Multispectral intravital microscopy for simultaneous bright-field and fluorescence imaging of the microvasculature.
Ontology highlight
ABSTRACT:
SUBMITTER: Janssen BGH
PROVIDER: S-EPMC8310548 | biostudies-literature | 2021 Jul
REPOSITORIES: biostudies-literature
Publications
Multispectral intravital microscopy for simultaneous bright-field and fluorescence imaging of the microvasculature.
Janssen Barry G H BGH Najiminaini Mohamadreza M Zhang Yan Min YM Omidi Parsa P Carson Jeffrey J L JJL
Applied microscopy 20210724 1
Intravital video microscopy permits the observation of microcirculatory blood flow. This often requires fluorescent probes to visualize structures and dynamic processes that cannot be observed with conventional bright-field microscopy. Conventional light microscopes do not allow for simultaneous bright-field and fluorescent imaging. Moreover, in conventional microscopes, only one type of fluorescent label can be observed. This study introduces multispectral intravital video microscopy, which com ...[more]
Similar Datasets
Project description:Intravital microscopy (IVM) emerged and matured as a powerful tool for elucidating pathways in biological processes. Although label-free multiphoton IVM is attractive for its non-perturbative nature, its wide application has been hindered, mostly due to the limited contrast of each imaging modality and the challenge to integrate them. Here we introduce simultaneous label-free autofluorescence-multiharmonic (SLAM) microscopy, a single-excitation source nonlinear imaging platform that uses a custom-designed excitation window at 1110 nm and shaped ultrafast pulses at 10 MHz to enable fast (2-orders-of-magnitude improvement), simultaneous, and efficient acquisition of autofluorescence (FAD and NADH) and second/third harmonic generation from a wide array of cellular and extracellular components (e.g., tumor cells, immune cells, vesicles, and vessels) in living tissue using only 14 mW for extended time-lapse investigations. Our work demonstrates the versatility and efficiency of SLAM microscopy for tracking cellular events in vivo, and is a major enabling advance in label-free IVM.
| S-EPMC5974075 | biostudies-literature
Project description:Ship ballast water contains high concentration of plankton, bacteria, and other microorganisms. If the huge amount of ballast water is discharged without being inactivated, it will definitely spell disaster to the marine environment. Microalgae is the most common species exiting in ballast water, so the detection of the concentration and viability of microalgae is a very important issue. The traditional methods of detecting microalgae in ballast water were costly and need the help of bulky equipment. Herein, a novel method based on microalgae cell intracellular chlorophyll fluorescence (CF) imaging combines with cell bright field (BF) microscopy was proposed. The geometric features of microalgae cells were obtained by BF image, and the cell viability was obtained by CF image. The two images were fused through the classic image registration algorithm to achieve simultaneous detection of the viability and concentration of microalgae cells. Furthermore, a low-cost, miniaturized CF/BF microscopy imaging prototype system based on the above principles was designed. In order to verify the effectiveness of the proposed method, four typical microalgae in ballast water (Platymonas, Pyramimonas sp., Chrysophyta, and Prorocentrum lima) were selected as the samples. The experimental results show that the self-developed prototype can quickly and accurately determine the concentration and the viability of microalgae cells in ship ballast water based on the dual images of BF and CF, and the detection accuracy is equivalent to that of commercial microscope. It was the first time to simultaneously detect the viability and concentration of microalgae cells in ship ballast water using the method that combining the fluorescence and bright field images; moreover, a miniaturized microscopic imaging prototype was developed. Those findings expected to contribute to the microalgae detection and ship ballast water management.
| S-EPMC8398499 | biostudies-literature
Project description:Advances in intravital microscopy (IVM) have enabled the studies of cellular organization and dynamics in the native microenvironment of intact organisms with minimal perturbation. The abilities to track specific cell populations and monitor their interactions have opened up new horizons for visualizing cell biology in vivo, yet the success of standard fluorescence cell labeling approaches for IVM comes with a "dark side" in that unlabeled cells are invisible, leaving labeled cells or structures to appear isolated in space, devoid of their surroundings and lacking proper biological context. Here we describe a novel method for "filling in the void" by harnessing the ubiquity of extracellular (interstitial) fluid and its ease of fluorescence labelling by commonly used vascular and lymphatic tracers. We show that during routine labeling of the vasculature and lymphatics for IVM, commonly used fluorescent tracers readily perfuse the interstitial spaces of the bone marrow (BM) and the lymph node (LN), outlining the unlabeled cells and forming negative contrast images that complement standard (positive) cell labeling approaches. The method is simple yet powerful, offering a comprehensive view of the cellular landscape such as cell density and spatial distribution, as well as dynamic processes such as cell motility and transmigration across the vascular endothelium. The extracellular localization of the dye and the interstitial flow provide favorable conditions for prolonged Intravital time lapse imaging with minimal toxicity and photobleaching.
| S-EPMC8341626 | biostudies-literature
Project description:Intraoperative applications of near-infrared (NIR) fluorescent contrast agents can be aided by instrumentation capable of merging the view of surgical field with that of NIR fluorescence. We demonstrate augmented microscopy, an intraoperative imaging technique in which bright-field (real) and electronically processed NIR fluorescence (synthetic) images are merged within the optical path of a stereomicroscope. Under luminance of 100,000 lx, representing typical illumination of the surgical field, the augmented microscope detects 189 nM concentration of indocyanine green and produces a composite of the real and synthetic images within the eyepiece of the microscope at 20 fps. Augmentation described here can be implemented as an add-on module to visualize NIR contrast agents, laser beams, or various types of electronic data within the surgical microscopes commonly used in neurosurgical, cerebrovascular, otolaryngological, and ophthalmic procedures.
| S-EPMC4881285 | biostudies-literature
Project description:SignificanceAutofluorescence characteristics of the reduced nicotinamide adenine dinucleotide and oxidized flavin cofactors are important for the evaluation of the metabolic status of the cells. The approaches that involve a detailed analysis of both spectral and time characteristics of the autofluorescence signals may provide additional insights into the biochemical processes in the cells and biological tissues and facilitate the transition of spectral fluorescence lifetime imaging into clinical applications.AimWe present the experiments on multispectral fluorescence lifetime imaging with a detailed analysis of the fluorescence decays and spectral profiles of the reduced nicotinamide adenine dinucleotide and oxidized flavin under a single excitation wavelength aimed at understanding whether the use of multispectral detection is helpful for metabolic imaging of cancer cells.ApproachWe use two-photon spectral fluorescence lifetime imaging microscopy. Starting from model solutions, we switched to cell cultures treated by metabolic inhibitors and then studied the metabolism of cells within tumor spheroids.ResultsThe use of a multispectral detector in combination with an excitation at a single wavelength of 750 nm allows the identification of fluorescence signals from three components: free and bound NAD(P)H, and flavins based on the global fitting procedure. Multispectral data make it possible to assess not only the lifetime but also the spectral shifts of emission of flavins caused by chemical perturbations. Altogether, the informative parameters of the developed approach are the ratio of free and bound NAD(P)H amplitudes, the decay time of bound NAD(P)H, the amplitude of flavin fluorescence signal, the fluorescence decay time of flavins, and the spectral shift of the emission signal of flavins. Hence, with multispectral fluorescence lifetime imaging, we get five independent parameters, of which three are related to flavins.ConclusionsThe approach to probe the metabolic state of cells in culture and spheroids using excitation at a single wavelength of 750 nm and a fluorescence time-resolved spectral detection with the consequent global analysis of the data not only simplifies image acquisition protocol but also allows to disentangle the impacts of free and bound NAD(P)H, and flavin components evaluate changes in their fluorescence parameters (emission spectra and fluorescence lifetime) upon treating cells with metabolic inhibitors and sense metabolic heterogeneity within 3D tumor spheroids.
| S-EPMC11440180 | biostudies-literature
Project description:In living cells, there are always a plethora of processes taking place at the same time. Their precise regulation is the basis of cellular functions, since small failures can lead to severe dysfunctions. For a comprehensive understanding of intracellular homeostasis, simultaneous multiparameter detection is a versatile tool for revealing the spatial and temporal interactions of intracellular parameters. Here, a recently developed time-correlated single-photon counting (TCSPC) board was evaluated for simultaneous fluorescence and phosphorescence lifetime imaging microscopy (FLIM/PLIM). Therefore, the metabolic activity in insect salivary glands was investigated by recording ns-decaying intrinsic cellular fluorescence, mainly related to oxidized flavin adenine dinucleotide (FAD) and the μs-decaying phosphorescence of the oxygen-sensitive ruthenium-complex Kr341. Due to dopamine stimulation, the metabolic activity of salivary glands increased, causing a higher pericellular oxygen consumption and a resulting increase in Kr341 phosphorescence decay time. Furthermore, FAD fluorescence decay time decreased, presumably due to protein binding, thus inducing a quenching of FAD fluorescence decay time. Through application of the metabolic drugs antimycin and FCCP, the recorded signals could be assigned to a mitochondrial origin. The dopamine-induced changes could be observed in sequential FLIM and PLIM recordings, as well as in simultaneous FLIM/PLIM recordings using an intermediate TCSPC timing resolution.
| S-EPMC4585718 | biostudies-literature
Project description:In transmitted optical microscopy, absorption structure and phase structure of the specimen determine the three-dimensional intensity distribution of the image. The elementary impulse responses of the bright field microscope therefore consist of separate absorptive and phase components, precluding general application of linear, conventional deconvolution processing methods to improve image contrast and resolution. However, conventional deconvolution can be applied in the case of pure phase (or pure absorptive) objects if the corresponding phase (or absorptive) impulse responses of the microscope are known. In this work, we present direct measurements of the phase point- and line-spread functions of a high-aperture microscope operating in transmitted bright field. Polystyrene nanoparticles and microtubules (biological polymer filaments) serve as the pure phase point and line objects, respectively, that are imaged with high contrast and low noise using standard microscopy plus digital image processing. Our experimental results agree with a proposed model for the response functions, and confirm previous theoretical predictions. Finally, we use the measured phase point-spread function to apply conventional deconvolution on the bright field images of living, unstained bacteria, resulting in improved definition of cell boundaries and sub-cellular features. These developments demonstrate practical application of standard restoration methods to improve imaging of phase objects such as cells in transmitted light microscopy.
| S-EPMC3928359 | biostudies-literature
Project description:Nanosecond temporal resolution enables new methods for wide-field imaging like time-of-flight, gated detection, and fluorescence lifetime. The optical efficiency of existing approaches, however, presents challenges for low-light applications common to fluorescence microscopy and single-molecule imaging. We demonstrate the use of Pockels cells for wide-field image gating with nanosecond temporal resolution and high photon collection efficiency. Two temporal frames are obtained by combining a Pockels cell with a pair of polarizing beam-splitters. We show multi-label fluorescence lifetime imaging microscopy (FLIM), single-molecule lifetime spectroscopy, and fast single-frame FLIM at the camera frame rate with 103-105 times higher throughput than single photon counting. Finally, we demonstrate a space-to-time image multiplexer using a re-imaging optical cavity with a tilted mirror to extend the Pockels cell technique to multiple temporal frames. These methods enable nanosecond imaging with standard optical systems and sensors, opening a new temporal dimension for wide-field low-light microscopy.
| S-EPMC6783475 | biostudies-literature
Project description:Deconvolution enhances contrast in fluorescence microscopy images, especially in low-contrast, high-background wide-field microscope images, improving characterization of features within the sample. Deconvolution can also be combined with other imaging modalities, such as confocal microscopy, and most software programs seek to improve resolution as well as contrast. Quantitative image analyses require instrument calibration and with deconvolution, necessitate that this process itself preserves the relative quantitative relationships between fluorescence intensities. To ensure that the quantitative nature of the data remains unaltered, deconvolution algorithms need to be tested thoroughly. This study investigated whether the deconvolution algorithms in AutoQuant X3 preserve relative quantitative intensity data. InSpeck Green calibration microspheres were prepared for imaging, z-stacks were collected using a wide-field microscope, and the images were deconvolved using the iterative deconvolution algorithms with default settings. Afterwards, the mean intensities and volumes of microspheres in the original and the deconvolved images were measured. Deconvolved data sets showed higher average microsphere intensities and smaller volumes than the original wide-field data sets. In original and deconvolved data sets, intensity means showed linear relationships with the relative microsphere intensities given by the manufacturer. Importantly, upon normalization, the trend lines were found to have similar slopes. In original and deconvolved images, the volumes of the microspheres were quite uniform for all relative microsphere intensities. We were able to show that AutoQuant X3 deconvolution software data are quantitative. In general, the protocol presented can be used to calibrate any fluorescence microscope or image processing and analysis procedure.
| S-EPMC3942261 | biostudies-literature
Project description:Rationale: Focused ultrasound-stimulated microbubbles have been shown to be capable of inducing blood flow shutdown and necrosis in a range of tissue types in an approach termed antivascular ultrasound or nonthermal ablation. In oncology, this approach has demonstrated tumor growth inhibition, and profound synergistic antitumor effects when combined with traditional platforms of chemo-, radiation- and immune-therapies. However, the exposure schemes employed have been broad and underlying mechanisms remain unclear with fundamental questions about exposures, vessel types and sizes involved, and the nature of bubble behaviors and their acoustic emissions resulting in vascular damage - impeding the establishment of standard protocols. Methods: Here, ultrasound transmitters and receivers are integrated into a murine dorsal window chamber tumor model for intravital microscopy studies capable of real-time visual and acoustic monitoring during antivascular ultrasound. Vessel type (normal and tumor-affected), caliber, and viability are assessed under higher pressure conditions (1, 2, and 3 MPa), and cavitation signatures are linked to the biological effects. Results: Vascular events occurred preferentially in tumor-affected vessels with greater incidence in smaller vessels and with more severity as a function of increasing pressure. Vascular blood flow shutdown was found to be due to a combination of focal disruption events and network-related flow changes. Acoustic emissions displayed elevated broadband noise and distinct sub- and ultra-harmonics and their associated third-order peaks with increasing pressure. Conclusions: The observed vascular events taken collectively with identified cavitation signatures provide an improved mechanistic understanding of antivascular ultrasound at the microscale, with implications for establishing a specific treatment protocol and control platform.
| S-EPMC9800738 | biostudies-literature