2.1.

Animal Preparation

Male Sprague–Dawley rats ($n=5$; 300 to 500 g) were anesthetized with 1% pentobarbital sodium (Sigma, P3761-25g). The toe-pinch test was used to ensure the animal was in an adequate state of anesthesia. The animal was placed in a stereotactic frame and a craniotomy and durotomy were performed to expose somatosensory cortex ($+2$ to $-3\mathrm{mm}$ anterior/posterior and 4 mm lateral to bregma). Mannitol (1.0 ml, 20% concentration) was given to prevent potential brain edema. Warm ($\sim 37°\mathrm{C}$) 1.5% agar in saline was used to stabilize the cortex and a glass coverslip was placed on the agar to create an optical imaging window ($\sim 4\times 3{\mathrm{mm}}^2$). Dental cement was used to seal the cranial window to the skull. All animal experimental procedures used in this study were approved by the Animal Care and Use Committee of Zhejiang University.

2.2.

Experimental System and Data Acquisition

Simultaneous OISI and OCT were performed on rat somatosensory cortex. Figure 1 shows the schematic of the coregistered INS and fOCT and OISI system. OCT is primarily based on a typical Fourier-domain configuration. The superluminesent diode had a radiant power of 10 mW, a central wavelength of 1325 nm, and a full-width at half-maximum bandwidth of 100 nm, theoretically offering an axial resolution of $\sim 7.6\mu \mathrm{m}$ in air. The output light was delivered into a $2\times 2$ fiber coupler and split into the reference and sample arms, respectively. The OCT detection unit was a high-speed spectrometer equipped with a fast line-scan InGaAs camera (Sensors Unlimited Inc., SU1024-LDH2, 92 kHz line-scan rate, and 1024 active pixels). The camera output was digitalized and fed to a custom-designed program in PC1. In the OCT sample arm, an $x\text{-}y$ galvanometer was adopted for three-dimensional (3-D) volume scanning. A scanning lens (Thorlabs, LSM03) with an effective focal length of 36 mm was used to collimate the detecting light on the sample, yielding a lateral resolution of $\sim 10\mu \mathrm{m}$. Prior to the scanning lens, a dichroic mirror (Semrock, FF700-SDi01) was employed to separate the light paths for coregistered OCT and OISI. The OISI system employed 540-nm green LED illumination for acquiring cortical blood vessel distribution map and 632-nm red LED illumination for observing changes in blood oxygen concentration. Light reflected from the cortex transmitted through the dichroic mirror and a tube lens onto a CCD camera (Photonfocus, MV1-D1312-160, $1312\times 1082\text{pixels}$). The coregistered OISI-OCT had a field of view of $\sim 3\times 2.5\mathrm{mm}$ in the $x-y$ plane. OCT offered an imaging range of 2.5 mm in the depth ($z$) direction. The INS-OCT and OISI system were synchronized by signal controller, which is triggered by PC2.

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.

In this study, the volumetric OCT dataset ($z-x-y$) was acquired with a raster scanning protocol. In the fast-scan ($x$) direction, 512 A-lines formed a B-frame. A total of 420 B-frames were acquired with equal interval in the slow-scan ($y$) direction. To record the time course of INS-evoked responses, OCT was then switched to a B-M mode scanning protocol (i.e., disabling the slow-scanner for repeated B-scan). In this study, the OCT B-frame rate was set to be 100 frames per second (fps), and OISI frame rate was set to be 20 fps. For temporal analysis (Fig. 4), the frame rate of both was increased to 240 and 60 fps, respectively.

2.3.

Laser Stimulation Protocol

INS was performed using a near-infrared diode laser at $1870\pm 20\mathrm{nm}$ wavelength (Cygnus Technology Inc., PG4000A). Such a wavelength was selected for reasonable energy transfer and reasonable tissue penetration of 300 to $600\mu \mathrm{m}$.⁴⁷ Light was delivered through a fiber ($200\mu \mathrm{m}$ diameter and 0.22 numerical aperture) onto the region of interest, yielding a focal stimulation. Each INS pulse had a $250\text{-}\mu \mathrm{s}$ pulse width and pulse trains were delivered at a repetition rate of 200 Hz. Each trial consisted of a 1-s prestimulus period, followed by 0.5 s INS (i.e., a train of 100 pulses) and a subsequent 18.5-s poststimulus period that allowed full recovery to baseline, as showed in the inset schematic in Fig. 1. A total of 15 trials were repeated. Four different radiant exposures (i.e., 0.3, 0.5, 0.7, and $1.0\mathrm{J}/{\mathrm{cm}}^2$ per pulse) were selected for radiance dependency study. In each experiment, an additional blank (no stimulation) condition was imaged.

2.4.

Data Analysis

The raw OCT cross-sectional images were reconstructed for each trial, which could be denoted as a 3-D array $I(z,x,t)$ with $z$ is the depth direction, $x$ is the lateral direction, and $t$ is the temporal dimension. The prestimulus baseline value $I_b$ is determined by averaging intensity over the prestimulus period $t_{1:N}$:

To rule out the spontaneous intensity fluctuations and improve the SNR of OCT signals, an adaptive processing algorithm⁴⁸ was employed to emphasize significant pixels. The pixel $(z,x,t_i)$ was a positive significant signal pixel if its intensity value was greater than the baseline value $I_b$ plus three times the prestim standard deviation (STD) $3{\sigma }_b(z,x)$ in five continuous frames:

Similarly, the pixel $(z,x,t_i)$ was a negative significant signal pixel if its intensity value was less than the baseline value $I_b$ minus $3{\sigma }_b$ in five continuous frames:

To further exclude the hemodynamic influence on fOCT signals, a binary vascular mask was applied to the OCT raw data. OCT angiogram was introduced in Figs. 2(a) and 2(b) to emphasize blood flow by mathematically analyzing the temporal dynamics induced by moving red blood cells (RBCs).⁴⁹ The cross-sectional angiogram was binarized to generate an avascular mask for each frame in one trial, where the vessel area was set to be zero and the surrounding tissue was one. The intersection of all single-frame avascular masks was taken as the final vascular mask, as showed in Fig. 2(c). Inter-B-scan decorrelation value was computed for velocity quantification on a basis of previous studies.⁵⁰ And we confirmed that the calculated decorrelation value monotonically increases with the velocity of flow phantoms within a range from 0 to $1.2\mathrm{mm}/\mathrm{s}$, as shown in Fig. 2(d). The preparation of the flow phantoms has been detailed in Ref. 51.

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.

After applying both the adaptive processing and the avascular masks, the fractional change of OCT scattering signal $dR/R$ is defined as the relative ratio of the poststimulus frames over prestimulus baseline value:

The fOCT signals were averaged over all the significant pixels within the whole cross-sectional frame ($3\times 2.5\mathrm{mm}$ size in the $x–z$ plane) and 15 repeated trials for noise reduction, and the pixels without sufficient intensity were excluded with an intensity mask using an intensity threshold of six times STD above the noise level. The filtered negative responses were inversed to be normalized with the positive ones and the final significant fOCT signals were generated by their averaging. To see the hemodynamic response to INS, the RBCs-induced interframe decorrelation value of OCTA was used as the velocity index of blood flow.⁴⁹

OISI and OCT were spatially coregistered using pial blood vessels as rough landmarks and shifting the ROI until Pearson’s correlation coefficient of OISI image (540-nm green LED illumination) and projection view of OCT reached the maximum. Accordingly, the functional OISI signals were calculated in a similar way yet without masks.⁴² Relative intensity changes to prestim baseline for each pixel were calculated and averaged within ROI as functional OISI signal in response to INS.

2.5.

Electrophysiology Recordings

Electrophysiology recording was also employed to validate that INS evoked neuronal activities successfully. Single-unit and multi-unit electrophysiology was utilized to evaluate the rat cortical neuronal responses within 30 trials of the INS stimulus protocol (i.e., 1-s prestimulus period, followed by 0.5 s INS and a subsequent 18.5-s poststimulus period). Glass-coated tungsten microelectrodes ($\sim 1.3\mathrm{M\Omega }$) were inserted into the rat cortex at depths of 50 to $750\mu \mathrm{m}$ in somatosensory regions of interest. The fiber optic was placed $\sim 500\mu \mathrm{m}$ away from the microelectrode. The sampling rate is 30 kHz. Signals were recorded using a Cerebus system (Blackrock Microsystem, LB-0028-14.00, 128 channels), and peristimulus time histograms (PSTHs) were generated and analyzed using Matlab software. The paired two-tailed $t$ test was employed to validate the statistical significance of the INS-evoked activation change.

3.1.

Spatial Analysis

To characterize the INS-evoked fOCT signal, we examined the spatial and temporal profiles of response, as well as its intensity dependence. As we have previously examined INS-induced neural response using 632-nm OISI, we used this as a standard of comparison. As shown in Figs. 3(a) and 3(c), INS ($1\mathrm{J}/{\mathrm{cm}}^2$) applied to rat cortex evokes a localized OISI signal (a darkening of tissue due to neural activity induced deoxygenation) roughly 0.4 mm in diameter. As shown in Fig. 3(e), maximal optical reflectance changes were observed at the INS center (ROI1). INS-induced response dropped by roughly a third near the periphery of the stimulation zone (ROI2) and was still near baseline outside (ROI3) the locus of stimulation. Moreover, the INS-induced response was in synchrony with the stimulus and reached a peak on the order of $\sim 0.2\%$ (relative intensity change), consistent with functional hemodynamic changes described in previous studies.⁴²

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 $100\mu \mathrm{m}$.

We then evaluated whether OCT could detect similar signatures of functional response. Figure 3(b) showed the projection view of OCT structural image, illustrating the well spatial registration [compare with Fig. 3(a)]. We observed that INS evoked a comparably localized, fOCT signal in rat cortex [images in Fig. 3(d) and quantified in Fig. 3(f)]. SOI 1 contained a maximal number of significant pixels ($\sim 449\text{pixels}$) that spanned approximately the same lateral extent as the OISI activation, SOI 2 exhibited a reduced number of pixels ($\sim 354\text{pixels}$), and SOI 3 exhibited no significant scattering response to INS. fOCT responses were consistent across trials (trial number $n=15$) and among different animals (animal number $n=5$). The fOCT signal mirrored the OISI signal with respect to spatial localization, averaged amplitude of all significant pixels, and the evoked time courses [compare Fig. 3(e) with Fig. 3(f)]. Furthermore, as shown in depth-resolved sequences [Fig. 3(g)], this temporal correspondence of fOCT signals was maintained up to a depth of $500\mu \mathrm{m}$ with no significant difference among cortical depths, consistent with the 300- to $600\text{-}\mu \mathrm{m}$ penetration depth of INS at 1870-nm wavelength.⁴²

3.2.

Temporal Analysis

The fOCT signal was also temporally wedded to the INS stimulation. For Fig. 4, we averaged over all the pixels with significant changes in a representative rat. Two sets of trials with 1 and 3-s prestim period were performed. As shown in Fig. 4(a), the rise and the fall of the OISI signal are closely associated with the onset and cessation of INS, respectively. Similar temporal coincidence in the fOCT signals can be shown in Fig. 4(b). The onset delay of OISI, fOCT, and electrophysiological signal was defined as the time-point when the signal power increased larger than three times STD above the baseline after the start of INS. The peak delay was defined when the signal power reached the maximum after the start of INS. They are quantified in Figs. 4(c) and 4(d), respectively. The fOCT onset time delayed by $\sim 30\mathrm{ms}$ after INS, which was ahead of the OISI response for $\sim 10\mathrm{ms}$. And the fOCT peak time delayed by $\sim 528\mathrm{ms}$ after INS, which was ahead of the OISI response for $\sim 5\mathrm{ms}$.

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.

3.3.

Radiant Exposure Dependence

Previous studies have shown that the fractional scattering signal would be linearly proportional to the membrane potential change.¹⁵ Figure 5 shows the response at different radiant exposures (0.3, 0.5, 0.7, $1.0\mathrm{J}/{\mathrm{cm}}^2$ per pulse). As expected, increased INS radiant exposure led to an increase in the fOCT signal magnitude [see Fig. 5(b)], exhibiting a linear fit with radiant exposure [Fig. 5(f)]. OISI signal also presented a similar intensity dependence [see Figs. 5(a) and 5(e)], which is in agreement with previous optical imaging studies in rat cerebral cortex.¹⁵

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.

Figure 5(c) shows the relative blood flow velocity changes in response to INS. The onset time of the velocity change was delayed by $\sim 1\mathrm{s}$ after INS, roughly consistent the measured $\sim 1\mathrm{s}$ in previous studies on cerebral RBC velocity response during rat forepaw electrical stimulation using functional ultrasound imaging,⁵² and those during hind paw electrical stimulation using the laser-Doppler flowmetry.^53,54 The maximal velocity change was also correlated to radiant exposure as showed in Fig. 5(g).

To test the hypothesis that the fractional changes in OISI and fOCT were based on INS-evoked neuronal activities, electrophysiological recording was employed. As shown in Fig. 5(d), the increase of the spike rate during INS period ($t=0$ to 0.5 s) indicates excitatory activation, which was determined to be statistically significant in comparison to 500 ms pre-INS and 500 ms post-INS period ($p<6.1\times {10}^{-6}$ and $p<3.7\times {10}^{-4}$, paired two-tail $t$-test). The spike rate was correlated to the radiant exposure [see Fig. 5(d) and 5(h)]. The onset time of the spike rate was delayed by $\sim 4\mathrm{ms}$ when the bin range is set to be 1 ms.