Figure 2.

Schematic of a basic multiphoton microscope. An ultra-fast pulsed, near-infrared laser beam is directed onto a pair of X/Y scanning mirrors, which deflect the laser beam in two dimensions, depending on the voltage applied to their galvanometric elements. The beam then passes through the scan and tube lenses. The lens combination determines two fundamental parameters of scanning microscopy: the expansion of the beam entering the objective lens, which controls the size of the beam focus; and the scan angle of the laser beam into the objective, which is responsible for the size of FOV. Next, the laser beam is directed into the objective by a dichroic mirror that reflects near-infrared but passes light in the visible range. The objective lens focuses the short laser pulses into a tiny spot at the sample, resulting in multiphoton excitation of fluorescent indicator molecules. The emitted photons pass the dichroic mirror before being split into two channels (here, red and green) and are detected by two dedicated photomultiplier tubes (PMTs).