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.