Photoacoustic microscopy

Optical & ultrasound (in vivo) · reads

Short laser pulses are absorbed mainly by haemoglobin in blood, briefly heating it so it emits ultrasound pressure waves. A detector picks up these generated waves, and their one-way acoustic travel time gives depth. In mouse cortex, imaged through intact skull, a cranial window, or noninvasively through scalp and skull, it reads vessel structure, diameter, blood oxygenation, flow and estimated oxygen metabolism, reaching about 0.7 mm through skull. These are haemodynamic measures, not direct neuronal readouts; neuronal calcium activity requires added fluorescence imaging.

Spatial resolution: 3 µm (point spread)
Temporal resolution: 2.5 ms (sample interval)
Field of view: 124 mm³ (acquired volume)
Reads: optical absorption by hemoglobin in red blood cells
Destructive?: minimally destructive
Prerequisites: anesthesia: Intraperitoneal α-chloralose, 100 mg/kg (MEMS-scanning functional PAM); other: Water-circulating heating pad at 37 °C; animal taped to holder; head fixed in stereotaxic frame (MEMS PAM); surgery: Scalp surgically removed, skull left intact; exposed skull cleaned with PBS (MEMS PAM); other: Acoustic coupling: ultrasound gel on skull plus de-ionized-water tank whose membrane gently contacts the gel (MEMS PAM); surgery: Cranial window for multiScope OR-PAM cortical imaging; other: Head fixation atop a circular treadmill (multiScope)

Description

Photoacoustic microscopy (PAM) is a focused-scanning form of photoacoustic imaging. Absorbed light, mainly by hemoglobin at 532 nm, becomes heat. The heat launches pressure waves that an ultrasonic transducer detects, and acoustic flight time gives depth, so each laser pulse yields a 1D depth profile. In the optical-resolution form, lateral resolution is set by the optical focus: ~3–7 μm, with ~15 μm axial resolution. Scanning methods include a MEMS mirror combined with a stage, a polygon scanner combined with a stage, and uniform rotary scanning. In mouse cortex, it measures vessel structure, diameter, perfusion, oxygen saturation, oxygen extraction, flow, and estimated oxygen metabolism. Depth reached ~0.7 mm through intact skull, and capillary resolution degrades with depth. Paired with fluorescence, it relates these signals to neuronal calcium activity. In the acoustic-resolution regime, the ultrasound detector sets resolution: a 20 MHz detector gave 70 μm lateral resolution. That system used raster scanning to track vessel oxygenation noninvasively through scalp and skull.

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