High resolution imaging with differential infrared absorption micro-spectroscopy

Abstract

Although confocal infrared (IR) absorption micro-spectroscopy is well established for far-field chemical imaging, its scope remains restricted since diffraction limits the spatial resolution to values a little above half the radiation wavelength. Yet, the successful implementations of below-the-diffraction limit far-field fluorescence microscopies using saturated irradiation patterns for example for stimulated-emission depletion and saturated structured-illumination suggest the possibility of using a similar optical patterning strategy for infrared absorption mapping at high resolution. Simulations are used to show that the simple mapping of the difference in transmitted/reflected IR energy between a saturated vortexshaped beam and a Gaussian reference with a confocal microscope affords the generation of high-resolution vibrational absorption images. On the basis of experimentally relevant parameters, the simulations of the differential absorption scheme reveal a spatial resolution better than a tenth of the wavelength for incident energies about a decade above the saturation threshold. The saturated structured illumination concepts are thus expected to be compatible with the establishment of point-like point-spread functions for measuring the absorbance of samples with a scanning confocal microscope recording the differential transmission/reflection.

Spatial-domain filter enhanced subtraction microscopy and application to mid-IR imaging

“…It is shown below that the weighted subtraction methods used earlier [2][3][4][5][6][7][8][9][10][11][12][13][14][15] induce significant intensity artefacts with the Gaussian and half-moon c-SIL images. Thus we are introducing a new subtraction method that aims at better matching the two PSFs at their periphery.…”

“…Along the axis normal to the direction of polarization, the width of the half-moon node is 1.25 µm, and we verified that an all optical spatial resolution <λ/2 is possible by resolving beads that are 1.4 ± 0.1 µm apart (λ/2.3). The weighted subtraction exploited in earlier studies when imaging with Gaussian and doughnut beams [2][3][4][5][6][7][8][9][10][11][12][13][14][15] generates significant intensity artefacts with the c-SIL PSFs. A new subtraction method is thus proposed where the Gaussian image is first filtered in the spatial-domain with a kernel optimized to match the Gaussian and half-moon PSFs at their periphery, thereby reducing the side-lobes and negativities responsible for these artefacts.…”

“…The subtraction was then computed from the simulated images according to Eq. (2), as well as according to the recently introduced intensity weighted subtraction [13] and to the constant weight scheme [2][3][4][5][6][7][8][9][10][11][12]14,17]. For IWS, following [13], the simulated images were normalized to one and the subtraction performed according to 1 .…”

“…Subtraction microscopy has been also theoretically proposed for mid-IR imaging [14,15]. These earlier proposals envisaged the exploitation of reflective objectives typically used for imaging in the mid-IR but for which the numerical apertures (NA) remain practically limited so that the PSF with a Gaussian beam exhibits a full-width at half maximum (FWHM) that is not narrower than ca.…”

“…Because the PSFs achieved with the c-SIL show strong side-lobes, a new subtraction method was also designed, where the Gaussian image is filtered in the spatial-domain prior to the subtraction, with a kernel matrix optimized so that the filtered Gaussian and doughnut PSFs match at their periphery. Because, it minimizes the side-lobes in the subtraction PSF, the spatial filtering of the Gaussian image leads to subtracted images that better preserve the relative intensities of the beads and that show higher spatial resolution, in comparison to the other subtraction methods [2][3][4][5][6][7][8][9][10][11][12][13][14][15].…”