High-Fidelity Micro- and Nano-Scale Infrared Spectroscopic Imaging

Label-free chemical imaging techniques are a powerful complement to other microscopy techniques in that molecular and morphological contrast is derived intrinsically from the sample rather than through attaching specific fluorescent probes to molecules or the use of non-specific dyes. Mid-infrared (IR) spectroscopic imaging promises the strongest optical molecular sensitivity[1] that arises from a large absorption cross-section. IR spectroscopic imaging combines an ability to record molecular content with the ability to visualize its spatial diversity. Given the need to record a significantly larger quantity of data than a typical microscopy image (MB vs. GB) and the extensive bandwidth of the spectra (~10 μm), trade-offs often have to be made. The closely related considerations of signal to noise ratio (SNR), spatial-spectral coverage, resolution and optical arrangements present a large design space but also a challenge to microanalysis methods.[2] Here, we present a path from rigorous theory to modeling and design to realizing the advantages offered by new ideas on fundamentally changing these trade-offs. We first describe a new microscope design for increased speed and rapid coverage that is useful for biomedical and clinical tissue imaging. Next, we describe a configuration that combines atomic force microscopy (AFM) detection with optical excitation to measure infrared spectra from nanoscale volumes.

We first present a far-field mid-IR spectroscopic microscope design[3,4] that relies on laser scanning with newly designed optics that re-imagine existing, all-reflective designs. Our refractive lens based scanning system simultaneously provides the high-throughput, low-noise, and fine-resolution. A ground-up design of the entire optical train utilizes a dual-axis galvo laser scanning capability to provide spatial quality at the theoretical limits. Simultaneously, large fields of view and high SNR data are recorded. We first report on the spatial and spectral performance of this microscope using standard benchmarks, providing at least a 5X improvement in speed compared to previous designs. Importantly, the design outperforms state of the art widefield Fourier transform infrared spectroscopic imaging,[4] even for wide spectral bandwidths. We demonstrate several examples of applying this technology to all-digital, label-free imaging of minimally processed tissue.

Near-field IR spectroscopic imaging techniques can potentially map both nanostructure and molecular composition simultaneously.[5] However, such scanning probe techniques are currently limited in measurement capability. For example, resonance enhanced (RE) AFM-IR imaging[6] can potentially offer high-sensitivity measurements. However. probe-sample mechanical coupling, nonmolecular optical gradient forces, and noise combine to produce signals that can overwhelm recorded chemical signals. Based on an understanding of the key factors by developing analytical models for the recorded signal, [7–9] we sought to understand the limiting factors in AFM-IR measurements. Of note was the idea that the probe deflection could be maintained at a controlled deflection by moving the sample in an opposite direction by real-time feedback control. This null-deflection technique allows for the recording of high- fidelity AFM-IR data. We further report high-sensitivity nanoscale IR imaging by combining null-deflection measurements with RE sensitivity. This null-deflection scanning probe IR (NDIR) spectroscopic imaging configurations provided ~24× improvement in SNR compared to present state of the art. Consequently, it fundamentally alters the ability to record data while reducing analytical variability to improve microanalysis. We measured chemical domains in 100-nm-thick sections of cellular acini of a prototypical cancer model cell line.