Benjamin E. Sherlock

Simultaneous, label-free, multispectral fluorescence lifetime imaging and optical coherence tomography using a double-clad fiber

We present a novel fiber-based imaging platform that allows simultaneous fluorescence lifetime imaging (FLIm) and optical coherence tomography (OCT) using a double-clad fiber. This platform acquires co-registered images showing structural and compositional contrast in unlabeled biological samples by scanning the fiber tip across the sample surface. In this Letter, we report a characterization of each modality and show examples of co-registered FLIm and OCT images acquired from a lemon segment and a section of human coronary artery. The close comparison between the combined FLIm and OCT images and a co-registered histology section provides a qualitative validation of the technique and highlights its potential for minimally invasive, multimodal imaging of tissue structure and composition.

Fiber-based fluorescence lifetime imaging of recellularization processes on vascular tissue constructs

New techniques able to monitor the maturation of tissue engineered constructs over time are needed for a more efficient control of developmental parameters. Here, a label-free fluorescence lifetime imaging (FLIm) approach implemented through a single fiber-optic interface is reported for nondestructive in situ assessment of vascular biomaterials. Recellularization processes of antigen removed bovine pericardium scaffolds with endothelial cells and mesenchymal stem cells were evaluated on the serous and the fibrous sides of the scaffolds, 2 distinct extracellular matrix niches, over the course of a 7 day culture period. Results indicated that fluorescence lifetime successfully report cell presence resolved from extracellular matrix fluorescence. The recellularization process was more rapid on the serous side than on the fibrous side for both cell types, and endothelial cells expanded faster than mesenchymal stem cells on antigen-removed bovine pericardium. Fiber-based FLIm has the potential to become a nondestructive tool for the assessment of tissue maturation by allowing in situ imaging of intraluminal vascular biomaterials.

Study of bovine pericardium biochemical and biomechanical properties during collagenase degradation using fluorescence lifetime imaging (Conference Presentation)

Bovine pericardium (BP) exhibits distinct biochemical and biomechanical properties that are dominant by the structural protein collagen. The enzymatic degradation of collagen molecules is critical for in vivo incorporation and remodeling of BP in tissue engineering applications. A non-destructive method for monitoring BP during degradation would provide a valuable tool for quantifying functional changes initially in vitro and ultimately in vivo. In this study, we demonstrated the sensitivity of multi-spectral fluorescence lifetime imaging system (ms-FLIm) developed by our group to collagen content and compressive modulus of BP during collagenase degradation. A pairwise study was performed using bacterial collagenase to partially digest BP. We measured the biomaterials properties with ms-FLIm and destructive conventional measurements including collagen assay, compressive test and histology. A single factor study design was utilized. Test group samples were digested by bacterial collagenase for 0, 8, 16 and 24 hours, while control group samples were prepared in the Hank’s balanced salt solution to control for time in solution. Statistical analysis was performed using the Kendall τB correlation test. The results demonstrate that fluorescence parameters measured by ms-FLIm are significantly correlated with collagen content and compressive modulus (|τB| > 0.45, p < 0.05). Based on these findings, we aim to predict BP’s collagen content and mechanical properties using fluorescence metrics, and ultimately apply ms-FLIm for non-destructively monitoring of in vivo remodeling of BP.

Label-Free Assessment of Collagenase Digestion on Bovine Pericardium Properties by Fluorescence Lifetime Imaging

The extracellular matrix architecture of bovine pericardium (BP) has distinct biochemical and biomechanical properties that make it a useful biomaterial in the field of regenerative medicine. Collagen represents the dominant structural protein of BP and is therefore intimately associated with the properties of this biomaterial. Enzymatic degradation of collagen molecules is critical for extracellular matrix turnover, remodeling and ultimately tissue regeneration. We present a quantitative, label-free and non-destructive method for monitoring changes in biochemical and biomechanical properties of BP during tissue degradation, based on multi-spectral fluorescence lifetime imaging (ms-FLIm). Strong correlations of fluorescence intensity ratio and average fluorescence lifetime were identified with collagen content, Young’s Modulus and Ultimate tensile strength during collagenase degradation, indicating the potential of optically monitoring collagen degradation using ms-FLIm. The obtained results demonstrate the value of ms-FLIm to assess the quality of biomaterials in situ for applications in regenerative medicine.

A strategy to measure electrophysiological changes with photoacoustic imaging (Conference Presentation)

Photoacoustic imaging is an emerging technology capable of both functional and structural biological imaging. Absorption and scattering in tissue limit the penetration depth of conventional microscopy techniques to <1mm. Photoacoustic imaging however, can offer high-resolution and contrast at depths of several centimeters. Though functional imaging of endogenous contrast agents, such as hemoglobin, is widely implemented, currently photoacoustic imaging is unable to functionally report electrophysiological changes within cells. We aim to develop photoacoustic contrast agents to fulfill this need. Cells throughout the brain and body create electrical signals using ion channel proteins. These proteins undergo structural changes to regulate the flux of salt ions into the cell. We have recently developed ion channel activity tracers that dissociate from ion channels after the protein changes structure. By conjugating the tracer to dyes that are sensitive to changes in their chemical environment, we can detect tracer dissociation and therefore ion channel activity. We are exploring whether a similar mechanism can create photoacoustic signal intensity changes. To test if the environmental sensitivity of the dye is photoacoustically distinguishable, we imaged the dye in different solvent backgrounds. We report that manipulation of the chemical environment of the contrast dye results in robust changes in photoacoustic properties. We are working to capture photoacoustic signal changes that occur when ion channel proteins activate using live cell imaging. This technology could permit photoacoustic imaging of electrophysiological dynamics in deep tissue, such as the brain. Further optimization of this technology could lead to concurrent imaging of neural activity and hemodynamic responses, a crucial step towards understanding neurovascular coupling in the brain.