The Journal of Neuroscience, May 11, 2005, ():
In Vivo Performance of Genetically Encoded Indicators of Neural Activity in Flies
J. Neurosci. Reiff et al.
25: 4766
Supplemental data
Files in this Data Supplement:
- supplemental material
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Text supplementary Fig. 1:
The fractional fluorescence change |*deltaup*|F/F (A and B) and the fractional ratio change |*deltaup*|R/R (C) were calculated for 20 boutons of YC3.3-expressing larval NMJs at rest. The |*deltaup*|F/F traces of the CFP (A) and the YFP (B) channel show different amounts of photo-bleaching. CFP is more photo-stable compared to its FRET-partner YFP. Thus the ratio also shows a negative change over time. However, the change in |*deltaup*|R/R is reduced to 50% compared to the signal change of YFP. Another advantage of ratiometric measurements is the reduction of different imaging artifacts. This phenomenon is visible in the increased homogeneity of the |*deltaup*|R/R signals
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Reiff et al., Text supplementary Fig. 2:
Different GECIs showed very different bleaching behavior. Here we show the benefit of two different procedures that aim to correct for the bleaching. In (A) the control without stimulus was subtracted from the stimulated ones (40 and 80 Hz). This procedure was only applicable to Inverse Pericam (IP). After subtraction of the control, the fluorescence change of IP is more clearly detectable and allows better quantification. (B) Fractional GCaMP1.3 fluorescence changes at different stimulus frequencies. After subtraction of the background |*deltaup*|F/F was calculated for the individual boutons (black traces). Double exponential bleach correction was done by deleting the stimulation period prior to fitting. The resulting function (red traces) perfectly matched the remaining fluorescence trace and was subsequently subtracted from the original |*deltaup*|F/F. The bleach corrected fluorescence is shown in blue. This method gave satisfying results up to 40Hz, at 80 Hz minor artifacts were introduced due to complex and more intense bleaching at high rates of neural activity. This problem was more severe in indicators like GCaMP1.6 that showed a lower photostability.
- supplemental material
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Reiff et al., Supplementary raw image and movie 1:
The movie shows the fractional change in FRET (|*deltaup*|R/R) observed in the presynaptic compartment of a YC3.3-expressing neuromuscular junction shown in Figure 2 of the manuscript. Presynaptic action potentials were evoked at a frequency of 80Hz for 2.2 seconds. The stimulation period is indicated by the flashing of the white box in the lower left of the movie. |*deltaup*|R/R is displayed in false color code (see right of movie) where darkest blue and white represent -2% and +7% |*deltaup*|R/R, respectively. For comparison also a raw image of the NMJ is shown in grayscale.
- supplemental material
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raw image of the YC3.3 expressing NMJ in movie 1
- supplemental material
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Reiff et al., Supplementary raw image and movie 2:
The movie shows the fractional change in fluorescence (|*deltaup*|F/F) observed in the presynaptic compartment of a GCaMP1.3-expressing neuromuscular junction. Fluorescence changes were evoked by three consecutive action potential volleys (80Hz for 1 second). The stimulation period is indicated by the flashing of the white box in the lower left of the movie. |*deltaup*|F/F is displayed in false color code (see right of movie) where darkest blue and white represent -2% and +7% |*deltaup*|F/F, respectively. The same experiment is shown in Figure 4A of the manuscript. For comparison also a raw image of the NMJ is shown in grayscale.
- supplemental material
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raw image of the GCaMP1.3- expressing NMJ in movie 2
- supplemental material
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Reiff et al., Supplementary raw image and movie 3:
The movie shows the fractional change in fluorescence (|*deltaup*|F/F) observed in the presynaptic compartment of a SpH-expressing neuromuscular junction. Fluorescence changes were evoked by three consecutive action potential volleys (80Hz for 1 second). The stimulation period is indicated by the flashing of the white box in the lower left of the movie. |*deltaup*|F/F is displayed in false color code (see right of movie) where darkest blue and white represent -2% and +14% |*deltaup*|F/F, respectively. The same experiment is shown in Figure 4C of the manuscript. For comparison also a raw image of the NMJ is shown in grayscale.
- supplemental material
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raw image of the SpH-expressing NMJ in movie3
