2.1.

Fiber Laser Excitation System

The design of the fiber laser was based on a master oscillator power amplifier (MOPA) configuration, operating at an emission wavelength of 1064 nm, which was then frequency doubled to 532 nm. The MOPA consisted of a seed laser, one preamplifier stage, and two high-power fiber amplifiers (see inset of Fig. 1). Polarization maintaining fiber was used throughout in order to provide a linearly polarized output as required for the final stage frequency doubling. The seed laser comprised a Fabry-Pérot laser diode that was injection locked using a distributed feedback laser diode in order to provide relatively high peak powers ($>100\mathrm{m}\mathrm{W}$) and a narrow linewidth ($<0.7\mathrm{nm}$). The latter is required for efficient second harmonic generation (SHG). The output of the seed laser was amplified by the preamplifier, which comprised a 6-m long Ytterbium-doped fiber with a $6\text{-}\mu \mathrm{m}$ core diameter that was core-pumped using a 978-nm CW single-mode laser diode in a backward configuration. The first high-power amplifier stage (Amp1) used a 6-m long Ytterbium-doped fiber with a $10\text{-}\mu \mathrm{m}$ core diameter and was cladding-pumped using a 915-nm CW laser diode in a forward configuration. Cladding pumping allows the use of high-power multimode laser diodes whose poor beam quality does not permit efficient launching into a single-mode core. The second high-power amplifier stage (Amp 2) used a 3-m long Ytterbium-doped fiber with a $25\text{-}\mu \mathrm{m}$ core diameter and was cladding-pumped using a 975-nm CW laser diode and free space optics in a backward configuration. Using a large core diameter ($25\mu \mathrm{m}$) fiber increases the power handling of the amplifier and the threshold for nonlinear effects. To ensure that only the fundamental mode propagated within the fiber, it was coiled around a 70-mm diameter cylinder, causing the higher order modes to leak out of the core. The output of the laser was then frequency doubled using a lithium triborate (LBO) nonlinear crystal. To achieve this, the beam was passed through a half wave plate to rotate the polarization of the light to match the optical axis of the crystal. The beam is then passed through a lens in order to focus it within the LBO crystal for SHG. The LBO crystal has a length of 25 mm and phase matching was implemented by tuning its temperature to 154°C. The second harmonic beam was collimated and separated from the residual fundamental beam after reflection from a dichroic mirror.

The system provided a PRF that was variable between 250 kHz and 2 MHz, a minimum pulse energy of 0.5 μJ at the laser output, pulse duration of 1.4 ns, and a high beam quality ($M^2<1.12$). The laser was designed to operate at a maximum PRF of 2 MHz, which corresponds to an RA limited maximum penetration depth of $750\mu \mathrm{m}$.

2.2.

Optical Scanning System

The scanning system is based on an asymmetric conjugate scanner specifically designed to provide both high speed and large field-of-view (FOV). The system comprises a pair of mutually orthogonal $x-y$ galvanometers (G1 and G2, Cambridge Technology Ltd.), which are used to raster scan the excitation beam over a pair of achromatic lenses (AC508-100-A, Thorlabs Ltd.) that focus the beam on to the sample; the focal length of the achromatic pair was 5 cm. The scanner has several specific design features. First, the collimated beam emitted by the laser is scanned by G1 over the surface of two off-axis concave mirrors (M1 and M2) and subsequently directed to a single point on G2. Rotation of G1, therefore, rotates the beam about a spatially invariant pivot-point on G2 thereby avoiding any beam shift, which would otherwise distort the focal spot, particularly when the beam is off-axis, which limits the FOV. Second, the focal lengths of the concave mirrors (M1 and M2) were chosen to be 5 and 10 cm, respectively, resulting in the initial 3-mm diameter beam provided by the fiber laser being expanded to twice its original diameter following its reflection from M2. This arrangement offers two advantages. First, the excitation beam that is incident on the achromatic doublet is of large diameter ($Ø6\mathrm{mm}$). This enables a reasonably small focal spot of $7\text{-}\mu \mathrm{m}$ diameter (FWHM)^5,14 to be achieved using a scan lens with a relatively long focal length so as to provide a large FOV ($>10\mathrm{mm}\times 10\mathrm{mm}$) with minimal vignetting. Second, using M1 and M2 to expand the beam diameter downstream of G1 means that the dimensions of G1 can be kept small ($5\mathrm{mm}\times 5\mathrm{mm}$). This minimizes inertia and thus maximizes speed, an important requirement for G1 since it provides scanning along the fast axis. By contrast, G2 is the slow-axis scanning galvanometer. Hence, a relatively large mirror ($10\mathrm{mm}\times 15\mathrm{mm}$) can be tolerated in order to accommodate the expanded 6-mm diameter beam reflected from M2.

The galvanometers were driven with a triangular waveform, continually scanning them back and forth. The oscillation frequency of the small mirror (G1) can reach 1 kHz depending on the scanning angles required. For example, when scanning over an angle of 1.4 deg, equivalent to a displacement of 1.25 mm on the tissue surface, the scanning speed of the mirror can be set to 1 kHz, corresponding to 2000 B-scans per second.

2.3.

Acoustic Detection and Signal Acquisition

The generated photoacoustic signals were detected using a planoconcave fiber-optic sensor that was acoustically coupled to the tissue sample using ultrasound gel. The design and acoustic performance of the sensor has been described previously.^16,17 Briefly, it comprised a $33.1\text{-}\mu \mathrm{m}$-thick planoconcave polymer cavity that acts as an optical microresonator deposited on to the tip of a single-mode fiber with a $10\text{-}\mu \mathrm{m}$ core diameter. An incident acoustic wave modulates the optical thickness of the cavity and thus its reflectivity. This is read-out using a CW 1550-nm laser beam that is coupled into the core of the fiber and tuned to a wavelength that corresponds to the edge of the cavity resonance. The sensor provides a high detection sensitivity ($\mathrm{NEP}<0.1\mathrm{kPa}$ over a 20-MHz measurement bandwidth¹⁶) and a broad bandwidth that extends to 60 MHz. An important characteristic of the sensor is the large acoustic acceptance angle ($\pm 90\mathrm{deg}$) it provides due to its small active element size ($\sim 10\mu \mathrm{m}$).^16,17 As a consequence, it provides a near omnidirectional response enabling photoacoustic waves generated over a large lateral FOV to be recorded without translating the sensor.

The photoacoustic signals recorded at each scan position were continuously streamed to a PC via a fast digitising card (PCI-5114 National Instruments) with a sampling rate of 50 MS/s and 8-bit resolution. A fast photodiode connected to the second channel of the digitizer was used to monitor the excitation laser pulses and remove any timing uncertainty due to jitter. To form an image, a maximum intensity projection (denoted ${\mathrm{MIP}}_{\mathrm{pk}\text{-}\mathrm{pk}}$) was produced by plotting the peak-to-peak amplitudes extracted from each recorded photoacoustic waveform as a function of $x–y$ position.

3.1.

Static Image Acquisition

As an initial evaluation of the system, and in order to provide a reference image to which subsequent images obtained when operating at a PRF of 2 MHz could be compared, the ear of a nude mouse was imaged while operating at 500 kHz PRF. The mouse was anaesthetized using a mixture of isoflorane and oxygen at a concentration of 4% for induction, and 1% to 2% for maintenance, with flow rates of 1 l/min. All experiments were performed in accordance with the UK Home Office Animals Scientific Procedures Act (1986). Images were acquired in both forward and backward modes. Figure 2(a) shows a schematic depicting the position of the sensor when operating in forward mode, and Fig. 2(b) shows the corresponding photoacoustic image of the mouse ear displayed as an ${\mathrm{MIP}}_{\mathrm{pk}-\mathrm{pk}}$. The imaged area was $8\mathrm{mm}\times 13\mathrm{mm}$ with 2- and $3.25\text{-}\mu \mathrm{m}$ step sizes in the $x$ and $y$ directions, respectively. At each scan point, a single A-line was acquired, thus a total of $N=16$ million A-lines were recorded. The acquisition time was 32 s and was limited by the PRF of the laser (500 kHz). The pulse energy of the fiber laser was 500 nJ, corresponding to an estimated fluence at the focus of $1.3\mathrm{J}/{\mathrm{cm}}^2$, which is comparable to that used in previous OR-PAM studies.^2,18–20 The microvasculature down to the level of individual capillaries can be clearly visualized with high contrast as shown in Fig. 2(b).

Fig. 2

Photoacoustic imaging of a mouse ear in forward and backward modes at 500,000 A-lines/s. (a) Schematic showing the position of the fiber-optic sensor when imaging in forward mode, (b) photoacoustic image obtained in vivo of a mouse ear while operating in forward mode, (c) schematic showing the position of the fiber-optic sensor when imaging in backward mode, and (d) photoacoustic image obtained in vivo of a mouse ear when operating in backward mode. In both cases, the scan area was $8\mathrm{mm}\times 13\mathrm{mm}$ with 2- and $\text{3.25-}\mu \mathrm{m}$ step size in the $x$ and $y$ directions, respectively, and the total number of A-lines acquired was $N=16$ million. The $x$ and $y$ galvanometers were driven at 62.5 and 0.015625 Hz, respectively. The PRF of the laser was 500 kHz and the pulse energy was 500 and 900 nJ for the forward and backward mode configurations, respectively. The position of the fiber sensor is indicated by the white cross in (b) and (d) and was located 1.5 mm above the sample for the forward mode configuration and directly adjacent to the sample for the backward mode configuration.

Figure 2(c) shows a schematic depicting the position of the sensor when operating in backward mode, which allows images to be acquired when access to both sides of the tissue is unavailable. The sensor was embedded in the substrate, by creating a groove in the substrate at a 30-deg angle where the sensor was placed. Figure 2(d) shows the acquired photoacoustic image of the mouse ear. As with the forward mode configuration, the microvasculature at the capillary level can be visualized. The imaging parameters were identical to those used in forward mode; other than the pulse energy was increased to 900 nJ to compensate for the greater acoustic propagation distance in the backward mode configuration compared to forward mode configuration.

Following the above initial evaluation of the system, the highest possible acquisition speed was achieved by increasing the PRF in order to acquire A-lines at a rate of 2 million per second. To achieve this, an area of $10\mathrm{mm}\times 10\mathrm{mm}$ was imaged in forward mode when operating the fiber laser at a PRF of 2 MHz, which corresponds to an RA limited depth of $750\mu \mathrm{m}$. The acquired photoacoustic image is shown in Fig. 3 and is of comparable quality to the images in Fig. 2. The photoacoustic image was formed of 16 million A-lines, which were acquired in 8 s, representing a factor of 4 increase in imaging speed compared to the imaging speed achieved in Figs. 2(b) and 2(d). Imaging in backward mode was not attempted at a PRF of 2 MHz due to the limited pulse energy provided by the fiber laser (only 270 nJ could be provided at the tissue surface) at this PRF.

Fig. 3

Photoacoustic image obtained in vivo of a mouse ear at 2 million A-lines/s. The scan area was $10\mathrm{mm}\times 10\mathrm{mm}$ with a $2.5\text{-}\mu \mathrm{m}$ step size and the total number of A-lines acquired was $N=16$ million. The $x$ and $y$ galvanometers were driven at 250 and 0.0625 Hz, respectively. The PRF of the laser was 2 MHz and the pulse energy was 270 nJ. The position of the fiber sensor is indicated by the white cross and was 1.5 mm above the sample.

3.2.

Dynamic Imaging

To illustrate the dynamic imaging capability of the system, a mouse ear was imaged at a frame rate of 2 fps over a period of 32 s while being continuously translated laterally using a manual translation stage. The imaged area was $5\mathrm{mm}\times 5\mathrm{mm}$ ($500\times 2000$ points), for which $N=1$ million A-lines were acquired per frame while operating the fiber laser at a PRF of 2 MHz. Figure 4 shows three individual frames obtained at times $t=\mathrm{0,7}$, and 13 s. The mouse ear was translated vertically in the $x$ direction. To highlight the motion, a common blood vessel is indicated by a yellow arrow in each of the three frames in Fig. 4. A video can also be viewed online (Video 1), which illustrates the motion in real time. The repetitive accordion type image deformation observed in the video is a consequence of insufficiently high frame rate and arises when the sample accelerates such that it moves an appreciable distance during the time taken to complete a single scan. The distortion is irregular and most pronounced at the start of each turn of the micrometer used to translate the stage as the sample is accelerating most rapidly at this time.

Fig. 4

Photoacoustic images of a mouse ear obtained at 2 fps while translating the mouse ear in the $x$ direction. (a)–(c) Individual frames obtained at times $t=\mathrm{0,7}$, and 13 s, respectively. A video can be viewed online (Video 1). The scan area was $5\mathrm{mm}\times 5\mathrm{mm}$ with a 2.5- and $10\text{-}\mu \mathrm{m}$ step size in the $x$ and $y$ directions, respectively, and the total number of A-lines acquired was $N=1$ million per frame. The $x$ and $y$ galvanometers were driven at 500 and 1 Hz, respectively. The PRF of the laser was 2 MHz and the pulse energy was 270 nJ. The position of the fiber sensor is indicated by a red cross and was 1.5 mm from the sample. The yellow arrows indicate a common feature in each frame. (Video 1, MP4, 2 MB [URL: https://doi.org/10.1117/1.JBO.23.12.126502.1]).

In a subsequent experiment, the mouse ear was imaged at a frame rate of 2 fps while remaining in a stationary position. The imaged area was $5\mathrm{mm}\times 5\mathrm{mm}$ ($500\times 500$ points), for which $N=0.25$ million A-lines were acquired per frame while operating the fiber laser at a PRF of 500 kHz. A total of 64 images were acquired at 2 fps with each image being represented as a single ${\mathrm{MIP}}_{\mathrm{pk}-\mathrm{pk}}$. All 64 ${\mathrm{MIP}}_{\mathrm{pk}-\mathrm{pk}}$ images were then combined to form a single 3-D dataset ($x,y$, and $T$), where $T$ corresponds to the time at which each frame was acquired. A maximum intensity projection (denoted ${\mathrm{MIP}}_T$) of this combined dataset was then formed by projecting the maximum values of the pixels lying along the temporal axis ($T$) on to the $x-y$ plane as shown in Fig. 5(a). In order to interpret this image, it is necessary to view video 2, which is available online. This video displays each of the 64 ${\mathrm{MIP}}_{\mathrm{pk}-\mathrm{pk}}$ frames sequentially in time and appears to show red blood cells (RBCs) appearing and disappearing in successive frames. This is shown in Figs. 5(b) and 5(c), which show two successive individual frames of a small area of the scanned region indicated by the solid square box in Fig. 5(a). The white arrows highlight features that could be single RBCs, which are present in (b) but not in (c), whereas the yellow arrows highlight single RBCs which are present in (c) but not in (b). When an ${\mathrm{MIP}}_T$ through the temporal axis of the entire data set (i.e., all 64 frames) is formed, as shown in Fig. 5(d), a network of capillaries becomes visible. Although the images shown in Fig. 5 are likely to be broadly representative of the distribution of individual RBCs within the capillaries, the RBCs in any given single frame [e.g., see Figs. 5(b) and 5(c)] seem to be more sparsely distributed than expected. Moreover, inspection of Video 2 suggests their motion is somewhat discontinuous. These effects could be due to the relatively low SNR provided by single RBCs. Small random fluctuations in signal amplitude due to variations in the degree of overlap of the focussed laser spot with individual RBCs, pulse energy or RBC orientation may then cause the signal to randomly drop below the noise floor resulting in a sparser than expected contrast distribution as well as the perception of disorganised motion. By contrast, the larger microvessels do not exhibit these effects due to their high RBC density, which results in a higher SNR and a more continuous contrast distribution.

Fig. 5

Dynamic photoacoustic imaging of a large FOV showing the motion of single RBCs in the capillaries of a mouse ear imaged in vivo at a frame rate of 2 fps. A video can be viewed online (Video 2). (a) ${\mathrm{MIP}}_T$ through the temporal axis ($T$) of a single 3-D dataset ($x,y$, and $T$) formed from 64 successive ${\mathrm{MIP}}_{\mathrm{pk}-\mathrm{pk}}$ frames, where $T$ corresponds to the time, in which each frame was acquired. (b) and (c) Two successive individual ${\mathrm{MIP}}_{\mathrm{pk}-\mathrm{pk}}$ frames of a region delineated in (a) by a solid square box. The white arrows highlight the presence of RBCs in (b) but not in (c) and the yellow arrows highlight the presence of RBCs in (c) but not in (b). (d) ${\mathrm{MIP}}_T$ through the temporal axis of the data set of the same region of interest as (b) and (c) shows delineation of capillaries vessels. The scan area of (a) was $5\mathrm{mm}\times 5\mathrm{mm}$ with a $10\text{-}\mu \mathrm{m}$ step size and the total number of A-lines acquired was $N=\mathrm{250,000}$. The $x$ and $y$ galvanometers were driven at 500 and 1 Hz, respectively. The PRF of the laser was 500 kHz and the pulse energy was 400 nJ. (Video 2, MP4, 2.4 MB [URL: https://doi.org/10.1117/1.JBO.23.12.126502.2]).

The video also shows transient variations in blood flow in larger vessels indicated in the video by arrows. To illustrate the flow in larger vessels in more detail, a second video (Video 3) was obtained at 2 fps when imaging a smaller area of $2.5\mathrm{mm}\times 2.5\mathrm{mm}$ ($500\times 500$ points) with a smaller scanning step size of $5\mu \mathrm{m}$. This area is delineated by the large dotted white box in Fig. 5(a) and the first frame of this video is shown in Fig. 6(a). The red and blue boxes show areas where variations in blood flow in specific vessels can be seen. Figure 6(b) shows individual frames of the region indicated by the blue box, taken at times $t=10.5$, 11.5, and 12.5 s. Similarly, Fig. 6(c) shows individual frames of the region indicated by the red box, taken at times $t=24.5$, 26.5, and 28 s. In both cases, the changes in contrast along the vessels are highlighted by an arrow, in each of the different frames. Videos of these two regions of interest are available online (Videos 4 and 5).

Fig. 6

Photoacoustic image obtained in vivo in a mouse ear at a frame rate of 2 fps showing variations in blood flow in selected regions. (a) The area delineated by the square dotted box in Fig. 5(a). A video can be viewed online (Video 3). (b) and (c) An expanded view of two areas of interest, where changes in blood flow in specific vessels (indicated by white arrows) are present, delineated in (a) by a solid red and a solid blue box, respectively. Videos can be viewed online (Videos 4 and 5). The scan area of (a) was $2.5\mathrm{mm}\times 2.5\mathrm{mm}$ with a $5\text{-}\mu \mathrm{m}$ step size and the total number of A-lines acquired was $N=\mathrm{250,000}$ per frame. The $x$ and $y$ galvanometers were driven at 500 and 1 Hz, respectively. The PRF of the laser was 500 kHz and the pulse energy was 400 nJ. (Video 3, MP4, 3 MB [URL: https://doi.org/10.1117/1.JBO.23.12.126502.3]); Video 4, MP4, 0.8 MB [URL: https://doi.org/10.1117/1.JBO.23.12.126502.4]); Video 5, MP4, 1.2 MB [URL: https://doi.org/10.1117/1.JBO.23.12.126502.5]).

To demonstrate the possibility of obtaining higher frame rates than 2 fps, a mouse ear was imaged at a frame rate of 10 fps over an area of $1.25\mathrm{mm}\times 1\mathrm{mm}$ ($250\times 200$ points) for a period of 32 s. The first frame of the dataset is shown in Fig. 7(a) and a video is available online (Video 6). Two haemodynamic events appear in this dataset. The first one appears in the region delineated by a red box, which shows a region of low contrast flowing along a vessel. Figure 7(b) shows individual frames of this occurrence at times $t=14.8$, 14.9, and 15 s, the region of low contrast being indicated by an arrow. The second event appears in the region delineated by a blue box, and once again a region of low contrast can be seen flowing along a vessel. Figure 7(c) shows individual frames of this occurrence at time $t=23.6$, 24, and 24.4 s, the region of low contrast being indicated by an arrow. Videos 7 and 8 show the complete time course of the events in Figs. 7(b) and 7(c).

Fig. 7

Photoacoustic imaging of a mouse ear at 10 fps showing transient hemodynamic events in selected vessels. (a) Photoacoustic image obtained in vivo in a mouse ear at a frame rate of 10 images per second showing blood flow variations in two different vessels. A video can be viewed online (Video 6). (b) Individual frames obtained at times $t=14.8$, 14.9, and 15 s of the region of interest delineated by the red box in (a). A video can also be viewed online (Video 7). (c) Individual frames obtained times $t=23.6$, 24, and 24.4 s of the region of interest delineated the blue box in (a). A video can also be viewed online (Video 8). The scan area of (a) was $1\mathrm{mm}\times 1.25\mathrm{mm}$ with a $5\text{-}\mu \mathrm{m}$ step size and the total number of A-lines acquired was $N=\mathrm{50,00}$ per frame. The $x$ and $y$ galvanometers were driven at 1000 and 5 Hz, respectively. The PRF of the laser was 500 kHz and the pulse energy was 200 nJ. (Video 6, MPEG, 2.5 MB [URL: https://doi.org/10.1117/1.JBO.23.12.126502.6]); Video 7, MPEG, 0.24 MB [URL: https://doi.org/10.1117/1.JBO.23.12.126502.7]); Video 8, MPEG, 0.9 MB [URL: https://doi.org/10.1117/1.JBO.23.12.126502.8]).