Chemical & Biomedical Imaging最新文献

The Raman fingerprint spectral region provides abundant structural information on molecules. However, analyzing vibrational images within this region using coherent Raman imaging remains challenging due to the small Raman cross section and congested spectral features. In this study, we combined ultrabroadband coherent anti-Stokes Raman scattering (CARS) microspectroscopy across the spectral range of 500–4000 cm^–1 with multivariate curve resolution-alternating least-squares (MCR-ALS) to reveal hidden Raman bands in the fingerprint region. Applying this method to mouse brain tissue, we extracted information on cholesterol and collagen, leveraging their distinctive molecular signatures, as well as on key molecules such as lipids, proteins, water, and nucleic acids. Moreover, the simultaneous detection of second harmonic generation facilitated label-free visualization of organelles, including arachnoid membrane and Rootletin filaments.

Elemental imaging in laser-induced breakdown spectroscopy is usually performed by placing laser shots adjacent to each other on the sample surface without spatial overlap. Seeing that signal intensity is directly related to the amount of ablated material, this restricts either spatial resolution (for a given excitation efficiency) or sensitivity (when reducing the laser spot size). The experimental applicability of a concept involving the spatial overlapping of shots on the sample surface is investigated and compared to the conventional approach. By systematic choice of spacing between laser shots, spatial resolution can be improved to the single digit micrometer range for a given laser spot size. Signal intensity is found to be linearly dependent on the area ablated per shot, facilitating larger signal-to-background ratios with increased spot sizes. Owing to this, the presented approach is also employed to enhance signal intensity, while preserving spatial resolution. The applicability of the method is explored by analyzing samples with distinct thickness of the surface layer, allowing for the assessment of the concept’s suitability for different sample types.

NMR hyperpolarization dramatically improves the detection sensitivity of magnetic resonance through the increase in nuclear spin polarization. Because of the sensitivity increase by several orders of magnitude, additional applications have been unlocked, including imaging of gases in physiologically relevant conditions. Hyperpolarized ¹²⁹Xe gas recently received FDA approval as the first inhalable gaseous MRI contrast agent for clinical functional lung imaging of a wide range of pulmonary diseases. However, production and utilization of hyperpolarized ¹²⁹Xe gas faces a number of translational challenges including the high cost and complexity of contrast agent production and imaging using proton-only (i.e., conventional) clinical MRI scanners, which are typically not suited to scan ¹²⁹Xe nuclei. As a solution to circumvent the translational challenges of hyperpolarized ¹²⁹Xe, we have recently demonstrated the feasibility of a simple and cheap process for production of proton-hyperpolarized propane gas contrast agent using ultralow-cost disposable production equipment and demonstrated the feasibility of lung ventilation imaging using hyperpolarized propane gas in excised pig lungs. However, previous pilot studies have concluded that the hyperpolarized state of propane gas decays very fast with an exponential decay T₁ constant of ∼0.8 s at 1 bar (physiologically relevant pressure); moreover, the previously reported production rates were too slow for potential clinical utilization. Here, we investigate the feasibility of high-capacity production of hyperpolarized butane gas via heterogeneous parahydrogen-induced polarization using Rh nanoparticle-based catalyst utilizing butene gas as a precursor for parahydrogen pairwise addition. We demonstrate a remarkable result: the lifetime of the hyperpolarized state can be nearly doubled compared to that of propane (T₁ of ∼1.6 s and long-lived spin-state T_S of ∼3.8 s at clinically relevant 1 bar pressure). Moreover, we demonstrate a production speed of up to 0.7 standard liters of hyperpolarized gas per second. These two synergistic developments pave the way to biomedical utilization of proton-hyperpolarized gas media for ventilation imaging. Indeed, here we demonstrate the feasibility of phantom imaging of hyperpolarized butane gas in Tedlar bags and also the feasibility of subsecond 2D ventilation gas imaging in excised rabbit lungs with 1.6 × 1.6 mm² in-plane resolution using a clinical MRI scanner. The demonstrated results have the potential to revolutionize functional pulmonary imaging with a simple and inexpensive on-demand production of proton-hyperpolarized gas contrast media, followed by visualization on virtually any MRI scanner, including emerging bedside low-field MRI scanner technology.

Essential metals such as iron, copper, and zinc are required for a wide variety of biological processes. For example, they act as cofactors in many proteins, conferring enzymatic activity or structural stability. Interactions between metals and proteins are often difficult to characterize due to the low concentration of metals in biological tissues and the sometimes labile nature of the chemical bonds involved. To better understand the cellular functions of essential metals, we correlate protein localization, using fluorescence light microscopy (FLM), and metal distribution with synchrotron X-ray fluorescence (SXRF), a high-sensitivity and high-spatial-resolution technique for metal imaging. Both chemical imaging modalities are implemented under cryogenic conditions to preserve native cell structure and chemical element distribution. As a proof of concept, we applied cryo-FLM and cryo-SXRF correlative imaging to cultured primary hippocampal neurons. Neurons were labeled under live conditions with fluorescent F-actin and tubulin dyes, then samples were flash-frozen and observed in a frozen hydrated state. This methodology, cryo-FLM combined to cryo-SXRF, revealed the distribution of iron, copper and zinc relative to F-actin and tubulin in the growth cones, dendrites, axons, and axonal en passant boutons of developing neurons.

We demonstrate low-frequency interferometric impulsive stimulated Raman scattering (ISRS) imaging with high robustness to distortions by optical scattering. ISRS is a pump-probe coherent Raman spectroscopy that can capture Raman vibrational spectra. Recording of ISRS spectra requires isolation of a probe pulse from the pump pulse. While this separation is simple in nonscattering specimens, such as liquids, scattering leads to significant pump pulse contamination and prevents the extraction of a Raman spectrum. We introduce a robust method for ISRS microscopy that works in complex scattering samples. High signal-to-noise ISRS spectra are obtained even when the pump and probe pulses pass through many scattering layers.

We demonstrate low-frequency interferometric impulsive stimulated Raman scattering (ISRS) imaging with high robustness to distortions by optical scattering. ISRS is a pump–probe coherent Raman spectroscopy that can capture Raman vibrational spectra. Recording of ISRS spectra requires isolation of a probe pulse from the pump pulse. While this separation is simple in nonscattering specimens, such as liquids, scattering leads to significant pump pulse contamination and prevents the extraction of a Raman spectrum. We introduce a robust method for ISRS microscopy that works in complex scattering samples. High signal-to-noise ISRS spectra are obtained even when the pump and probe pulses pass through many scattering layers.

Measuring chemical concentrations at the surface of implanted medical devices is important for elucidating the local biochemical environment, especially during implant infection. Although chemical indicator dyes enable chemical measurements in vitro, they are usually ineffective when measuring through tissue because the background obscures the dye signal and scattering dramatically reduces the spatial resolution. X-ray excited luminescent chemical imaging (XELCI) is a recent imaging modality which overcomes these limitations using a focused X-ray beam to excite a small spot of red light on scintillator-coated medical implants with well-defined location (because X-rays are minimally scattered) and low background. A spectrochemical indicator film placed over the scintillator layer, e.g., a polymer film containing pH-indicator dyes, absorbs some of the luminescence according to the local chemical environment, and this absorption is then detected by measuring the light intensity/spectrum passing through the tissue. A focused X-ray beam is used to scan point-by-point with a spatial resolution mainly limited by the X-ray beam width with minimum increase from X-ray absorption and scattering in the tissue. X-ray resolution, implant surface specificity, and chemical sensitivity are the three key features of XELCI. Here, we study spatial resolution using optically absorptive targets. For imaging a series of lines, the 20–80% knife-edge resolution was ∼285 (±15) μm with no tissue and 475 ± 18 and 520 ± 34 μm, respectively, through 5 and 10 mm thick tissue. Thus, doubling the tissue depth did not appreciably change the spatial resolution recorded through the tissue. This shows the promise of XELCI for submillimeter chemical imaging through tissue.

Chromatin organization plays a key role in gene regulation throughout the cell cycle. Understanding the dynamics governing the accessibility of chromatin is crucial for insight into mechanisms of gene regulation, DNA replication, and cell division. Extensive research has been done to track chromatin dynamics to explain how cells function and how diseases develop, in the hope of this knowledge leading to future therapeutics utilizing proteins or drugs that modify the accessibility or expression of disease-related genes. Traditional methods for studying the movement of chromatin throughout the cell relied on cross-linking spatially adjacent sections or hybridizing fluorescent probes to chromosomal loci and then constructing dynamic models from the static data collected at different time points. While these traditional methods are fruitful in understanding fundamental aspects of chromatin organization, they are limited by their invasive sample preparation protocols and diffraction-limited microscope resolution. These limitations have been challenged by modern methods based on high- or super-resolution microscopy and specific labeling techniques derived from gene targeting tools. These modern methods are more sensitive and less invasive than traditional methods, therefore allowing researchers to track chromosomal organization, compactness, and even the distance or rate of chromatin domain movement in detail and real time. This review highlights a selection of recently developed methods of chromatin tracking and their applications in fixed and live cells.

Chromatin organization plays a key role in gene regulation throughout the cell cycle. Understanding the dynamics governing the accessibility of chromatin is crucial for insight into mechanisms of gene regulation, DNA replication, and cell division. Extensive research has been done to track chromatin dynamics to explain how cells function and how diseases develop, in the hope of this knowledge leading to future therapeutics utilizing proteins or drugs that modify the accessibility or expression of disease-related genes. Traditional methods for studying the movement of chromatin throughout the cell relied on cross-linking spatially adjacent sections or hybridizing fluorescent probes to chromosomal loci and then constructing dynamic models from the static data collected at different time points. While these traditional methods are fruitful in understanding fundamental aspects of chromatin organization, they are limited by their invasive sample preparation protocols and diffraction-limited microscope resolution. These limitations have been challenged by modern methods based on high- or super-resolution microscopy and specific labeling techniques derived from gene targeting tools. These modern methods are more sensitive and less invasive than traditional methods, therefore allowing researchers to track chromosomal organization, compactness, and even the distance or rate of chromatin domain movement in detail and real time. This review highlights a selection of recently developed methods of chromatin tracking and their applications in fixed and live cells.