C.D.

Moss F.J.

Cohen B.N.

Lester H.A. Nicotine normalizes intracellular subunit stoichiometry of nicotinic receptors carrying mutations linked to autosomal dominant nocturnal frontal lobe epilepsy. Srinivasan et al., 2011 Srinivasan R.

Pantoja R.

Moss F.J.

Mackey E.D.

Son C.D.

Miwa J.

Lester H.A. Nicotine up-regulates alpha4beta2 nicotinic receptors and ER exit sites via stoichiometry-dependent chaperoning. We measured FRET by two independent techniques: donor dequenching (DD) and sensitized emission (SE). The methods for pixel-by-pixel FRET from sensitized emission and donor dequenching have been described in detail (). For cell culture experiments, FRET was examined 40–64 hr after transfection, while for experiments in acute slices FRET was examined 18–22 days following AAV injections in vivo.

The excitation wavelength used for NAPA-a or GFP was 488 nm, and fluorescence was detected at 505–525 nm (Image G ), while for NAPA-n or mCherry the excitation wavelength was 543 nm and fluorescence was detected at 560–600 nm (Image C ). For SE experiments, we measured an additional component, which is the total signal emitted at 560–600 nm when excited at 488 nm (Image F ). The photomultiplier voltages, gains, offsets, scan speeds, and pinhole diameters were constant for all similar experiments (cultured cells or brain tissue). During image acquisition, the focal plane was adjusted to between 15 and 30 μm beneath the surface of the slice or at the widest diameter of the imaged cell. In the cell culture experiments using SE, cells transfected with CMV mCherry or eGFP were included in every imaging session. For similar experiments in brain slices, control animals were injected with either NAPA-a or NAPA-n AAVs to determine spectral bleed through in slices.

DA represents the pixel intensity (I) of GFP in the presence of mCherry and I D represents GFP emission intensity after mCherry was photobleached. E D D = 1 − I D A I D (Equation 1)

We determined FRET efficiency for DD experiments in a cell by stepwise photobleaching of NAPA-n or mCherry using 543 nm wavelength laser light in 2 min increments. Only those cells that showed no changes in morphology were analyzed. We quantified the average change in GFP and mCherry fluorescence across all photobleaching steps and plotted increased GFP fluorescence versus the decrease in mCherry fluorescence. We calculated FRET efficiency from these plots for each ROI by determining the slope of the line using linear regression. We calculated FRET efficiency according to the following formula where Irepresents the pixel intensity (I) of GFP in the presence of mCherry and Irepresents GFP emission intensity after mCherry was photobleached.

F series. This subtraction is necessary, because there are three components that contribute to the apparent FRET image. First, the emission of GFP detected at 560–600 nm, i.e., the donor bleed through coefficient, BT D , multiplied by the pixel intensity of GFP in the Image G series, I D . Second, direct excitation of mCherry at 488 nm, i.e., the acceptor crosstalk coefficient, BT A , multiplied by the pixel intensity of mCherry in Image C series, I A . Third, the FRET component, I netFRET . For all experiments involving calculation of SE. FRET, the PixFRET ImageJ Plug-in was used to determine GFP (BT D ) and mCherry (BT A ) and to calculate the FRET efficiency values at each pixel ( Gordon et al., 1998 Gordon G.W.

Berry G.

Liang X.H.

Levine B.

Herman B. Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. I n e t F R E T = I F R E T − ( I D × B T D ) − ( I A × B T A ) (Equation 2)

For calculation of FRET by SE we assessed FRET efficiency by subtracting donor bleedthrough and acceptor crosstalk from the Imageseries. This subtraction is necessary, because there are three components that contribute to the apparent FRET image. First, the emission of GFP detected at 560–600 nm, i.e., the donor bleed through coefficient, BT, multiplied by the pixel intensity of GFP in the Imageseries, I. Second, direct excitation of mCherry at 488 nm, i.e., the acceptor crosstalk coefficient, BT, multiplied by the pixel intensity of mCherry in Imageseries, I. Third, the FRET component, I. For all experiments involving calculation of SE. FRET, the PixFRET ImageJ Plug-in was used to determine GFP (BT) and mCherry (BT) and to calculate the FRET efficiency values at each pixel ().

DA is the fluorescence of the donor in the presence of the acceptor and I D is the intensity of the donor in the absence of the acceptor ( Kenworthy, 2001 Kenworthy A.K. Imaging protein-protein interactions using fluorescence resonance energy transfer microscopy. Wouters et al., 1998 Wouters F.S.

Bastiaens P.I.

Wirtz K.W.

Jovin T.M. FRET microscopy demonstrates molecular association of non-specific lipid transfer protein (nsL-TP) with fatty acid oxidation enzymes in peroxisomes. D ) was determined by the addition of I DA and the calculated value, I netFRET , which was calculated by determining the additive bleed through components in the above equation ( Zal and Gascoigne, 2004 Zal T.

Gascoigne N.R. Photobleaching-corrected FRET efficiency imaging of live cells. I D = I D A + I n e t F R E T (Equation 3)

To calculate FRET efficiency from sensitized emission images, we used a method similar to that for acceptor photobleaching experiments where Iis the fluorescence of the donor in the presence of the acceptor and Iis the intensity of the donor in the absence of the acceptor (). However, for sensitized emission we used a modification, whereby the intensity of the donor in the absence of the acceptor (I) was determined by the addition of Iand the calculated value, I, which was calculated by determining the additive bleed through components in the above equation (). Therefore, we substituted the following into our equation for FRET calculation by donor dequenching:

G , Image F ), acceptor stacks (Image C , Image F ), and sample image stacks (Image G , Image C , Image F ) prior to image processing. E S E = 1 − I D A I D A + I n e t F R E T (Equation 4)

With the background and bleed through components determined, the FRET efficiency value for each pixel was calculated and the data were presented as 32-bit images with the FRET efficiency displayed on a LUT scale. Prior to analysis, we acquired independent images of mCherry and GFP (NAPA-n and NAPA-a) containing samples for bleed through calibration. Images were compiled into donor stacks (Image, Image), acceptor stacks (Image, Image), and sample image stacks (Image, Image, Image) prior to image processing.

E = R 0 6 / ( R 0 6 + r 6 ) (Equation 5)

Using the efficiency values, we then estimated the inter-molecular distance (r) for the mCherry and GFP fusion construct using the Förster equation.

0 includes terms for the donor quantum efficiency (φ D ), the solvent refractive index (n), overlap of the donor emission and acceptor absorption spectra (J DA ), and the orientation factor (κ2), which are described using the equation below. R 0 6 = ( 8.79 × 10 23 ) κ 2 n − 4 ø D J D A (Equation 6)

In Equation (5) , Rincludes terms for the donor quantum efficiency (φ), the solvent refractive index (n), overlap of the donor emission and acceptor absorption spectra (J), and the orientation factor (κ), which are described using the equation below.

2) at 2/3 assumes random orientations of the FPs, but could range from 0 to 4 depending on the orientation of the fluorophore dipoles as being perpendicular (0) or parallel (4) ( Lakowicz, 2006 Lakowicz J.R. Principles of Fluorescence Spectroscopy. 2 value of 2/3 is parsimonious and in accord with past guidance ( Grecco and Verveer, 2011 Grecco H.E.

Verveer P.J. FRET in cell biology: still shining in the age of super-resolution?. Lakowicz, 2006 Lakowicz J.R. Principles of Fluorescence Spectroscopy. Importantly, the term for the fluorophore dipole orientation factor (κ) at 2/3 assumes random orientations of the FPs, but could range from 0 to 4 depending on the orientation of the fluorophore dipoles as being perpendicular (0) or parallel (4) (). Given that we do not know the relative orientation of the FPs on astrocyte processes and synapses, assuming a κvalue of 2/3 is parsimonious and in accord with past guidance ().