Single-cell analysis, or cytometry, is a ubiquitous tool in the biomedical sciences. Whereas most cytometers use fluorescent probes to ascertain the presence or absence of targeted molecules, biophysical parameters such as the cell density, refractive index, and water content are difficult to obtain. We present quantitative phase imaging as an effective technique to quantify the absolute intracellular water content in single cells at video rate, using an assumption of a spherical cellular geometry. Our study demonstrates the utility of QPI for rapid intracellular water quantification and shows a path forward for identifying biophysical mechanisms using label-free imaging. We further demonstrate the use of two complementary techniques - quantitative phase imaging and Brillouin spectroscopy—as a label-free image cytometry platform capable of measuring more than a dozen biophysical properties of individual cells simultaneously. Our system will unlock new avenues of research in biophysics, cell biology, and medicine.
We apply our recently developed QPI methodologies, which quantify and track intracellular water content, to investigate the role of aquaporin (AQP) proteins in pulsed electric field (PEF) induced water uptake in cells. QPI imaging is performed on Jurkat and glial cells subjected to PEF simulation. The effects on the transmembrane water flux of the cells are investigated when solutions contain mercury, a known broad spectrum AQP blocker, or commercially available AQP blockers. Our study demonstrates the utility of QPI for rapid intracellular water quantification and shows a path forward for identifying biophysical mechanisms using label-free imaging.
Petroleum products such as gasoline or oils tend to age over time with iterative thermocycles, leading to a degradation in quality. To investigate this aging process, spectroscopy techniques involving nonlinear four-wave mixing have been recently used to shed light on the viscoelastic properties of these materials. Impulsive stimulated Brillouin scattering is an emerging spectroscopy technique for monitoring changes in the mechanical properties of materials by using a transient laser grating to create acoustic waves within the sample. A probe beam then diffracts off of this standing acoustic wave and yields in a frequency shift detected using optical heterodyning. Impulsive stimulated Brillouin scattering was used to examine mineral oils, motor oils, and a variety of different gasoline grades. The gasoline samples underwent thermocycling to 70°C and back to room temperature to observe viscoelastic differences and noticeable hysteresis.
Brillouin spectroscopy has recently emerged as a valuable tool for assessing microscopic viscoelastic properties in biological tissues and cells. For many practical biomedical applications, the viscoelastic measurement techniques should be sensitive to low sample concentrations in biological media. In this report, we assess the sensitivity of a recently improved impulsive stimulated Brillouin scattering (ISBS) setup. We explored biologically relevant solutions in distilled water using citric acid, glycine, and sucrose, for which we performed Brillouin measurements. We detailed the peak fitting methodology and analyzed the Brillouin shift and linewidth as a function of concentration. We discuss the sensitivity of the ISBS setup to low concentration measurements and its implications to biological applications.
Dysfunctions in the endothelial cell lining of the vascular endothelium are linked with human pathogenesis of atherosclerosis, venous thrombosis, and several human viral infections. These diseases typically originate from abnormalities resulting from poor structural integrity of the tunica intima of the vascular endothelium. In this report, impulsive stimulated Brillouin scattering spectroscopy was used to assess viscoelastic properties of cells in a microfluidic chip which was designed to mimic the vascular endothelium tunica intima. Brillouin spectroscopy method enabled non-invasive data acquisition of viscoelastic measurements to understand the role of collagen type I on the anchoring of endothelial cells to the extracellular matrix.
Photobiomodulation (PBM) describes the enhancement of cellular functions following exposure to low irradiance visible or NIR light. Although these effects are not well understood, PBM has been shown to enhance the synthesis of ATP so the mitochondrion is the hypothesized target for the processes of photobiomodulation. More specifically, cytochromecontaining enzyme complexes of the electron transport chain (ETC) in the mitochondria are expected to be the primary photoabsorbers of the light thought to induce PBM. Recently, our group found light-induced changes in the activity of complex III (cytochrome c reductase) in isolated mitochondria. In this study, we use femtosecond transient absorption spectroscopy (TAS) to study the excited state dynamics of the electronic transitions in complex III as well as reduced cytochrome c. To investigate the potential for inducing PBM effects in these proteins, TAS experiments are performed without, and with, low irradiance light exposures during the scanning procedure choosing from blue (450 nm), red (635 nm), and near-infrared (808 nm) laser diodes. The TAS experiments with and without light exposures during the procedure are compared to determine if PBM effects were induced. Understanding illumination induced changes in the excited state dynamics of proteins can help to better characterize the molecular processes caused by PBM and lead to a more optimized treatment for the enhancement of human performance and therapy.
Raman imaging continues to grow in popularity as a label-free technique for characterizing the underlying chemical structure of biological materials, both in-vitro and in-vivo. While Raman spectra demonstrate high chemical specificity, spontaneous Raman scattering is an inherently weak process and requires prohibitively long acquisition times. When Raman is utilized to image highly scattering cellular environments, integration times can be on the order of several minutes to hours. Recently developed compressed sensing techniques can greatly improve hyperspectral Raman acquisition times by randomly under-sampling the spatial dimensions. A digital micromirror device (DMD) is used to spatially encode the image plane. The encoded image is then propagated to a spectrometer where the spectral components are produced by shearing one spatial dimension. Several reconstruction algorithms have been developed that can then be used to return the original. Here, we will present single-shot, 2D Raman imaging of CHO cells using compressed hyperspectral Raman microscope. This system provides an order of magnitude improvement on traditional hyperspectral acquisition rates. Single-shot compressed hyperspectral Raman images can reveal biochemical changes due to short lifetime dynamic processes. These improvements will allow imaging of samples that metabolize quickly, rapidly oxidize, or are physically altered under experimental conditions.
Scanning confocal Raman spectroscopy was applied for detecting and identifying topically applied ocular pharmaceuticals on rabbit corneal tissue. Raman spectra for Cyclosporin A, Difluprednate, and Dorzolamide were acquired together with Raman spectra from rabbit corneas with an unknown amount of applied drug. Kernel principle component analysis (KPCA) was then used to explore a transform that can describe the acquired set of Raman spectra. Using this transform, we observe some spectral similarity between cornea spectra and Cyclosporin A, with little similarity to Dorzolamide and Difluprednate. Further investigation is needed to identify why these differences occur.
Photobiomodulation (PBM) is a biological outcome of exposure to low-level light in the red and near-infrared (NIR) wavelengths. Current literature has attributed beneficial effects to PBM, to include improved wound healing, enhanced mitochondrial function, functional enhancements in patients suffering from stroke, and improved cognitive function in a murine model for traumatic brain injury. Cytochrome c oxidase, also named complex IV (C-IV) in the electron transport chain (ETC), is the expected primary chromophore for the red and NIR exposures. The direct evidence that PBM is a consequence of absorption by C-IV is incomplete. Recently, our lab has found metabolic perturbations in cells and isolated mitochondria from low-level exposures to blue and green light as well. To study the immediate and early events of PBM we used a combination of fluorescence microscopy, resonance Raman spectroscopy, Fourier transformed IR (FTIR) spectroscopy, and ultrafast transient absorption spectroscopy (TAS) on cells, isolated mitochondria, and purified ETC enzymes. In this paper, we show that FTIR spectroscopy is useful in determining substrate-dependent, steady-state rates of CO2 production by the tricarboxylic acid (TCA) cycle. The method allows for determinations of wavelength-specific changes in metabolic rate in real time with low-level light exposures. These data will help determine if any mitochondrial components have absorption spectra that correlate with the global PBM response in the literature.
Photobiomodulation, also known as low level laser therapy (LLLT), is a technique that uses light in the red and near infrared (NIR) range (600-900 nm) to elicit a clinically beneficial physiological change in tissue. This physiological change is thought to begin in the mitochondria by altering the metabolic rate for the electron transport chain (ETC). Resonance Raman spectroscopy at 532 nm was used to determine the reduction/oxidation (redox) state of cytochrome c in isolated mitochondria after undergoing LLLT. Mitochondria from hTERT-RPE1 cells were isolated and placed in glutamate buffer and then exposed to violet (405 nm) or red (635 nm) light. The resonance Raman spectrum of the cytochrome c redox state before and after light illumination was measured. This gives us an insight into the types of metabolic changes that occurs within the mitochondria while being illuminated by light during photobiomodulation.
We performed Transient Absorption Spectroscopy (TAS) on samples of cytochrome c in its reduced and oxidized states as well as mitochondria, and we use kinetic analysis to determine the decay rates of the transients. We also tested the samples following red light exposures to determine if there were any changes in transient decay rates. Mitochondria were isolated from hTERT-RPE1 cells and were tested in a glutamate buffer solution while cytochrome c was tested in a Sodium Phosphate solution (Na2PO4). Femtosecond TAS was performed with a pump pulse with wavelength centered at 418 nm and a supercontinuum probe pulse with a spectrum between 440 and 760 nm. Red light illuminations were performed with a 635 nm continuous wave light source at an intensity of 1.6 mW/cm2 for 0.5 hours. We find transients and kinetic decay rates for reduced and oxidized cytochrome c that are consistent with previous literature. However, we do not find any transients for mitochondria under our current TAS testing parameters, and we expect that this may be due to low Signalto- Noise ratio or choice of pump wavelength.
To successfully develop and manufacture a generic drug product, the latter is expected to be bioequivalent to its referencelisted drug, i.e. to show no significant difference in the rate and extent of absorption of the active pharmaceutical ingredient. Optical spectroscopy methods based on vibrational spectroscopy imaging have recently attracted significant attention as potentially viable approaches to quantify drugs’ pharmacokinetics in vivo. However, substantial hurdles still exist due to signal interference from surrounding tissues, significant attenuation of signal in the depth of tissue to optical absorption and scattering, and a lack of quantifiable ways of assessing the signal generated from a drug compound in the depth of a tissue. In this report, we evaluated the major challenges of quantification and sensitive and reproducible analysis of drug distribution in tissues using Raman spectroscopy. Specific attention is given to the noise assessment, which affects both the sensitivity and reproducibility of data.
Understanding the optical properties of water is critical to both laser-tissue interactions as well as setting ocular laser safety standards. The nonlinear properties of water are responsible for supercontinuum generation; however, these effects are poorly understood for wavelengths longer than 1064 nm. A previous study suggested that the supercontinuum generation may convert retinal-safe femtosecond near-infrared pulses with wavelengths longer than 1064 nm into visible wavelength pulses that are above the maximum permissible exposure limit as defined by ANSI Z136.1-2014. To address this knowledge gap, we extend the Z-scan technique in distilled water to wavelengths between 1150 nm to 1400 nm, where linear absorption is strong. Utilizing wavelength tunable, nominally 100 fs laser pulses, we observe wavelength dependence of the nonlinear optical properties of water. The nonlinear refractive index at 1150 nm was consistent with measurements taken at 532 nm in previous studies, and was observed to increase at longer wavelengths. The nonlinear absorption was positive for wavelengths between 1150 nm and 1350 nm and reversed to saturable absorption at 1400 nm. Saturable absorption poses a previously unanticipated eye safety risk as current ocular laser safety standards assume strong absorption at 1400 nm. These results expand our current understanding of the nonlinear optical properties of water to wavelengths in the 1150 nm to 1400 nm region, and inform efforts to revise national and international exposure limits to account for retinal hazards due to nonlinear effects.
Filamentation in air is a profound effect caused by high energy photons. While it has been studied in a wide-range of laser systems, there still exist wavelength regimes where filamentation hasn’t been created, due to lack of sources. Using a tunable near-infrared femtosecond laser, we generated filamentation in air by wavelengths from 1.2 to 2.5 µm. The observed filaments produced harmonic and continuum generation well into the visible spectrum; a rainbow of colors.
Zinc Selenide (ZnSe) has long been recognized as a nonlinear optical material and is used in many optoelectronic devices such as light emitting diodes. ZnSe is known for its remarkably wide transmission range for infrared radiation leading to its use in infrared laser applications. In this report, we discuss higher order harmonic generation when exposing ZnSe to tunable femtosecond mid-IR laser pulses with wavelengths ranging from 2.7 μm to 8.0 μm and pulse energies between 3 and 17 μJ. Higher order harmonic generation was in some instances strong enough to be directly seen by the unaided eye. We also compare these results with measurements taken for other optical materials.
We study supercontinuum (SC) generation in large-mode-area (LMA) photonic crystal fibers with various core sizes and lengths, pumped by a picosecond Nd:YVO4 laser. Micro-joule level SC pulse energy is achieved, and the spectrum extends beyond 1600 nm, corresponding to an effective Raman detection range over 3000 wavenumbers. A multiplex CARS setup based on the SC source is constructed, and we demonstrate CARS acquisition in air, and compare the signal obtained with different LMA fiber parameters.
Calcium fluoride, BK7 and fused silica are common optical materials used in lenses and windows. In this report, we discuss supercontinuum generation using tunable femtosecond mid-IR laser pulses with wavelengths ranging from 2.7 μm to 7.0 μm and pulse energies between 3 and 18 microjoules. We observed harmonic generation in fused silica and BK7, but not supercontinuum generation. Other borosilicate targets generated supercontinuum in both visible and near infrared regions of the spectrum. The visible supercontinuum was, in some instances, strong enough to be observed directly by the human eye. These results contribute to ongoing work being done to refine eye safety standards for femtosecond lasers.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.