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An optical-resolution photoacoustic microscope (OR-PAM) with capability of fast axial-scanning was developed by using a tunable acoustic gradient (TAG) lens. The TAG lens was designed to continuously changing the focal plane of OR-PAM by modulating its refractive power with fast-changing ultrasonic standing wave. The performance was shown by imaging a carbon fiber. We achieved a DoF of about 750 μm. The head of a zebrafish was also imaged to further demonstrate the feasibility of our method.
© 2017 Optical Society of America
1. Introduction
Photoacoustic imaging is a promising technique that combines optical contrast with ultrasonic detection to map the distribution of the absorbing pigments in biological tissues [1–4]. It has been widely used in biological researches, such as structural imaging of vasculature [5], brain structural and functional imaging [6], and tumor detection [7]. Considering the lateral resolution of photoacoustic microscopy (PAM), it can be classified into two categories: optical-resolution (OR-) and acoustic-resolution (AR-) PAM [8, 9]. In AR-PAM, the spatial resolution is determined by the acoustic focus, since the laser light is weekly or even not focused on the sample. Conversely, in the OR-PAM, the laser light is tightly focused into the sample to achieve sharp excitation. However, in OR-PAM, the depth of field (DoF) is quite limited, for it is determined by the optical focus. The short DoF hinders OR-PAM from high-quality three-dimensional volumetric imaging or acquiring dynamic information in depth direction.
To address this issue, depth scanning using motorized stage is commonly used as it is the most convenient approach [10, 11]. However, this method limits the volumetric imaging speed with its slow mechanical adjustment. Therefore, several methods engineering the illumination have been proposed to improve the imaging speed. The DoF can be doubled for thin samples by illuminating from both top and bottom sides simultaneously, whereas unavailable for thick samples [12]. Utilizing chromatic aberration of non-achromatic objective, multi-wavelength laser can generate multi-focus along the depth direction [13]. However, this method sacrifices the capability of functional imaging. Non-diffraction beam inherently own a large DoF. Bessel beam based PAM can extend the DoF while high lateral resolution is preserved, at a price that additional process must be made to suppress the artifacts introduced by the side lobes of the Bessel beam [14, 15], which increases the complexity of the imaging. Electrically tunable lens (ETL) has also been introduced in OR-PAM [16]. This resulted in a focus-shifting time of about 15 ms. It is fast enough for pulsed lasers with a repetition rate of tens of hertz, while being quite slow for those lasers with repetition rate of kilos to hundreds of kilohertz, which has been widely used in the OR-PAM.
In this manuscript, we report a novel method to rapidly scan the optical focus along the depth direction in OR-PAM by employing a high-speed TAG lens. The laser pulse, TAG lens, and scanning stage were synchronized for the imaging. The high-speed varifocal performance and DoF of the system were estimated with a phantom made by a carbon fiber. A zebrafish was also imaged in vivo to demonstrate the feasibility of the system in biomedical imaging.
2. Methods
2.1 System setup
Figure 1 shows the scheme of OR-PAM with TAG lens. The system is equipped with an Nd:YLF laser (IS8II-E, EdgeWave GmbH) that irradiate laser light at the wavelength of 523 nm and a repetition rate of 1 kHz. The laser beam is reshaped by an iris with a diameter of 0.8 mm. Then, it is focused by a plano-convex lens L1, and collimated by a plano-convex lens L2. A 50 μm-diameter pinhole (PH) is used as a spatial filter. An objective (4 × Olympus objective, N. A. 0.1) is used to form a focus on the tip of a single-mode fiber. The distal end of the fiber is inserted into a fiber port (PAFA-X-4-A, Thorlabs) which collimates the guided laser beam with an output 1/e2 waist diameter of 0.65 mm. Then the optical axis of the laser beam is turned to vertical to reduce the gravity induced Y coma aberration of the TAG lens. The home-made TAG lens used in our system consists of a cylindrical piezoelectric shell (PZT-8, Boston Piezo Optics, inner diameter = 16mm, outer diameter = 20mm, length = 20mm), filled with a transparent silicone oil (100 cS, Sigma-Aldrich) with a refractive index of 1.403 and a speed of sound of 1000 m s−1. When the TAG lens is driven by a sinusoidal radio frequency signal, the lens power is modulated to be proportional to the potential difference between the outer and inner sides of the shell [18].The light comes from TAG is delivered into a beam expander formed by plano-convex lenses L4 (f = 18 mm) and L5 (f = 150 mm) with a magnification factor of 8.3, and finally focused on the sample by an objective (5 × Mitutoyo objective, N. A. 0.14). The beam expander also conjugates the TAG lens and the pupil plane of the objective. A home-made acoustic lens (NA = 0.5) is glued on an ultrasonic transducer (central frequency 50 MHz, V214-BB-RM, Olympus) to form a focused detection [17]. The sample is placed on a glass slide. To couple the photoacoustic signals, we use a water tank. Both of sample and water tank are mounted on a three-dimensional scanning stage which is assembled by a two-dimensional linear stage (ANT95-XY, Aerotech) and a lifting stage (M-Z01.5G0, Physik Instrumente). The ultrasonic detection is coaxial and confocal with the optical excitation to achieve high detection efficiency. The two-dimensional linear stage is used for the raster scanning of the sample. Photoacoustic signals detected by the ultrasonic transducer is amplified (AU-1291, MITEQ) and acquired by a data acquisition card (ATS9350, Alazartech).
Fig. 1 Scheme of the system. AL, acoustic lens; ASC, axial scanning circuit; BS, beam sampler; DAQ, data acquisition card ;FP, fiber port; FG, function generator; GS, glass slide; M1 and M2, mirrors; L1, L2, L3, L4 and L5, optical lenses; Obj1 and Obj2, objectives; PD, photodiode; PH, pinhole; PSO, position synchronized output signal; S, sample; SH, sample holder; SMF, single mode fiber; TAG, TAG lens; UT, ultrasonic transducer; W, water tank; WS, work station.
Depth scanning during cross-sectional B scan is achieved by a home-made circuit, which is sketched in Fig. 2 and indicated by the red rectangle in Fig. 1. The circuit is designed to change the depth position of the focal spot periodically for each A line by cycling the selection of reference voltage. In the B-scan process, the linear stage moves at a constant speed, generating a position synchronization output (PSO) pulse every pixel pitch. The PSO signal is a square wave. Its rising edge triggers the DAQ card for acquisition, as well as the laser pulse when the driving signal of TAG lens reaches the reference voltage, which corresponds to a certain depth position. As long as the acquisition for each A-line is completed, the synchronization signal from the DAQ card will trigger the dual D-type edge-triggered flip-flop (74LS74, Texas Instruments) to control the multiplexer (ADG408, Analog Devices), and the next reference voltage will be picked from the queue. Thus, the laser would be fired at another depth for the next A line. In this research, three input voltages are selected as the references. The circuit inherently introduces 12 ns delay as we measured with respect to the driving signal, which should be taking into account. The diagram of the position of the focal spot in the scanning is illustrated in Fig. 3.
Fig. 2 Schematic diagram of the axial scanning circuit. D, D-type flip-flop; DD, dual D-type edge-triggered flip-flop; MUX, multiplexer; VC, voltage comparator; VON, voltage offset unit.
Fig. 3 The focal plane shifts periodically with each step in a B scan. The red and blue dots represent maximum and minimum focal shift, respectively.
2.2 Performance of TAG lens
As shown in Fig. 4, we validated the TAG lens with a similar paradigm used for B scan, except that the PSO signal of the scanner is replaced with 1 kHz square wave from function generator FG2 (TFG2006V, SUING). And the clock of the D-type flip-flop (SN74AUC1G74, Texas Instruments), is replaced with the synchronous TTL square wave that is obtained from the Sync output of the function generator FG1 (DS345, Stanford Research Systems). During the measurement, a 707 kHz sinusoidal signal generated by FG1 drives the TAG lens, makes the PZT shell in the lens to resonance. A wavefront sensor (WFS150-5C, Thorlabs) was placed 50 mm after the output window of TAG lens to measure the beam wavefront. The lens power can be calculated according to its correlation with distance and radius of curvature (RoC) afterwards [18]. By changing the delay time of the digital delay and pulse generator with the step of 100 ns, time-depended lens power is profiled.
Fig. 4 Schematic diagram for determining lens power of TAG lens using a wavefront sensor. DG, digital delay and pulse generator; D, D type flip-flop; FG1 and FG2, function generator; M, mirror; TAG, tunable acoustic gradient index of refraction lens; WS, wavefront sensor.
2.3 System performance
We then measured the lateral resolution of our system by imaging a sharp bar edge of an USAF 1951 resolution target (Edmund Optics). The TAG lens was off during the measurement. The system performance of DoF was demonstrated by using a vertically tilted carbon fiber with a diameter of 6 μm. We used a glass slide, and several fragments of cover glass to make the carbon fiber vertically tilted. Each cover glass was 170 μm thick. And several pieces of glass were glued onto a glass slide to form a raised platform with a height of about 800 μm. One end of a straightened carbon fiber was fixed on the glass slide, while the other end was fixed to the surface of glass slide. The depth information of the carbon fiber could be obtained by raster scanning with a fixed height. And the carbon fiber was kept linear during the scanning. We imaged the phantom with TAG lens on and off, related to a driving signal with 7 V peak to peak voltage and no driving signal, respectively. The driving signal is biased + 3.5 V to above zero by the VON unit. The input voltage 1, 2 and 3 in Fig. 2 is equal to 2.3 V, 3.5 V and 4.6 V, respectively. Thus, a time delay sequence of 84 ns, 12 ns, and −67 ns relative to the off state of the TAG lens was chosen.
2.4 In vivo imaging
To demonstrate in vivo imaging capability of our system, a 30-day-old zebrafish (AB strain) was chosen. Before the imaging, a culture dish was coated with a thin layer of low-melting-point agarose (A-4018, Sigma-Aldrich), which was dissolved in 40 °C deionized water (1.2% w/v). When the temperature of the agarose dropped to 37 °C, the zebrafish was placed and oriented in the culture dish so that it was lying on its back, lightly covered with the melted liquid agarose, waiting for solidification. After the solidification, some deionized water was pour into the culture dish. The temperature was kept around 25 °C during the imaging. All parameters were the same with the system performance test.
3. Results
3.1 Performance of TAG lens
Figure 5 shows the lens power of TAG lens as a function of time with a 10 V driving signal at a frequency of 707 kHz. We obtained the lens power from delay time 0 ns to 11200 ns. A two-parameter (amplitude and phase) sinusoidal fit results in 8 cycles with a period of 1400 ns. The lens power of the TAG lens varies between a maximum of 0.94 m−1 and a minimum of −0.94 m−1.
Fig. 5 Lens power of TAG lens as a function of time for a 10 V driving signal at a frequency of 707 kHz. The solid curve shows a two-parameter (amplitude and phase) sinusoidal fit to the data.
3.2 System Performance
The sharp bar edge of an USAF 1951 resolution target indicated in Fig. 6(a) was imaged with a step size of 0.5 μm to obtain edge spread function (ESF), by summing the acquired 3D data along the y-axis and then projecting along the depth direction by maximum amplitude projection (MAP). We used an error function to fit the profile of the measured ESF. The lateral resolution is defined as the full width at half maximum (FWHM) of the line spread function (LSF) that can be calculated as the first-order derivative of the ESF. As shown in Fig. 6(b), the lateral resolution is 3.3 μm.
Fig. 6 Lateral resolution of the system. (a) PA image of a bar edge on the resolution target. (b) Edge spread function (ESF) and line spread function (LSF) extracted from (a). NPA, normalized photoacoustic amplitude.
Figures 7(a) and 7(b) are the MAP images when TAG lens is on and off, respectively. Figures 7(d) and 7(e) show the variations of lateral resolution along the z direction when TAG lens is on and off, respectively. We acquired the FWHM of the profile of carbon fiber by utilizing Gaussian fit to each line in Figs. 7(a) and 7(b). The data in Figs. 7(d) and 7(e) show some oscillation as the FWHM extraction is quite sensitive to noise. Since the lateral resolution of the focal plane is the best, we can conclude that there are three focal planes exist (f1, f2 and f3) in Fig. 7(d). While there is only one focus (f2’) exist in Fig. 7(e). The position in depth of each focus is indicated by the red dashed lines in Fig. 7(d). The depth difference Δz1 between f1 and f2 is about 231.6μm, and Δz2 between f2 and f3 is about 222 μm. The middle focuses f2 and f2’ have a depth difference Δz (~40. 8μm), which is introduced by the time delay of home-made axial scanning circuit. The time delay of the axial scanning circuit was measured to be about 12ns, which can theoretically cause about 38 μm of the focus shift, close to the measured Δz. The lateral resolution varies from 4μm to 8μm over the depth range of about 750μm. In this manuscript, we defined the DOF as the depth range in which the FWHM of the fiber broadened to twice the narrowest one. Thus, we can estimate that the DOF is about 750 μm with TAG on.
Fig. 7 Distribution of lateral resolution along the depth direction. (a) and (b) are MAP images of a vertically tilted carbon fiber when TAG lens is on and off, respectively. (c) Linear relation between vertical distance Δz and planar distance Δy. (d) and (e), Distribution of the lateral resolution along the depth direction of (a) and (b), respectively. f1, f2, f2’ and f3, focal planes indicated by white arrows ; The yellow dashed line in (b) indicates the position of the focus f2; NPA, normalized photoacoustic amplitude.
3.3 In vivo imaging of the head of a zebrafish
Figures 8(a) and 8(b) show MAP images of the head of a zebrafish obtained when TAG lens was on and off, respectively. Owing to the larger DoF of the system when TAG lens on, more details from different depths can be observed. Figures 8(c) and 8(d) are close-up images of the small areas indicated by the yellow dashed rectangles in Figs. 8(a) and 8(b), respectively. Figures 8(e) and 8(f) are close-up images of the small areas indicated by the white dashed rectangles in Figs. 8(a) and 8(b), respectively. The pigment was clearly visualized when TAG lens was on, whereas quite blurred when TAG lens was off. Figures 8(g)-8(i) show cross-sectional B images through the yellow, green and white dashed lines in Fig. 8(a), respectively. The profile of pigments from bottom to top can be distinguished. Figures 8(j)-8(l) show cross-sectional B images through the yellow, green and white dashed lines in Fig. 8(b), respectively. Only the middle part was in the DoF, the others were out of the DoF. It is worth noting that, since the surface of the head has a lot of melanin, which will absorb most laser energy with 523nm, the signal of blood vessels under the surface will be covered by the signal from melanin.
Fig. 8 Images of the head of a zebrafish acquired with our system. (a) and (b) are the MAP images of the head of a zebrafish when TAG lens was on and off, respectively. (c) and (d) are close-up images of the areas indicated by the yellow dashed rectangles in (a) and (b), respectively; (e) and (f) are close-up images of the areas indicated by the white dashed rectangles in (a) and (b), respectively. (g)–(i) are cross-sectional B images through the yellow, green and white dashed lines in (a), respectively; (j)–(l) show cross-sectional B images through the yellow, green and white dashed lines in (b), respectively. NPA, normalized photoacoustic amplitude.
4. Conclusion
By using a TAG lens, we achieved fast axial scanning in an OR-PAM system, which shown an extended DoF. In our experiments, the axial scanning time is as low as 1 ms, which is at least an order faster than any other axial scanning method in OR-PAM, to the best of our knowledge. However, the capability of the TAG lens is not completely explored in our system, which is limited by the repetition rate of our pulsed laser. Actually, the scanning time could be about 1 μs as long as we have a laser with repetition of 1 MHz. Our current setup can be further improved. First, the scanning scheme along the lateral direction is still depend on scanning stage. Combing the MEMS mirror [10], which can achieve high-speed raster scanning, the acquisition speed of three-dimensional volumetric data could be vastly improved. Second, our system can be upgrade to the reflection mode to extend the application scope in biomedical researches. And the reflection mode can be realized by adopting an optical-acoustic combiner to transmit the laser pulse and collect ultrasound [17]. Our system could be used for various applications, including the in vivo imaging of non-flat thick biological tissues and samples subject to breathing movements.
Funding
Science Fund for Creative Research Group of China (Grant No. 61421064); National Natural Science Foundation of China (NSFC) (Grants No. 91442201); Director Fund of WNLO.
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