High-speed Multispectral Imaging Research Articles

A study on surface-temperature measurement by means of a vacuum-arc cathode under different current conditions

Accurate detection and analysis of the cathode surface temperature delivers an important reference value as regards the prediction of electrode life and corrosion degree, a thorough study of the vacuum-breakdown phenomenon, design and application of the vacuum-arc ion source and vacuum switch. In this paper, multispectral true temperature measurement T of the surface temperature field of pulsed vacuum arc cathode materials under different discharge currents is carried out. Firstly, aiming at solving the problem of synchronous imaging measurement of the temperature field across the cathode of pulsed vacuum arc, a multi-wavelength monochromatic image-acquisition optical path is designed, based upon the image acquisition module of a high-speed monochrome camera. Also, multiple monochromatic spectral radiation images of the target are synchronously acquired on the focal plane of the same monochrome camera detector. As a result, the spectral-radiation image information of multiple wavelengths can be obtained in a single exposure time, which greatly shortens the temperature-field image acquisition time. Secondly, based upon the multispectral true-temperature calculation method, high-speed multispectral imaging measurement of the target temperature field is realized. Finally, the two-dimensional true temperature distribution of the cathode temperature field under different current conditions was studied by setting up an experimental platform for measuring the cathode temperature of the vacuum arc under different current conditions.

High-speed multi-spectral imaging of the hypergolic ignition of ammonia borane

Recently, ammonia borane (a hydrogen-dense solid fuel) has received increased attention as a potential fuel for use in hybrid rocket systems due to its potential for higher performance. Ammonia borane (AB) is also highly hypergolic with white fuming nitric acid (WFNA) while lacking the health hazards associated with more conventional hypergolic fuels (e.g. hydrazine and its derivatives). To date, there remains a lack of information regarding the combustion behavior of ammonia borane. This work makes use of simultaneous visible and infrared multi-spectral imaging to study the combustion behavior of AB during a hypergolic ignition event (with reagent grade nitric acid droplets applied to the AB as the oxidizer). Using optical filters corresponding to previously reported spectral bands, emission from BO, BO2, HBO2, and the B-H stretch mode of AB were selectively imaged. A two-step ignition process consisting of an initial gas evolution step followed by a separate, seemingly premixed flame propagation step was observed. Observation of HBO2 emission during the pre-ignition/gas evolution phase suggests that rapid formation of HBO2 occurs even at the lower temperatures present prior to ignition. This initial formation of HBO2 (and the subsequent heat release associated with it) may be a major driver of AB/nitric acid decomposition. Emission intensity profiles obtained after the AB samples began burning with the ambient air suggests some degree of HBO2 consumption within the flame front, seemingly counter to the current understanding that HBO2 is a stable product species. This may indicate that boron combustion via boranes may proceed to more complete combustion than is typical for more conventional boron fuels. The ignition dynamics and sequence of boron product formation was well-captured by the multi-spectral imaging technique applied to this reaction system for the first time. This technique could be applied for further study of other boron systems.

Abstract 4250: Multispectral imaging of brightfield multiplex immunohistochemistry

Abstract Introduction. Brightfield microscopy is the current gold standard for tissue-based cancer diagnoses and has long been the choice of pathologists. Accordingly, the clinical use of brightfield-based immunohistochemistry (IHC) far exceeds immunofluorescence (IF), except in multiplexing applications for which IF is predominately used. Brightfield multiplex imaging is currently limited by 1) the broad spectral absorbance of conventional chromogens and 2) color cameras that only have three broad and overlapping color channels. In this work, we combine narrowband covalently deposited chromogens (CDCs; Day et al., Lab Invest 2017 vol.97 p.104) with matched narrow illumination channels and monochrome imaging to demonstrate that high-order multiplexing is possible in brightfield. Material and Methods. Multiplex IHC was performed using 4 or 5 CDCs and hematoxylin (htx) nuclear staining on formalin-fixed paraffin-embedded (FFPE) tissue sections. Spectral widths of 5 CDC absorbance bands ranged from 69 to 85 nm (FWHM), and 150 nm for a 6th broader CDC. Illumination was provided with a tungsten lamp/filter wheel combination (average bandwidth = 28nm) or filtered light emitting diodes (LEDs; average bandwidth = 23nm). Images were recorded on microscopes fitted with either illumination source and monochrome CCD or CMOS cameras. Furthermore, a custom-designed 12 wavelength LED illuminator facilitated high-speed multispectral imaging with short exposure times (average Δt=10ms). On-slide spectra of all chromogens were measured. Spectral unmixing was used to calculate protein abundances, which were pseudo-colored to render a visualization. Results. Multiple assays were tested on FFPE sections of lung, prostate, and colon tumors with up to 5 IHC markers (5 CDCs + htx). Spectral cross-talk between chromogen signals was significantly reduced with spectral unmixing (SNR≈30). Biomarker concentrations were pseudo-colored to produce customizable visualizations of each assay. Brightfield renditions of each assay were nearly identical to a traditional visualization. Multiplex staining results were concordant with single stain DAB IHC of serial sections. Automated control and image acquisition using the 12 LED illuminator permitted acquisition of all 12 wavelengths in 700 ms per 1 mm2 of tissue. Conclusion. Feasibility of brightfield IHC multiplexing was achieved by matching narrowband CDCs with specific light channels to enable detection of up to six analytes (5 CDCs plus nuclear stain), approaching the capabilities of IF. Our 12-color system is expandable to even higher-order multiplexing and boasts imaging speed roughly comparable to that of a clinical brightfield scanner. This system presents an attractive alternative to IF by combining high-order multiplexing with the speed, pathologist familiarity, and broadly established clinical utility of brightfield microscopy. Citation Format: Daniel R. Bauer, Mark Lefever, Torsten Leibold, Lauren Behman, Esteban Roberts, Julia Ashworth-Sharpe, Larry Morrison. Multispectral imaging of brightfield multiplex immunohistochemistry [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4250.

Focus on analytical equipment, sensors, and instrumentation

Focus on analytical equipment, sensors, and instrumentation

Fast, in vivo voltage imaging using a red fluorescent indicator.

Genetically encoded voltage indicators (GEVIs) are emerging optical tools for acquiring brain-wide cell-type-specific functional data at unparalleled temporal resolution. To broaden the application of GEVIs in high-speed multispectral imaging, we used a high-throughput strategy to develop voltage-activated red neuronal activity monitor (VARNAM), a fusion of the fast Acetabularia opsin and the bright red fluorophore mRuby3. Imageable under the modest illumination intensities required by bright green probes (<50 mW mm-2), VARNAM is readily usable in vivo. VARNAM can be combined with blue-shifted optical tools to enable cell-type-specific all-optical electrophysiology and dual-color spike imaging in acute brain slices and live Drosophila. With enhanced sensitivity to subthreshold voltages, VARNAM resolves postsynaptic potentials in slices and cortical and hippocampal rhythms in freely behaving mice. Together, VARNAM lends a new hue to the optical toolbox, opening the door to high-speed in vivo multispectral functional imaging.

Contribution of Imaging Flow Cytometry to Storage Lesion Assessment: Identification of a Sub-Population of Morphologically Altered Erythrocytes

The storage lesion encompasses a series of biochemical and molecular modifications that alter erythrocytes during hypothermic storage, reducing transfusion yield. Indeed, in humans, up to 25% of transfused erythrocytes are cleared from the circulation in a few hours (Luten et al., 2008). The mechanisms underlying this rapid clearance are not fully elucidated but it is reasonable to assume that these erythrocyte alterations are sensed by the spleen, resulting in retention. Among those, membrane shedding may have a major impact on post-transfusion clearance of erythrocytes since it causes a progressive decrease in the surface-volume ratio of the cell, leading to the loss of its flexible biconcave shape. The proportion of "damaged" erythrocytes cleared in the hours following transfusion increases with the duration of storage while the deformability measured by ektacytometry (Frank et al., 2013) progressively decreases during this period. To characterize the morphological alteration of stored erythrocytes, we used imaging flow cytometry (Imagestream X Mark II, AMNIS°). This technology allows a simultaneous high-speed multispectral imaging of cells in brightfield, darkfield, and 9 channels of fluorescence. It combines the ability of conventional flow cytometry to analyze a very high number of events with a powerful exploration of cell morphology.We analyzed the morphological, biochemical, metabolic, and bio-mechanical characteristics of erythrocytes stored in optimal blood bank conditions for 6 donors, at Day 3, 21, 28, 35 and 42 of the storage period. This longitudinal study of parameters such as mean corpuscular volume, intracellular ATP level, hemolysis, osmotic fragility, deformability and the plasma levels of ions and metabolites has highlighted a great inter-donor variability in the storage lesion process. Moreover, Imagestream analysis of front views of sharp, single cells revealed a subpopulation of small erythrocytes. The "projected surface area" distribution on normalized frequency plots was bimodal in 5 of 6 concentrates, showing a well-demarcated subpopulation of less than 62 µm2. The proportion of this sub-population increased with storage from 0.5-3.4% at D3 to 4-23.5% at D42 (p<0.05). These cells displayed a low fluorescence staining in the EMA-binding test, a diagnostic test for hereditary spherocytosis. With a more detailed morphological analysis we could determine that this sub-population corresponds to a mix of echinocytes III, spherocytes and sphero-echinocytes (Bessis classification). These results have been confirmed by differential interference phase contrast microscopy (DIC) observations, carried out in parallel, as a gold standard of our imaging flow cytometry study. Indeed, we found a very good correlation between the proportion of small erythrocytes detected in imaging flow cytometry and the echinocytes III, sphero- and spheroechino-cytes detected by DIC (correlation coefficient=0.84).These morphological alterations have been considered irreversible (Berezina et al., 2002) and are reminiscent of those associated with mechanical clearance of erythrocytes in the spleen of patients with hereditary spherocytosis (Mohandas et al., 2008). We hypothesize that these storage-induced small erythrocytes correspond to the subpopulation of "damaged" erythrocytes that are rapidly cleared after transfusion. Confirmation of these findings using ex-vivo perfusion of human spleens and observational studies in transfused patients would support the use of imaging flow cytometry to predict transfusion yield. DisclosuresNo relevant conflicts of interest to declare.

A novel image-based quantitative method for the characterization of NETosis

NETosis is a newly recognized mechanism of programmed neutrophil death. It is characterized by a stepwise progression of chromatin decondensation, membrane rupture, and release of bactericidal DNA-based structures called neutrophil extracellular traps (NETs). Conventional ‘suicidal’ NETosis has been described in pathogenic models of systemic autoimmune disorders. Recent in vivo studies suggest that a process of ‘vital’ NETosis also exists, in which chromatin is condensed and membrane integrity is preserved. Techniques to assess ‘suicidal’ or ‘vital’ NET formation in a specific, quantitative, rapid and semiautomated way have been lacking, hindering the characterization of this process. Here we have developed a new method to simultaneously assess both ‘suicidal’ and ‘vital’ NETosis, using high-speed multi-spectral imaging coupled to morphometric image analysis, to quantify spontaneous NET formation observed ex-vivo or stimulus-induced NET formation triggered in vitro. The use of imaging flow cytometry allows automated, quantitative and rapid analysis of subcellular morphology and texture, and introduces the potential for further investigation using NETosis as a biomarker in pre-clinical and clinical studies.

High-speed multispectral imaging of nanoplasmonic array

A multispectral microscopy imaging system is developed for the single-particle scattering spectroscopy of many individual plasmonic nanostructures simultaneously. The system dispenses with the need for the mechanical scanning of sample stage and thus enables high-speed plasmon resonance imaging of nanostructure arrays. The darkfield scattering intensity images of nanoplasmonic structures at individual wavelengths are acquired with a spectral resolution of 2 nm in the wavelength range from 500 nm to 800 nm, and a frame rate of 2 seconds/wavelength. The images are processed afterwards and the plasmon resonance wavelength of every nanostructure within the field of view can be obtained at once. The plasmon resonance wavelengths of more than 1000 Au colloidal nanoparticles and a nanofabricated Au nanowire array are measured within 5 minutes. The presented high-speed spectral imaging system promises the practical application of large-scale high-density nanoplasmonic sensor arrays for label-free biomolecular detections in the near future.