Frequency domain optical resolution photoacoustic and fluorescence microscopy using a modulated laser diode

In this paper a multimodal optical-resolution photoacoustic and fluorescence microscope in frequency domain is presented. Photoacoustic waves and modulated fluorescence are generated in chromophores by using a modulated diode laser. The photoacoustic waves, recorded with a hydrophone, and the fluorescence signals, acquired with an avalanche photodiode, are simultaneously measured using a lock-in technique. Two possibilities to optimize the signal-to-noise ratio are discussed. The first method is based on the optimization of the excitation waveform and it is argued why square-wave excitation is best. The second way to enhance the SNR is to optimize the modulation frequency. For modulation periods that are much shorter than the relaxation times of the excited chromophores, the photoacoustic signal scales linearly with the modulation frequency. We come to the conclusion that frequency-domain photoacoustic microscopy performed with modulation frequencies in the range of 100 MHz can compete with time-domain photoacoustic microscopy regarding the signal-to-noise ratio. The theoretical predictions are confirmed by experimental results. Additionally, images of stained and unstained biological samples are presented in order to demonstrate the capabilities of the multimodal imaging system.