Cell mechanical properties may undergo changes as a response to external stimuli or altered environment. Being able to map the mechanical properties over an entire cell in a contact-free and label-free manner is crucial to avoid affecting the measurements. Brillouin imaging is a non-invasive all-optical modality that can provide a distribution map of high-frequency elastic modulus and viscosity. In this report we first provide an overview of prior Brillouin cell imaging studies. We then demonstrate viscoelastic 2D and 3D cell imaging capabilities of a molecular filter-based Brillouin imaging system, with a first-time imaging application of multi-excitation (ME) Brillouin elastography.
Nanosecond electric pulses (nsEPs) are known to cause a variety of effects on mammalian cells, ranging from destabilization of cell membranes to changes in cytoskeleton and elastic moduli. Measurement of a cells mechanoelastic properties have previously been limited to only invasive and destructive techniques such as atomic force microscopy or application of optical tweezers. However, due to recent advances, Brillouin spectroscopy has now become viable as a non-contact, non-invasive method for measuring these properties in cells and other materials. Here, we present progress toward applying Brillouin spectroscopy using a unique confocal microscope system for measuring changes in CHO-K1 cells when exposed to nsEPs of 600ns pulse duration with intensity of 10-50 kV/cm. Successful measurement of mechanoelastic changes in these cells will demonstrate Brillouin spectroscopy as a viable method for measuring changes in elastic properties of other cells and living organisms.
The presented work shows a novel approach to correct the undesired background in Brillouin spectra. Specifically, we have developed a Brillouin spectroscopy modification suitable for correction of distortions caused by a molecular filter’s absorption, fluorescent background, or ambient room light. Due to the weak intensity of the Brillouin signal, a distortion of the baseline or a partial absorption of Brillouin peak can have strong impact on data analysis. In the worst case, such as investigation of strongly scattering biological material with simultaneous fluorescence measurements, these perturbations can make it impossible to accurately determine the Brillouin shift. A new Sequentially-Shifted Excitation (SSE) Brillouin spectroscopy method has been developed to allow acquisition of quality Brillouin spectra in most challenging of conditions. The idea behind this method is the observation that the Brillouin and elastically scattered light strongly depend on the wavelength of the incident light, and the location of their respective peaks in the final spectrum changes in response to the smallest change in excitation wavelength. On the other hand, the fluorescence background or distortions due to molecular filter absorption remain the same for small, ~1 pm, changes in incident wavelength. SSE Brillouin spectroscopy involves acquiring multiple Brillouin spectra using slightly offset excitation wavelengths, and computationally separating the signal and distortion/background components, thus recovering the Brillouin signal. The application of SSE Brillouin spectroscopy in highly-scattering sample is presented experimentally using sample of cream.
Raman and Brillouin spectroscopy are powerful tools for non-invasive and non-destructive investigations of material chemical and mechanical properties. In this study, we use a newly developed custom-built dual Raman-Brillouin microspectroscopy instrument to build on previous works studying in-vivo stress response of live plants using only a Raman spectroscopy system. This dual Raman-Brillouin spectroscopy system is capable of fast simultaneous spectra acquisition from single-point locations. Shifts and changes in a samples Brillouin spectrum indicate a change in the physical characteristics of the sample, namely mechano-elasticity; in measuring this change, we can establish a relationship between the mechanical properties of a sample and known stress response agents, such as reactive oxygen species and other chemical constituents as indicated by peaks in the Raman spectra of the same acquisition point. Simultaneous application of these spectroscopic techniques offers great promise for future development and applications in agricultural and biological studies and can help to improve our understanding of mechanochemical changes of plants and other biological samples in response to environmental and chemically induced stresses at microscopic or cellular level.
We propose that antihistamine desloratadine affects lipid content of tissues, a change that can be accessed with Raman and Brillouin spectroscopies. Antihistamines are commonly prescribed to alleviate allergy symptoms. However, reports indicate increase in appetite and weight gain among their possible side effects. This study examines the relationship between the antihistamines’ use and obesity. Four groups of rats consumed regular or high-lipid diets while daily receiving either desloratadine or placebo. We analyzed changes in the chemical composition and local elasticity of skin and adipose tissue samples using Raman and Brillouin spectroscopy respectively. Both the medicated regular-diet group and the non-medicated high-lipid-diet group showed an increase in samples’ elasticity and lipid content compared to the control group that received placebo. Interestingly, the adipose tissues’ elasticity was significantly lower in the high-lipid-diet group receiving daily desloratadine compared to other groups. Raman and Brillouin spectroscopy demonstrated that desloratadine does affect tissues’ lipid content. Antihistamines may contribute to weight gain as shown by an increased lipid content in the medicated regular-diet group. However, it remains unclear why a combination of antihistamines and a high-lipid diet decreased the elasticity of adipose. This observation may indicate a change in the adipose tissue’s density or lipid absorption.
Burns are thermal injuries that can completely damage or at least compromise the protective function of skin, and affect the ability of tissues to manage moisture. Burn-damaged tissues exhibit lower elasticity than healthy tissues, due to significantly reduced water concentrations and plasma retention. Current methods for determining burn intensity are limited to visual inspection, and potential hospital x-ray examination. We present a unique confocal microscope capable of measuring Raman and Brillouin spectra simultaneously, with concurrent fluorescence investigation from a single spatial location, and demonstrate application by investigating and characterizing the properties of burn-afflicted tissue on chicken skin model. Raman and Brillouin scattering offer complementary information about a material's chemical and mechanical structure, while fluorescence can serve as a useful diagnostic indicator and imaging tool. The developed instrument has the potential for very diverse analytical applications in basic biomedical science and biomedical diagnostics and imaging.
Nanosecond electric pulses (nsEPs) are known to cause a variety of effects on mammalian cells, ranging from destabilization of cell membranes to changes in cytoskeleton and elastic moduli. Measurement of a cells mechanoelastic properties have previously been limited to only invasive and destructive techniques such as atomic force microscopy or application of optical tweezers. However, due to recent advances, Brillouin spectroscopy has now become viable as a non-contact, non-invasive method for measuring these properties in cells and other materials. Here, we present progress toward applying Brillouin spectroscopy using a unique microscopy system for measuring changes in CHO-K1 cells when exposed to nsEPs of 600ns pulse duration with intensity of 50kV/cm. Successful measurement of mechanoelastic changes in these cells will demonstrate Brillouin spectroscopy as a viable method for measuring changes in elastic properties of other cells and living organisms.
Brillouin microscopy is an emerging technique in biomedical imaging capable of non-invasive assessing viscoelastic properties on a microscopic scale. In this report, we outline the latest developments in Brillouin spectroscopy instrumentation and applications in an attempt to anticipate the future impact areas of this new imaging modality.
Obesity and overweight are accompanied by an enlargement of adipocytes, which is commonly related to the increasing number or size of lipid droplets within the cells. Some studies have shown that the accumulation of lipid droplets within adipocytes results in their increased stiffness. Recently, Brillouin microspectroscopy has been introduced as a nondestructive method of imaging the elasticity of cells. Unlike other imaging modalities, it is capable of assessing the elastic properties on both tissue- and cell levels. In this study, Brillouin spectroscopy was used to measure the elasticity changes in response to accumulation of lipid droplets within adipocyte during adipogenesis. The cell line used in the study is 3T3-L1, with chemically-induced differentiation from pre-adipocytes to mature adipocytes. The Brillouin shift measurements of the cells before and after differentiation indicate that the stiffness of adipocytes increases due to accumulation of lipid droplets. The results are in agreement with previous atomic force microscopy (AFM) nanoindentation studies. Brillouin microspectroscopy is a technique suitable for measuring the changes of elasticity of adipocytes in response to lipid droplet accumulation.
Changes in mechanical properties represent one of the driving factors behind cell differentiation during embryonic
development. However, measuring these changes without disrupting the normal progression of morphogenesis or
destroying the developing organism is not trivial. Brillouin microspectroscopy has been shown to be capable of nocontact,
non-destructive and non-disruptive assessment of elastic properties in developing zebrafish embryos. The
present study builds upon the previous work, and observes the changes in elasticity during the development of heart and
brain in zebrafish embryos from 8 to 28 hpf (hours post-fertilization) at regular intervals. Brillouin microspectroscopy
has proved to be a suitable technique to continuously monitor tissue differentiation and the development of individual
organs with high spatial resolution without harming the developing organism.
The purpose of this study is to demonstrate the efficacy of using Brillouin spectroscopy for differentiation between healthy and cancerous tissues. Previous studies of various cancers indicate that elasticity of the tumor differs from that of the surrounding tissue. We hypothesize that it is possible to distinguish between normal and malignant areas based on their Brillouin measurements. Brillouin spectroscopy is an emerging spectroscopic technique capable of assessing the local elasticity of samples by measuring the Brillouin shift. In the present study, we have used malignant melanoma tissue samples from Sinclair miniature swine to demonstrate the validity of our proposed application. We performed Brillouin measurements on healthy tissue, normal tumor and regressing tumor (as indicated by depigmentation of tissue). Overall, the tumors were found to be stiffer than the surrounding healthy tissue. However, the regressing tumor displayed the elastic properties closer to that of the healthy tissue. Based on the Brillouin measurements, we have successfully differentiated between the tumor and healthy tissues with a high degree of confidence (p<104 for normal tumor, p<0.05 for regressing tumor). Our results indicate that Brillouin spectroscopy is an appropriate tool to not only pinpoint tumor boundaries, but also to monitor tumor growth or evaluate its response to treatment.
The purpose of this study is to evaluate the effect of a high-lipid diet on elasticity of adipose tissue. We employed dual Raman/Brillouin microspectroscopy to analyze brown and white adipose tissues obtained from adult rats. The rats were divided into two groups, one of which received a high-fat feed, while the other served as a control. We hypothesized that the changes in the elasticity of adipose tissues between the two groups can be successfully assessed using Brillouin spectroscopy. We found that the brown adipose tissue possessed a lesser Brillouin shift than the white adipose within each group and that the elastic modulus of both adipose tissues increases in the high-fat diet group. The Raman spectra provided supplementary chemical information and indicated an increase in the lipid-to-protein ratio in the brown adipose, but not in the white adipose.
It has been shown that exposure of live neurons to a low-intensity pulsed infrared light can be used to excite action potentials. Infrared pulsed laser coupled to an optical fiber can be utilized to create a rapid localized increase in temperature in the vicinity of the cell. The resulting temperature gradient leads to an increase in membrane fluidity and permeability, causing depolarization of the target cell. In order to characterize the fluidity of the cell membrane at various temperatures with and without pulsed IR light exposure, we used a polarity-sensitive fluorescent probe di-4- ANEPPDHQ. This dye exhibits a fluorescent shift between the disordered and ordered phases of the membrane, and can be used to quantitatively evaluate the state of the membrane by calculating the generalized polarization (GP) value. Using high-speed imaging of cells exposed to a IR light of varying pulse width, it was determined that a longer pulse width leads to a greater change in the GP value. Comparison of GP values of cells at different ambient temperatures without the pulsed IR light exposure and cells exposed to pulsed IR light indicated that a rapid temperature gradient caused by the exposure to pulsed light induces a larger change in GP value than the ambient temperature increase alone, indicating a greater disruption of membrane fluidity and permeability.
Resonance Raman spectroscopy is an emerging spectroscopy tool capable of highly specific and highly sensitive
analysis of biological molecules in solutions. The complexity of experimental set-up and reliance on laser sources with
very short life-time and very high maintenance requirement were always considered the major bottle-neck problem on
the way of wide spread of applications of deep-UV Raman spectroscopy in biology and medicine. In this report, we
present the design of a very inexpensive system based on a diode-pumped solid-state laser system capable of performing
Raman analysis in the deep UV.
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