Zachary J. Smith

Cell-Phone-Based Platform for Biomedical Device Development and Education Applications

In this paper we report the development of two attachments to a commercial cell phone that transform the phones integrated lens and image sensor into a 350× microscope and visible-light spectrometer. The microscope is capable of transmission and polarized microscopy modes and is shown to have 1.5 micron resolution and a usable field-of-view of 150×150 with no image processing, and approximately 350×350 when post-processing is applied. The spectrometer has a 300 nm bandwidth with a limiting spectral resolution of close to 5 nm. We show applications of the devices to medically relevant problems. In the case of the microscope, we image both stained and unstained blood-smears showing the ability to acquire images of similar quality to commercial microscope platforms, thus allowing diagnosis of clinical pathologies. With the spectrometer we demonstrate acquisition of a white-light transmission spectrum through diffuse tissue as well as the acquisition of a fluorescence spectrum. We also envision the devices to have immediate relevance in the educational field.

Single exosome study reveals subpopulations distributed among cell lines with variability related to membrane content

Current analysis of exosomes focuses primarily on bulk analysis, where exosome-to-exosome variability cannot be assessed. In this study, we used Raman spectroscopy to study the chemical composition of single exosomes. We measured spectra of individual exosomes from 8 cell lines. Cell-line-averaged spectra varied considerably, reflecting the variation in total exosomal protein, lipid, genetic, and cytosolic content. Unexpectedly, single exosomes isolated from the same cell type also exhibited high spectral variability. Subsequent spectral analysis revealed clustering of single exosomes into 4 distinct groups that were not cell-line specific. Each group contained exosomes from multiple cell lines, and most cell lines had exosomes in multiple groups. The differences between these groups are related to chemical differences primarily due to differing membrane composition. Through a principal components analysis, we identified that the major sources of spectral variation among the exosomes were in cholesterol content, relative expression of phospholipids to cholesterol, and surface protein expression. For example, exosomes derived from cancerous versus non-cancerous cell lines can be largely separated based on their relative expression of cholesterol and phospholipids. We are the first to indicate that exosome subpopulations are shared among cell types, suggesting distributed exosome functionality. The origins of these differences are likely related to the specific role of extracellular vesicle subpopulations in both normal cell function and carcinogenesis, and they may provide diagnostic potential at the single exosome level.

Development of a time-gated system for Raman spectroscopy of biological samples.

A time gating system has been constructed that is capable of recording high quality Raman spectra of highly fluorescing biological samples while operating below the photodamage threshold. Using a collinear gating geometry and careful attention to power conservation, we have achieved all-optical switching with a one picosecond gating time and 5% peak gating efficiency. The energy per pulse in this instrument is more than 3 orders of magnitude weaker than previous reports. Using this system we have performed proof-of-concept experiments on a sample composed of perylene dissolved in toluene, and the stem of a Jasminum multiflorum plant, the latter case being particularly important for the study of plants used in production of cellulosic biofuels. In both cases, a high SNR spectrum of the high-wavenumber region of the spectrum was recorded in the presence of an overwhelming fluorescence background.

Validation of an integrated Raman- and angular-scattering microscopy system on heterogeneous bead mixtures and single human immune cells

A microscopy system has been constructed that is capable of simultaneously acquiring both Raman spectra and angle-resolved elastic-scattering patterns in either epi- or transillumination modes with a 7 mum spot size. The benefits and drawbacks of the epi- and transillumination modalities are discussed. Validation studies have been performed on single beads of a few micrometers in size, as well as on ensembles of submicrometer particles. In addition, transilluminated Raman and elastic-scattering spectra were obtained from single granulocytes and peripheral blood monocytes. Both the Raman- and the elastic-scattering channels show clear differences between the two types of immune cells.

Integrated Raman- and angular-scattering microscopy

A microscopy system has been constructed that is capable of simultaneously acquiring both traditional Raman spectra as well as angle-resolved elastic-scattering patterns using a single focused laser spot less than 10 mum wide. The elastic-scattering signal was analyzed by generalized Lorenz-Mie theory, representing what we believe to be the first experimental validation of the theorys prediction of angular backscatter from single spheres. The microscope system exhibits 3 nm precision in predicting sphere diameters, while simultaneously yielding high-quality Raman signals. Applications to single cell analysis are envisioned.

Surface-sensitive polarized Raman spectroscopy of biological tissue

In a two-layer diffusing medium, polarized light directly backscattering off the superficial layer will partially retain its sense of polarization, whereas deeper-probing light will be increasingly depolarized by diffusion. This effect has been studied in both elastic scattering and fluorescence contexts. We apply this method to Raman scattering in two two-layer models with a highly diffusing lower layer of glucose powder and an upper layer of either clear plastic or chicken skin. We employ detection of orthogonal polarization states to generate a Raman spectrum of only the superficial layer by combining the orthogonal signals.

Integrated Raman and angular scattering microscopy reveals chemical and morphological differences between activated and nonactivated CD8+ T lymphocytes

Integrated Raman and angular-scattering microscopy (IRAM) is a multimodal platform capable of noninvasively probing both the chemistry and morphology of a single cell without prior labeling. Using this system, we are able to detect activation-dependent changes in the Raman and elastic-scattering signals from CD8+ T cells stimulated with either Staphylococcal enterotoxin B (SEB) or phorbol myristate acetate (PMA). In both cases, results obtained from the IRAM instrument correlate well with results obtained from traditional fluorescence-based flow cytometry for paired samples. SEB-mediated activation was distinguished from resting state in CD8+ T cells by an increase in the number and mean size of small ( approximately 500-nm) elastic scatterers as well as a decrease in Raman bands, indicating changes in nuclear content. PMA-mediated activation induced a different profile in CD8+ T cells from SEB, showing a similar increase in small elastic scatterers but a different Raman change, with elevation of cellular protein and lipid bands. These results suggest the potential of this multimodal, label-free optical technique for studying processes in single cells.

Raman scattering in pathology

Raman scattering is the inelastic scattering of light by chemical bonds, and can therefore show molecular specificity. It can be used both in pure spectroscopy mode, and in imaging mode. While many applications of Raman spectroscopy and imaging in the biomedical field have been so far demonstrated, the use of this technology for pathology applications is still in early stages. In this paper we review some of the most important recent developments in this field, including a description of relevant technologies, applications to molecular sensing, characterization of cells and tissues of interest, and disease detection via Raman scattering.

Nanometer-scale sizing accuracy of particle suspensions on an unmodified cell phone using elastic light scattering.

We report on the construction of a Fourier plane imaging system attached to a cell phone. By illuminating particle suspensions with a collimated beam from an inexpensive diode laser, angularly resolved scattering patterns are imaged by the phones camera. Analyzing these patterns with Mie theory results in predictions of size distributions of the particles in suspension. Despite using consumer grade electronics, we extracted size distributions of sphere suspensions with better than 20 nm accuracy in determining the mean size. We also show results from milk, yeast, and blood cells. Performing these measurements on a portable device presents opportunities for field-testing of food quality, process monitoring, and medical diagnosis.

Multivariate optical computing using a digital micromirror device for fluorescence and Raman spectroscopy.

A multivariate optical computer has been constructed consisting of a spectrograph, digital micromirror device, and photomultiplier tube that is capable of determining absolute concentrations of individual components of a multivariate spectral model. We present experimental results on ternary mixtures, showing accurate quantification of chemical concentrations based on integrated intensities of fluorescence and Raman spectra measured with a single point detector. We additionally show in simulation that point measurements based on principal component spectra retain the ability to classify cancerous from noncancerous T cells.