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The ability to characterize the combined structural, functional, and thermal properties of biophysically dynamic samples is needed to address critical questions related to tissue structure, physiological dynamics, and disease progression. Towards this, we have developed an imaging platform that enables multiple nonlinear imaging modalities to be combined with thermal imaging on a common sample. Here we demonstrate label-free multimodal imaging of live cells, excised tissues, and live rodent brain models. While potential applications of this technology are wide-ranging, we expect it to be especially useful in addressing biomedical research questions aimed at the biomolecular and biophysical properties of tissue and their physiology.

Post-surgical hypoparathyroidism and hypocalcemia are known to occur after nearly 50% of all thyroid surgeries as a result of accidental disruption of blood supply to healthy parathyroid glands, which are responsible for regulating calcium. However, there are currently no clinical methods for accurately identifying compromised glands and the surgeon relies on visual assessment alone to determine if any gland(s) should be excised and auto-transplanted. Here, we present Laser Speckle Contrast Imaging (LSCI) for real-time assessment of parathyroid viability. Taking an experienced surgeon’s visual assessment as the gold standard, LSCI can be used to distinguish between well vascularized (n = 32) and compromised (n = 27) parathyroid glands during thyroid surgery with an accuracy of 91.5%. Ability to detect vascular compromise with LSCI was validated in parathyroidectomies. Results showed that this technique is able to detect parathyroid gland devascularization before it is visually apparent to the surgeon. Measurements can be performed in real-time and without the need to turn off operating room lights. LSCI shows promise as a real-time, contrast-free, objective method for helping reduce hypoparathyroidism after thyroid surgery.

Breast conserving surgery is the preferred treatment for women diagnosed with early stage invasive breast cancer. To ensure successful breast conserving surgeries, efficient tumour margin resection is required for minimizing tumour recurrence. Currently surgeons rely on touch preparation cytology or frozen section analysis to assess tumour margin status intraoperatively. These techniques have suboptimal accuracy and are time-consuming. Tumour margin status is eventually confirmed using postoperative histopathology that takes several days. Thus, there is a need for a real-time, accurate, automated guidance tool that can be used during tumour resection intraoperatively to assure complete tumour removal in a single procedure. In this paper, we evaluate feasibility of a 3-dimensional scanner that relies on Raman Spectroscopy to assess the entire margins of a resected specimen within clinically feasible time. We initially tested this device on a phantom sample that simulated positive tumour margins. This device first scans the margins of the sample and then depicts the margin status in relation to an automatically reconstructed image of the phantom sample. The device was further investigated on breast tissues excised from prophylactic mastectomy specimens. Our findings demonstrate immense potential of this device for automated breast tumour margin assessment to minimise repeat invasive surgeries.

Developing clinical tools that assess bone matrix quality could improve the assessment of a person’s fracture risk. To determine whether Raman spectroscopy (RS) has such potential, we acquired Raman spectra from human cortical bone using microscope- and fiber optic probe-based Raman systems and tested whether correlations between RS and fracture toughness properties were statistically significant. Calculated directly from intensities at wavenumbers identified by second derivative analysis, Amide I sub-peak ratio I1670/I1640, not I1670/I1690, was negatively correlated with Kinit (N = 58; R2 = 32.4%) and J-integral (R2 = 47.4%) when assessed by Raman micro-spectroscopy. Area ratios (A1670/A1690) determined from sub-band fitting did not correlate with fracture toughness. There were fewer correlations between RS and fracture toughness when spectra were acquired by probe RS. Nonetheless, the I1670/I1640 sub-peak ratio again negatively correlated with Kinit (N = 56; R2 = 25.6%) and J-integral (R2 = 39.0%). In best-fit general linear models, I1670/I1640, age, and volumetric bone mineral density explained 50.2% (microscope) and 49.4% (probe) of the variance in Kinit. I1670/I1640 and v1PO4/Amide I (microscope) or just I1670/I1640 (probe) were negative predictors of J-integral (adjusted-R2 = 54.9% or 37.9%, respectively). While Raman-derived matrix properties appear useful to the assessment of fracture resistance of bone, the acquisition strategy to resolve the Amide I band needs to be identified.

Monitoring cervical structure and composition during pregnancy has high potential for prediction of preterm birth (PTB), a problem affecting 15 million newborns annually. We use in vivo Raman spectroscopy, a label-free, light-based method that provides a molecular fingerprint to non-invasively investigate normal and impaired cervical remodeling. Prostaglandins stimulate uterine contractions and are clinically used for cervical ripening during pregnancy. Deletion of cyclooxygenase-1 (Cox-1), an enzyme involved in production of these prostaglandins, results in delayed parturition in mice. Contrary to expectation, Cox-1 null mice displayed normal uterine contractility; therefore, this study sought to determine whether cervical changes could explain the parturition differences in Cox-1 null mice and gestation-matched wild type (WT) controls. Raman spectral changes related to extracellular matrix proteins, lipids, and nucleic acids were tracked over pregnancy and found to be significantly delayed in Cox-1 null mice at term. A cervical basis for the parturition delay was confirmed by other ex vivo tests including decreased tissue distensibility, hydration, and elevated progesterone levels in the Cox-1 null mice at term. In conclusion, in vivo Raman spectroscopy non-invasively detected abnormal remodeling in the Cox-1 null mouse, and clearly demonstrated that the cervix plays a key role in their delayed parturition.