In the scheme depicted in Fig. 1(a), an Nd:YVO4 laser (Spot 10-200-532, Elforlight, Daventry, United Kingdom) generates a train of 1.6-ns pulses at 532 nm with a 2.35 kHz pulse repetition rate. To allow per-pulse energy variation, the laser beam is passed through an electro-optic modulator (350-50, Conoptics, Danbury, Connecticut), which is controlled by LabVIEW (National Instruments, Austin, Texas) and synchronized with the laser trigger. The beam is then spatially filtered, partially sampled by a photodiode (S1226–18BK, Hamamatsu, Bridgewater, New Jersey), and focused onto the sample using an infinity corrected objective (NA 1.20, , UPlanSApo, Olympus, Center Valley, Pennsylvania). The focus is precisely adjusted using a piezoelectric actuator (PAS080, Thorlabs, Newton, New Jersey). The laser pulse energy at the target ranges from 0.5 to 5 nJ for imaging melanoma cells, 20 to 100 nJ for imaging mitochondria, and 0.01 to 0.25 nJ for imaging gold nanoparticles. A train of four pulses with varied pulse energies is fired for each pixel. The photodiode is used to accurately measure the energy of each pulse for pulse-to-pulse signal calibration. The PA signal is detected by an ultrasonic transducer (40 MHz central frequency and NA 0.5). After amplification, the PA signal is digitized at a sampling rate of 500 MHz using a data acquisition card (ATS9350, Alazartech, Pointe-Claire, Quebec, Canada). A piezoelectric scanning stage (NPXY400A, nPoint, Middleton, Wisconsin) raster scans the objective lens and the ultrasonic transducer with a step size of 25 nm in the -plane. Coefficients are extracted by fitting the PA dependence on pulse energy with a polynomial. Both conventional PAM and PA nanoscopy images were processed the same way by passing the raw data (for conventional PAM) or the high-order coefficient images (for PA nanoscopy) through a low-pass Wiener filter to remove high-frequency noise. In the current embodiment of PA nanoscopy, the acquisition time for an image with is .