In Chapter 17 we noted that conventional microscopes are ineffective at rejecting out-of-focus light, either scattered or emitted as fluorescence, and are consequently limited to imaging thin samples. We also reviewed some methods to achieve optical sectioning in microscopic imaging by spatial gating, which requires the illumination source to be spatially coherent, and, for some methods, may also require phasefront-preserving optics for the detection stage. The other general class of methods that can effect sectional imaging is based on temporal gating of back-reflected light. Conceptually, this is simple: if one can generate a very short pulse of light – a 10-fs pulse, for example, would span ∼3 μm in the axial direction – and measure precisely the arrival time of a back-reflected pulse, a time gate could (theoretically) reject light that reflects from shorter or longer distances. In principle, this is what pulse-echo ultrasound imaging achieves: an ultrasound imaging system determines the depth of imaged objects by timing the arrival of sound pulses that have reflected from discontinuities in the tissue density (or acoustic characteristic impedance) associated with structures in the body (see Section 16.1.2). Since the speed of sound (in water) is ∼1500 m/s, the timing precision needed for, say, 100 μm in axial resolution would be of the order of 100 ns, which is comfortably within the capability of common electronic timing circuitry. In Chapter 16 (Section 16.4.3), we saw that for hybrid photoacoustic imaging, the spatial (lateral) resolution of the ultrasound imaging component can approach microscopic dimensions.

In a purely optical system, however, even though ultrashort laser pulses are available, microscopic axial resolution of, say, 3 μm would require a temporal resolution for detection of the order of 10−14 s, which is far shorter than the response time of any electronic sensor. Optical methods, nonetheless, offer us ways to get around this limitation. Early approaches, in a manner conceptually analogous to the sectioning capability of second-harmonic-generation microscopy (as detailed in Section 17.4.2), invoked time gating by correlating the overlap of the sample signal with a reference signal in a nonlinear medium. For example, Fujimoto et al. (1986) first demonstrated the potential for precision “time-of-flight” ranging of distances in biological structures by use of a nonlinear-optical cross-correlation time-gate based on second-harmonic generation, with the harmonic signal separated angularly between the crossed beams from the sample and reference.