Image enhancement in two-photon laser scanning microscopy

Two-photon laser scanning microscopy (TPLSM) offers many advantages for exploring biological systems, allowing one to image living cells with less photo-bleaching and -toxicity than is possible with confocal laser scanning microscopy (CLSM). The authors investigated and compared the signal-to-noise ratio (SNR) and the resolution of TPLSM and CLSM on biological specimens. The S-D point spread functions (PSFs) of TPLSM at different excitation intensities demonstrate that resolution decreases with increased excitation intensity; for example, the lateral resolution is 12% worse and axial resolution is 30% worse at nearly 8-fold more intensity. The 3-D PSFs of CLSM, measured at different pinhole sizes, reveal that resolution improves at the smaller pinhole size, while it´s optimal SNR lies at 35 μm-pinhole. Image-deconvolution techniques improved the resolution of TPLSM from 400 nm to 240 nm in the lateral direction, and from 1080 nm to 540 nm in the axial direction. When combined with its ability to image at increased depth in living biological tissue, TPLSM offers a powerful tool for analyzing biological structures and mechanisms