FIGURE 1.6. Bleaching and hemodynamic artifacts in fluorescence signals imaged in vivo.
FIGURE 1.6. Bleaching and hemodynamic artifacts in fluorescence signals imaged in vivo.

FIGURE 1.6

Bleaching and hemodynamic artifacts in fluorescence signals imaged in vivo. (A) Grayscale map of the change in fluorescence (ΔF) elicited by 2-hexanone (single trial) in a spH-expressing mouse, imaged from the dorsal olfactory bulbs through thinned bone. The response map is scaled from 95% to −30% of the maximal ΔF. Two regions from which the traces in (B) and (C) were derived are indicated. (B) Time course of the fluorescence decrease due to photobleaching (gray trace) taken from a no-stimulus trial, superimposed on the raw traces from two glomeruli (black traces) during a hexanone trial. (C) Time course of the odorant-evoked spH signal from the same two glomeruli after correction for photobleaching (subtraction of gray trace from panel B). Gray box demarcates the image frames used to create the map of activation in panel B. Data were collected at 7 Hz and are presented with no temporal filtering. (From Bozza, T., McGann, J.P., Mombaerts, P., and Wachowiak, M., Neuron, 42: 9–21, 2004.) (D) Fluorescence signal imaged from the dorsal olfactory bulb of a mouse in which olfactory receptor neurons were loaded with Alexa Fluor 488 dextran and the overlying bone removed. Signal is the spatial average of a small (~100 μm) region adjacent to a blood vessel. The large signal fluctuations are due to blood vessel movement and brain pulsation associated with heartbeat. (E) Resting fluorescence and reponse maps on the dorsal olfactory bulbs after loading with Alexa Fluor 488 dextran as in (D). The bone is intact in this preparation. Outlines of the bulbs are indicated by white solid line. The optical signal evoked by hexanone reveals a strong decrease in fluorescence associated with blood vessels (seen as dark lines) and a smaller, spatially diffuse fluorescence decrease which is difficult to distinguish in the map. The image is scaled from 0% to −70% of the maximum ΔF/F. Scale bar, 500 μm. (F) Traces showing fluorescence signals from two regions, one from a blood vessel (4) and another from the underlying bulb (3). The signal appears as a slow fluorescence decrease which is larger over regions associated with blood vessels. (G) Time course of the intrinsic hemodynamic signal elicited by 2-s pulses of two different concentrations of hexanal. The amplitude was concentration dependent and showed a similar time course to the spH signal (compare with panel C). (From Bozza, T., McGann, J.P., Mombaerts, P., and Wachowiak, M., Neuron, 42: 9–21, 2004.)

From: Chapter 1, Optical Imaging of Brain Activity In Vivo Using Genetically Encoded Probes

Cover of In Vivo Optical Imaging of Brain Function
In Vivo Optical Imaging of Brain Function. 2nd edition.
Frostig RD, editor.
Boca Raton (FL): CRC Press/Taylor & Francis; 2009.
Copyright © 2009, Taylor & Francis Group, LLC.

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