SHG imaging

Figure 1 shows the experimental setup of the inverted SHG microscope. We used a mode-locked Cr:Forsterite laser (CrF-65P, Avesta Ltd., Moscow, Russia, centre wavelength 1250 nm, pulse duration 70 fs, repetition rate 73 MHz) to enhance the depth of penetration in SHG imaging.¹⁴ The mean power of the laser output light was adjusted to 20 mW at the sample surface by using a combination of a half-wave plate (HWP; CWO-1250-02-10, Lattice Electro Optics Inc., California, retardation tolerance = λ/500) and a polariser (P; PGL8610, CASIX Inc., Fujian, China, extinction ratio < 1 × 10^-6). Its linear polarisation was converted into circular polarisation by a quarter-wave plate (QWP; CWO-1250-04-10, Lattice Electro Optics Inc., retardation tolerance = λ/500) in order to cancel the polarisation dependence of the SHG efficiency of the orientated collagen fibres. The focal point of the laser beam was two-dimensionally scanned on a sample by a combination of a galvanometer mirror (GM), relay lenses (RL1 and RL2), and an objective lens (OL; CFI Plan 50 × H, Nikon Corp., Tokyo, Japan, magnification ×50, numerical aperture 0.9, working distance 350 µm, oil-immersion type). Backscattered SHG light was collected by the same OL, was reflected by a harmonic separator (HS; LWP-45-Runp625-Tunp1250-B-1013, Lattice Electro Optics Inc., Fullerton, California, reflected wavelength 625 nm), passed through an optical filter with a sharp pass-band (BPF; 625/26 nm BrightLine, Semrock Inc., Rochester, New York, pass wavelength 612 nm to 638 nm), and was then detected by a photon-counting photomultiplier with Peltier cooling (PMT; H7421-40, Hamamatsu Photonics K.K., Hamamatsu, Japan). Using the above GM optics, SHG images of a 400 µm × 400 µm region, composed of 256 pixels × 256 pixels, were acquired at a rate of 0.5 images/second. The range of probing depth in the present system was 0 ~ 200 µm from the sample surface, which was limited by the working distance (350 µm) of the OL and the thickness (120 ~ 170 µm) of a cover glass between the OL and the sample, and not limited by the penetration depth (~several hundred µm) of the 1250 nm laser light. To expand the lateral imaging region, we scanned the sample position at intervals of 400 µm using a stepping-motor-driven 3-axis translation stage every time an SHG image was obtained by the GM. Finally, we obtained a large-area SHG image with a size of 3.2 mm × 3.2 mm by stitching together 64 SHG images in a matrix of eight rows and eight columns. The total image acquisition time was around 15 minutes for one sample. During the experiment for SHG imaging, the sample was immersed in the physiological saline solution to avoid tissue dehydration. Although the sample temperature was not controlled, we consider the assessment to be unaffected. There was no load on the tendon when SHG imaging was performed. For calculation of the mean SHG light intensity, we used the large-area SHG images without contrast enhancement. In addition, image analysis based on 2D Fourier transform was performed using the magnified SHG images without or with contrast enhancement. The presence or absence of contrast enhancement did not influence results because we used the shape of the 2D Fourier transform spectrum.

Diagram showing the experimental setup of an inverted SHG microscope (HWP, half-wave plate; P, Polariser; QWP, quarter-wave plate; GM, galvanometer mirror; RL1 and RL2, relay lenses; HS, harmonic separator; OL, objective lens; BPF, optical band-bass filter; PMT, photon-counting photomultiplier).