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 laserscanning 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.

Chromato-axial memory effect through a forward-scattering slab

“…Optimization can be achieved in an iterative way [1] or by prior measurement of the transmission matrix [2]. Furthermore, nonlinear imaging by two-photon fluorescence excitation [3], second-harmonic generation [4], sum frequency generation [5], or coherent anti-Stokes Raman scattering [6], especially efficient for deep imaging, requires using broadband ultrashort pulses. In this case, not only the spatial profile of the beam must be controlled but also its spectral phase, in order to control the pulse shape.…”

Focusing large spectral bandwidths through scattering media

“…Many nonlinear optical interactions used for tissue imaging are however more complex than SHG since they involve polychromatic configurations with more than one beam, sometimes in a broadband ultra-short pulsed regime. The most frequent examples are multicolour multiphoton fluorescence imaging [10] [11], four wave mixing, coherent anti stokes Raman scattering (CARS) [12], and sum frequency generation (SFG) [13]. These situations are very challenging for wavefront shaping since coherent manipulation of waves through a scattering medium is only applicable within a wavelength range below the spectral bandwidth of the medium [14][ [15] [16] [17].…”

Biophotonics feature: introduction

“…In this feature issue of Biomedical Optics Express, we have collected several papers [3][4][5] that represent some of the technologies discussed at the NTM symposium that moved the field, both in terms of the technology and also the biological applications.…”