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Cancer is a complex disease, characterized by alteration of different physiological molecular processes and cellular features. Keeping this in mind, the possibility of early identification and detection of specific tumor biomarkers by non-invasive approaches could improve early diagnosis and patient management.Different molecular imaging procedures provide powerful tools for detection and non-invasive characterization of oncological lesions. Clinical studies are mainly based on the use of computed tomography, nuclear-based imaging techniques and magnetic resonance imaging. Preclinical imaging in small animal models entails the use of dedicated instruments, and beyond the already cited imaging techniques, it includes also optical imaging studies. Optical imaging strategies are based on the use of luminescent or fluorescent reporter genes or injectable fluorescent or luminescent probes that provide the possibility to study tumor features even by means of fluorescence and luminescence imaging. Currently, most of these probes are used only in animal models, but the possibility of applying some of them also in the clinics is under evaluation.The importance of tumor imaging, the ease of use of optical imaging instruments, the commercial availability of a wide range of probes as well as the continuous description of newly developed probes, demonstrate the significance of these applications. The aim of this review is providing a complete description of the possible optical imaging procedures available for the non-invasive assessment of tumor features in oncological murine models. In particular, the characteristics of both commercially available and newly developed probes will be outlined and discussed.
Optical imaging (OI) techniques such as bioluminescence and fluorescence imaging have been widely used to track diseases in a non-invasive manner within living subjects. These techniques generally require bioluminescent and fluorescent probes. Here we demonstrate the feasibility of using radioactive probes for in vivo molecular OI.
In principle, the millisecond emission lifetimes of lanthanide chelates should enable their ultrasensitive detection in biological systems by time-resolved optical microscopy. In practice, however, lanthanide imaging techniques have provided no better sensitivity than conventional fluorescence microscopy. Here, we identified three fundamental problems that have impeded lanthanide microscopy: low photon flux, inefficient excitation, and optics-derived background luminescence. We overcame these limitations with a new lanthanide imaging modality, transreflected illumination with luminescence resonance energy transfer (trLRET), which increases the time-integrated signal intensities of lanthanide lumiphores by 170-fold and the signal-to-background ratios by 75-fold. We demonstrate that trLRET provides at least an order-of-magnitude increase in detection sensitivity over that of conventional epifluorescence microscopy when used to visualize endogenous protein expression in zebrafish embryos. We also show that trLRET can be used to optically detect molecular interactions in vivo. trLRET promises to unlock the full potential of lanthanide lumiphores for ultrasensitive, autofluorescence-free biological imaging.
Fluorescence properties of a molecule can be used to study the structural and functional nature of biological processes. Physical properties, including fluorescence lifetime, emission spectrum, emission polarization, and others, help researchers probe a molecule, produce desired effects, and infer causes and consequences. Correlative imaging techniques such as hyperdimensional imaging microscopy (HDIM) combine the physical properties and biochemical states of a fluorophore. Here we present a fiber-based imaging system that can generate hyper-dimensional contrast by combining multiple fluorescence properties into a single fluorescence lifetime decay curve. Fluorescence lifetime imaging microscopy (FLIM) with controlled excitation polarization and temporally dispersed emission can generate a spectrally coded, polarization-filtered lifetime distribution for a pixel. This HDIM scheme generates a better contrast between different molecules than that from individual techniques. This setup uses only a single detector and is simpler to implement, modular, cost-efficient, and adaptable to any existing FLIM microscope. We present higher contrast data from Arabidopsis thaliana epidermal cells based on intrinsic anthocyanin emission properties under multiphoton excitation. This work lays the foundation for an alternative hyperdimensional imaging system and demonstrates that contrast-based imaging is useful to study cellular heterogeneity in biological samples.
Time stretch imaging offers real-time image acquisition at millions of frames per second and subnanosecond shutter speed, and has enabled detection of rare cancer cells in blood with record throughput and specificity. An unintended consequence of high throughput image acquisition is the massive amount of digital data generated by the instrument. Here we report the first experimental demonstration of real-time optical image compression applied to time stretch imaging. By exploiting the sparsity of the image, we reduce the number of samples and the amount of data generated by the time stretch camera in our proof-of-concept experiments by about three times. Optical data compression addresses the big data predicament in such systems.
Direct visualization of metabolic dynamics in living animals with high spatial and temporal resolution is essential to understanding many biological processes. Here we introduce a platform that combines deuterium oxide (D2O) probing with stimulated Raman scattering (DO-SRS) microscopy to image in situ metabolic activities. Enzymatic incorporation of D2O-derived deuterium into macromolecules generates carbon-deuterium (C-D) bonds, which track biosynthesis in tissues and can be imaged by SRS in situ. Within the broad vibrational spectra of C-D bonds, we discover lipid-, protein-, and DNA-specific Raman shifts and develop spectral unmixing methods to obtain C-D signals with macromolecular selectivity. DO-SRS microscopy enables us to probe de novo lipogenesis in animals, image protein biosynthesis without tissue bias, and simultaneously visualize lipid and protein metabolism and reveal their different dynamics. DO-SRS microscopy, being noninvasive, universally applicable, and cost-effective, can be adapted to a broad range of biological systems to study development, tissue homeostasis, aging, and tumor heterogeneity.
Two-photon imaging has become a useful tool for optical monitoring of neural circuits, but it requires high laser power and serial scanning of each pixel in a sample. This results in slow imaging rates, limiting the measurements of fast signals such as neuronal activity. To improve the speed and signal-to-noise ratio of two-photon imaging, we introduce a simple modification of a two-photon microscope, using a diffractive optical element (DOE) which splits the laser beam into several beamlets that can simultaneously scan the sample. We demonstrate the advantages of DOE scanning by enhancing the speed and sensitivity of two-photon calcium imaging of action potentials in neurons from neocortical brain slices. DOE scanning can easily improve the detection of time-varying signals in two-photon and other non-linear microscopic techniques.
The spatial resolution of light microscopy is limited by the wavelength of visible light (the 'diffraction limit', approximately 250 nm). Resolution of sub-cellular structures, smaller than this limit, is possible with super resolution methods such as stochastic optical reconstruction microscopy (STORM) and super-resolution optical fluctuation imaging (SOFI). We aimed to resolve subcellular structures (axons, myelin sheaths and astrocytic processes) within intact white matter, using STORM and SOFI.
Optical multiplexing has a large impact in photonics, the life sciences and biomedicine. However, current technology is limited by a 'multiplexing ceiling' from existing optical materials. Here we engineered a class of polyyne-based materials for optical supermultiplexing. We achieved 20 distinct Raman frequencies, as 'Carbon rainbow', through rational engineering of conjugation length, bond-selective isotope doping and end-capping substitution of polyynes. With further probe functionalization, we demonstrated ten-color organelle imaging in individual living cells with high specificity, sensitivity and photostability. Moreover, we realized optical data storage and identification by combinatorial barcoding, yielding to our knowledge the largest number of distinct spectral barcodes to date. Therefore, these polyynes hold great promise in live-cell imaging and sorting as well as in high-throughput diagnostics and screening.
We report the construction and testing of a simple and versatile optical trapping apparatus, suitable for visualizing individual microtubules (∼25 nm in diameter) and performing single-molecule studies, using a minimal set of components. This design is based on a conventional, inverted microscope, operating under plain bright field illumination. A single laser beam enables standard optical trapping and the measurement of molecular displacements and forces, whereas digital image processing affords real-time sample visualization with reduced noise and enhanced contrast. We have tested our trapping and imaging instrument by measuring the persistence length of individual double-stranded DNA molecules, and by following the stepping of single kinesin motor proteins along clearly imaged microtubules. The approach presented here provides a straightforward alternative for studies of biomaterials and individual biomolecules.
High-resolution colposcopic optical coherence tomography (OCT) provides key anatomical measures, such as thickness and minor traumatic injury of vaginal epithelium, of the female reproductive tract noninvasively. This information can be helpful in both fundamental investigations in animal models and disease screenings in humans. We present a fiber-based visible-light OCT and two probe designs for colposcopic application. One probe conducts circular scanning using a DC motor, and the other probe is capable of three-dimensional imaging over a 4.6 × 4.6 - mm 2 area using a pair of galvo scanners. Using this colposcopic vis-OCT with both probes, we acquired high-resolution images from whole isolated macaque vaginal samples and identified biopsy lesions.
Platelets are specialized anucleate cells that play a major role in hemostasis following vessel injury. More recently, platelets have also been implicated in innate immunity and inflammation by directly interacting with immune cells and releasing proinflammatory signals. It is likely therefore that in certain pathologies, such as chronic parasitic infections and myeloid malignancies, platelets can act as mediators for hemostatic and proinflammatory responses. Fortunately, murine platelet function ex vivo is highly analogous to human, providing a robust model for functional comparison. However, traditional methods of studying platelet phenotype, function and activation status often rely on using large numbers of whole isolated platelet populations, which severely limits the number and type of assays that can be performed with mouse blood. Here, using cutting edge 3D quantitative phase imaging, holotomography, that uses optical diffraction tomography (ODT), we were able to identify and quantify differences in single unlabeled, live platelets with minimal experimental interference. We analyzed platelets directly isolated from whole blood of mice with either a JAK2V617F-positive myeloproliferative neoplasm (MPN) or Leishmania donovani infection. Image analysis of the platelets indicates previously uncharacterized differences in platelet morphology, including altered cell volume and sphericity, as well as changes in biophysical parameters such as refractive index (RI) and dry mass. Together, these data indicate that, by using holotomography, we were able to identify clear disparities in activation status and potential functional ability in disease states compared to control at the level of single platelets.
Imaging the spatial distribution of biomolecules is at the core of modern biology. The development of fluorescence techniques has enabled researchers to investigate subcellular structures with nanometer precision. However, multiplexed imaging, i.e. observing complex biological networks and interactions, is mainly limited by the fundamental 'spectral crowding' of fluorescent materials. Raman spectroscopy-based methods, on the other hand, have a much greater spectral resolution, but often lack the required sensitivity for practical imaging of biomarkers. Addressing the pressing need for new Raman probes, herein we present a series of Raman-active nanoparticles (Rdots) that exhibit the combined advantages of ultra-brightness and compact sizes (~20 nm). When coupled with the emerging stimulated Raman scattering (SRS) microscopy, these Rdots are brighter than previously reported Raman-active organic probes by two to three orders of magnitude. We further obtain evidence supporting for SRS imaging of Rdots at single particle level. The compact size and ultra-brightness of Rdots allows immunostaining of specific protein targets (including cytoskeleton and low-abundant surface proteins) in mammalian cells and tissue slices with high imaging contrast. These Rdots thus offer a promising tool for a large range of studies on complex biological networks.
Vascular leakage in the brain is a major complication associated with brain injuries and certain pathological conditions due to disruption of the blood-brain barrier (BBB). We have developed an optical imaging method, based on excitation and emission spectra of Evans Blue dye, that is >1000-fold more sensitive than conventional ultraviolet spectrophotometry. We used a rat thromboembolic stroke model to validate the usefulness of our method for vascular leakage. Optical imaging data show that vascular leakage varies in different areas of the post-stroke brain and that administering tissue plasminogen activator causes further leakage. The new method is quantitative, simple to use, requires no tissue processing, and can map the degree of vascular leakage in different brain locations. The high sensitivity of our method could potentially provide new opportunities to study BBB leakage in different pathological conditions and to test the efficacy of various therapeutic strategies to protect the BBB.
Lacrimal canaliculus (LC) has a key role in tear drainage, but it is difficult to evaluate the LC in detail, using the existing examinations. In this study, our novel LC imaging technique provided the high-resolution images of LC in a non-invasive manner. Three-dimensional images of LC were acquired via the palpebral conjunctiva from 20 healthy volunteers (20 eyes) and 10 patients with various lacrimal disorders (10 eyes), using optical coherence tomography (OCT) dacryography (OCTD). The LC images showed morphological differences between the vertical and horizontal segments. The function of LC could be evaluated by measuring the intralumen signal intensity over time after instillation of a contrast agent (2% rebamipide ophthalmic suspension). OCTD clearly visualised the blind extremity of the LC in four patients with punctal obstruction, which was useful for deciding the punctal incision location. In one patient with canalicular obstruction, contrast agent successfully highlighted the LC that had become narrow toward the site of obstruction. Significant differences were not found in the function and morphology of LC between the patients with NLDO and the healthy subjects. OCTD may be a useful tool for LC imaging, because it facilitates quantitative and simultaneous evaluation of LC morphology and function.
It is shown that using elliptically polarized light permits selecting well-defined subsurface volumes in a turbid medium. This suggests the possibility of probing biological tissues at specific depths. First, we present the method and preliminary results obtained on an Intralipid phantom. We next report on the method's performance on a biological phantom (chicken breast) and, finally, on the exposed cortex of an anesthetized rat.
We introduce multi-directional optical coherence tomography (OCT), a technique for investigation of the scattering properties of directionally reflective tissue samples. By combining the concepts of multi-channel and directional OCT, this approach enables simultaneous acquisition of multiple reflectivity depth-scans probing a mutual sample location from differing angular orientations. The application of multi-directional OCT in retinal imaging allows for in-depth investigations on the directional reflectivity of the retinal nerve fiber layer, Henle's fiber layer and the photoreceptor layer. Major ophthalmic diseases (such as glaucoma or age-related macular degeneration) have been reported to alter the directional reflectivity properties of these retinal layers. Hence, the concept of multi-directional OCT might help to gain improved understanding of pathology development and progression. As a first step, we demonstrate the capabilities of multi-directional OCT in the eyes of healthy human volunteers.
Whole-animal fluorescence imaging with recombinant or fluorescently-tagged pathogens or cells enables real-time analysis of disease progression and treatment response in live animals. Tissue absorption limits penetration of fluorescence excitation light, particularly in the visible wavelength range, resulting in reduced sensitivity to deep targets. Here, we demonstrate the use of an optical fiber bundle to deliver light into the mouse lung to excite fluorescent bacteria, circumventing tissue absorption of excitation light in whole-animal imaging. We present the use of this technology to improve detection of recombinant reporter strains of tdTomato-expressing Mycobacterium bovis BCG (Bacillus Calmette Guerin) bacteria in the mouse lung. A microendoscope was integrated into a whole-animal fluorescence imager to enable intravital excitation in the mouse lung with whole-animal detection. Using this technique, the threshold of detection was measured as 103 colony forming units (CFU) during pulmonary infection. In comparison, the threshold of detection for whole-animal fluorescence imaging using standard epi-illumination was greater than 106 CFU.
To understand how the body of plants is made, it is essential to observe the morphology, structure and arrangement of constituent cells. However, the opaque nature of the plant body makes it difficult to observe the internal structures directly under a microscope. To overcome this problem, we developed a reagent, ClearSee, that makes plants transparent, allowing direct observation of the inside of a plant body without inflicting damage on it, e.g. through physical cutting. However, because ClearSee is not effective in making some plant species and tissues transparent, in this study, we further improved its composition to prevent oxidation, and have developed ClearSeeAlpha, which can be applied to a broader range of plant species and tissues. Sodium sulfite, one of the reductants, prevented brown pigmentation due to oxidation during clearing treatment. Using ClearSeeAlpha, we show that it is possible to obtain clear chrysanthemum leaves, tobacco and Torenia pistils and fertilized Arabidopsis thaliana fruits-tissues that have hitherto been challenging to clear. Moreover, we show that the fluorescence intensity of purified fluorescent proteins emitting light of various colors was unaffected in the ClearSeeAlpha solution; only the fluorescence intensity of TagRFP was reduced by about half. ClearSeeAlpha should be useful in the discovery and analysis of biological phenomena occurring deep inside the plant tissues.
Proliferative diabetic retinopathy (PDR) is a major cause of blindness in diabetic individuals. Optical coherence tomography (OCT) and OCT-angiography (OCTA) are noninvasive imaging techniques useful for the diagnosis and assessment of PDR. We aim to review several recent developments using OCT and discuss their present and potential future applications in the clinical setting. An electronic database search was performed so as to include all studies assessing OCT and/or OCTA findings in PDR patients published from 1 January 2020 to 31 May 2021. Thirty studies were included, and the most recently published data essentially focused on the higher detection rate of neovascularization obtained with widefield-OCT and/or OCTA (WF-OCT/OCTA) and on the increasing quality of retinal imaging with quality levels non-inferior to widefield-fluorescein angiography (WF-FA). There were also significant developments in the study of retinal nonperfusion areas (NPAs) using these techniques and research on the impact of PDR treatment on NPAs and on vascular density. It is becoming increasingly clear that it is critical to use adequate imaging protocols focused on optimized segmentation and maximized imaged retinal area, with ongoing technological development through artificial intelligence and deep learning. These latest findings emphasize the growing applicability and role of noninvasive imaging in managing PDR with the added benefit of avoiding the repetition of invasive conventional FA.
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