Separation of hemodynamic signals from GCaMP fluorescence measured with wide-field imaging

Abstract

Wide-field calcium imaging is often used to measure brain dynamics in behaving mice. With a large field of view and a high sampling rate, wide-field imaging can monitor activity from several distant cortical areas simultaneously, revealing cortical interactions. Interpretation of wide-field images is complicated, however, by the absorption of light by hemoglobin, which can substantially affect the measured fluorescence. One approach to separating hemodynamics and calcium signals is to use multiwavelength backscatter recordings to measure light absorption by hemoglobin. Following this approach, we develop a spatially detailed regression-based method to estimate hemodynamics. This Spatial Model is based on a linear form of the Beer–Lambert relationship but is fit at every pixel in the image and does not rely on the estimation of physical parameters. In awake mice of three transgenic lines, the Spatial Model offers improved separation of hemodynamics and changes in GCaMP fluorescence. The improvement is pronounced near blood vessels and, in contrast with the Beer–Lambert equations, can remove vascular artifacts along the sagittal midline and in general permits more accurate fluorescence-based determination of neuronal activity across the cortex. NEW & NOTEWORTHY This paper addresses a well-known and strong source of contamination in wide-field calcium-imaging data: hemodynamics. To guide researchers toward the best method to separate calcium signals from hemodynamics, we compare the performance of several methods in three commonly used mouse lines and present a novel regression model that outperforms the other techniques we consider.

Miniaturized head-mounted device for whole cortex mesoscale imaging in freely behaving mice

“…The Mesoscope performs reflectance measurements at a single wavelength using a green LED to correct for hemodynamic effects. Multiple wavelength reflectance measurements allow for a more accurate correction of hemodynamic effects 38,55 . In future versions, an additional red LED could be incorporated to obtain reflectance measurements at two wavelengths.…”

“…Mesoscale imaging of calcium activity using GCaMP6f indicators is affected by the absorption of light by hemoglobin in the blood 37,38 , which can in turn significantly affect the measured fluorescence signal. Hemodynamic changes can be corrected using reflectance imaging.…”

Sources of widefield fluorescence from the brain

“…The concentrating effect of scattering is lost when angles at the brain surface are randomized, such as by overlying skull. In mouse, widefield imaging is often performed through intact skull ( Mohajerani et al, 2013 ; Silasi et al, 2016 ; Allen et al, 2017 ; Makino et al, 2017 ; Gilad and Helmchen, 2020 ; Valley et al, 2020 ). Mouse skull is ~150–300 µm thick and transparent but strongly scattering ( Soleimanzad et al, 2017 ; Wang et al, 2018 ).…”

“…Blood vessels with circular cross-sections of radius 100 and 250 µm were simulated as regions in which all photons were absorbed. From the Beer-Lambert law, 500 µm of blood transmits <<1% of incident light but 200 µm of blood transmits ~6% (2.2 × 10 −3 mol/L hemoglobin, 50% hemoglobin oxygenation, hemoglobin molar extinction coefficient 27,895 cm −1 M −1 ; Valley et al, 2020 ). Likely this simple simulation slightly overestimates the effects of vessels, particularly small vessels.…”

Dopamine D1 receptor signalling in dyskinetic Parkinsonian rats revealed by fiber photometry using FRET-based biosensors

“…Given that D1R agonists can impact blood flow 58–60 we hypothesized that this effect was caused by alterations in local hemodynamics around the optical probe. Interference from local hemodynamics in fluorescent imaging has been previously reported both with FRET-based and single fluorophore, intensity-based biosensors, and experimental and computational approaches have been proposed to address this confounding factor 53,61,62 . In our experiments, the decrease in fluorescence signal caused by the presumed hemodynamic changes led to a reduction in FRET ratios, which could mask the effects of D1R activation.…”