Spatially Resolved Two-Dimensional Infrared Spectroscopy via Wide-Field Microscopy

Abstract

We report the first wide-field microscope for measuring two-dimensional infrared (2D IR) spectroscopic images. We concurrently collect more than 16 000 2D IR spectra, made possible by a new focal plane array detector and mid-IR pulse shaping, to generate hyperspectral images with multiple frequency dimensions and diffraction-limited spatial resolution. Both frequency axes of the spectra are collected in the time domain by scanning two pairs of femtosecond pulses using a dual acousto-optic modulator pulse shaper. The technique is demonstrated by imaging a mixture of metal carbonyl absorbed polystyrene beads. The differences in image formation between FTIR and 2D IR microscopy are also explored by imaging a patterned USAF test target. We find that our 2D IR microscope has diffraction-limited spatial resolution and enhanced contrast compared to FTIR microscopy because of the nonlinear scaling of the 2D IR signal to the absorptivity coefficient for the vibrational modes. Images generated using off-diagonal peaks, created from vibrational anharmonicities, improve the molecular discrimination and eliminate noise. Two-dimensional wide-field IR microscopy provides information on vibrational lifetimes, molecular couplings, transition dipole orientations, and many other quantities that can be used for creating image contrast to help disentangle and interpret complex and heterogeneous samples. Such experiments made possible could include the study of amyloid proteins in tissues, protein folding in heterogeneous environments, and structural dynamics in devices employing mid-IR materials.

Spatially-resolved fluorescence-detected two-dimensional electronic spectroscopy probes varying excitonic structure in photosynthetic bacteria

“…Several phase-cycling approaches to F-2DES have been demonstrated 33 , 35 , 36 . To date, phase-cycling methods have been employed in spatially resolved 2D-infrared (2DIR) vibrational spectroscopy in the ground electronic state of a metal-carbonyl stained polystyrene bead 37 , 38 . Very recently, Goetz et al 39 performed phase-cycling-based F-2DES in a microscope.…”

Amyloid found in human cataracts with two-dimensional infrared spectroscopy

“…FTIR or Raman microscopy might give more conclusive results where spatial resolution can provide an internal calibration. The 2DIR imaging is also under development, which might provide both high sensitivity and spatial resolution (38,39).…”

Self-Phase-Stabilized Heterodyne Vibrational Sum Frequency Generation Microscopy

“…16 By spatially resolving interfaces and domains, microscopy could obtain domain specific knowledge and mitigate ensemble averaging. [17][18][19][20][21] Yet, in traditional optical microscopy, signals of non-centrosymmetric molecular interfaces and domains are often overwhelmed by those of isotropic bulk materials. Even-order nonlinear optical microscopy, such as second harmonic generation (SHG) microscopy, 22 has been a popular technique to image non-centrosymmetric systems and is widely implemented in biophysics.…”