Two-photon photoacoustic microscopy (TP-PAM) can visualize deep structures in living tissues with high spatial resolution determined by the volume of nonlinear absorption. Generally, the out-of-focus background fluorescence limits the imaging depth in nonlinear optical microscopies. In this study, to overcome this drawback that is also expected to exist in TP-PAM, we propose TP-PAM with spatial overlap modulation using femtosecond optical pulse train. Because the modulation depth of the spatial overlap in the focal region is much greater than those in out-of-focus regions, the out-of-focus background is effectively rejected by extracting the modulated photoacoustic signals.
Photoacoustic microscopy (PAM) is a biological visualization technique that can provide high spatial resolution and high contrast images of deep structures in living tissues. However, because of the spherical aberration of the objective lens and the wavefront distortion due to the surface shape and light scattering of the specimen, obtained photoacoustic images in deep tissues are sometimes blurred or distorted. In order to solve this problem, we have developed a PAM using a transmissive liquid-crystal adaptive optics (AO) element. The transmissive and thin structure of the AO element can be easily installed in the PAM system. Using photoacoustic images of a USAF 1951 resolution test target measured through the glass substrate (thickness; 1.5-mm), the lateral resolutions in PAM were estimated with and without the AO element, when a flashlamp-pumped nanosecond pulse laser (pulse width, 5-ns; wavelength, 500-nm) and water-immersion objective lens (NA = 0.8) were employed. The lateral resolution of PAM at the depth of 1.5-mm was improved from 1.04 ± 0.04 μm to 0.53 ± 0.10 μm by optimizing AO corrections. We have also visualized small blood vessels in mouse ear in vivo by PAM with AO correction. Thus, by optimizing the AO correction according to the imaging depth, our proposed PAM improves the spatial resolution in biological tissues.
Photoacoustic microscopy (PAM) is a biological visualization technique that can provide high spatial resolution and high contrast images of deep structures in living tissues. In PAM, the lateral resolution is determined by the size of the focus spot. Generally, because the wavefront aberration, due to the difference of refractive index between samples and air (water) and the shape of samples, enlarges the focus spot, obtained deep images are blurred or distorted. In order to solve this problem, we corrected the wavefront aberration occurring in samples using a transmissive liquid-crystal adaptive optics (AO) element. Our AO element consists of three liquid-crystal layers which have different ITO (indium tin oxide) patterns and are controlled independently. Their patterns are designed to correct the wavefront aberration suitable for a 40X waterimmersion objective lens. The AO element with transmissive and thin structure is easily installed in the PAM system. Also, our AO element is inexpensive and has low power consumption. In this study, we compared photoacoustic images obtained without and with the AO element for a USAF test target, polystyrene beads diffused in glycerol and various tissue specimens. As a result, we found that the use of transmissive AO element improves the lateral resolution and signal-tonoise ratio in PAM.
Conventional one-photon photoacoustic microscopy (PAM) utilizes high-frequency components of generated photoacoustic waves to improve the depth resolution. However, to obtain optically-high resolution in PAM in the depth direction, the use of high-frequency ultrasonic waves is to be avoided. It is because that the propagation distance is shortened as the frequency of ultrasonic waves becomes high. To overcome this drawback, we have proposed and developed two-photon photoacoustic microscopy (TP-PAM). Two-photon absorption occurs only at the focus point. TPPAM does not need to use the high-frequency components of photoacoustic waves. Thus, TP-PAM can improve the penetration depth while preserving the spatial resolution. However, the image acquisition time of TP-PAM is longer than that of conventional PAM, because TP-PAM needs to scan the laser spot both in the depth and transverse directions to obtain cross-sectional images. In this paper, we have introduced a focus-tunable electrically-controlled liquid lens in TP-PAM. Instead of a mechanical stepping-motor stage, we employed electrically-controlled liquid lens so that the depth of the focus spot can be quickly changed. In our system, the imaging speed of TP-PAM using the liquid lens and one-axis stepping-motor stage was 10 times faster than that using a two-axis stepping-motor stage only. TP-PAM with focus-scanning head consisting of the liquid lens and stepping-motor stage will be a promising method to investigate the inside of living tissues.
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