The refractive index (RI) of biological materials is a fundamental parameter for the optical characterization of living systems. Numerous light scattering technologies are grounded in a quantitative knowledge of the refractive index at cellular and subcellular scales. Recent work in quantitative phase microscopy (QPM) has called into question the widely held assumption that the index of the cell nucleus is greater than that of the cytoplasm, a result which disagrees with much of the current literature. In this work, we critically examine the measurement of the nuclear and whole-cell refractive index using QPM, validating that nuclear refractive index is lower than that of cytoplasm in four diverse cell lines and their corresponding isolated nuclei. We further examine Mie scattering and phase-wrapping as potential sources of error in these measurements, finding they have minimal impact. Finally, we use simulation to examine the effects of incorrect RI assumptions on nuclear morphology measurements using angle-resolved scattering information. Despite an erroneous assumption of the nuclear refractive index, accurate measurement of nuclear morphology was maintained, suggesting that light scattering modalities remain effective.

There have been sustained efforts on the part of cell biologists to understand the mechanisms by which cells respond to mechanical stimuli. To this end, many rheological tools have been developed to characterize cellular stiffness. However, measurement of cellular viscoelastic properties has been limited in scope by the nature of most microrheological methods, which require direct mechanical contact, applied at the single-cell level. In this article, we describe, to our knowledge, a new analysis approach for quantitative phase imaging that relates refractive index variance to disorder strength, a parameter that is linked to cell stiffness. Significantly, both disorder strength and cell stiffness are measured with the same phase imaging system, presenting a unique alternative for label-free, noncontact, single-shot imaging of cellular rheologic properties. To demonstrate the potential applicability of the technique, we measure phase disorder strength and shear stiffness across five cellular populations with varying mechanical properties and demonstrate an inverse relationship between these two parameters. The existence of this relationship suggests that predictions of cell mechanical properties can be obtained from examining the disorder strength of cell structure using this, to our knowledge, novel, noncontact technique.

We present a unique confocal microscope capable of measuring the Raman and Brillouin spectra simultaneously from a single spatial location. Raman and Brillouin scattering offer complementary information about a material's chemical and mechanical structure, respectively, and concurrent monitoring of both of these spectra would set a new standard for material characterization. We achieve this by applying recent innovations in Brillouin spectroscopy that reduce the necessary acquisition times to durations comparable to conventional Raman spectroscopy while attaining a high level of spectral accuracy. To demonstrate the potential of the system, we map the Raman and Brillouin spectra of a molded poly(ethylene glycol) diacrylate (PEGDA) hydrogel sample in cyclohexane to create two-dimensional images with high contrast at microscale resolutions. This powerful tool has the potential for very diverse analytical applications in basic science, industry, and medicine.

Bacterial meningitis is a disease of pronounced clinical significance, especially in the developing world. Immediate treatment with antibiotics is essential, and no single test can provide a conclusive diagnosis. It is well established that elevated total protein in cerebrospinal fluid (CSF) is associated with bacterial meningitis. Brillouin spectroscopy is a widely used optical technique for noninvasive determination of the elastic moduli of materials. We found that elevated protein levels in CSF alter the fluid elasticity sufficiently to be measurable by Brillouin spectroscopy, with model healthy and diseased fluids distinguishable to marked significance (P = 0.014), which increases with sample concentration by dialysis. Typical raw output of a 2-stage VIPA Brillouin spectrometer: inelastically scattered Brillouin peaks (arrows) and elastically scattered incident radiation (center cross).

Emerging medical devices which employ shape memory polymers (SMPs) require precise measurements of the glass transition temperature (T) to ensure highly controlled shape recovery kinetics. Conventional techniques like differential scanning calorimetry (DSC) and dynamic mechanical analysis (DMA) have limitations that prevent utilization for certain devices, including limited accuracy and the need for sacrificial samples. In this report, we employ an approach based on Brillouin spectroscopy to probe the glass transition of SMPs rapidly, remotely, and nondestructively. Further, we compare the T obtained from Brillouin scattering with DMA- and DSC-measured T to demonstrate the accuracy of Brillouin scattering for this application. We conclude that Brillouin spectroscopy is an accurate technique for obtaining the glass transition temperature of SMPs, aligning closely with the most common laboratory standards while providing a rapid, remote, and nondestructive method for the analysis of unique polymeric medical devices.