On the fundamental imaging-depth limit in two-photon microscopy
One of the principle advantages of two-photon microscopy over one-photon techniques is that it can provide high-resolution images from very deep within living tissue. While imaging depths of 500 micron in brain tissue have become standard performance, larger depths have been inaccessible mainly due to the power limitation of current femto-second laser sources. Here we investigate strategies to improve the imaging depth in two-photon microscopy. In particular, we show that the two-photon imaging depth can be significantly improved using optically amplified femto-second laser pulses. Using a regenerative amplifier as the excitation source we obtained images of stained vasculature and GFP-labeled neurons down to a depth of about 1000 micron below the brain surface in the cortex of mice in vivo. The maximum imaging depth was now limited by out-of-focus background fluorescence and not by the available excitation power. In order to provide a quantitative description of this behavior, we have investigated the effects of scattering on fluo-rescence excitation and detection. The most prominent parameters that influence the maximum two-photon imaging depth are the excitation numerical aperture and the sample staining charac-teristics. The largest depths can be achieved with the largest excitation numerical aperture and the lowest out-of-focus volume staining.
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