In this work, we developed a new multimodal technology that can simultaneously measure a subset of mechanical, optical, and acoustical properties of the sample, which is based on the integration of Brillouin and photoacoustic (PA) microscopy. Notably, this integration offers a novel approach to probing the refractive index of the sample, which is inaccessible through each technique alone. As a proof-of-concept, we demonstrated the feasibility of the combined setup to obtain the co-registered Brillouin and time-resolved PA signals in a synthetic phantom made of kerosene and CuSO4 aqueous solution. In addition, we measured the refractive index of saline solutions and validated the result in comparison with previously reported data.
Brillouin microscopy provides a non-invasive method for quantifying the mechanical properties of biological materials with diffraction-limited resolution. Since the signal of spontaneous Brillouin scattering is very weak, high laser power and ultrasensitive detection are usually required. Here, we report a new approach to enhance the signal of Brillouin microscopy by recycling the power of the illumination beam. With a multipass illumination geometry, we obtained about 3.75 times enhancement of the Brillouin signal under the same input laser power. We will present the details of the optical design as well as the experiment.
The mechanisms involved in neural tube formation are complex and can be easily disrupted. Neurulation is one such process, governed by mechanical forces where tissues physically fold and fuse. When neural tube folding and closure fail to complete during neurulation, it results in structural and functional abnormalities of the brain and spinal cord. Thus, it is important to understand the interplay between forces and tissue stiffness during neurulation. Brillouin microscopy is an all-optical, noninvasive, high-resolution imaging technique capable of mapping tissue stiffness, but it cannot provide structural information, resulting in “blind” imaging. To overcome this limitation, we have combined a Brillouin microscopy system with optical coherence tomography (OCT) in one synchronized and co-aligned instrument to provide structural guidance when mapping the biomechanical properties of neural tube formation in mouse embryos. We developed custom instrumentation control software that utilizes the OCT structural image to guide Brillouin imaging. We acquired first 3D OCT images and then 2D structural and mechanical maps of mouse embryos at embryonic day (E) 8.5, 9.5, and 10.5. Brillouin microscopy showed the cell-dense layer of neural plate derived from the ectoderm at E 8.5, which was unable to be distinguished with OCT. At E 9.5 and 10.5, the neuroepithelium could be clearly seen by Brillouin microscopy with a greater stiffness than the surrounding tissue. Our results show the capability of the co-aligned and synchronized Brillouin-OCT system to map tissue stiffness of murine embryos using OCT-guided Brillouin microscopy.
Neural tube closure, or neurulation, has been studied across a range of vertebrates as it is the basis of embryonic development. Closure failure can lead to severe congenital malformations. Current technologies require fixed specimens and physical contact to extract a modulus. Here we investigate the mechanical changes of the neural plate from formation to closure within intact live chick embryos using time-lapse Brillouin imaging and ex-ovo culture. We observed an increase in the Brillouin modulus of the neural plate as the embryo develops in ex-ovo culture. By quantifying the timing and the extent of the forces that drive neural tube closure, we can more accurately identify when and why neural tube defects occur.
Retinal diseases such as diabetic retinopathy and glaucoma are leading cause of blindness in the world. Assessing biomechanical properties of retina is very crucial since it is constantly under stress due to the vitreous humor and eye movements. Characterizing biomechanical properties of retina noninvasively is a challenge due to its location inside the eye-globe, fragility, and thin geometry. Brillouin microscopy is a noninvasive, all optical imaging technique to qualitatively map the biomechanical properties of tissues. In this work, we mapped the layer by layer distribution of biomechanical properties of retinas using Brillouin microscopy. We found that the nuclear layer was stiffer compared to other layers. Furthermore, we observed fixing the retinas with paraformaldehyde increased the retinal stiffness compared to the fresh retinas.
The nucleus is the largest and probably stiffest organelle of eukaryotic cells. As such, its mechanical properties are tightly related to various cell functions. However, the mechanical behavior of the nucleus within intact cells is much less know because it is embedded in the cytoplasm thus not directly accessible by the existing contact-based technique. In this work, the regulation of nuclear mechanics was investigated by all-optical Brillouin microscopy and the results were found to be consistent with three-dimensional chemomechanical modeling. We found the intact nuclear mechanics is regulated by both the cytoskeletal networks and internal nanostructure.
One challenge of common confocal Brillouin microscopy is the relatively slow mapping speed, which is due to both the intrinsically weak signal of spontaneous Brillouin scattering and the point-by-point mapping strategy. We previously have proposed a multiplex mapping idea called line-scanning Brillouin microscopy and demonstrated its capability for fast imaging. In this work, we adapt this idea into a new setup for biological application by redesigning the whole optical configuration. We will evaluate the performance of the new setup and show the feasibility for rapid biomechanical mapping.
The biomechanical properties of the crystalline lens play a crucial role in its visual function. Assessing biomechanical properties of the lens may help with early disease detection and robust assessment of therapeutic interventions. However, measuring the biomechanical properties of the lens is a challenge due to its location inside the eye-globe. In this study, we demonstrate the combination of optical coherence elastography (OCE) and Brillouin microscopy to evaluate the stiffness of porcine lenses ex vivo (N=6). Brillouin microscopy can map the Brillouin-derived longitudinal modulus of the whole lens, but imaging times are lengthy. OCE can provide quantitative measurements of viscoelasticity rapidly, but the limited scattering of the lens limits its in-depth measurements. By combining these two techniques, we show a strong correlation between the Brillouin modulus and OCE-measured Young’s modulus in the lens, enabling depth-wise mapping of the Young’s modulus. The correlation coefficient between the two measurements was R=0.89. Using this correlation, the elasticity of the anterior lens was 2.72±0.89 kPa, and the mean Young’s modulus of the nucleus was 12.92±2.75 kPa. Similarly, the elasticity of the posterior lens was 3.80±1.25 kPa. While both techniques can evaluate the stiffness of the biological tissues separately, our work demonstrates that combining these techniques could enable mapping of the Young’s modulus completely noninvasively in non-scattering tissues such as the crystalline lens.
KEYWORDS: Principal component analysis, Sensors, Signal to noise ratio, Terahertz radiation, Gallium arsenide, Signal detection, Antennas, Superlattices, Terahertz spectroscopy, Spectroscopy
Terahertz (THz) spectroscopy with high sensitivity is essential for biological application considering the strong absorption and scattering effects therein. As the most commonly used THz detector, the photoconductive antenna’s (PCA) response greatly relies on the properties of the substrate’s material. THz detection properties of the PCAs fabricated on low-temperature-grown GaAs (LT-GaAs) and ErAs:GaAs superlattices were compared at the sub-THz band. The detection efficiency of the PCAs with regard to incident laser power was characterized. In addition, using the PCAs as detectors, the signal-to-noise ratio (SNR) and dynamic range (DR) of a terahertz time-domain spectroscopy were quantified. The result indicates that the PCA detector with LT-GaAs has higher efficiency than the one with ErAs:GaAs. Consequently, the corresponding THz spectrometer has better SNR and DR. This result is contrary to the previous report, in which enhanced detection efficiency was observed with ErAs:GaAs-based PCA, which is probably due to the different structures of ErAs:GaAs superlattices used in the experiment.
Embryonic development involves the interplay of driving forces that shape the tissue and the mechanical resistance that the tissue offers in response. While increasing evidence has suggested the crucial role of physical mechanisms underlying embryo development, tissue biomechanics is not well understood due to the lack of techniques that can quantify the stiffness of tissue in-situ with 3D high-resolution and in a non-contact manner. In this work, we used two all-optical technique, optical coherence tomography (OCT) and Brillouin microscopy, to map the longitudinal modulus of the neural tube tissue of mouse embryo in-situ. We found the tissue stiffens significantly after the closure of the neural tube at cranial regions by comparing embryos at E 8.5 and E 9.5. In addition, we observed that the region of fusion following neural tube closure is softer than the adjacent neural folds, and the neural folds show a modulus gradient along dorsal-ventral direction. Furthermore, we found the overlaying ectoderm is much softer and more pliable than the closed neural tube, and thus can be distinguished based on its mechanical properties. In conclusion, we demonstrated the capability of OCT and Brillouin microscopy to quantify tissue modulus of mouse embryos in-situ, and observed a distinct change of tissue modulus during the closure of cranial neural tube, suggesting this method could be helpful in investigating the role of tissue biomechanics in the regulation of embryo development.
The nucleus is the largest and stiffest organelle of eukaryotic cells, and as such, its mechanical properties are tightly related to various cell functions. Many efforts have been devoted to characterize the mechanical properties of nucleus, but the current techniques generally need physical contact of the cell and staining of the nucleus and thus cannot acquire the mechanical information directly. Brillouin microscope is an integration of a confocal microscope and a Brillouin spectrometer, which measures the spectral shift due to the spontaneous Brillouin light scattering, and from that the longitudinal modulus of the sample can be quantified. In this work, by combining the standard Brillouin microscope with the microfluidic technique, we developed a Brillouin flow cytometry that can quantify the mechanical properties of the intact cellular nucleus in a non-contact and label-free manner. As cell flows through a microfluidic channel, its mechanical property at different regions will be sampled by a sub-micron beam spot of the Brillouin microscope. The mechanical information of the nucleus from the cell population can then be identified and extracted via data post-processing, which is further confirmed by co-registering Brillouin data with fluorescence data from the same cell. Currently, the overall throughput of this technique is about 200 cells per hour, mainly relies on the acquisition speed of the spectrometer, which could be readily improved with available technology. We verified the capability of this all-optical technique by distinguishing the stiffness changes of the nucleus that are relevant to physiological and pathological phenomena.
Brillouin spectroscopy is able to measure material’s mechanical properties by analyzing the optical spectrum of acoustically-induced light scattering within a sample. In the past decade, the development of high-resolution Brillouin spectrometers based on virtually-imaged phased array (VIPA) has greatly increased the spectral detection efficiency thus enabling mechanical characterization of biological tissue and biomaterials. Further improvements in spectrometer performances have enabled in vivo measurements at safe power levels and 2D/3D imaging of biological cells. However, it remains a slow technique compared to other imaging modalities, because only one point of the sample can be measured by the traditional backward-scattering configuration at a time. In this work, we demonstrate a parallel detection configuration with 90-degree geometry where the Brillouin shift of hundreds of points in a line can be measured simultaneously. In a 1.1mm-by-1.5mm samples, this novel configuration effectively shortens the acquisition time of 2D Brillouin imaging from hours to ~30 seconds with spatial resolution of ~3um, thus making it a powerful technology for label-free mechanical characterization of tissue and biomaterials.
Embryos undergo dramatic changes in size, shape, and mechanical properties during development, which is regulated by both genetic and environmental factors. Quantifying mechanical properties of different embryonic tissues may represent good metrics for the embryonic health and proper development. Alternations and structure coupled with biomechanical information may provide a way for early diagnosis and drug treatment of various congenital diseases. Many methods have been developed to determine the mechanical properties of the embryo, such as atomic force microscopy (AFM), ultrasound elastography (UE), and optical coherent elastography (OCE). However, AFM is invasive and time-consuming. While UE and OCE are both non-invasive methods, the spatial resolutions are limited to mm to sub-mm, which is not enough to observe the details inside the embryo. Brillouin microscopy can potentially enable non-invasive measurement of the mechanical properties of a sample by measuring the spectra of acoustically induced light scattering therein. It has fast speed (~0.1 second per point) and high resolution (sub-micron), and thus has been widely investigated for biomedical application, such as single cell and tissue. In this work, we utilized this technique to characterize the mechanical property of an embryo. A 2D elasticity imaging of the whole body of an E8 embryo was acquired by a Brillouin microscopy, and the stiffness changes between different organs (such as brain, heart, and spine) were shown. The elasticity maps were correlated with structural information provided by OCT.
Brillouin spectroscopy allows non-invasive measurement of the mechanical properties of a sample by measuring the spectra of acoustically induced light scattering therein, and thus has been widely investigated for biomedical application. Recently, the development of fast Brillouin spectrometry based on virtually-imaged phased array (VIPA) has made in-situ measurement of biomedical sample possible. However, one limitation of current Brillouin technique is the low spectral extinction, which limits the measurement to nearly transparent sample. In order to measure turbid sample, multistage VIPA can be cascaded to gain spectral extinction. For example, spectral extinction of ~80 dB was achieved using three-stage VIPA; however, this approach significantly sacrificed measurement throughput. In this work, we develop a novel spectrometer that achieves high extinction without significant signal loss. To achieve this goal, we combine a two-stage VIPA spectrometer with a triple-pass Fabry-Perot interferometer. The triple-pass Fabry-Perot interferometer acts as a band-pass filter with ~3 GHz bandwidth and ~35-dB spectral extinction. Therefore, the overall extinction of this spectrometer greatly surpasses 80 dB with only ~20% excess loss. We demonstrated the performance of this spectrometer measuring background-free Brillouin spectra from Intralipid solutions and within chicken tissue.
In order to obtain an accurate Avogadro constant with a relative uncertainty of 1×10-8 to redefine the kilogram, the diameter of a perfect single crystal silicon sphere is required with the measurement uncertainty of 0.3 nm using the X-ray
crystal density method. To achieve this, phase-shifting interferometers have been developed. A laser frequency tuning system calibrated by a Fabry-Perot cavity is proposed to improve the laser wavelength and the phase-shift accuracy. The laser frequency standard deviation of the beat frequency is 85 kHz with a gate time of 0.1 s. The gap distances in the diameter determination interferometer are measured based on the laser tuning system, which are 275.3 nm and 110.5 nm, respectively.
A frequency measurement system for dual frequency He-Ne lasers is set up based on an external cavity diode laser
locked to fiber femtosecond optical frequency comb using an Rb clock as a frequency standard. The frequencies of the
Zeeman split orthogonal polarized lasers are measured by beating with the locked diode laser at the same time. Locking
the diode laser to the 1 894 449th comb tooth, the absolute frequency of the diode laser is 473 612 190 000.0 (2.4) kHz,
with a relative frequency uncertainty of 5.1×10-12. A commercial dual frequency He-Ne laser is measured to test the
system, and the results show that the mean absolute frequencies of the horizontal polarized laser and the vertical
polarized laser are 473 612 229 934 kHz and 473 612 232 111 kHz, respectively, with a relative Allan deviation of 5.2×
10-11 at 1 024 s, and the mean split frequency is 2.177 MHz with a standard deviation of 2 kHz.
The Avogadro constant NA is used as one of the several possible routes to redefinition of the kilogram in metrology
today. Usually in order to accurately determine NA, the volume of a perfect single crystal silicon sphere of nearly 1 kg
mass should be measured with a high relative uncertainty, i.e. about 1×10-8. However, the oxide layer grown on the
surface of the silicon sphere causes a remarkable systematic difference between the measured and real diameters. A novel
ellipsometer has been developed to determine the thickness of the oxide layer accurately and automatically. The
arrangement of this instrument is suitable for measuring the layer on the sphere surface. What's more, the measuring is
faster by optimizing the parameters and developing the algorithm of calculating the thickness and refractive index of the
oxide layer. The preliminary simulation result has present. Thus, the uncertainty of the diameter measurement caused by
the oxide layer can be observably reduced. And the further improving of this ellipsometer is discussed in the end.
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