Figure 3: SHG microscopy with axially extended depth of field.
From: Efficient second-harmonic imaging of collagen in histological slides using Bessel beam excitation
(A–C) Images of collagen fibers within a 15 μm thick histological section of mouse kidney were recorded with Gaussian (0.7 μm × 0.3 μm) and Bessel (7 μm × 0.3 μm) excitations (800 nm). (A,B) Gaussian 2D images recorded at different depths. (C) 2D image of the same field recorded with Bessel excitation. (D–F) Comparison of SHG imaging contrast using Bessel and Gaussian excitations. (D) image of collagen fibers with 3D-scanned Gaussian excitation (0.7 μm × 0.3 μm) and sum projection of a Z-series. (E), corresponding field recorded with 2D-scanned Bessel excitation (23 μm × 0.3 μm) and off-axis detection. (F) Ratio of Bessel-to-Gaussian images calculated assuming similar signals for the smallest fibers. The corresponding histogram is shown on the right. Values superior to 1 reflect the coherent enhancement obtained with extended-depth Bessel excitation. Pixel dwell times, excitation powers after the objective, and maximum signal levels: (A,B) 65 μs, 43 mW, 250 photons; (C) 330 μs, 35 mW, 70 photons; (D) 5 μs *20, 12 mW, 250 photons/plane; (E) 20 μs, 45 mW, 230 photons.
