Kyona Schacht, AG’25; Jose Rodriguez Sanchez, E'26; and Malika Zakarina, EG’1G

As part of a recent lab project, we explored the use of frequency-domain near-infrared spectroscopy (FD-NIRS) to characterize the optical properties of a diffuse optical phantom, simulating tissue-like scattering and absorption behavior. This work aimed to validate a multi-distance scanning configuration for accurately recovering absorption (μₐ) and reduced scattering (μ’s) coefficients across two near-infrared wavelengths (690 nm and 830 nm), relevant to biomedical optical imaging.

We implemented a linear slope method to retrieve optical properties by analyzing the amplitude (AC) and phase of diffuse reflectance measurements as a function of source-detector distance. These measurements were then linearized, and the resulting slope values were used to extract absolute optical properties. Absorption coefficients were found to be higher at 830 nm (μₐ ≈ 0.00110 mm⁻¹) compared to 690 nm (μₐ ≈ 0.00074 mm⁻¹), consistent with typical hemoglobin absorption behavior. Conversely, scattering coefficients were higher at 690 nm (μ’s ≈ 0.4914 mm⁻¹) than at 830 nm (μ’s ≈ 0.3529 mm⁻¹), reflecting expected wavelength-dependent tissue scattering trends.

All data analysis, modeling, and visualization were performed using custom MATLAB scripts, which processed raw measurement data to generate linear fits, extract optical parameters, and visualize diffuse reflectance. This workflow not only validated the FD-NIRS approach for optical property quantification but also provided hands-on experience in signal processing, light-tissue interaction modeling, and computational data analysis.

This project deepened my understanding of optical imaging physics, spectroscopy, and the practical application of computational tools for biomedical research. It demonstrates the potential of FD-NIRS as a non-invasive technique for probing tissue structure and function, with applications in medical diagnostics and tissue monitoring.

Kyona Schacht, AG’25; Jose Rodriguez Sanchez, E'26; and Malika Zakarina, EG’1G

This research laboratory paper was completed for BME-0156: Biophotonics Laboratory at Tufts University under the supervision of Maria Savvidou, Ph.D., and in collaboration with Kyona Schacht, AG’25, and Malika Zakarina, EG’1G. Our objective was to compare brightfield, phase contrast, and fluorescence microscopy in imaging biological samples, with a focus on assessing their respective advantages and limitations in contrast generation, resolution, and visualization.

We examined stained histology slides, live GFP+ and RFP+ cells, Convallaria rhizome, and 0.5 μm fluorescent beads. Brightfield microscopy provided strong contrast for stained samples but lacked effectiveness for unstained specimens. Phase contrast microscopy enhanced visualization of live, transparent cells by detecting refractive index differences, making it especially useful for imaging GFP+ and RFP+ cells. Fluorescence microscopy enabled specific imaging of cellular structures using targeted fluorophores, including DAPI for nuclei, GFP for protein fluorescence, and TRITC as a vascular or cell wall marker. However, interpreting composite fluorescence images required caution—overlapping signals sometimes obscured features, such as green fluorescence masking red emission in Figure 7f.

We also observed unexpected variations in bead size measurements at 40x magnification, likely due to focus-related limitations. Overall, our findings emphasize the trade-offs of each technique: brightfield excels with stained specimens, phase contrast enhances unstained cell visibility but can introduce halo artifacts, and fluorescence microscopy offers molecular specificity while requiring careful staining and attention to photobleaching and signal overlap. This project reinforced the importance of selecting the appropriate microscopy method based on the biological sample and research objective.