Simultaneous dual-wavelength imaging of nonfluorescent tissues with 3D subdiffraction photothermal microscopy.

Multi-wavelength microscopic imaging is essential to visualize a variety of nanoscale cellular components with high specificity and high spatial resolution. However, previous techniques are based on fluorescence, and thus cannot visualize nonfluorescent species, which are much less suffered from photodamage or photobleaching and hence are intrinsically useful in wider range of optical microscopy. Here, we show that simultaneous multi-wavelength imaging of nonfluorescent species can be achieved with the use of a photothermal microscope. Dual-wavelength subdiffraction imaging of biological tissues stained with hematoxylin and eosin is demonstrated. Three-dimensional label-free imaging of mouse melanoma tissue section is also presented to demonstrate the effectiveness of the enhanced spatial resolution. Our technique can be implemented using cost-effective and compact laser diodes and is applicable for various types of both fluorescent and nonfluorescent tissues.

"Optimal detection angle in sub-diffraction resolution photothermal microscopy: application for high sensitivity imaging of biological tissues": reply.

The differences between our model and existing models are rationalized in terms of the experimental conditions. The theory in [Opt. Express 22(16), 18833-18842 (2014)] is applicable when the temperature increase is moderate (~1 K) and the spatial extend of refractive index being modulated is comparable to or smaller than the wavelength, which are in accordance with our experiment.

Sensitivity enhancement of photothermal microscopy with radially segmented balanced detection.

A novel detection method is proposed for highly sensitive photothermal microscopic imaging. This method is based on the characteristics of an angular-dependent photothermal signal; it improves signal intensity by up to two times and rejects the intensity noise of the probe beam. The subdiffraction resolution photothermal imaging of mouse skin melanoma is demonstrated using a laser diode-based photothermal microscopy system to evaluate this method. We confirm that the signal intensity is enhanced 1.7 times compared with the conventional detection method. Moreover, the intensity noise of the laser diode used for the probe beam is effectively reduced by approximately 31 dB, even for a sample with non-uniformity of the refractive index and stationary absorption. This method is implemented by means of a commonly used balanced detector and is thus potentially useful for high-speed imaging.

Optimal detection angle in sub-diffraction resolution photothermal microscopy: application for high sensitivity imaging of biological tissues.

We evaluated the optimal detection angle for maximizing the signal to noise ratio (SNR) in sub-diffraction resolution photothermal microscopy. The angular dependent photothermal signal was calculated based on scattering theory using the temporally modulated Yukawa potential, and its detection angle and modulation frequency dependencies were analyzed. We verified the theoretical findings by imaging gold nanoparticles using laser diode based photothermal microscopy with balanced detection scheme. High-sensitivity (SNR ~40) photothermal biological imaging of a mouse brain was also demonstrated.

Resolution improvement in laser diode-based pump-probe microscopy with an annular pupil filter.

We experimentally demonstrate the use of annular beams to improve lateral resolution in laser-diode-based pump-probe microscopy. We found that the width of the point-spreading function in the case of the annular pump-probe beams is 162 nm, which is 30% smaller than that of the circular beams (232 nm). Furthermore, side lobes were efficiently suppressed at the focal plane since the pump-probe signal is proportional to the product of the two beam intensities. This scheme is demonstrated for the photothermal signal of nonfluorescent gold nanoparticles and the stimulated emission signal of fluorescence beads.