INS-fOCT: a label-free, all-optical method for simultaneously manipulating and mapping brain function

Abstract

Significance: Current approaches to stimulating and recording from the brain have combined electrical or optogenetic stimulation with recording approaches, such as two-photon, electrophysiology (EP), and optical intrinsic signal imaging (OISI). However, we lack a label-free, all-optical approach with high spatial and temporal resolution. Aim: To develop a label-free, all-optical method that simultaneously manipulates and images brain function using pulsed near-infrared light (INS) and functional optical coherence tomography (fOCT), respectively. Approach: We built a coregistered INS, fOCT, and OISI system. OISI and EP recordings were employed to validate the fOCT signals. Results: The fOCT signal was reliable and regional, and the area of fOCT signal corresponded with the INS-activated region. The fOCT signal was in synchrony with the INS onset time with a delay of $\sim 30\mathrm{ms}$ . The magnitude of fOCT signal exhibited a linear correlation with the INS radiant exposure. The significant correlation between the fOCT signal and INS was further supported by OISI and EP recordings. Conclusions: The proposed fiber-based, all-optical INS-fOCT method allows simultaneous stimulation and mapping without the risk of interchannel cross-talk and the requirement of contrast injection and viral transfection and offers a deep penetration depth and high resolution.

Keywords: functional imaging; functional optical coherence tomography; infrared neural stimulation.

Fig. 1

Schematic of the coregistered INS and fOCT and OISI system. L, lens; C, collimator; D1, InGaAs camera; D2, CCD camera; DC, dispersion compensator; PC, personal computer; FPC, fiber polarization controller; FL, Fourier lens; TL, tube lens; SL, scanning lens; SLD, super luminescent diode; DM, dichroic mirror; LED, $540/632\text{-}\mathrm{nm}$ LED. Inset: The INS schematic. Each trial is composed of 1-s prestimulus, 0.5-s stimulation, and 18.5-s poststimulus. INS pulse parameters: $\text{pulse width}=250\mu \mathrm{s}$, pulse $\text{repetition rate}=200\mathrm{Hz}$. A total of 15 trials were applied.

Fig. 2

Binary vascular mask processing and flow velocity measurement. (a) Projection view of OCT angiogram. The dashed yellow line indicates the locus of fOCT scan. (b) Cross-sectional OCT angiogram (gray) along the dashed line in (a). Bold arrows indicate the large blood vessels, which are mainly located in the superficial pial layer. Thin arrows indicate the capillary blood vessels, which are mainly located in the cortical layer. (c) Vascular binarization mask (white areas represent blood vessels and tail artifacts) superimposed with OCT cross-sectional activation map (color) from INS. (d) Plot of the measured decorrelation values versus the velocity of the flow phantom. The phantom velocity is controlled by a syringe pump.

Fig. 3

Spatial correspondence of INS-evoked fOCT signals in rat cortex. (a) OISI blood vessel map with 540-nm green light illumination. (b) Projection view of the 3-D OCT structural image. Pearson’s correlation coefficient of (a) and (b)  = 0.85. Yellow arrows in (a), (b) indicate pial blood vessels. Red circles in (a), (b) indicate site of INS stimulation. ROI means region of interest in OISI. SOI means section of interest in OCT. ROI 1 and SOI 1, sites at INS center; ROI 2 and SOI 2: sites near INS edge; ROI 3 and SOI 3, sites distant from INS. The closer to the INS center, the stronger the signals. The yellow dashed lines indicate SOIs 1 to 3 in fOCT. (c) OISI activation map in response to INS with 632-nm red light illumination at time window $t=0.5\mathrm{s}$. Darkening indicates activation. The yellow boxes indicate ROIs 1 to 3 ($20\times 20\text{pixels}$) in OISI. (d) fOCT cross-sections (green-red color scale) superimposed with the OCT anatomical image (gray scale) at time window $t=0.5\mathrm{s}$. (e) Averaged OISI time-courses from ROIs 1 to 3 in (c). For ROI 1, $dR/R=0.12\pm 0.018\%$ ($\text{mean}\pm \mathrm{STD}$ of 15 trial) at 0.5 s. (f) Averaged fOCT time-courses from SOIs 1 to 3 in (d). For SOI 1, $dR/R=2.5\pm 0.52\%$ ($\text{mean}\pm \mathrm{STD}$ of 15 trial) at 0.5 s. (g) Depth-resolved, averaged fOCT time-courses from depths 1to 6, i.e., 0 to $600\mu \mathrm{m}$ under the superficial cortex, of SOI 1 in (d). Each depth contains a thickness of $100\mu \mathrm{m}$.

Fig. 4

Temporal coincidence of INS-evoked fOCT signals. Time courses of (a) OISI and (b) fOCT of a representative rat, respectively. Temporal resolution is 17 and 4.2 ms for OISI and fOCT, respectively. Solid and dashed curves indicate a 1- and 3-s prestim period, respectively. Measurement of (c) the response onset delay and (d) the peak delay in fOCT and OISI, respectively. Averaged by $3\times 2.5\mathrm{mm}$ B-frame size in the $x-z$ plane and 15 trials. Error bar: STD of five rats.

Fig. 5

fOCT signal is positively correlated with INS radiant exposure. Time courses of (a) OISI and (b) fOCT signal for different radiant exposures. (c) The time course of flow velocity (derived from interframe decorrelation, 240 fps) in response to INS with radiant levels of 0 (blank), 0.5, 0.7, and $1.0\mathrm{J}/{\mathrm{cm}}^2$. (d) Representative PSTH from a cortical layer with radiant levels of 0.5 and $1.0\mathrm{J}/{\mathrm{cm}}^2$. Tungsten microelectrodes ($\sim 1.3\mathrm{M\Omega }$) were inserted into the somatosensory cortex at depths of 50 to 750 and $\sim 500\mu \mathrm{m}$ away from the fiber tip. Threshold of spike was set to $-41\mathrm{mV}$. Radiant exposure versus peak amplitude of the (e) OISI fractional signal, (f) fOCT fractional signal, (g) velocity, and (h) peak spike rates.