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Abstract
The range of imaging depth in optical resolution photoacoustic microscopy (PAM) is limited by the short depth of focus of high-numerical aperture objective lenses. In this paper, focus tunable lens modulation has been employed at the resonant frequency of focus tunable lenses in order to enhance both the range of imaging depth and the scanning speed. By electrically controlling the focal length in the axial direction of the sample, the range of imaging depth was extended approximately 1.22 times and the scanning speed was enhanced by approximately 7.40 times, in comparison to corresponding values of conventional PAM systems.
© 2017 Optical Society of America
1. Introduction
Photoacoustic microscopy (PAM) is capable of non-invasive and non-labeling functional biomedical imaging [1]. The PAM modality provides high-resolution and high-optical absorption contrast; however, many studies have been conducted to improve its imaging resolution and speed [2–5]. Optical resolution PAM (OR-PAM) systems with high numerical aperture (NA) objective lenses possess enhanced lateral resolution. The transmission-type OR-PAM has been developed with a lateral resolution of 220 nm [6]. By using several lateral scanning modalities, OR-PAM systems have substantially improved scanning speed and range. Many lateral scanning methods have been reported for PAM imaging systems, such as the combination of a two-dimensional (2D) galvanometer and flat-type ultrasonic transducer [7, 8], combination of a voice-coil stage and focused-type ultrasonic transducer [9], and a 1- or 2-axis Microelectromechanical systems (MEMS) scanner [10, 11]. An interrogation laser beam scanning method has also been reported [12]. However, a high-NA lens equipped OR-PAM has a limited range of imaging depth owing to the short depth of focus (DOF) of the objective lens. To extend the range of the imaging depth, various axial scanning methods have been implemented for use in high-NA OR-PAM systems. Double-illumination PAM systems have been developed to improve the range of imaging depth without requiring axial beam scanning [13]. However, providing double-illumination on the bottom and top layers requires the use of an additional laser beam path and guiding optics, which increases system complexity. Mechanical scanning in the axial direction has been reported to extend the imaging range, but scanning systems are slow and have low axial accuracy. These limitations prevent this technique from being applied to OR-PAM systems [14, 15].
Recently, focus tunable lenses (FTLs) have been used to implement the method of axial scanning without mechanical movement in an optical imaging system [16–18]. FTLs can tune their focal length by changing the index or shape of the lens [19, 20]. Optical scanning with an FTL can reduce unwanted motion artifacts, and shape-changing FTLs have been applied in various optical imaging applications, including three-dimensional (3D) light-sheet microscopy [16], two-photon microscopy [17], and variable focus photoacoustic microscopy [18]. Although the response time of an FTL is relatively fast, biomedical imaging systems require faster scanning speeds to reduce the image acquisition time. It has been found that an FTL equipped with a large-sized membrane filled with liquid crystals exhibits an over-damped response [21]. If the current applied to an FTL changes before the FTL attains a steady state, the focal length is unable to follow the changing current, which limits the FTL tuning range. When a PAM system was developed regardless of the response properties of the FTL, it was difficult for the system to achieve optimum performance in terms of the axial scanning speed and tunable depth range.
In this paper, we propose to apply modulation at the resonant frequency of an FTL to enhance the range of imaging depth and scanning speed of a PAM system. When the modulation frequency of an applied current change is close to the resonant frequency of the FTL, the tunable range of the FTL is maximized. When this technique was employed in the experiments described in this paper, the resultant scanning speed of the OR-PAM system was found to be approximately 7.40 times faster and the range of imaging depth was increased approximately 1.22 times compared to conventional PAM systems.
2. Methods and materials
2.1. PAM system with the FTL
Figure 1 demonstrates a schematic of the proposed PAM system. In the figure, the optical source is a diode-pumped solid state pulsed laser (FQS-200-1-Y-532, Elforlight Ltd.) with a maximum pulse repetition rate of 10 kHz at a wavelength of 532 nm and a pulse energy of 5 µJ. The repetition rate of the laser is controlled by a function generator (AFG3022B, Tektronix Co. Ltd). A 25-µm pinhole (P25C, Thorlabs, Inc.) is located between the two convex lenses for spatial filtering and a neutral density filter is used to attenuate the intensity of the laser. A convex lens is used to collimate the attenuated beam and the laser beam is expanded to fully exploit the aperture of an objective lens, which is then focused by the shape-changing FTL (EL-10-30-C, Optotune Inc.). A function generator is employed to electrically control the focal length of the FTL, and a tube lens is used to maintain the NA of the objective lens while changing the focal length of the FTL [16]. The samples are scanned using a 3-axis motorized stage (PAP3D100, Em4sys Co. Ltd).
Fig. 1 Schematic of the proposed PAM, where FG is the function generator; CL is a convex lens; ND is the neutral density filter; PH is the pin hole; FTL is the focus tunable lens; TL is the tube lens; OL is the objective lens; WT is the water tank; UT is the ultrasonic transducer; LPF is the low pass filter; and LNA is the low noise amplifier.
The generated photoacoustic (PA) signals are detected by a focused-type ultrasonic transducer (V324-SM, Olympus Inc.) with a center frequency of 25 MHz and 20-mm focal length. After passing through the low pass filter with a 55-MHz cut-off frequency to reduce the high frequency noise, the PA signal was amplified by a low noise amplifier (ZFL-500LN + , Minicircuits) before being acquired by a high-speed digitizer (ATS9350, Alazartech Inc.) at 100 MS/s.
2.2. Resonance modulation of the FTL
In the proposed PAM system, an FTL is used to change the focal length in the axial direction. The FTL consists of a container filled with an optical fluid and sealed with an elastic polymer membrane [21]. The focal length of the FTL is controlled by a drive current, and can be adjusted to any value between 80 and 200 mm. The drive current is typically maintained at either a low or high value. When a step function change in the current is applied to the FTL, it is necessary to wait for the FTL to stabilize before applying additional changes. If the current changes before the FTL reaches a stable state, the FTL focal length does not attain the desired value. The required waiting time is then referred to as the 'settling time', and is defined as the time required for the over-damped response to disappear from the response curve and the focal length to attain a steady state, in the event of which, the vibrations are reduced to less than 2%. In this experiment, setting time of the FTL was determined to be approximately 15 ms. On the other hand, when the current was modulated in a sinusoidal form, the tunable range of the FTL exhibited different properties as the modulation frequency increased. The modulation frequency and corresponding FTL response determine the axial scanning range of an FTL. Note that the FTL response is slightly over-damped [21]. The settling frequency is defined as
Figure 2 captures scanning ranges for each modulation frequency (fm). At low modulation frequencies, the lens reaches the expected focal length and attains a stable state. If the modulation frequency is lower than the settling frequency, the scanning range becomes independent of the modulation frequency, but depends on the drive current of the FTL instead, as shown in Fig. 2(a). If the modulation frequency is higher than the settling frequency and lower than the resonant frequency (fr), as shown in Fig. 2(b), the scanning range becomes highly dependent on the modulation frequency. In this case, the drive current changes are rapid, and the current reverses before the FTL has time to attain the steady state. Note that the scanning range of the FTL is reduced owing to the over-damped nature of the response. In our experiments, we used a modulation frequency equal to the FTL resonant frequency, as shown in Fig. 2(c). As the modulation frequency was close to the resonant frequency of the FTL, drive modulation on the FTL response was synchronized with damping properties of the FTL, and the FTL scanning speed was optimal with respect to the expected scanning range of the FTL. When the modulation frequency is maintained the same as the resonant frequency, the mechanical response due to the external driving force is maximized [22]. The scanning range of the FTL is also maximized at the resonant frequency. If the modulation frequency of the FTL exceeds its resonant frequency, as shown in Fig. 2(d), the scanning range is reduced, as shown in Fig. 2(b). In general, the higher the modulation frequency, the smaller will be the scanning range.
Fig. 2 Relationship between the scanning range and modulation frequency. (a) The modulation frequency of the FTL is lower than the settling frequency. (b) The modulation frequency of the FTL is higher than the settling frequency and lower than the resonant frequency. (c) The resonant frequency is used as the modulation frequency. (d) The modulation frequency of the FTL is higher than the resonant frequency.
In order to improve the scanning range and maximize modulation speed of a PAM system that includes an FTL, it is necessary to determine the resonant frequency of the system. However, it is difficult to directly monitor the focal position changes of a PAM system with an FTL at a high modulation frequency. Therefore, the following method is proposed to measure the resonant frequency of an FTL in a PAM system. The detailed measurement procedure is as follows.
- 1. Position the sample diagonally.
- 2. Measure the PA signal as the focal length of the FTL changes while maintaining the NA of the objective lens at X1.
- 3. Record the maximum PA signal value for each depth.
- 3. Measure the maximum PA signal value again after moving the sample along the x-axis.
- 4. Determine the envelope from the collected maximum PA signal values for each x-position.
- 5. Measure the range of imaging depth by finding the full width at half maximum (FWHM) of the envelope of PA signals.
- 6. Repeat steps 2 to 5 in order to determine the range of imaging depth as the modulation frequency changes.
Figure 3 shows the method of measuring PA signals to determine the resonant frequency. The PA signals are maximized when the sample is positioned at a point within the focusing range of the laser beam. To accurately verify this phenomenon, the sample is positioned diagonally, as shown in Fig. 3(a). If the laser beams X2, X3, and X4 are in focus, the maximum PA signal values are strong. However, the maximum PA signal values begin to decrease once the sample is placed outside the scanning range of the FTL, such as at positions X1 and X5. Even though the sample is now placed out of the focusing range, sufficient energy of the laser beam still remains to produce a weak PA signal. An envelope comprising the maximum PA signals is generated by interlinking the values at each sample position and frequency, as shown in Fig. 3(b). The envelope line provides characteristics of the maximum range of imaging depth, which is extended by the resonance. The range of imaging depth is defined as that position where the depth of the PA signal drops by 3 dB. As shown in Fig. 3(b), the range of imaging depth can be found by measuring FWHM values of PA signals at each modulation frequency. The resonant frequency of the FTL is defined as the frequency at which the PA range of imaging depth is at its maximum.
Fig. 3 Measurement method used to determine the PA range of imaging depth as the modulation frequency changes. (a) PA signals are measured at each focal position by moving the sample along the x-axis. Laser beams are in focus and the PA signals are strong at positions X2, X3, and X4. Sample positions X1 and X5 are out of focus and weakly illuminated. (b) Envelope of the PA signals is generated by interlinking the values at each sample position. The range of imaging depth is defined as the FWHM of the envelope.
Data in the experiment were obtained using the measurement method shown in Fig. 3. Figure 4 (a) shows the envelope of the collected maximum PA signals at modulation frequencies of 50, 200, and 370 Hz, respectively. A 10x objective lens (NA 0.25) was used to observe the PA range of imaging depth of the FTL. The envelope width was found to have decreased more at a modulation frequency of 200 Hz than at 50 Hz, as shown in Fig. 4(a). When the modulation frequency was increased to 370 Hz, the envelope width was also found to have increased. The changes in FTL response were measured as the modulation frequency varied between 50 and 800 Hz, as shown in Fig. 4(b). The range of imaging depth was computed based on the FWHM of the width of the envelope. Between 360 and 380 Hz, the range of imaging depth was maximized and the FTL exhibited a resonance response. However, it should be noted that an FTL does not consist of a single layer and material, and the lens structure is complex and includes a membrane and a volume of liquid material. The complexity of this structure makes it difficult to isolate a single frequency responsible for the resonance response of the lens.
Fig. 4 (a) Envelopes of the collected maximum PA signals at modulation frequencies of 50, 200, and 370 Hz, respectively. (b) Range of imaging depth at modulation frequencies ranging from 50 to 800 Hz.
3. Results
3.1. Resolution evaluation and measurement
To verify the performance of the PAM system with a resonance modulated FTL, the lateral resolution was measured by moving the edge of a sharp blade along the x-axis in steps of 0.5 μm, as shown in Fig. 5(a). The edge spread function (ESF) was fitted based on the blade PA amplitude along the x-axis. The first derivative of ESF creates a line spread function (LSF), and the FWHM of the LSF was considered to be the lateral resolution. The measured lateral resolution was estimated as 3.8 μm (NA 0.4) at a wavelength of 532 nm, as shown in Fig. 5(a). To measure the axial resolution, a tungsten wire of 10 µm thickness was placed in a water tank, and the PA signal was measured with a laser beam focused on the wire. The LSF for the tungsten wire was fitted by a Gaussian function, and the FWHM was utilized as the axial resolution. As shown in Fig. 5(b), the axial resolution was estimated to be 60.1 μm. A theoretical axial resolution was determined based on the bandwidth of the ultrasonic transducer [4], as follows. Ra ≈0.88c/B = 52.8 μm, where Ra is the axial resolution, c is the speed of sound, and B is the acoustic −6 dB bandwidth.
Fig. 5 Lateral and axial resolutions of the proposed PAM system. (a) ESF fitted to experimental data, and the corresponding LSF based on the first derivative of the ESF. (b) LSF fitted from experimental data.
3.2. Top and bottom illumination using the FTL
We monitored the range of imaging depth for each modulation frequency using the measurement method shown in Fig. 3. In order to measure the range of imaging depth of the proposed PAM system, a black human hair was placed diagonally with respect to the x- and z-axes. The sample was sequentially illuminated by the laser beam at the longest (the top region of the sample) and shortest focal lengths (the bottom region of the sample) of the FTL, while scanning the sample in the x- and y-directions. Two layers of PA images were obtained with 1600 pixel 60 pixel resolution along the x and y axes. The B-scan range used to acquire the PA images was found to be 16 mm with a step size of 10 μm.
Figures 6(a–c) show the B-scan PA images of the side-view of the human hair with top and bottom illumination. We merged the two PA images of the top and bottom layers. The PA signals were converted into PA images via Hilbert transformation. Median filtering was applied during image processing to reduce the amount of noise. The final PA images were volumetrically rendered using a digital imaging software (Amira, FEI, Inc.). The range of imaging depth was measured at each modulation frequency. In Fig. 6(a), we confirmed that the range of imaging depth was narrow and the axial scanning speed was slow due to the low modulation frequency. The modulation frequency of 200 Hz provided a faster axial scanning speed than that of 50 Hz but had the narrowest range of imaging depth as shown in Fig. 6(b). At higher modulation frequencies, the axial scanning speed was reduced. However, no losses in range of imaging depth were encountered and a fast axial scanning speed was realized by using the resonant frequency. A 370-Hz signal was used as the resonant frequency because it offered a wider range of imaging depth than other frequencies shown in Fig. 6(c).
Fig. 6 B-scan PA image of a human hair with top (green) and bottom (orange) illumination at 50 Hz (a), 200 Hz (b), and 370 Hz (c).
To determine the range of imaging depth, the PA amplitudes represent the maximum values of the x–y positions for each depth. Figure 7(a) shows the normalized PA amplitude for the top and bottom illuminations at the resonant frequency of the FTL. The range of imaging depth for the single layer PA image (0.42 mm) was greater than the extended scanning range (0.39 mm) of the FTL at the resonant frequency. The use of dual-layer scanning makes it possible to maximize the range of imaging depth. Figure 7(b) depicts PA amplitudes of the top and bottom illuminations on the sample at modulation frequencies of 50, 200, and 370 Hz. The range of imaging depth was found to be 0.63, 0.57, and 0.77 mm, respectively, for these cases. The expanded ratio of the range of imaging depth demonstrated results similar to those shown in Fig. 4(b).
Fig. 7 Maximum PA amplitude for top and bottom illumination in the axial position. (a) Normalized PA amplitudes for the top illumination (green dots) and bottom illumination (orange dots) at the resonant frequency. Each dot indicates the maximum value of the acquired PA signals. (b) Normalized PA amplitudes at each modulation frequency. The extended imaging depth was about 0.77 mm beyond merging point of the top and bottom PA signals at the resonant frequency.
The measurement times for a single B-scan PA image were 32.00 s and 4.32 s at modulation frequencies of 50 Hz and 370 Hz. The motorized stage speeds were 0.5 mm/s and 3.7 mm/s, respectively. Imaging data were found to be obtained 7.40 times faster at the resonant frequency than at the modulation frequency of 50 Hz.
3.3. In vivo imaging
To demonstrate in vivo PA images generated by the proposed axial scanning method, the microvasculature of a living nude mouse ear (BALB/c Nude) was imaged. All experimental procedures were carried out in accordance with the protocols approved by the Animal Care and Ethics Committees of the Gwangju Institute of Science and Technology (GIST) (accession number: GIST-IACUC-2015-89). Approximately 106 MAT B-III cells were injected into the mouse ear to increase the density of the micro vessels. The lateral scanning range was 8 × 4 mm2 and the PA image had 400 800 400 pixels. The motorized stage speeds were 3.7 mm/s and the measurement times for a single B-scan PA image were 1.08 s when using the resonant frequency. The red rectangular region of the photograph of the mouse ear 6 days after the tumor cell injection was imaged, as shown in Fig. 8(a). The region of interest (ROI) included part of the neo-vascular area affected by tumor injection.
Fig. 8 In vivo PA images of the ear of a mouse. (a) photograph of the ear, (b) PA image with top illumination, (c) side-view image indicated by the dashed line box in (b), (d) PA image with bottom illumination, (e) side-view image indicated by the dashed line box in (d), (f) merged PA image of the top (a) and bottom (b) illuminations, and (g) side-view image indicated by the dashed line box in (f).
The microvasculature PA images were constructed using laser illumination on the top layer of the mouse ear. A maximum amplitude projection (MAP) image was projected onto the x–y plane. The fine erratic patterns of blood vessels were imaged, as shown on the right side of the dashed line indicated by the arrow (T) in Fig. 8(b). However, the left side, indicated by arrow (B) in the PA image, resulted in a blurred vessel image because the ear of the mouse was not flat on the laser focal plane, and the high NA 0.4 objective lens had a short DOF. Likewise, the MAP image was constructed by bottom layer illumination, as shown in Fig. 8(d). In the region of arrow (B), the neo-vascular area was matched with the depth of the focal plane, and the PA images demonstrated high resolution of the microvasculature. The side-view PA images were obtained from the dashed line region shown in Figs. 8(b) and 8(d). Depending on the focal plane, the PA amplitudes were higher than those in the defocused area, as shown in Figs. 8(c) and 8(e), respectively. We selected higher resolution PA signals between two illumination cases and merged the two PA images, as shown in Fig. 8(f), without compromising the contrast and resolution of the image. If a merged MAP image was constructed from the sum of the two PA signals without considering the relative amplitude difference between the PA signals, the merged image would have the lower of the two resolutions from amongst the PA images. High contrast amplitudes were obtained from the side-view of the merged PA image without depending on the imaging depth, as shown in Fig. 8(g).
4. Discussions and conclusion
Axial scanning speed and range of imaging depth of an OR-PAM system has been shown to be enhanced by the application of FTL modulation at the resonant frequency. To verify resonance properties of the FTL, a method to determine the PA range of imaging depth as the modulation frequency of the FTL in the PAM system changes has been proposed. Other methods of measuring the axial scanning range utilize an astigmatic lens approach with a cylinder lens, quadrant diode [23], and fluorescence imaging with a high-speed camera [17]. In contrast, the proposed method does not require additional optical and measurement components to determine the axial scanning range of the FTL. It is found that the FTL response changes as the modulation frequency is varied from 50 Hz to 800 Hz. It is also determined that when the modulation frequency equals the resonant frequency, the range of imaging depth is maximized. The resonant frequency of the FTL used in our experiments was found to be approximately 370 Hz. Our results demonstrate that when the proposed method of modulation is applied, the scanning speed is found to have enhanced 7.40 times. Additionally, the range of imaging depth is found to have been extended approximately 1.22 times. In addition, the use of a fully electrical control method for varying the focal length of the objective lens with an FTL potentially minimizes unwanted motion artifacts for an inertia-free fast focus scanning. A high NA objective lens (NA 0.4) has been for in vivo imaging, and the scanning range of the FTL was found to be about 0.6 mm. The range of imaging depth of a single layer was found to be larger than the extended axial scanning range of the FTL at the resonant frequency. Dual-layer scanning at the longest and shortest focal lengths of the FTL is available to achieve the maximum range of imaging depth, because the top and bottom layer PA signals overlap before the PA signal amplitude drops to one-half of its maximum value. In case of NA 0.25 objective lens, the scanning range was found to be about 1.5 mm. If we apply an objective lens with lower NA to extend the imaging depth, three or more layers can be scanned by using the PAM system.
The proposed method has been demonstrated using a black hair phantom and in vivo mouse ear PA images. The merged PA image of the in vivo study demonstrates that the neo-vascular structural details in the region of a tumor could be clearly distinguished from normal blood vessels over the entire depth of the ear. The dual focusing PAM system with resonance modulated FTL has been shown to shorten the acquisition time when determining the functional activity and dynamics of a tumor structure.
Disclosures
The authors declare that there are no conflicts of interest related to this article.
Funding
GIST–Caltech Research Collaboration Project (K07160); ;GIST Research Institute (GRI) in 2017; Industrial Technology Innovation Program (No. N0002310, 10063062, and 10047943) of the Ministry of Trade, Industry and Energy of Korea.
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