Brillouin microscopy is a label-free and noncontact optical method to measure fundamental viscoelastic properties of biological systems. One of the main challenges in Brillouin microscopy is given by the strong Rayleigh background light overcoming the Brillouin peaks that are spectrally located at a few GHz from the laser frequency. Here, we demonstrate a common-path and broadband notch filter providing an unprecedented 65 dB extinction ratio. We demonstrate the filter by acquiring Brillouin spectra of highly turbid samples, such as pure milk and unprocessed vertebra bone tissues.
Non-linear optical (NLO) microscopy techniques like coherent anti-Stokes Raman scattering (CARS) are highly effective tools for label-free vibrational imaging, allowing for chemical analysis of biological samples in their native state. We introduce video-rate wide-field CARS microscopy over a vast field of view (tens of micrometers) to enable real-time analysis of fast biological dynamics with down to few-millisecond time resolution. We generate stable broadband Stokes pulses in a YAG crystal using an amplified ytterbium laser source delivering 260-fs high-energy (μJ-level) pulses in the near-infrared. Our system combines fast and tunable single-wavelength real-time imaging with the acquisition of hypercubes, chemically more information-rich.
Coherent anti-Stokes Raman scattering (CARS) is a highly effective Non-Linear Optical (NLO) microscopy technique for label-free vibrational imaging of unperturbed samples. We present a novel scheme for video-rate wide-field CARS microscopy over a wide (up to 100-μm diameter) field of view, based on an Ytterbium amplified laser followed by white-light generation and optical parametric amplification. It records the full fingerprint region of the molecular vibrational spectrum and enables chemically-specific real-time characterization of fast dynamics with down to few-milliseconds time resolution. The system can perform either real-time imaging at single Raman shifts or acquire three-dimensional hypercubes, chemically richer in information.
Stimulated Raman Histology (SRH) can be used for label-free histological tissue analysis. However the current approaches are limited to two Raman peaks, thus restricting chemically-specific insights. We present a broadband coherent Raman platform (CRP), which overcomes these limitations combining an all-fiber dual output laser and a multichannel lock-in amplifier. This enables the simultaneous detection of 38 channels across the entire CH spectrum (2800-3100 cm-1) in parallel, facilitating chemometric and multimodal tissue analysis. By incorporating AI-driven virtual H&E staining and tissue segmentation for diagnostic purposes, it advances SRH towards clinical-diagnostic.
Image classification using Deep Ensemble Learning and Transfer Learning methods is performed on a small, labeled dataset of multimodal nonlinear optical microscopy images coming from Stimulated Raman Scattering, Two Photon Excited Fluorescence and Optical Transmission, to differentiate proliferating cancer cells from senescent ones, a peculiar phenotype following an anti-cancer treatment responsible for tumour relapse. The superior performances of the Deep Ensemble Transfer Learning approach are compared with other less complex neural network architectures. Ultimately, the predictions of the neural network are evaluated using the Grad-CAM visualization approach, which allows highlighting the most important features in the input images responsible for the labels assigned by the network.
Recent oncology research highlights that senescence, once deemed beneficial in cancer treatments, can contribute to cancer relapse. Detecting therapy-induced senescent cells is challenging due to their complexity and lack of specific markers. Nonlinear optical (NLO) microscopy provides a fast, non-invasive, label-free detection solution. To distinguish between senescent and proliferating cells, here we present the development of a deep learning architecture based on multimodal NLO microscopy images coming from Stimulated Raman Scattering, Two Photon Excited Fluorescence and Optical Transmission. Despite limited labeled data, Transfer Learning, Data Augmentation, and Ensemble Learning techniques allowed us to achieve an accuracy over 90%. Ultimately, the predictions of the neural network are evaluated using the Grad-CAM visualization approach, which allows highlighting the most important features in the input images responsible for the labels assigned by the network. This work reveals the effectiveness of deep learning in senescence classification, potentially advancing treatment strategies.
We present label-free morpho-chemical cancer cell phenotyping by combining confocal Raman micro-spectroscopy and three-dimensional holotomography. Observing colon cancer cell types with different progression stages from adenoma to metastasis, we demonstrate the advantage of a multimodal approach for rapid and accurate cancer cell phenotyping. We introduce data processing pipelines to decode and comprehensively interpret molecular and structural information from hyperspectral Raman images and co-registered refractive index tomograms. Finally, we investigate and discuss any unique or shared information that combined Raman spectroscopy and holotomography can provide when characterizing the same sample, and how this synergy advances cell type differentiation.
In this novel multimodal wide-field Raman microscope, spectra are obtained by the time-domain Fourier-transform method. The wide-field approach enables faster collection of Raman maps, while the time-domain method disentangles fluorescence and Raman signals. This is obtained by choosing a proper sampling of the interferogram, thanks to the use of an ultrastable common-path birefringent interferometer. Validation of the system is performed on plastic microbeads; multimodality is demonstrated by fluorescence and Raman maps of a few-layers transition metal dichalcogenide sample.
We combine an all-fiber dual wavelength, self-synchronized laser and a dedicated multi-channel detection unit to perform state-of-the-art multiplex Stimulated Raman Scattering (SRS) microscopy. The system covers the full CH spectrum in 1 μs reaching shot-noise limited performances with 25 μW per detection channel. This all-inone solution is based on a passively synchronized dual-wavelength laser source with shot-noise limited relative intensity noise from 600 kHz and a modular multi-channel lock-in detection unit. The synergistic design between laser source and detection system simplifies multiplex SRS implementation for real-time full-chemical imaging.
We present a novel wide-field Raman microscope, based on the time-domain Fourier-transform method. This enables parallel acquisition of Raman spectra on all the pixels of the 2D detector; the resulting wide-field approach allows faster collection of Raman maps with respect to standard raster-scanning methods. In addition, the time-domain method disentangles fluorescence and Raman signals. The system is robust and stable, thanks to the use of an ultrastable common-path birefringent interferometer. Validation of the system is performed on plastic microbeads and on a few-layers transition metal dichalcogenide sample.
Recently, anticancer treatments were discovered to induce cell senescence other than death, a critical phenotype driving tumor recurrence. This calls for the development of safe, precise, and rapid tools to reveal critical therapy-induced senescence (TIS). Here, we present label-free multimodal nonlinear optical (NLO) microscopy as a powerful technique to spot early TIS. We home-built a microscope including different NLO modalities: Stimulated Raman Scattering (SRS), forward and epi-detected Coherent Anti-Stokes Raman Scattering (CARS and E-CARS), and Two-Photon Excited Fluorescence (TPEF). The infrared laser source outputs synchronized narrowband 780 nm pump pulses and 950-1050 nm tunable Stokes pulses, so to match the CH-stretching region of the Raman spectrum. Thanks to the co-registration of these NLO signals from label-free TIS cells and controls, we unveiled quantitative all-optical traits of early-stage TIS, monitored over 72 hours of treatment. TPEF from metabolic coenzymes combined with E-CARS from cardiolipin and cytochrome C indicated an shrinking of mitochondrial networks. CARS and SRS revealed lipid vesicles accumulation in cytoplasms. Nuclei enlarged irregularly, visualized via subtraction of SRS signals of proteins and lipids, and CARS from deoxyribose. We believe our results will strongly influence anticancer pre-clinical studies and translated clinical applications, constituting a quick, non-invasive, and accurate aid to expose TIS manifestation in tumors.
Coherent anti-Stokes Raman Scattering (CARS) microscopy is a label-free vibrational imaging technique that delivers chemical maps of cells and tissues. CARS employs two narrowband picosecond pulses (pump and Stokes) that are spatiotemporally superimposed at the sample plane to probe a single vibrational mode. Broadband CARS (BCARS) combines narrowband pump pulses with broadband Stokes pulses to record broad vibrational spectra. Despite many technological advancements, BCARS microscopes still struggle to image biological samples spanning the entire Raman active region of biological samples (400-3100 cm-1). Here, we demonstrate a novel BCARS method to answer this need. Our experimental setup is based on a femtosecond fiber laser at 1035 nm and 2 MHz repetition rate, thus delivering high energy pulses used for generating sub-20 fs broadband Stokes pulses by white-light continuum in a bulk YAG crystal, a compact and alignment-insensitive technique. Combining them with narrowband picosecond pulses, we can generate a CARS signal with high (< 10 cm-1) spectral resolution in the entire Raman window exploiting both two-color and three-color excitation mechanisms. The system is equipped with a home-made transmission microscope to image cells and tissue at high-speed (< 3 ms) and large field of views. Using a post-processing pipeline, we deliver high-quality chemical maps, identifying the main chemical compounds in cancer cells and discriminating tumorous from healthy regions in liver slices of mouse models, unveiling the path for applications in histopathological settings.
Recent studies have shown that common anticancer treatments can induce cell senescence rather than death, a critical phenotype governing tumor recurrence. This calls for the urgent development of safe, precise, and quick tools to unveil critical Therapy-Induced Senescence (TIS). Merging different coherent Raman and multiphoton techniques, we present label-free multimodal nonlinear optical (NLO) microscopy as a powerful tool to spot early TIS. We home-built a microscope including different NLO modalities: Stimulated Raman Scattering (SRS), forward and epi-detected Coherent Anti-Stokes Raman Scattering (CARS and E-CARS), and Two-Photon Excited Fluorescence (TPEF). The infrared laser source outputs synchronized narrowband 780 nm pump pulses and 950-1050 nm tunable Stokes pulses, so to match the CH-stretching region of the Raman spectrum. Thanks to the co-registration of these diverse techniques applied on label-free TIS cells and controls, we exposed quantitative hallmarks of early TIS, confirmed by comparing different optical signals monitored over 72 hours of treatment. TPEF from metabolic coenzymes combined with E-CARS from cardiolipin and cytochrome C indicated an early shrinking of mitochondria. CARS and SRS revealed lipid vesicles overproduction and accumulation. Nuclei enlarged irregularly, visualized via subtraction of SRS signals of proteins and lipids, and CARS from deoxyribose. We consider our results will strongly influence anticancer pre-clinical studies and translated clinical applications, helping to identify quickly, non-invasively, and quantitatively TIS in human tumors.
Cancer research recently revealed that anticancer therapies can cause cell senescence instead of death, a phenotype governing tumor relapse. Developing safe, quick, and precise tools to spot such therapy-induced senescence (TIS) is an urgency.
We present multimodal coherent Raman and multiphoton nonlinear optical microscopy as powerful to unveil TIS, via a home-built microscope including forward-detected Stimulated Raman Scattering, forward and epi-detected Coherent Anti-Stokes Raman Scattering, Two-Photon Excited Fluorescence and Second-Harmonic Generation modalities. We exposed early TIS in human cancer cells, confirmed comparing diverse signals during therapy period.
We consider our findings will strongly influence anticancer practices, helping prevent tumor recurrence.
Coherent Raman scattering (CRS) microscopy is a powerful third-order nonlinear optical technique for label-free chemical identification of molecules based on their intrinsic vibrational spectrum. It can reach video-rate imaging speed and detect very dilute species. I will review the working principles and state of the art of this technique, presenting our recent results and perspectives. I will also show how artificial intelligence methods can be employed effectively to increase the signal-to-noise ratio and extract the relevant information from the large experimental hyperspectral datacubes.
Coherent anti-Stokes Raman scattering is an extremely powerful non-linear optical (NLO) microscopy technique for label-free vibrational imaging allowing for chemical characterization of biological samples in their native state. We introduce here video-rate wide-field signal generation and acquisition over a large field of view (tens of micrometers) allowing for real-time investigation of fast biological dynamics. To this aim, our innovative approach employs an amplified femtosecond ytterbium laser source delivering high energy (≈ µJ) pulses in the near-infrared at 1035-nm central wavelength and 2-MHz repetition rate from which pump and Stokes beams are generated. Narrowband pump pulses with ≈1.1 nm bandwidth (10 cm−1) guarantee sufficient spectral resolution for the vibrational Lorentian peaks. Broadband Stokes pulses in the 1100 to 1500 nm range are produced via supercontinuum generation in 10-mm YAG crystal and amplified in a non-collinear optical parametric amplifier (NOPA). This allows us to acquire hypercubes that cover the entire fingerprint region of the molecular vibrational spectrum, the richest in chemical information. Our results pave the way for future clinical diagnostics applications with video-rate imaging capabilities.
We propose a novel approach to broadband coherent anti-Stokes Raman scattering (B-CARS) based on a femtosecond laser at 1035 nm and 2 MHz repetition rate. These features of the driving laser enable white-light continuum generation in bulk media, employed as broadband Stokes. In this way, we demonstrate state-of-the-art acquisition speed (<1 ms/pixel) with unprecedented sensitivity (≈14.1 mmol/L) when detecting dimethyl-sulfoxide in water, covering the whole fingerprint region. To further enhance the performance of the system, we designed an innovative spectral denoiser based on a convolutional neural network, coupled with a post-processing pipeline to distinguish different chemical species of biological tissues.
We present a novel broadband coherent anti-Stokes Raman scattering (B-CARS) approach, employing a femtosecond fiber laser at 1035 nm and 2 MHz repetition rate. These characteristics of the driving laser enable to generate broadband Stokes pulses through white-light supercontinuum in bulk media and to use higher pulse energy to shine the sample, thus producing stronger signals. In this way, we demonstrate state-of-the-art acquisition speed (<1 ms/pixel) with unprecedented sensitivity (≈14.1 mmol/L), covering the whole fingerprint vibrational region. The B-CARS microscope, coupled with a deep learning-based post-processing pipeline, delivers high-quality hyperspectral images identifying the main Raman features of heterogeneous biological tissues.
We present several approaches to high-speed broadband stimulated Raman scattering (SRS) and broadband coherent anti-Stokes Raman scattering (CARS) spectroscopy and microscopy for label-free chemical identification of molecules through their intrinsic vibrational spectrum. (1) A 32-channels lock-in amplifier for parallel detection of broadband SRS spectra. (2) A broadband CARS microscope covering the entire fingerprint with down to 1-ms pixel dwell time, based on a low-repetition-rate amplified laser allowing white-light supercontinuum generation in bulk media. (3) Artificial-intelligence-based methods to enhance the signal-to-noise ratio and remove the non-resonant background signal from CARS spectra. (4) Video-rate wide-field CARS microscopy over the fingerprint region.
We present a novel hyperspectral imaging system working in the visible and in the short-wave infrared (SWIR) spectral region based on a Fourier-transform (FT) approach. The technology presents high light throughput and spatial resolution, a software adjustable spectral resolution, and a wide versatility of use. It employs a common-path interferometer, generating two replicas of the image with controllable delay and remarkable accuracy and stability. The monochromatic camera (CMOS, CCD, or InGaAs bidimensional sensor) does not require any relative movement with respect to the sample. The absence of gratings and slits guarantees an exceptional throughput that ensures high-quality data even at the lowest light dose, making this technology particularly suitable in fluorescence studies, or where low-illuminance conditions are recommended in order not to damage the samples. We will show several examples of the use in remote sensing and microscopy.
Spontaneous Raman spectroscopy is a powerful label-free and non-invasive imaging technique for mapping cells and tissues, delivering relevant biochemical information. However, in its standard implementation, the spontaneous Raman cross sections are too low, thus preventing high-speed microscopy, especially in the more informative low wavenumber region, also known as fingerprint region. Coherent Raman microscopy overcomes this hurdle providing several orders of magnitude higher speed thanks to the coherent excitation of the vibrational modes in the laser focus. In this work, we present a novel approach to broadband coherent anti-Stokes Raman scattering (B-CARS) that allows acquiring the entire fingerprint vibrational response of the sample at an unprecedented speed. The system is based on an amplified Ytterbium laser at 2 MHz repetition rate, that provides sufficient pulse energies to generate broadband near-infrared white-light supercontinuum in bulk media that we employ as broadband Stokes pulses. Coupled to narrowband pump pulses at 1035 nm, we demonstrate B-CARS microscopy down to 1 ms pixel dwell time with a diffraction-limited spatial resolution over large field of views. To extract the maximum amount of information, we enhance the signal to noise ratio of the vibrational spectra via artificial intelligence-based methods. In particular, we developed a convolutional neural network trained on data-augmented experimental input-output pairs of B-CARS spectra. Traditional algorithms are then used to remove the non-resonant background, extrapolating the pure vibrational response, and to perform chemometric analysis on the hypercubes. We test the setup performances by imaging heterogeneous biological systems, such as tissue slices of murine spine.
We developed an innovative multimodal nonlinear microscope, that operates in four different modalities. Using this tool on murine models, we set procedures and protocols to understand the role of dipeptidyl peptidase 3 in skeletal diseases.
The investigation of artworks of cultural heritage is generally aimed at the characterization of the constituent materials and the evaluation of their state of conservation. Research may shed light on the pigments and their potential deterioration mechanisms, and on the conservation treatments. Laboratory analysis on micro-samples taken from the artwork is still an invaluable practice for a deep understanding of the paint layer composition. In this context, a powerful technique is spectral microscopy, which acquires the spectrum for each point in the image of a sample. To acquire a continuous spectrum, one very efficient method is based on Fourier-transform (FT) spectroscopy as it allows massive parallelization on all the image pixels. Here we introduce a hyperspectral microscope based on an innovative FT spectrometer; the device is compact, robust, with high throughput and broad spectral coverage. In our microscope, light is collected by an infinity-corrected objective, propagates in the innovative spectrometer and is imaged on a silicon monochrome CMOS camera by a tube lens. The typical spectral resolution of the microscope, which can be flexibly adjusted for each measurement, is 3 THz (4 nm at 600 nm). We show very compact implementations of the hyperspectral microscope and their use for wide-field imaging of reflection, fluorescence and, interestingly, fluorescence-free Raman spectra. Thanks to the high throughput, the acquisition time of our microscope is significantly shorter than traditional raster-scanning approaches.
We introduce a Fourier-transform hyperspectral microscope based on an ultrastable birefringent interferometer. The microscope enables wide field acquisition with broad spectral coverage, tunable spectral resolution, high sensitivity and short acquisition time. We present the prototype of an add-on to a commercial microscope. We provide examples of applications in biology and solid state physics. The microscope is suited for fluorescence and Raman imaging.
The detection of biomarkers by means of Surface Enhanced Raman Spectroscopy (SERS) is foreseen to became a very important tool in the clinical practice because of its excellent sensitivity and potential for the simultaneous detection of multiple biomarkers. In the present paper we describe how it was possible to build a sensor for the detection of genetic biomarkers involved in acute myeloid leukemia. The assay is based on the use of a specifically designed SERS substrate made of a 2D crystal structure of polymeric pillars embedded in a gold layer. This substrate is characterized by good enhancing properties coupled with an excellent homogeneity. The SERS substrate was conjugated with DNA probes complementary to a target sequence and used in a sandwich assay with gold nanoparticles labeled with a second DNA probe and a Raman reporter. The so developed assay allowed the detection of a leukemia biomarker (WT1 gene) and an housekeeping gene with low picomolar sensitivity. At last, we optimized the assay in order to tackle one of the main limitations of SERS based assay: the loss of signal that is observed when the Raman spectra are collected in liquid. Combining a preferential functionalization on the polymeric pillars with a different height of the polymer pillars from the gold layer the assay demonstrated its effectiveness even when measured in buffer.
Acute myeloid leukemia (AML) is a proliferative neoplasm, that if not properly treated can rapidly cause a fatal
outcome. The diagnosis of AML is challenging and the first diagnostic step is the count of the percentage of blasts
(immature cells) in bone marrow and blood sample, and their morphological characterization. This evaluation is still
performed manually with a bright field light microscope. Here we report results of a study applying Raman spectroscopy
for analysis of samples from two patients affected by two AML subtypes characterized by a different maturation stage in
the neutrophilic lineage. Ten representative cells per sample were selected and analyzed with high-resolution confocal
Raman microscopy by scanning 64x64 (4096) points in a confocal layer through the volume of the whole cell. The
average spectrum of each cell was then used to obtain a highly reproducible mean fingerprint of the two different AML
subtypes. We demonstrate that Raman spectroscopy efficiently distinguishes these different AML subtypes. The
molecular interpretation of the substantial differences between the subtypes is related to granulocytic enzymes (e.g.
myeloperoxidase and cytochrome b558), in agreement with different stages of maturation of the two considered AML
subtypes . These results are promising for the development of a new, objective, automated and label-free Raman based
methods for the diagnosis and first assessment of AML.
The Wilms tumor gene (WT1) is a biomarker overexpressed in more than 90% of acute myeloid leukemia patients. Fast and sensitive detection of the WT1 in blood samples would allow monitoring of the minimal residual disease during clinical remission and would permit early detection of a potential relapse in acute myeloid leukemia. In this work, Surface Enhanced Raman Spectroscopy (SERS) based detection of the WT1 sequence using bifunctional, magnetic core – gold shell nanoparticles is presented. The classical co-precipitation method was applied to generate magnetic nanoparticles which were coated with a gold shell after modification with aminopropyltriethoxy silane and subsequent deposition of gold nanoparticle seeds. Simple hydroquinone based reduction procedure was applied for the shell growing in water based reaction mixture at room temperature. Thiolated ssDNA probes of the WT1 sequence were immobilized as capture oligonucleotides on the gold surface. Malachite green was applied both for testing the amplification performance of the core-shell colloidal SERS substrate and also as label dye of the target DNA sequence. The SERS enhancer efficacy of the core-shell nanomaterial was compared with the efficacy of classical spherical gold particles produced using the conventional citrate reduction method. The core-shell particles were found not only to provide an opportunity for facile separation in a heterogeneous reaction system but also to be superior regarding robustness as SERS enhancers.
Surface Enhanced Raman Spectroscopy is a powerful analytical technique that combines the excellent chemical specificity of Raman spectroscopy with the good sensitivity provided by the enhancement of the signal observed when a molecule is located on (or very close to) the surface of suitable nanostructured metallic materials. The availability of cheap, reliable and easy to use SERS substrates would pave the road to the development of bioanalytical tests that can be used in clinical practice. SERS, in fact, is expected to provide not only higher sensitivity and specificity, but also the simultaneous and markedly improved detection of several targets at the same time with higher speed compared to the conventional analytical methods. Here, we present the SERS activity of 2-D plasmonic crystals made by polymeric pillars embedded in a gold matrix obtained through the combination of soft-lithography and plasma deposition techniques on a transparent substrates. The use of a transparent support material allowed us to perform SERS detection from support side opening the possibility to use these substrates in combination with microfluidic devices. In order to demonstrate the potentialities for bioanalytical applications, we used our SERS active gold surface to detect the oxidation product of apomorphine, a well-known drug molecule used in Parkinson’s disease which has been demonstrated being difficult to study by traditional HPLC based approaches.
Surface Enhanced Raman Spectroscopy (SERS) is a popular method in bio-analytical chemistry and a potentially powerful enabling technology for in vitro diagnostics. SERS combines the excellent chemical specificity of Raman
spectroscopy with the good sensitivity provided by enhancement of the signal that is observed when a molecule is
located on (or very close to) the surface of nanostructured metallic materials. Star-like gold nanoparticles (SGN) are a new class of multibranched nanoparticles that in the last few years have attracted the attention of SERS community for their plasmonic properties. In this work we present a new method to prepare star-like gold nanoparticles with a simple one step protocol at room temperature using hydroquinone as reducing agent. Besides we compare the enhancement of Raman signal of malachite green, a dye commonly employed as label in biological studies, by star-like gold nanoparticles having different size, directly in liquid. This study shows that SGN provide good enhancement of Raman signal and that the effect of their dimension is strongly dependent on the wavelength used. Moreover preliminary results suggest that SGN produced using this method are characterized by good physical-chemical properties and they can be functionalized using the standard thiol chemistry. Overall, these results suggest that star-like gold nanoparticles produced through this method could be used for the further development of highly specific and sensitive SERS-based bio-analytical tests.
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