Femtosecond photoacoustics: integrated two-photon fluorescence and photoacoustic microscopy

Conventional photoacoustic imaging systems excite a photoacoustic wave by illuminating an area on the order of square centimeters with millijoule laser pulses. Spatial resolution is then determined by the ultrasound transducer and is typically on the order of 100 μm. We report on a system that focuses femtosecond, nanojoule pulses to a spot with a diameter of ~ 1 μm to perform laser-scanning photoacoustics with micrometer resolution. Near-infrared femtosecond laser pulses with a pulse energy of 2.4 nanojoules excite a train of photoacoustic waves at the repetition rate of the pulsed laser (80 MHz). These photoacoustic waves are detected by an unfocused single-element ultrasound transducer tuned to 80 MHz. A radiofrequency lock-in amplifier recovers the amplitude of the frequency component of the photoacoustic signal at the pulse repetition frequency. This amplitude is an indicator of the absorption coefficient of the sample at the laser focus and at the laser wavelength. Initial experiments using a graphite rod as absorber reproducibly yield signals in the 0.2 - 2 microvolt range with a signal-to-noise ratio of 18 dB, recovered from 10 mV of broadband noise. The photoacoustic imaging system is integrated in a commercial laser-scanning two-photon fluorescence microscope, enabling simultaneous three-dimensional fluorescence- and photoacoustic imaging. One major application will be to image both morphology and oxygen saturation of microvasculature in the cerebral cortex of anesthetized rodents in vivo in the context of tumor angiogenesis. In this paper we describe the physics of femtosecond photoacoustics and demonstrate initial results.