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Optical detection of ultrasound for photoacoustic imaging provides a large bandwidth and high sensitivity at high acoustic frequencies. Therefore, higher spatial resolutions can be achieved using Fabry-Pérot cavity sensors than conventional piezoelectric detection. However, fabrication constraints during the deposition of the sensing polymer layer require precise control of the interrogation beam wavelength to provide optimal sensitivity. This is commonly achieved by employing slowly tunable narrowband lasers as interrogation sources, hence limiting the acquisition speed. We propose instead to use a broadband source and a fast-tunable acousto-optic filter to adjust the interrogation wavelength at each pixel within a few microseconds. We demonstrate the validity of this approach by performing photoacoustic imaging with a highly inhomogeneous Fabry-Pérot sensor.
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An ultra-thin multimode fiber is an ideal platform for minimally invasive microscopy with the advantages of a high density of modes, high spatial resolution, and a compact size. In practical applications, the probe needs to be long and flexible, which unfortunately destroys the imaging capabilities of a multimode fiber. In this work, we propose and experimentally demonstrate sub-diffraction imaging through a flexible probe based on a unique multicore-multimode fiber. A multicore part consists of 120 Fermat's spiral distributed single-mode cores. Each of the cores offers stable light delivery to the multimode part, which provides optimal structured light illumination for sub-diffraction imaging. As a result, perturbation-resilient fast sub-diffraction fiber imaging by computational compressive sensing is demonstrated.
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We report a bending-insensitive multi-core fiber (MCF) for lensless endoscopy imaging with modified fiber geometry that enables optimal light coupling in and out of the individual cores. In a previously reported bending insensitive MCF (twisted MCF), the cores are twisted along the length of the MCF allowing for the development of flexible thin imaging endoscopes with potential applications in dynamic and freely moving experiments. However, for such twisted MCFs the cores are seen to have an optimum coupling angle which is proportional to their radial distance from the center of the MCF. This brings coupling complexity and potentially degrades the endoscope imaging capabilities. In this study, we demonstrate that by introducing a small section (1 cm) at two ends of the MCF, where all the cores are straight and parallel to the optical axis one can rectify the above coupling and output light issues of the twisted MCF, enabling the development of bend-insensitive lensless endoscopes.
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A stimulated Raman microscope is conventionally performed by modulating either the pump or Stokes beam and demodulating the other. Here, we propose a double modulation scheme that modulates both beams at fm and 2fm. Exploiting aliasing and reduction of the repetition rate, we show that the proposed double modulation scheme amplifies the signal amplitude by a factor of 1.5, 2, and 4 for different modulation frequencies and experimental realizations for the same average power at the sample. By deriving the noise power for different sources, we show that the double modulation scheme can perform stimulated Raman scattering (SRS) imaging with an up to 16-fold speed improvement as compared with single beam modulation.
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Two-photon excited fluorescence (2PEF) microscopy is the most popular non-linear imaging method of biomedical samples. State-of-the art 2PEF microscopes use multiple detectors and spectral filter sets to discriminate different fluorophores based on their distinct emission behavior (emission discrimination). One drawback of 2PEF is that fluorescence photons outside the filter transmission range are inherently lost, thereby reducing the imaging efficiency and speed. Furthermore, emission discrimination of different fluorophores may fail if their emission profiles are too similar. Here, we present an alternative 2PEF method that discriminates fluorophores based on their excitation spectra (excitation discrimination). For excitation we use two lasers of different wavelengths (ω1, ω2) resulting in excitation energies at 2ω1, 2ω2, and the mixing energy ω1+ω2. Both lasers are frequency encoded (FE) by an intensity modulation at distinct frequencies while all 2PEF emission is collected on a single detector. The signal is fed into a lock-in-amplifier and demodulated at various frequencies simultaneously. A customized nonnegative matrix factorization (NNMF) then generates fluorescence images that are free of cross talk. Combining FE-2PEF with multiple detectors has the potential to enable the simultaneous imaging of an unprecedented number of fluorophores.
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We implement a near-infrared (NIR) version of compressive Raman imaging that incorporates a digital micromirror device (DMD) and a single-pixel detector for fast chemometric analysis and microscopic imaging. The NIR compressive Raman system is successfully used to detect and image active pharmaceutical ingredients exhibiting polymorphism within compact pharmaceutical tablets. We report the chemical imaging of a mixture of two clopidogrel polymorphs and three excipients in solid tablets with a pixel dwell time of 2.5 ms (0.5 ms per species). These results open the road to fast pharmaceutical tablet quality control imaging using compressive Raman technology.
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Excipientes , Análise Espectral Raman , Excipientes/análise , Análise Espectral Raman/métodos , Comprimidos/química , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Controle de Qualidade , Tecnologia Farmacêutica/métodosRESUMO
Impulsive stimulated Raman scattering (ISRS) is a robust technique for studying low frequency (<300 cm-1) Raman vibrational modes, but ISRS has faced difficulty in translation to an imaging modality. A primary challenge is the separation of the pump and probe pulses. Here we introduce and demonstrate a simple strategy for ISRS spectroscopy and hyperspectral imaging that uses complementary steep edge spectral filters to separate the probe beam detection from the pump and enables simple ISRS microscopy with a single-color ultrafast laser source. ISRS spectra are obtained that span from the fingerprint region down to <50 cm-1 vibrational modes. Hyperspectral imaging and polarization-dependent Raman spectra are also demonstrated.
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We report on the use of a thin diffuser placed in the close vicinity of a camera sensor as a simple and effective way to superlocalize plasmonic nanoparticles in 3D. This method is based on holographic reconstruction via quantitative phase and intensity measurements of a light field after its interaction with nanoparticles. We experimentally demonstrate that this thin diffuser can be used as a simple add-on to a standard bright-field microscope to allow the localization of 100â nm gold nanoparticles at video rate with nanometer precision (1.3â nm laterally and 6.3â nm longitudinally). We exemplify the approach by revealing the dynamic Brownian trajectory of a gold nanoparticle trapped in various pockets within an agarose gel. The proposed method provides a simple but highly performant way to track nanoparticles in 3D.
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We present and model a dark-field illumination scheme for coherent anti-Stokes Raman scattering (DF-CARS) that highlights the interfaces of an object with chemical sensitivity. The proposed DF-CARS scheme uses dedicated arrangements of the pump kp1, Stokes kS and probe kp2 beams' k-wave-vectors to address the sample's interfaces along the x, y or z axis. The arrangements of the incident k-wave-vectors are derived from the Ewald sphere representation of the outgoing anti-Stokes radiation and the effective CARS excitation wave-vector keff = kp1 + kp2 - kS under the intention to avoid probing the object frequency K(0,0,0), i.e., the contribution of a homogeneous sample (dark-field configuration). We suggest a possible experimental realization using simple masks placed in the back pupil of the excitation microscope objective lens. Applying a full vectorial model, the proposed experimental implementation is numerically investigated on grounds of the Debye-Wolff integral and dynadic Green function to confirm the predicted chemical interface contrast.
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Fourier ptychography tomography (FPT) is a novel computational technique for coherent imaging in which the sample is numerically reconstructed from images acquired under various illumination directions. FPT is able to provide three-dimensional (3D) reconstructions of the complex sample permittivity with an increased resolution compared to standard microscopy. In this work, FPT is applied to coherent anti-Stokes Raman scattering (CARS) imaging. We show on synthetic data that complex third-order susceptibilities can be reconstructed in 3D from a limited number of widefield CARS images. In addition, we observe that the non-linear interaction increases significantly the potential of CARS-FPT compared to linear FPT in terms of resolution. In particular, with a careful choice of the pump and Stokes beam directions, CARS-FPT is able to provide optical sectioning even in transmission configuration.
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In this Letter, we report a high-efficiency, miniaturized, ultra-fast coherent beam, combined with 3D-printed micro-optics directly on the tip of a multicore fiber bundle. The highly compact device footprint (180 µm in diameter) facilitates its incorporation into a minimally invasive ultra-thin nonlinear endoscope to perform two-photon imaging.
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Endoscópios , Endoscopia , Endoscopia Gastrointestinal , Óptica e Fotônica , Fótons , Impressão TridimensionalRESUMO
We report a shot noise limited high-speed stimulated Raman microscopy platform allowing to acquire molecular vibrational spectra over 200 cm-1 in 12 µs at a scan rate of 40kHz. Using spectral focusing together with optimized acousto-optics programmable dispersive filters, the designed low noise imaging platform performs chemical imaging of dynamical processes such as Mannitol crystal hydration and reaches a signal to noise ratio sufficient to perform label free histological imaging on frozen human colon tissue slides.
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Neoplasias do Colo/química , Análise Espectral Raman/métodos , Diagnóstico por Imagem , Humanos , Manitol/química , Azeite de Oliva/química , Polimetil Metacrilato/química , Poliestirenos/química , Sensibilidade e Especificidade , Soroalbumina Bovina/química , Razão Sinal-Ruído , TriazinasRESUMO
The fidelity of stimulated Raman scattering (SRS) microscopy images is impaired by artifacts such as thermal lensing, cross-phase modulation and multi-photon absorption. These artifacts affect differently the stimulated Raman loss (SRL) and stimulated Raman gain (SRG) channels making SRL and SRG image comparisons attractive to identify and correct SRS image artifacts. To provide answer to the question: "Can I trust my SRS images?", we designed a novel, but straightforward SRS scheme that enables the dectection of the stimulated Raman gain and loss (SRGAL) simultaneously at the same pixel level. As an advantage over the conventional SRS imaging scheme, SRGAL doubles the SRS signal by acquiring both SRL as well as SRG and allows for the identification of SRS artifacts and their reduction via a balanced summation of the SRL and SRG images.
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Stimulated Raman Scattering (SRS) imaging can be hampered by non-resonant parasitic signals that lead to imaging artifacts and eventually overwhelm the Raman signal of interest. Stimulated Raman gain opposite loss detection (SRGOLD) is a three-beam excitation scheme capable of suppressing this nonlinear background while enhancing the resonant Raman signal. We present here a compact electro-optical system for SRGOLD excitation which conveniently exploits the idler beam generated by an optical parametric oscillator (OPO). We demonstrate its successful application for background suppressed SRS imaging in the fingerprint region. This system constitutes a simple and valuable add-on for standard coherent Raman laser sources since it enables flexible excitation and background suppression in SRS imaging.
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We report a line-scanning imaging modality of compressive Raman technology with a single-pixel detector. The spatial information along the illumination line is encoded onto one axis of a digital micromirror device, while spectral coding masks are applied along the orthogonal direction. We demonstrate imaging and classification of three different chemical species.
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We present a theoretical and numerical study of coherent anti-Stokes Raman scattering Fourier ptychography microscopy (CARS-FPM), a scheme that has not been considered so far in the previously reported CARS wide-field imaging schemes. In this approach, the distribution of the Raman scatterer density of the sample is reconstructed numerically from CARS images obtained under various angles of incidences of the pump or Stokes beam. Our inversion procedure is based on an accurate vectorial model linking the CARS image to the sample and yields both the real and imaginary parts of the susceptibility, the latter giving access to the Raman information, with an improved resolution.
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We report a line scanning imaging modality of compressive Raman technology with spatial frequency modulated illumination using a single pixel detector. We demonstrate the imaging and classification of three different chemical species at line scan rates of 40 Hz.
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Real-time vibrational microscopy has been recently demonstrated by various techniques, most of them utilizing the well-known schemes of coherent anti-stokes Raman scattering and stimulated Raman scattering. These techniques readily provide valuable chemical information mostly in the higher vibrational frequency regime (>400 cm-1). Addressing the low vibrational frequency regime (<200 cm-1) is challenging due to the usage of spectral filters that are required to isolate the signal from the Rayleigh scattered excitation field. In this Letter, we report on rapid, high-resolution, low-frequency (<130 cm-1) vibrational microscopy using impulsive coherent Raman excitation. By combining impulsive excitation with a fast acousto-optic delay line, we detect the Raman-induced optical Kerr lensing and spectral shift effects with a 25 µs pixel dwell time to produce shot-noise limited, low-frequency hyper-spectral images of various samples.
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Combining stimulated emission depletion and fluorescence correlation spectroscopy (STED-FCS) provides a powerful and sensitive tool for studying the molecular dynamics in live cells with high spatio-temporal resolution. STED-FCS gives access to molecular diffusion characteristic at the nanoscale occurring within short period of times. However due to the incomplete suppression of fluorescence in the STED process, the STED-FCS point spread function (PSF) deviates from a Gaussian shape and challenges the analysis of the auto-correlation curves obtained by FCS. Here, we model the effect of the incomplete fluorescence suppression in STED-FCS experiments and propose a new fitting model improving the accuracy of the diffusion times and average molecule numbers measurements. The implementation of a STED module with pulsed laser source on a commercial confocal/FCS microscope allowed us to apply the STED-background corrected model to fit the STED-FCS measurements. The experimental results are in good accordance with the theoretical analysis both for the number of molecules and the diffusion time which decrease accordingly with the STED power.
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Processamento de Imagem Assistida por Computador/métodos , Microscopia Intravital/métodos , Modelos Químicos , Espectrometria de Fluorescência/métodos , Citoesqueleto de Actina/metabolismo , Animais , Células COS , Chlorocebus aethiops , Difusão , Fluorescência , Microscopia Intravital/instrumentação , Citometria de Varredura a Laser/instrumentação , Citometria de Varredura a Laser/métodos , Lasers , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Software , Espectrometria de Fluorescência/instrumentaçãoRESUMO
We experimentally implement a compressive Raman technology (CRT) that incorporates chemometric analysis directly into the spectrometer hardware by means of a digital micromirror device (DMD). The DMD is a programmable optical filter on which optimized binary filters are displayed. The latter are generated with an algorithm based on the Cramer-Rao lower bound. We compared the developed CRT microspectrometer with two conventional state-of-the-art Raman hyperspectral imaging systems on samples mimicking microcalcifications relevant for breast cancer diagnosis. The CRT limit of detection significantly improves, when compared to the CCD based system, and CRT ultimately allows 100× and 10× faster acquisition speeds than the CCD- and EMCCD-based systems, respectively.