Search

We present the use of stimulated Brillouin scattering spectroscopy to achieve rapid measurements of cell biomechanics in a flow cytometer setup. Specifically, our stimulated Brillouin scattering flow cytometry can acquire at a rate of 200 Hz, with a spectral acquisition time of 5 ms, which marks a 10x improvement compared to previous demonstrations of spontaneous Brillouin scattering flow cytometry. We experimentally validate our stimulated Brillouin scattering flow cytometer by measuring cell populations of normal breast epithelial cells and metastatic breast epithelial cancer cells., Competing Interests: None., (© 2024 Optica Publishing Group.)

Brillouin spectrometers, used for characterizing material mechanical properties, traditionally employ etalons such as Fabry-Pérot interferometers and virtually imaged phased arrays (VIPA) that use spatial dispersion of the spectrum for measurement. Here, we introduce what we believe to be a novel approach to Brillouin spectroscopy using hot atomic vapors. Using laser induced circular dichroism of the rubidium D2 line in a ladder-type configuration, we developed a narrow-band monochromator for Brillouin analysis. Unlike etalon-based spectrometers, atomic line monochromators operate in free-space, facilitating Brillouin spectroscopy integration with microscopy instruments. We report the transmission and spectral resolution performances of the spectrometer and demonstrate Brillouin spectra measurements in liquids.

Spectral imaging techniques extract spectral information using dispersive elements in combination with optical microscopes. For rapid acquisition, multiplexing spectral information along one dimension of imaged pixels has been demonstrated in hyperspectral imaging, as well as in Raman and Brillouin imaging. Full-field spectroscopy, i.e., multiplexing where imaged pixels are collected in 2D simultaneously while spectral analysis is performed sequentially, can increase spectral imaging speed, but so far has been attained at low spectral resolutions. Here, we extend 2D multiplexing to high spectral resolutions of ∼80 MHz (∼0.0001 nm) using high-throughput spectral discrimination based on atomic transitions.

Corneal biomechanics play a critical role in maintaining corneal shape and thereby directly influence visual acuity. However, direct corneal biomechanical measurement in-vivo with sufficient accuracy and a high spatial resolution remains an open need. Here, we developed a three-dimensional (3D) motion-tracking Brillouin microscope for in-vivo corneal biomechanics mapping. The axial tracking utilized optical coherence tomography, which provided a tracking accuracy better than 3 µm. Meanwhile, 10 µm lateral tracking was achieved by tracking pupils with digital image processing. The 3D tracking enabled reconstruction of depth-dependent Brillouin distribution with a high spatial resolution. This superior technical performance enabled the capture of high-quality mechanical mapping in vivo even while the subject was breathing normally. Importantly, we improved Brillouin spectral measurements to achieve relative accuracy better than 0.07% verified by rubidium absorption frequencies, with 0.12% stability over 2000 seconds. These specifications finally yield the Brillouin measurement sensitivity that is required to detect ophthalmology-relevant corneal biomechanical properties., Competing Interests: The authors declare no conflicts of interest., (© 2022 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement.)

To understand the dynamics of tissue stiffness during neural tube formation and closure in a murine model, we have developed a multimodal, coaligned imaging system combining optical coherence tomography (OCT) and Brillouin microscopy. Brillouin microscopy can map the longitudinal modulus of tissue but cannot provide structural images. Thus, it is limited for imaging dynamic processes such as neural tube formation and closure. To overcome this limitation, we have combined Brillouin microscopy and OCT in one coaligned instrument. OCT provided depth-resolved structural imaging with a micrometer-scale spatial resolution to guide stiffness mapping by Brillouin modality. 2D structural and Brillouin frequency shift maps were acquired of mouse embryos at gestational day (GD) 8.5, 9.5, and 10.5 with the multimodal system. The results demonstrate the capability of the system to obtain structural and stiffness information simultaneously.

Spectral contrast, the ability to measure frequency components of vastly different intensity, is critical in optical spectroscopy. For high spectral contrast at high spectral resolution, scanning etalons are generally used, as they allow cascading multiple dispersive elements. However, scanning instruments are inherently limited in terms of acquisition speed. Here we report a single-shot cascaded spectrometer design, in which light is dispersed along a single dispersion direction at every stage and thus can be recirculated in the same etalon multiple times. Using this design principle, we demonstrate single-shot spectral measurements at sub-gigahertz resolution and unprecedented spectral contrast (∼80 d B ).

Assessing the biomechanical properties of the crystalline lens can provide crucial information for diagnosing disease and guiding precision therapeutic interventions. Existing noninvasive methods have been limited to global measurements. Here, we demonstrate the quantitative assessment of the elasticity of crystalline lens with a multimodal optical elastography technique, which combines dynamic wave-based optical coherence elastography (OCE) and Brillouin microscopy to overcome the drawbacks of individual modalities. OCE can provide direct measurements of tissue elasticity rapidly and quantitatively, but it is a challenge to image transparent samples such as the lens because this technique relies on backscattered light. On the other hand, Brillouin microscopy can map the longitudinal modulus with micro-scale resolution in transparent samples. However, the relationship between Brillouin-deduced modulus and Young's modulus is not straightforward and sample dependent. By combining these two techniques, we can calibrate Brillouin measurements with OCE, based on the same sample, allowing us to completely map the Young's modulus of the crystalline lens. The combined system was first validated with tissue-mimicking gelatin phantoms of varying elasticities (N = 9). The OCE data was used to calibrate the Brillouin shift measurements and subsequently map the Young's modulus of the phantoms. After validation, OCE and Brillouin measurements were performed on ex-vivo porcine lenses (N = 6), and the Young's modulus of the lenses was spatially mapped. The results show a strong correlation between Young's moduli measured by OCE and longitudinal moduli measured by Brillouin microscopy. The correlation coefficient R was 0.98 for the phantoms and 0.94 for the lenses, respectively. The mean Young's modulus of the anterior and posterior lens was 1.98 ± 0.74 kPa and 2.93 ± 1.13 kPa, respectively, and the Young's modulus of the lens nucleus was 11.90 ± 2.94 kPa. The results presented in this manuscript open a new way for truly quantitative biomechanical mapping of optically transparent (or low scattering) tissues in 3D., Competing Interests: The authors declare that there are no conflicts of interest related to this article., (© 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreement.)

In Brillouin microscopy, absorption-induced photodamage of incident light is the primary limitation on signal-to-noise ratio in many practical scenarios. Here we show that 660 nm may represent an optimal wavelength for Brillouin microscopy as it offers minimal absorption-mediated photodamage at high Brillouin scattering efficiency and the possibility to use a pure and narrow laser line from solid-state lasing medium. We demonstrate that live cells are ~80 times less susceptible to the 660 nm incident light compared to 532 nm light, which overall allows Brillouin imaging of up to more than 30 times higher SNR. We show that this improvement enables Brillouin imaging of live biological samples with improved accuracy, higher speed and/or larger fields of views with denser sampling., Competing Interests: The authors declare that there are no conflicts of interest related to this article.

Two-stage cross axis VIPA spectrometers have been widely used in Brillouin microscopy since they provide single shot spectral measurements at high throughput and extinction. However, this spectrometer configuration presents challenges such as size, cost and alignment difficulties between the two cascaded etalons. Here, we present a cross-axis VIPA spectrometer that implements a single etalon, using a light recirculation architecture to achieve the multistage configuration., Competing Interests: The authors declare that there are no conflicts of interest related to this article.

Adaptive optics systems have been integrated in many imaging modalities in order to correct for aberrations that are introduced by samples and optical elements. Usually, the optical system has access to a guide star (i.e., a point-like structure that is smaller than the diffraction limit). This guide star can be used as a beacon for adaptive optics enhancement. In contrast, for spectroscopy and densely-labeled fluorescent samples, the signal is diffused throughout the entire beam path and is not confined to a well-defined point-like structure. Here, we show analytically and experimentally that, in these scenarios, adaptive optics systems are expected to yield significantly lower signal enhancement than when a guide star is available. We discuss adaptive optics' performance degradation for different imaging modalities (e.g., confocal, multi-photon microscopy) and identify solutions to overcome low signal enhancements.

VIPA (virtually imaged phase array) spectrometers have enabled rapid Brillouin spectrum measurements and current designs of multi-stage VIPA spectrometers offer enough spectral extinction to probe transparent tissue, cells and biomaterials. However, in highly scattering media or in the presence of strong back-reflections, such as at interfaces between materials of different refractive indices, VIPA-based Brillouin spectral measurements are limited. While several approaches to address this issue have recently been pursued, important challenges remain. Here we have adapted the design of coronagraphs used for exosolar planet imaging to the spectral domain and integrated it in a double-stage VIPA spectrometer. We demonstrate that this yields an increase in extinction up to 20 dB, with nearly no added insertion loss. The power of this improvement is vividly demonstrated by Brillouin imaging close to reflecting interfaces without index matching or sample tilting.

Brillouin imaging of turbid biological tissues requires an effective rejection of the background noise due to elastic scattering of probe laser light. We have developed a narrowband spectral notch filter based on a pair of a free-space Fabry-Perot etalon and a mirror. The etalon filter in a 4-pass configuration is able to suppress elastically-scattered laser light with a high extinction ratio of > 40 dB and transmit inelastically-scattered light in a frequency shift range of 2-14 GHz with only 2 dB insertion loss. We also describe a simple etalon that enables us to use semiconductor diode laser sources for Brillouin microscopy by removing spontaneous emission noise. Using a clinically-viable Brillouin microscope employing these filters, we demonstrate the first Brillouin confocal imaging of the sclera and conjunctiva of the porcine eye.

Several phenomena have been recently exploited to circumvent scattering and have succeeded in imaging or focusing light through turbid layers. However, the requirement for the turbid medium to be steady during the imaging process remains a fundamental limitation of these methods. Here we introduce an optical imaging modality that overcomes this challenge by taking advantage of the so-called shower-curtain effect, adapted to the spatial-frequency domain via speckle correlography. We present high resolution imaging of objects hidden behind millimeter-thick tissue or dense lens cataracts. We demonstrate our imaging technique to be insensitive to rapid medium movements (> 5 m/s) beyond any biologically-relevant motion. Furthermore, we show this method can be extended to several contrast mechanisms and imaging configurations.

Brillouin spectroscopy has been used to characterize shear acoustic phonons in materials. However, conventional instruments had slow acquisition times over 10 min per 1 mW of input optical power, and they required two objective lenses to form a 90° scattering geometry necessary for polarization coupling by shear phonons. Here, we demonstrate a confocal Brillouin microscope capable of detecting both shear and longitudinal phonons with improved speeds and with a single objective lens. Brillouin scattering spectra were measured from polycarbonate, fused quartz, and borosilicate in 1-10 s at an optical power level of 10 mW. The elastic constants, phonon mean free path and the ratio of the Pockels coefficients were determined at microscopic resolution.

Two-photon polymerization has enabled precise microfabrication of three-dimensional structures with applications spanning from photonic microdevices, drug delivery systems, and cellular scaffolds. We present two-photon collagen crosslinking (2P-CXL) of intact corneal tissue using riboflavin and femtosecond laser irradiation. Collagen fiber orientations and photobleaching were characterized by second harmonic generation and two-photon fluorescence imaging, respectively. Measurement of local changes in longitudinal mechanical moduli with confocal Brillouin microscopy enabled the visualization of the cross-linked pattern without perturbation of the surrounding non-irradiated regions. 2P-CXL induced stiffening was comparable to that achieved with conventional one-photon CXL. Our results demonstrate the ability to selectively stiffen biological tissue in situ at high resolution with broad implications in ophthalmology, laser surgery, and tissue engineering.

We report a novel configuration of a two-stage virtually imaged phased array spectrometer that enables high-throughput sub-GHz spectroscopy at a high finesse (>750). Two etalons with different free spectral range and different dispersion are arranged in an orthogonal direction and spread the spectrum across two dimensions, with a greatly improved rejection ratio of white-light background noise. A proof-of-concept application for Brillouin spectroscopy is demonstrated.

The mechanical stability of the cornea is critical for maintaining its normal shape and refractive function. Here, we report an observation of the mechanical resonance modes of the cornea excited by sound waves and detected by using phase-sensitive optical coherence tomography. The cornea in bovine eye globes exhibited three resonance modes in a frequency range of 50-400 Hz. The vibration amplitude of the fundamental mode at 80-120 Hz was ~8 µm at a sound pressure level of 100 dB (2 Pa). Vibrography allows the visualization of the radially symmetric profiles of the resonance modes. A dynamic finite-element analysis supports our observation.

We demonstrate sub-Rayleigh limit imaging of an object via speckle illumination. Imaging beyond the conventional Rayleigh limit is achieved by illuminating the object with pseudothermal light that exhibits a random speckle pattern. An object image is reconstructed from the second-order correlation measurement and the resolution of the image, which exceeds the Rayleigh limit, is shown to be related to the size of the speckle pattern that is tied to the lateral coherence length of the pseudothermal light.

We report the first Brillouin measurement of the human eye in vivo. We constructed a Brillouin optical scanner safe for human use by employing continuous-wave laser light at 780 nm at a low power of 0.7 mW. With a single scan along the optic axis of the eye, the axial profile of Brillouin frequency shift was obtained with a pixel acquisition time of 0.4 s and axial resolution of about 60 μm, showing the depth-dependent biomechanical properties in the cornea and lens.

We demonstrate a high-resolution high-extinction parallel spectrometer for Brillouin spectroscopy of turbid samples. Cascading multiple VIPA etalons in the cross-axis configuration allowed us to achieve a high extinction ratio of up to 80 dB with sub-GHz resolution. Using a three-stage VIPA, we obtained the Brillouin spectra from Intralipid solutions at concentrations up to 10%.

We demonstrate a method of cascading multiple diffractive elements for improving the purity of spectral dispersion. The cross-axis cascade was implemented in a two-stage grating spectrograph, resulting in a hundredfold reduction of stray light and a high dynamic range up to -75 dB. The technique can be used for parallel spectral measurements and processing.

We describe fluorescence imaging using the second-order correlation of entangled photon pairs. The proposed method is based on the principle that one photon of the pair carries information on where the other photon has been absorbed and has produced fluorescence in a sample. Because fluorescent molecules serve as "detectors" breaking the entanglement, multiply-scattered fluorescence photons within the sample do not cause image blur. We discuss experimental implementations.