2.1.

Five-Wavelength Pulsed Laser

The five-wavelength pulsed laser needs to have sufficient pulse energy for each wavelength and nanosecond wavelength switching time. Figure 1 shows a schematic of the laser and the imaging probe. We use a pulsed laser (532-nm wavelength, VPFL-G-20, Spectra-Physics) to pump the other wavelengths via the SRS effect in optical fibers. The Raman frequency shift of the silica fiber is $\sim 13.2\mathrm{THz}$, corresponding to a 13-nm wavelength shift near 532 nm. The five wavelengths are 532, 545, 558, 570, and $620/640\mathrm{nm}$. The five pulses are temporally separated with different optical delays in the fiber. The pulse energy on the sample surface for each wavelength is above 90 nJ.

Fig. 1

Schematic of five-wavelength OR-PAM. AL, acoustic lens; BS, beam splitter; R/T, 10/90; ${\mathrm{DM}}_1$, ${\mathrm{DM}}_2$, 565-nm long-pass dichroic mirror; ${\mathrm{DM}}_3$, 600-nm short-pass dichroic mirror; ${\mathrm{DM}}_4$, 550-nm long-pass dichroic mirror; FC, fiber coupler; ${\mathrm{HWP}}_{1-6}$, half-wave plate; LPF, 537-nm long-pass filter; ${\mathrm{M}}_{1-10}$, mirror; MMF, graded-index multimode fiber; ${\mathrm{NDF}}_{1-5}$, neutral density filter; OL, optical lens; ${\mathrm{PBS}}_{1-2}$, polarizing beam splitter; PM-SMF, polarization-maintaining single-mode fiber; SPF, 580-nm short-pass filter; UST, ultrasonic transducer; WT, water tank.

In the SRS shifter, two polarizing beam splitters (${\mathrm{PBS}}_1$ and ${\mathrm{PBS}}_2$, PBS251, Thorlabs Inc.) and one 10/90 beam splitter (BS, BSN10, Thorlabs Inc.) split the pump laser beam into four paths, i.e., a 532-nm direct path, a 545-nm Raman path, a $620/640\text{-}\mathrm{nm}$ Raman path, and a 558/570-nm Raman path. The pump pulse width is 7 ns. The total pump energy is $23.5\mu \mathrm{J}$ per pulse. The incident pulse energies for the split paths are $0.25\mu \mathrm{J}$ for the 532-nm path, $2.25\mu \mathrm{J}$ for the 545-nm path, $8\mu \mathrm{J}$ for the $620/640\text{-}\mathrm{nm}$ path, and $13\mu \mathrm{J}$ for the 558/570-nm path. Half-wave plates (${\mathrm{HWP}}_{1-6}$, WPH10E-532, Thorlabs Inc.) are used to control the polarization so that the SRS efficiency can be adjusted. Neutral density filters (${\mathrm{NDF}}_{1-5}$, NDC-50C-2, Thorlabs Inc.) are used to adjust the laser pulse energies.

The 532-nm path is coupled into the OR-PAM probe with minimal delay. The 545-nm path uses a 25-m polarization-maintaining single-mode fiber (PM-S405-XP, Nufern) to shift the wavelength and delay the pulse by 121 ns. A long-pass filter (LPF, RET537lp, Chroma) is used to select the 545-nm wavelength. In the $620/640\text{-}\mathrm{nm}$ Raman path, a 50-m fiber (PM-S405-XP, Nufern) is used to generate the $620/640\text{-}\mathrm{nm}$ wavelength and delay it by 243 ns. In the 558/570-nm Raman path, the pump beam is coupled into a 100-m graded-index multimode fiber (MMF) [GIMMSC (50/125)HT, Fibercore] to generate the 558- and 570-nm wavelengths and delay them by 486 ns. Because the multimode fiber has a larger core diameter ($50\mu \mathrm{m}$), it has a higher SRS threshold and thus can output higher pulse energy than a single-mode fiber at the same length.⁴⁵ Furthermore, the MMF can generate the SRS wavelengths with at least 75% output energy in the fundamental mode.⁴⁶ A long-pass dichroic mirror (${\mathrm{DM}}_1$, T565lpxr-UF1, Chroma) is used to separate the 558- and 570-nm wavelengths. Another 25-m fiber (PM-S405-XP, Nufern) is used to delay the 570-nm pulse by 121 ns. A short-pass filter (SPF, 580SP, Omega) is placed at the output end of the 25-m fiber to select the 570 nm wavelength.

The five wavelengths are combined using four beam combiners. First, a long-pass dichroic mirror (${\mathrm{DM}}_2$, T565lpxr-UF1, Chroma) is used to combine the 558- and 570-nm paths. Second, the $558/570\text{-}\mathrm{nm}$ beam is combined with the $620/640\text{-}\mathrm{nm}$ beam via a short-pass dichroic mirror (${\mathrm{DM}}_3$, #69-204, Edmund Optics). Third, a long-pass dichroic mirror (${\mathrm{DM}}_4$, T550lpxr, Chroma) combines the 545-nm path and the 558/570/620/640-nm path. Last, the four SRS wavelengths are combined with the direct 532-nm path via a 10/90 BS and are coupled into the OR-PAM probe via a 2-m single-mode fiber (P1-460B-FC-2, Thorlabs Inc.).

The coupling efficiencies for all single-mode fibers are above 50%. Because the 558-nm beam has high-order modes, when coupling it to the 2-m single-mode fiber, the coupling efficiency is $\sim 30\%$. In the 532-, 545-, 558-, and 570-nm light paths, the pulse energies on the sample surface are above 90 nJ. The pulse energy for the $620/640\text{-}\mathrm{nm}$ wavelength can reach 300 nJ on the sample surface. Detailed pulse energies and time delays for all wavelengths are summarized in Table 1. The shortest wavelength switching time is 121 ns, which corresponds to $\sim 200\text{-}\mu \mathrm{m}$ imaging depth. This is acceptable for superficial imaging. For future deeper in vivo application, we may consider separating mixed signals from different wavelengths. For example, a deconvolution method has been developed to separate two overlapping signals generated from ultrafast dual-wavelength excitation.⁴⁷

Table 1

Pulse energy, delay time, pulse energy fluctuation, and drift of the five wavelengths. C&T represents airflow isolation and temperature control.

In the OR-PAM probe, the laser beam from the 2-m fiber is focused by a pair of achromatic doublets (AC064-013-A, Thorlabs Inc.). The focused optical beam is reflected on an optical/acoustic beam combiner, is transmitted through a planoconcave lens (45-697, Edmund Optics), then illuminates the sample. Induced ultrasonic waves are collimated by the planoconcave lens, transmitted through the optical/acoustic beam combiner, and detected by a 50-MHz broadband piezoelectric transducer (V214-BC-RM, Olympus). To optimize the detection sensitivity, the focused optical beam is coaxially and confocally aligned with the focused ultrasonic detection beam. The detailed information about the OR-PAM probe can be referred to in previous publications.^46,48–50

2.2.

Laser Stability and Specifications

Fiber coupling and SRS efficiency may be affected by airflow and temperature changes, causing random errors and long-term drift in the pulse energy. To improve the pulse-energy stability, we isolate the airflow using a cover and maintain the temperature fluctuation within $\pm 0.1°\mathrm{C}$.

As shown in Table 1, we measured the pulse energy fluctuation and drift at each wavelength for one hour. The fluctuation is calculated from the standard deviation of the pulse energy. The drift is determined from the averaged pulse energy difference between the first and the last minute in the hour. With airflow isolation and temperature control, the pulse energy fluctuations at all wavelengths are reduced by 4% to 20%, and the drifts for all wavelengths are reduced by 11% to 23%. Because the pulse energy ratios among 532, 545, 558, and 570 nm are used in the calculation of blood flow and ${\mathrm{sO}}_2$, we list the relative pulse-energy fluctuations, i.e., the standard deviations of the pulse energy ratios of 545/532, 558/532, and 570/532, in Table 1. The relative pulse energy fluctuations of 545, 558, and 570 nm are consistently smaller than their absolute pulse fluctuations. Because the MMF has a large mode area and is more sensitive to perturbation, the pulse energy fluctuations and drifts are larger in the multimode fiber (558 and 570 nm) than in single-mode fibers (545 and $620/640\mathrm{nm}$). With the airflow cover and temperature control, the pulse energy fluctuations of the SRS wavelengths (relative for 545, 558, and 570 nm and absolute for $620/640\mathrm{nm}$) are 26% to 41% higher than the pump wavelength, and the drifts are 20% to 75% higher than the pump one.

We measured the imaging depths for each wavelength by obliquely inserting a black human hair into fresh chicken breast tissue. The imaging depths are 0.75, 0.76, 0.77, 0.78, and 0.81 mm for 532, 545, 558, 570, and $620/640\mathrm{nm}$, respectively, as shown in the Figs. 2(a)–2(e). The lateral resolutions for the five wavelengths are measured by scanning the stainless-steel blade and measuring the amplitude profiles across the sharp edges. By fitting the PA signals to an edge spread function (ESF), we can obtain the line spread functions (LSFs) and the full widths at half maximum (FWHMs) as the lateral resolutions, as shown in Figs. 2(f)–2(j). The lateral resolutions are 3.12, 3.25, 3.37, 3.51, and $3.94\mu \mathrm{m}$ for 532, 545, 558, 570, and $620/640\mathrm{nm}$, which agree with the simulation results shown as Fig. S1 in the Supplementary Material. We also measured the axial resolutions by scanning a $10\text{-}\mu \mathrm{m}$-diameter tungsten filament. The FWHMs of a Hilbert-transformed A-line are shown in Fig. 2(k). The axial resolutions for all wavelengths are $38\mu \mathrm{m}$.

Fig. 2

(a)–(e) Measured penetration depths with 6-dB SNR for 532-, 545-, 558-, 570-, and $620/640\text{-}\mathrm{nm}$ wavelengths. (f)–(j) Measured lateral resolutions of the five wavelengths. (k) Axial resolution measured via imaging a $10\text{-}\mu \mathrm{m}$-diameter tungsten filament.

2.3.

Multi-Contrast Imaging

Figure 3(a) shows the laser spectrum measured with an optical spectrometer (USB 2000+, Ocean Optics). The linewidths of the SRS wavelengths are within $\pm 1.5\mathrm{nm}$.^46,48 We use the molar extinction coefficients at the peak wavelengths for ${\mathrm{sO}}_2$ calculation. Figure 3(b) shows the absorption spectra of oxyhemoglobin, deoxyhemoglobin, and Evans blue (EB).⁵¹ The hemoglobin molecules and the EB dye have distinct absorption coefficients at 620 to 640 nm. With EB dye uptake, the lymphatic vessels can be imaged with the $620/640\text{-}\mathrm{nm}$ wavelength. The other wavelengths are used to image the $C_{\mathrm{Hb}}$, ${\mathrm{sO}}_2$, and flow speed in the blood vessels.

Fig. 3

(a) Spectrum of the five-wavelength stimulated-Raman-scattering laser. (b) Absorption spectra of ${\mathrm{HbO}}_2$, HbR, and EB.

Under thermal and stress confinements, the PA amplitude in the blood vessel can be approximated as

At the 532-nm wavelength, ${\epsilon }^{\mathrm{oxy}}$ and ${\epsilon }^{\mathrm{de}}$ are almost the same, thus ${\mu }_a$ is not sensitive to the ${\mathrm{sO}}_2$ change.⁵² We can determine the relative change of $C_{\mathrm{Hb}}$ from the PA amplitude at 532 nm or the absolute $C_{\mathrm{Hb}}$ after calibration.

The 545- and 532-nm wavelengths are used to measure the blood flow speed using a dual-pulse photoacoustic flowmetric method that is published in Ref. 46. When we excite two PA signals with a sub-microseconds delay, the residual heat from the first pulsed excitation elevates the second PA amplitude via the Grüneisen relaxation effect.⁵³ Blood flow may accelerate the heat dissipation and thus alter the second PA amplitude as follows:

Using 532-, 545-, 558-, and 570-nm wavelengths, we can measure oxygen saturation. Considering the Grüneisen relaxation effect among successive pulse excitations, a PA amplitude is affected by previous excitations. Because the 620/640-nm PA signal (the third pulse) from the blood vessel is only $\sim 2.5\%$ of those by other pulses, we neglect the heating by the third pulse. The PA amplitudes for the first, second, fourth, and fifth pulses can be written as

3.1.

In Vivo Imaging of Blood and Lymphatic Vessels

Four-week-old female ICR mice were used in experiments. All procedures involving animals were approved by the animal ethical committee of the City University of Hong Kong. The mice were anesthetized by inhaling isoflurane gas. EB dye solution (0.5%, mass fraction, $20\mu L$) was injected into the ear tip to label the lymphatic vessels. The mouse ear was placed on a flat holder and was imaged in 5 min after EB injection. The laser repetition rate is 4 kHz for each wavelength. The pulse energy is 160 nJ for the $620/640\text{-}\mathrm{nm}$ wavelength and 90 nJ for the other four wavelengths. The scanning step size is $2.5\mu \mathrm{m}$. The imaging area is $2.5\mathrm{mm}\times 2.5\mathrm{mm}$. It takes 250 s to acquire a volumetric image. Maximum-amplitude-projected images are shown in Fig. 4. Figure 4(a) is the ${\mathrm{sO}}_2$ image. The ${\mathrm{sO}}_2$ images without and with compensation for the Grüneisen relaxation effect are shown as Fig. S2 in the Supplementary Material. The average ${\mathrm{sO}}_2$ values calculated with and without compensation for the Grüneisen relaxation effect are significantly different ($P<0.001$). With compensation, the ${\mathrm{sO}}_2$ values in the arteries and veins are closer to their normal physiological values.^19,20,23,46 The compensation for the Grüneisen relaxation improves the averaged ${\mathrm{sO}}_2$ values by 4% to 17% in the arteries and by 21% to 24% in the veins. The ${\mathrm{sO}}_2$ in the veins is corrected more than that in the arteries. The possible reason is that the vein has slower flow speed and thus has a more severe Grüneisen relaxation effect than the arteries. Due to the same reason, the ${\mathrm{sO}}_2$ is corrected more in the small arteries than in the big ones. Because the flow speed and the Grüneisen relaxation effect in the veins are relatively uniform, the ${\mathrm{sO}}_2$ correction shows no obvious difference in the big and small venous vessels. Figure 4(b) shows the blood flow speed image. Figure 4(d) depicts the average ${\mathrm{sO}}_2$ and flow speeds in different vessel segments. From the root to the tip of the mouse ear, the averaged blood flow speed in the trunk arteries decreases from $5.8\pm 0.24$ (SD) to $5.0\pm 0.21$ (SD) mm/s. In the small arterial branches, the averaged blood flow speed decreases to $3.4\pm 0.36$ (SD) mm/s. The averaged blood flow speed in the veins maintains at $\sim 1.9\pm 0.18\mathrm{mm}/\mathrm{s}$. After measuring the blood vessel diameter, we can further compute the flow rate. In Fig. 4(b), the profiles across five arteries and six veins are labeled with white dashed lines as ${\mathrm{A}}_{1-5}$, and ${\mathrm{V}}_{1-6}$, respectively. As shown in Fig. 4(e), the vessel diameters are 86, 74, 58, 47, and $43\mu \mathrm{m}$ for ${\mathrm{A}}_{1-5}$ and 189, 112, 97, 84, 48, and $123\mu \mathrm{m}$ for ${\mathrm{V}}_{1-6}$. The corresponding flow rates are 1.03, 0.65, 0.41, 0.25, and $0.15\mu L/\min$ for ${\mathrm{A}}_{1-5}$ and 1.68, 0.63, 0.42, 0.28, 0.14, and $0.68\mu L/\min$ for ${\mathrm{V}}_{1-6}$. The veins ${\mathrm{V}}_2$, ${\mathrm{V}}_3$, and ${\mathrm{V}}_6$ are converged to ${\mathrm{V}}_1$, and the total flow rate in ${\mathrm{V}}_2$, ${\mathrm{V}}_3$, and ${\mathrm{V}}_6$ matches the flow rate in ${\mathrm{V}}_1$. The total flow rate in ${\mathrm{V}}_4$ and ${\mathrm{V}}_5$ is approximately equal to the one in ${\mathrm{V}}_3$. Similarly, the arteries ${\mathrm{A}}_2$ and ${\mathrm{A}}_3$ are the branches of ${\mathrm{A}}_1$, and thus the total flow rate in ${\mathrm{A}}_2$ and ${\mathrm{A}}_3$ matches the one in ${\mathrm{A}}_1$. The total flow rate in ${\mathrm{A}}_4$ and ${\mathrm{A}}_5$ matches the flow rate in ${\mathrm{A}}_3$. Four pairs of arteries and veins (${\mathrm{A}}_{2-5}$ and ${\mathrm{V}}_{2-5}$) are compared. The flow rates in the arteries match the ones in the veins. The flow rates in ${\mathrm{A}}_1$ and ${\mathrm{V}}_1$ are not very close. We guess the possible reason might be that the two vessels are different in shape, and the measured diameter cannot be used to accurately estimate the cross-sectional area. Figures 4(c) and 4(d) show the blood and lymphatic vessels and the relative lymphatic concentration in vessel segments. With distinct absorption spectra, the blood and the dye-labeled lymphatic vessels can be identified with high contrast. The highest SNRs (signal amplitude over noise standard deviation) of the blood and lymphatic vessels are 20.1 and 22.3 dB.

Fig. 4

Five-wavelength OR-PAM of the blood and lymphatic vessels in the mouse ear. The imaging area is $2.5\mathrm{mm}\times 2.5\mathrm{mm}$. (a) Compensated oxygen saturation. (b) Blood flow speed determined with the dual-pulse method. The profiles of five arteries and six veins are labeled with white dashed lines. (c) PAM imaging of the blood and lymphatic vessels. (d) Variation of oxygen saturation, blood flow speed, and relative lymphatic concentration from the root to the tip of the mouse ear. A, arteries; V, veins; L, lymphatic vessels. Along the arteries and veins, the first value is compensated oxygen saturation, and the second value is blood flow speed (mm/s). (e) The averaged diameter ($\mu \mathrm{m}$) of arteries and veins profiles labeled in (b). (f) The averaged flow rate ($\mu L/\min$) of arteries and veins profiles labeled in (b). Data are presented as $\text{mean}\pm \mathrm{SD}$, and the mean values are labeled in the figure.

3.2.

In Vivo Imaging of Tumor, Lymphatic Clearance, and Brain Function

To demonstrate the potential applications, we first conducted in vivo early cancer detection experiments using five-wavelength OR-PAM. Before tumor implantation, the multifunctional vascular images of hemoglobin concentration, compensated oxygen saturation, and blood flow speed were acquired in the mouse ear and are shown as Fig. S3 in the Supplementary Material. Then, 4T1 breast cancer cells were injected to the mouse ear. After the tumor developed for 5 days, the same region was imaged to demonstrate the changes of the aforementioned multi-functional parameters. As a hallmark of cancer,^57,58 obvious angiogenesis is observed in Fig. 5(a). Figures 5(b) and 5(c) show the ${\mathrm{sO}}_2$ and blood flow speed in the tumor region. Compared with the baseline, the averaged ${\mathrm{sO}}_2$ in the tumor vessels increases by 45% to 51%, and the blood flow speed increases by 32% to 37%, indicating hyperoxia and elevated blood perfusion in the early-stage tumor due to fast growing.^55,57,58 Figure 5(d) shows pseudo-color-encoded vascular depth, manifesting height changes in the tumor region. Figures 5(e) and 5(f) show the vessel dilation and vessel tortuosity in the tumor region. Compared with the non-tumor region, the average diameter and tortuosity of the venules are increased by 48% to 52% and 10% to 12% in the tumor region, which has been validated in other reports.^57,58 Detailed quantification methods and comparisons are shown as Fig. S4 in the Supplementary Material.

Fig. 5

(a)–(f) OR-PAM of hemoglobin concentration, oxygen saturation, blood flow speed, depth, diameter, and tortuosity in the tumor region. (g)–(i) Simultaneous imaging of hemoglobin and dye concentrations at 0, 10, and 20 min after EB dye injection. (j)–(l) In vivo brain imaging of hemoglobin concentration, oxygen saturation, and blood flow speed.

Enabled by the new wavelength, five-wavelength OR-PAM can image the dye-removal processes in lymphatic vessels. Figures 5(g)–5(i) show the simultaneous imaging of the blood and the dye-labeled lymphatic vessels in the mouse ear at 0, 10, and 20 min after injecting EB dye solution (0.5%, mass fraction, $20\mu L$). The normalized concentrations of hemoglobin and EB are encoded with red and blue colors, respectively. After 20 min, the average EB concentration decreases by 87% in the lymphatic vessels, indicating the waste clearance ability of the local lymphatic vessels. Taking advantage of simultaneous multi-contrast imaging, five-wavelength OR-PAM can realize the simultaneous in vivo dye-labeled lymphatic and multi-functional vascular imaging, which is of great importance to imaging the dynamic blood perfusion and lymphatic circulation.

We also demonstrated five-wavelength OR-PAM in multi-contrast brain imaging. A $2\mathrm{mm}\times 2\mathrm{mm}$ window was opened in the mouse skull after the mouse was anesthetized. Using 60-nJ pulse energy, we acquired functional brain images and quantified normalized hemoglobin concentration, ${\mathrm{sO}}_2$, and blood flow speed, as shown in Figs. 5(j)–5(l). In the arteries and veins, the average ${\mathrm{sO}}_2$ values are $0.95\pm 0.06$ (SD) and $0.68\pm 0.08$ (SD); the average blood flow speeds are $7.2\pm 0.62$ (SD) mm/s and $3.9\pm 0.45$ (SD) mm/s, respectively. Because the new five-wavelength system has red light, other exogenous photoacoustic contrast agents can be used in the future to label neurons or tumors in the brain.