2.1.

fPAM

Two 532-nm picosecond-pulsed lasers [laser 1 in Fig. 1(a): Olive-1064-4 BW, Huaray Precision Laser; laser 2 in Fig 1(a): APL-4000-1064, Attodyne Lasers] were employed in this dual-wavelength fPAM system, one each for the 558-nm path and the 532-nm path, respectively. In the 558-nm path, the pump laser beam was loosely focused (LA1461-A, Thorlabs; focal length: 250 mm) into the 30-mm-long potassium gadolinium tungstate [$\mathrm{KGd}{({\mathrm{WO}}_4)}_2$, KGW] crystal (KGW-702, EKSMA OPTICS; b-cut), and the stimulated Raman scattering (SRS) effect partially converted the 532-nm pump photons to the first Stokes line at 558 nm [Fig. 1(b)]. We employed a picosecond laser here because it provided greater instantaneous power compared with a nanosecond laser of the same pulse energy, making it more easily cause the SRS effect.²⁶ Since the Raman gain coefficient of KGW is dependent on the polarization of the pump laser, a zero-order half-wave plate (WPH05M-532, Thorlabs) was used to align the pump laser’s polarization with the $a$ axis of the KGW crystal. The output beam was collimated by another lens and filtered by a band-pass filter (575/25 nm BrightLine, Semrock) to selectively pass the first Stokes line. In the 532-nm path, the laser beam was expanded and collimated by a pair of convex lenses. The 558-and 532-nm laser beams were combined by a dichroic mirror (Dichroic Laser Beam Combiner #86-393, Edmund Optics), focused by an achromatic lens (AC127-025-A-ML, Thorlabs), and reflected into the tissue by the lab-made MEMS scanner. PA waves excited by the laser pulses were also reflected by the MEMS scanner and detected by a polyvinylidene fluoride (PVDF) ultrasonic transducer (custom made by CAPISTRANO LABS: 11-mm diameter, 14-mm focal length, 40-MHz central frequency, 110% $-6\mathrm{dB}$ bandwidth). PVDF transducers match better in acoustic impedance with the tissue coupling water than do ceramic transducers.²⁷ Instead of using a complex optical-acoustic splitter, which has a low acoustic transmission efficiency, as in previous PAMs,^22,28,29 we maintained the confocal configuration of the laser beams and the PA waves by passing the laser beams directly through the central hole of this doughnut-shaped PVDF transducer. Both the MEMS scanner and the transducer were immersed in deionized water in a water tank for acoustic coupling. Several critical improvements that have been made since the previous fPAM²² are described in the Appendix.

Fig. 1

fPAM of the mouse brain. (a) Schematic of the fPAM system: $\lambda /2$, half-wave plate; BPF, band-pass filter; DAQ, data acquisition unit; and UT, ultrasonic transducer. (b) Spectrum of the Raman laser plotted on the background of hemoglobin absorption spectra. ${\mathrm{HbO}}_2$ and HbR: oxy- and deoxy-hemoglobin, respectively. (c) Scheme for the functional study. Imaging is performed continuously over an area in the somatosensory region illustrated by the blue square in (d), while the mouse receives impulse stimulation to either one of its forepaws alternatingly. The stimulation-induced ${\mathrm{sO}}_2$ response starts with a sharp decrease, the initial dip, followed by an overshoot [inset in (c)]. (e) A representative MAP image of the somatosensory region. (f) The depth-encoded image of (e).

The PA signals acquired by the transducer were then amplified by a pair of radio frequency amplifiers in tandem (ZFL-500LN+, Mini-Circuits) and digitized by a fast data acquisition unit (ATS9350, Alazar Tech) at a 250-MHz sampling rate. For laser pulse energy calibration, a photodiode (PDA36A, Thorlabs) was used to sample the laser beams, which were split by a wedged optical window (34-245, Edmunds Optics). The MEMS scanner was connected to a stepper motor, and the mouse was supported by a motor-driven translation stage. For raster scanning, fast line scans were performed by the MEMS scanner (Fig. S1 in the Supplementary Material) at a 1-kHz rate (500-Hz resonance frequency), whereas slow orthogonal scans were provided by one of the two motors. The motor stage supporting the mouse was used for the wide-field-of-view (FOV) scanning [Fig. 2(a)], and the stepper motor supporting the MEMS scanner was used for the narrow-FOV scanning [Fig. 2(c)]. Therefore, the mouse remained in a natural motionless state in the latter case.

Fig. 2

fPAM of cerebral vascular response to electrical stimulations. (a) ${\mathrm{sO}}_2$ mapping of the somatosensory area. The blue rectangle illustrates the region of interest for the subsequent functional imaging studies. (b) Oxygen release in resting state (blue) and 0.4 s poststimulusly (red) along a microvessel marked by the white dashed line in (a). It is observed that the capillary had the greatest change between the two states. A plateau, indicated by the arrow, is the site where the blood from another capillary flowed into this vessel. (c) Timing of the initial dip onset, defined by $3\sigma$, superimposed onto the grayscale structure image. (d) Timing of the initial dip trough. (e) Fractional change of the trough of the initial dip. (f) Timing of the hyperemic response onset. Data in (b)–(f) are averaged over five trials.

To separate the two PA signals from the 532- and 558-nm laser pulses, laser 2 was triggered $0.5\mu \mathrm{s}$ later than laser 1. This $0.5\text{-}\mu \mathrm{s}$ interval allowed the earlier PA wave to travel $\sim 0.75\mathrm{mm}$ (greater than the maximum penetration depth of this fPAM system), and such a short interval also ensured that the two laser beams sufficiently overlapped in the focal plane during scanning.

2.2.

Data Processing

We employed the Hessian-based vasculature enhancement filter³⁰ to extract the blood vessels from our fPAM images. The enhanced image was used to create a binary mask of vessels, which was applied to the raw images to remove nonvessel structures, such as residual blood stains on the brain.

The relative concentrations of oxy- and deoxy-hemoglobin are calculated by spectral analysis of the PA images acquired at 532 nm, a near isosbestic wavelength of hemoglobin, and at 558 nm, a nonisosbestic wavelength [Fig. 1(b)]. Details of this method are described in Ref. 25. In short, the PA signal amplitude is linearly proportional to the local concentrations of oxy- and deoxy-hemoglobins, as described here:

Fractional changes in HbT are extracted from the images acquired at 532 nm (Fig. S2 in the Supplementary Material). Vessel diameters were measured as the shortest line across a vessel at different angles, with vessel boundaries identified in the unmasked image using the threshold of three standard deviations ($3\sigma$) of the background noise.

2.3.

Experimental Animals

Female Swiss Webster mice (Hsd:ND4, Envigo; 6- to 8-weeks old) were used for the animal experiment. The laboratory animal protocols were approved by the Institutional Animal Care and Use Committee of California Institute of Technology. First, the mouse was anesthetized with isoflurane and then taped to an animal holder with its head tightly fixed by a stereotaxic frame. Its body temperature was kept at 37°C by a heating pad. Second, the scalp was surgically removed, and the skull above the area roughly corresponding to the forepaw of the somatosensory cortex (S1FL region) was carefully thinned to a thickness of $\sim 50\mu \mathrm{m}$. Ultrasound gel was then applied on the brain to retain moisture and couple the acoustic waves. After the surgery, anesthesia was transferred to $\alpha$-chloralose by an intraperitoneal injection of a dosage at 50 mg per kg body weight every 2 h. Anesthesia depth was carefully controlled by monitoring the heart rate, respiration rate, and hindpaw pinch reflex. Next, a water tank filled with deionized water was placed on top of the mouse head. The plastic membrane at the bottom of the water tank was in gentle contact with the ultrasound gel applied on the brain. Finally, the mouse was placed under the fPAM for imaging.

2.4.

Electrical Stimulation Protocol

Electrical stimulations were introduced by two pairs of needle electrodes inserted under the skin of the right and left forepaws. The electrodes were connected to a stimulator (Isolated Pulse Stimulator Model 2100, A-M Systems) for providing the electrical stimuli. A stimulation sequence consisted of a 60-s rest period, a single strong but brief electrical pulse to one of the forepaws, and a 50-s rest period. This electrical stimulus had an amplitude of $\sim 2\mathrm{mA}$ and a pulse width of 40 ms [Fig. 1(c)]. Next, the same stimulation was applied to the other forepaw. This sequence was repeated five times, with a 1.0- to 1.5-min gap in between. The stimulation period and intensity were controlled without inducing noticeable motions. We employed a single brief stimulus instead of a pulse train since the initial dip is a fast response that can be confounded by multiple stimuli. Moreover, this stimulation scheme is also used in some other initial dip studies.^14,31

3.1.

System Performance

First, we characterized the performance of the fPAM system. The 558-nm Raman laser output has a linewidth of $\sim 0.5\mathrm{nm}$ [Fig. 1(b)], which was measured by a spectrometer (AvaSpec-ULS2048XL-EVO, AVANTES). And its relative pulse energy deviation was measured to be 1.64%, when producing $\sim 150\mathrm{nJ}$ pulses at a 1-MHz pulse repetition rate (Fig. S3 in the Supplementary Material), indicating good monochromaticity and stability. This energy fluctuation, monitored by the photodiode during imaging, was compensated for using Eq. (1) in ${\mathrm{sO}}_2$ calculation. Due to the improved sensitivity of our ultrasonic detection scheme, fPAM has achieved a signal-to-noise ratio of 33.2 dB while imaging single RBCs in the cerebral vasculature through a thinned skull (Fig. S4 in the Supplementary Material).

3.2.

Spatial Profile of the Initial Dip

Using our fPAM, we studied the mouse brain hemoglobin response to a single 40-ms-long impulse stimulus by monitoring both ${\mathrm{sO}}_2$ and HbT simultaneously. Here we define impulse as a stimulus with a duration much shorter than the response delay. First, we acquired volumetric images of the somatosensory cortex with an imaging depth of up to 0.5 mm in a single raster scan [Figs. 1(d)–1(f) and Fig. S5, experimental parameters summarized in Table S1 in the Supplementary Material]. The pulse energy used for imaging was $\sim 150\mathrm{nJ}$, which was not strong enough to result in optical absorption saturation.²² By fixing the optical focus at $\sim 200\mu \mathrm{m}$ below the skull, the structure and oxygenation of microvessels as deep as layer III of the cortex were clearly mapped [Fig. 2(a)]. In the resting state, the ${\mathrm{sO}}_2$ in the microvessels decreased by $\sim 15\%$ per $100\mu \mathrm{m}$ along the vessel [Fig. 2(b)], consistent with literature reports.³² Next, a region of interest, illustrated by the blue box in Fig. 2(a), was monitored at a 6-Hz 3-D imaging rate while the mouse received an impulse electrical stimulus at its forepaw. Our high-sensitivity and high-speed fPAM successfully resolved subtle and transient responses from microvessels. Identification of individual mircovascular compartments is shown in Fig. S6 in the Supplementary Material. Upon contralateral stimulation, the ${\mathrm{sO}}_2$ from capillaries and venules exhibited a two-phase response: a sharp and short decrease, then a recovery and a small overshoot [Fig. 3]. The fast decrease of ${\mathrm{sO}}_2$, the initial dip, started shortly after the stimulus nearly simultaneously across all microvascular compartments [Fig. 2(c) and Fig. S7 in the Supplementary Material], but the trough of the initial dip demonstrated a wave pattern from upstream arterioles propagating through capillaries and further down to venules [Figs. 2(d) and 4]. Similarly, the overshoot from capillaries notably preceded that of the downstream venules. The initial dip at capillaries had a greater fractional change than those from larger vessels [Fig. 2(e)]: $\sim 8\%$ versus $\sim 2\%$ to 6%, due to capillaries being the dominant site of oxygen exchange. The trough of the initial dip was followed by the hyperemia resulting from neurovascular coupling, and its spatiotemporal propagation closely resembled that of the overshoot phase in ${\mathrm{sO}}_2$ [Figs. 2(f) and 4]. We also observed some arterial dilation co-localized with the hyperemia (Fig. S8 in the Supplementary Material).

Fig. 3

A still image of Video 1 showing the ${\mathrm{sO}}_2$ response to the single-impulse stimulation. (a) ${\mathrm{sO}}_2$ mapping recorded at 6 Hz and (b) fractional change of ${\mathrm{sO}}_2$ superimposed onto the grayscale structural imaging. The curve at the bottom left shows the ${\mathrm{sO}}_2$ temporal profile of the area indicated by the arrow in (a) (Video 1, MPEG, 221 kB [URL: https://doi.org/10.1117/1.JBO.25.6.066501.1]).

Fig. 4

A still image of Video 2 showing the ${\mathrm{sO}}_2$ and HbT response to the single-impulse stimulation. The video starts at the beginning of the 40-ms-long impulse stimulus. (a) Response of ${\mathrm{sO}}_2$ superimposed onto the structural image. The pseudocolor shows the timing of the trough and peak of the ${\mathrm{sO}}_2$ change. (b) Response of HbT. The pseudocolor shows the timing of the peak of the HbT change (Video 2, MPEG, 1.26 MB [URL: https://doi.org/10.1117/1.JBO.25.6.066501.2]).

3.3.

Temperal Profile of the Initial Dip

Next, we performed fast line scans (1 kHz) for 2-D imaging across representative vessel segments from different microvascular compartments [arrows in Fig. 2(a)] to acquire finer temporal profiles of the impulse response (Fig. 5). Figure 5(a) shows a representative single-trial ${\mathrm{sO}}_2$ response to an impulse somatic stimulation. The initial dip started first in capillaries [Figs. 5(b) and 5(c)], with the onset time averaged over five mice being $0.13\pm 0.01\mathrm{s}$ (all time points are relative to the beginning of the stimulus). The initial dip onset in other microvascular compartments occurred only up to 0.05 s later than that in capillaries, and it reached the trough at 0.4 to 0.7 s, with the trough from venules arriving $\sim 0.3\mathrm{s}$ later than that from arterioles [Fig. 5(c)]. The capillary has a trough fractional change of up to 8%, which is more than twice that of the arteriole but only $\sim 20\%$ greater than that of the postcapillary venules [Fig. 5(d)]. Second-stage venules, the venules joined by postcapillary venules, also had an initial dip with a significantly longer duration than capillaries and arterioles, probably due to the out-of-phase influx of deoxygenated blood from different capillaries. Following the trough, the ${\mathrm{sO}}_2$ levels quickly recovered, and capillaries and venules manifested a weak and flat overshoot, which was insignificant in arterioles [Fig. 5(c)]. This overshoot phase lasted up to 2 s in venules. The hyperemia started first at the arteriole around 0.4 s, slightly ahead of the trough of the initial ${\mathrm{sO}}_2$ dip in all compartments of the vessel [Fig. 5(b) and Fig. S9 in the Supplementary Material]. Its peak amplitudes at the arteriole and capillary were similar, while venules had a weaker response but of a longer duration. The tail of hyperemia closely coincided with that of the overshoot phase of ${\mathrm{sO}}_2$ response [Fig. 5(b)], indicating a strong relation between them.

Fig. 5

Temporal profiles of the vascular response. (a) A representative single-trial capillary ${\mathrm{sO}}_2$ time course in response to an impulse stimulus. The raw data (blue line) is filtered with a Bessel low-pass filter (25-Hz cutoff frequency) to produce the orange curve. We chose the Bessel filter because it preserves the wave shape of the filtered signal in the passband and therefore maintains causality. (b) Comparison of the averaged ${\mathrm{sO}}_2$ and HbT time courses at the capillaries. By applying the threshold of $3\sigma$, the onset times of the ${\mathrm{sO}}_2$ and HbT responses are calculated as 0.13 and 0.46 s, respectively. (c) Time courses of the ${\mathrm{sO}}_2$ fractional changes in four vessel segments representative of the different microvascular compartments. An example of these representative segments is illustrated by the color-coded arrows in Fig. 2(a). (d) Initial dip amplitude versus vessel diameter for arterioles, capillaries, and postcapillary venules. Data in (b) and (c) are averaged over five trials on each of the five mice; data in (d) are averaged over five trials; the stimulus is illustrated by the small red bar on the horizontal axis; venule 1 and venule 2 denote postcapillary and second-stage venules, respectively; error bar shows standard error.

Meanwhile, the ipsilateral stimulation resulted in only a hyperoxic response in ${\mathrm{sO}}_2$ and a hyperemia of a smaller amplitude, which occurred at a time point similar to that resulting from contralateral stimulations (Fig. S10 in the Supplementary Material).