Hyperspectral imaging with laser-scanning sum-frequency generation microscopy

Abstract

Vibrationally sensitive sum-frequency generation (SFG) microscopy is a chemically selective imaging technique sensitive to non-centrosymmetric molecular arrangements in biological samples. The routine use of SFG microscopy has been hampered by the difficulty of integrating the required mid-infrared excitation light into a conventional, laser-scanning nonlinear optical (NLO) microscope. In this work, we describe minor modifications to a regular laser-scanning microscope to accommodate SFG microscopy as an imaging modality. We achieve vibrationally sensitive SFG imaging of biological samples with sub-μm resolution at image acquisition rates of 1 frame/s, almost two orders of magnitude faster than attained with previous point-scanning SFG microscopes. Using the fast scanning capability, we demonstrate hyperspectral SFG imaging in the CH-stretching vibrational range and point out its use in the study of molecular orientation and arrangement in biologically relevant samples. We also show multimodal imaging by combining SFG microscopy with second-harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) on the same imaging platfrom. This development underlines that SFG microscopy is a unique modality with a spatial resolution and image acquisition time comparable to that of other NLO imaging techniques, making point-scanning SFG microscopy a valuable member of the NLO imaging family.

Keywords: (110.3080) Infrared imaging; (170.0180) Microscopy; (180.4315) Nonlinear microscopy; (190.4223) Nonlinear wave mixing.

Fig. 1

Schematic of the SFG microscope. Galvanometric mirrors are part of an Olympus Fluoview 300 laser scanner, and excitation and collection optics are part of an Olympus IX71 frame. PMT: photomultiplier, BF: bandpass filter, Obj: microscope objective, DM: dichroic mirror.

Fig. 2

SHG and SFG imaging of rat tail tendon, a collagen I rich tissue. a) SHG image obtained with the polarization orientation of the incident beam parallel to the long axis of the collagen fibers. b) SHG image similar to a), but with the polarization orientation rotated by 90°. c) SHG polarization plot taken in the red region of interest shown in a). Direction refers to the polarization orientation of the incident beam. d) SFG image at 2945 cm⁻¹ of the same sample and with the polarization orientation of both beams aligned with the main axis of the collagen tissue. e) SFG image similar to e), but with the polarization orientation rotated by 90°. f) SFG spectra extracted from the hyperspectral data stack. Red spectrum refers to the region of interest in d) and blue spectrum refers to the region of interest indicated in e). Scale bar is 15 μm.

Fig. 3

Multivariate analysis of collagen rich tissue. a) Vertex component analysis (VCA) image showing three end-members in red, green and blue, based on a hyperspectral SFG data stack. The arrow indicates the polarization direction of the incident beams. b) Corresponding end-member SFG spectra extracted from the VCA.

Fig. 4

Hyperspectral SFG imaging of a cellulose fiber. a) SFG image taken at 2945 cm⁻¹. b) Corresponding SHG image. c) VCA image of the SFG hyperspectral data stack, showing three end-members in red, green and blue. d) SFG end-member spectra obtained from the VCA. Scale bar is 10 μm.

Fig. 5

SFG imaging of cholesterol microcrystals. a) SFG image at 2845 cm⁻¹. Scale bar is 10 μm. b) SHG polar plots obtained in the red and blue boxed regions of interest of the image in a). c) SFG spectra extracted from the regions of interest in image a). The red spectrum is obtained from the red box, whereas the blue spectrum is obtained from the blue box. d) Composite SFG image formed by overlaying images taken at 2955 cm⁻¹ (blue), 2925 cm⁻¹ (green), and 2845 cm⁻¹ (red).

Fig. 6

Cholesterol microcrystals visualized with a) SFG and b) CARS. Vibrational driving frequency in both images is set at 2845 cm⁻¹. Scale bar is 10 μm.