Two-Photon Autofluorescence

Two-photon excitation by femtosecond NIR laser pulses can be used to obtain clear images of tissue layers as deep as 1 mm [132, 278, 279, 344, 462, 495, 534]. The efficiency of two-photon excitation depends on the square of the power density. It therefore works with noticeable efficiency only in the focus of the laser beam. With a microscope lens of high numerical aperture a lateral resolution around 300 nm and a longitudinal resolution of about 1 pm is obtained. Two-photon laser scanning microscopy has therefore become a standard technique of tissue microscopy. Two-photon laser scanning can be combined with 

Dual-wavelength TCSPC detection in two-photon laser scanning microscopes is relatively simple [37]. Multispectral TCSPC detection in a two-photon laser scanning microscope requires a suitable relay optics between the objective lens and the polychromator [35, 60]. Details are described under TCSPC Laser Scanning Microscopy . 

Applications of single-wavelength TCSPC imaging to autofluorescence of tissue are described in [281, 282, 283, 428]. A commercial instrument for skin inspection has been designed by Jenlab, Jena, Germany. The Dermainspect is based on a Ti Sapphire laser, a fast optical scanner, and multidimensional TCSPC. The instrument is shown in Fig. 5.66. 

A typical result is shown in Fig. 5.67. It shows autofluorescence lifetime images of stratum comeum (upper row, 5 pm deep), and stratum spinosum (lower row, 50 mm deep). The multiexponential decay was approximated by a doubleexponential model, and the decay parameters determined by a Levenberg-Marquardt fit. The colour represents the fast lifetime component, Ti, the slow lifetime component, i2, the ratio of lifetime components, Zi /12, and the ratio of the amplitudes of the components, fli / fl2- The brightness of the pixels represents the intensity. 

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Fig. 5.78 Two-photon autofluorescence lifetime image of aortic tissue. Top Intensity image, lifetime image of average lifetime, lifetime distribution. Bottom Fluorescence decay in indicated pixel and double exponential fit with 81% of 294 ns and 18.7% of 2.26 ns. From [39]...

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