Ultraviolet (UV) spectroscopy is a powerful tool for quantitative (bio)chemical analysis, but its application to molecular imaging and microscopy has been limited. Here we introduce ultraviolet hyperspectral interferometric (UHI) microscopy, which leverages coherent detection of optical fields to overcome significant challenges associated with UV spectroscopy when applied to molecular imaging. We demonstrate that this method enables quantitative spectral analysis of important endogenous biomolecules with subcellular spatial resolution and sensitivity to nanometer-scaled structures for label-free molecular imaging of live cells.

We present a novel light source specifically tailored for stimulated Raman scattering–spectroscopic optical coherence tomography (SRS-SOCT), which is, to the best of our knowledge, a novel molecular imaging method that combines the molecular sensitivity of SRS with the spatial and spectral multiplexing capabilities of SOCT. The novel laser consists of an 8 W, 450 fs Yb:KGW oscillator, with a repetition rate of 40 MHz, which delivers the Stokes beam for SRS-SOCT and also pumps and amplifies an optical parametric oscillator (OPO). The output of the amplified OPO is then frequency doubled and coherently broadened using a custom-made tapered fiber that generates bandwidth pulses >40 nm, compressible to <50 fs, with the average power over 150 mW, near the shot-noise limit above 250 kHz. The broadened and compressed pulse simultaneously serves as the pump beam and SOCT light source for SRS-SOCT. This light source is assessed for SRS-SOCT, and its implications for other imaging methods are discussed.

Purpose: We analyze melanin structure and biochemical composition in conjunctival melanocytic lesions using pump-probe microscopy to assess the potential for this method to assist in melanoma diagnosis. Methods: Pump-probe microscopy interrogates transient excited-state photodynamic properties of absorbing molecules, which yields highly specific molecular information with subcellular spatial resolution. This method is applied to analyze melanin in 39 unstained, thin biopsy specimens of melanocytic conjunctival lesions. Quantitative features of the biochemical composition and structure of melanin in histopathologic specimens are assessed using a geometric representation of principal component analysis (PCA) and principles of mathematical morphology. Diagnostic power is determined using a feature selection algorithm combined with cross validation. Results: Conjunctival melanomas show higher biochemical heterogeneity and different overall biochemical composition than primary acquired melanosis of the conjunctiva (PAM) without severe atypia. The molecular signatures of PAMs with severe atypia more closely resemble melanomas than other types of PAMs. Pigment organization in the tissue becomes more disorganized as diagnosis of the lesions worsen, but nevi are more inconsistent biochemically and structurally than other lesions. Relatively high sensitivity (SE) and specificity (SP) is achieved for differentiating between various melanocytic lesions, particularly PAMs without severe atypia and melanomas (SE = 89%; SP = 87%). Conclusions: Pump-probe microscopy is a powerful tool that can identify quantitative, phenotypic differences between various types of conjunctival melanocytic lesions. Translational Relevance: This study further validates the use of pump-probe microscopy as a potential diagnostic aid for histopathologic evaluation of conjunctival melanocytic lesions.

There is currently no low-cost method to quantitatively assess the contents of a blood bag without breaching the bag and potentially damaging the sample. Towards this end, we adapt oblique back-illumination microscopy (OBM) to rapidly, inexpensively, and non-invasively screen blood bags for red blood cell (RBC) morphology and white blood cell (WBC) count. OBM has been recently introduced as a tomographic technique that produces high-resolution wide-field images based on phase-gradient and transmission. Here we modify this technique to include illumination at dual wavelengths to facilitate spectral analysis for cell classification. Further, we apply a modified 2D Hilbert transform to recover the phase information from the phase-gradient images for facile cell segmentation. Blood cells are classified as WBCs and RBCs, and counted based on shape, absorption spectrum, and phase profile using an automated algorithm. This work has important implications for the non-invasive assessment of (1) cell viability in storage bags for transfusion applications and (2) suitability of a cord blood collection bag for stem cell therapy applications.