Two-photon excitation microscopy is one of the key techniques used to observe three-dimensional (3-D) structures in biological samples. We utilized a visible-wavelength laser beam for two-photon excitation in a multifocus confocal scanning system to improve the spatial resolution and image contrast in 3-D live-cell imaging. Experimental and numerical analyses revealed that the axial resolution has improved for a wide range of pinhole sizes used for confocal detection. We observed the 3-D movements of the Golgi bodies in living HeLa cells with an imaging speed of 2 s per volume. We also confirmed that the time-lapse observation up to 8 min did not cause significant cell damage in two-photon excitation experiments using wavelengths in the visible light range. These results demonstrate that multifocus, two-photon excitation microscopy with the use of a visible wavelength can constitute a simple technique for 3-D visualization of living cells with high spatial resolution and image contrast.

We present three-dimensional (3-D) high-resolution spectral-domain optical coherence microscopy (SD-OCM) by using a supercontinuum (SC) fiber laser source with 300-nm spectral bandwidth (full-width at half-maximum) in the 1700-nm spectral band. By using low-coherence interferometry with SC light and a confocal detection scheme, we realized lateral and axial resolutions of 3.4 and 3.8  μm in tissue (n  =  1.38), respectively. This is, to the best of our knowledge, the highest 3-D spatial resolution reported among those of Fourier-domain optical coherence imaging techniques in the 1700-nm spectral band. In our SD-OCM, to enhance the imaging depth, a full-range method was implemented, which suppressed the formation of a coherent ghost image and allowed us to set the zero-delay position inside the samples. We demonstrated the 3-D high-resolution imaging capability of 1700-nm SD-OCM through the measurement of an interference signal from a mirror surface and imaging of a single 200-nm polystyrene bead and a pig thyroid gland. Deep tissue imaging at a depth of up to 1.8 mm was also demonstrated. This is the first demonstration of 3-D high-resolution SD-OCM in the 1700-nm spectral band.