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Calibration of a spectral microscope system. ( a ) The multi-ion discharge lamp spectrum measured with a spectral epi fl uorescent microscope system ( red line ) aligned with the spectrum measured with the spectrometer ( black line ), con fi rming that the wavelength calibration of the HSi-300 AOTF (Chromodynamics, Inc.) was accurate. ( b ) Known spectrum of the NIST-traceable light source (input). ( c ) Spectrum of the NIST-traceable light source measured using a hyperspectral epi fl uorescent microscope system (output). ( d ) The correction coef fi cient used to achieve a fl at spectral response is calculated by dividing the known spectrum by the measured spectrum (input/output)
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In recent years a variety of fluorescent probes for measurement of cGMP signals have been developed (Nikolaev et al., Nat. Methods 3:23-25, 2006; Honda et al., Proc Natl Acad Sci USA 98:2437-42, 2001; Nausch et al., Proc Natl Acad Sci USA 105:365-70, 2008). The probes are comprised of known cGMP binding sites-e.g., from phosphodiesterase type 5 (PD...
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Context 1
... con fi rm the factory calibration of the AOTF (or other spectral fi lter system), a multi-ion discharge lamp is placed on the microscope stage and a spectral image stack is obtained. The spectrum of the image stack is then compared to the spectrum of the multi-ion lamp, as measured using a spectrometer or other appropriate NIST- calibrated device (Fig. 3a ). Details are provided in [ 9, 11 ] ...
Context 2
... the spec- tral information in each image stack acquired to compensate for wavelength-dependent attenuation ( see Note 15 ). The correction coef fi cient used to achieve a fl at spectral response is calculated by dividing the known spectrum by the mea- sured spectrum (input/output) (i) A NIST-traceable light source is used as the known input (Fig. 3b ...
Citations
... Herein, we report on hyperspectral fluorescence imaging and spectral unmixing applied to whole animal cryo-imaging. Although this methodology has been deployed in a variety of other applications, including imaging of living small animals [36,37], surgical guidance [40,41,45], microscopy [46][47][48], tomographic imaging [38,49] and clinical point probe measurements [50,51], all demonstrating improvements in the ability to isolate the specific signal-of-interest, this study is the first to evaluate the technique in whole body cryo-imaging. In this configuration, the hyperspectral information reduced the detection limit thresholds by 2-5 times, which can be important for applications in which reporter signal is relatively low compared to other contaminating signals. ...
Whole-animal fluorescence cryo-imaging is an established technique that enables visualization of the biodistribution of labeled drugs, contrast agents, functional reporters and cells in detail. However, many tissues produce endogenous autofluorescence, which can confound interpretation of the cryo-imaging volumes. We describe a multi-channel, hyperspectral cryo-imaging system that acquires densely-sampled spectra at each pixel in the 3-dimensional stack. This information enables the use of spectral unmixing to isolate the fluorophore-of-interest from autofluorescence and/or other fluorescent reporters. In phantoms and a glioma xenograft model, we show that the approach improves detection limits, increases tumor contrast, and can dramatically alter image interpretation.
... Unmixed HIFEX and confocal data were used to estimate FRET efficiencies and/or indices of manually selected cells as previously described. 6,11,[15][16][17] ...
Förster resonance energy transfer (FRET) is a valuable tool for measuring molecular distances and the effects of biological processes such as cyclic nucleotide messenger signaling and protein localization. Most FRET techniques require two fluorescent proteins with overlapping excitation/emission spectral pairing to maximize detection sensitivity and FRET efficiency. FRET microscopy often utilizes differing peak intensities of the selected fluorophores measured through different optical filter sets to estimate the FRET index or efficiency. Microscopy platforms used to make these measurements include wide-field, laser scanning confocal, and fluorescence lifetime imaging. Each platform has associated advantages and disadvantages, such as speed, sensitivity, specificity, out-of-focus fluorescence, and Z-resolution. In this study, we report comparisons among multiple microscopy and spectral filtering platforms such as standard 2-filter FRET, emission-scanning hyperspectral imaging, and excitation-scanning hyperspectral imaging. Samples of human embryonic kidney (HEK293) cells were grown on laminin-coated 28 mm round gridded glass coverslips (10816, Ibidi, Fitchburg, Wisconsin) and transfected with adenovirus encoding a cAMP-sensing FRET probe composed of a FRET donor (Turquoise) and acceptor (Venus). Additionally, 3 FRET "controls" with fixed linker lengths between Turquoise and Venus proteins were used for inter-platform validation. Grid locations were logged, recorded with light micrographs, and used to ensure that whole-cell FRET was compared on a cell-by-cell basis among the different microscopy platforms. FRET efficiencies were also calculated and compared for each method. Preliminary results indicate that hyperspectral methods increase the signal-to-noise ratio compared to a standard 2-filter approach.
... Hyperspectral image stacks were unmixed using a linear unmixing algorithm implemented in MATLAB (Mathworks). FRET levels and cAMP concentrations were calculated as described elsewhere (13,(15)(16)(17). Localized signals were estimated as described in the Results and Discussion, below. ...
Ca2+ and cAMP are ubiquitous second messengers known to differentially regulate a variety of cellular functions over a wide range of timescales. Studies from a variety of groups support the hypothesis that these signals can be localized to discrete locations within cells, and that this subcellular localization is a critical component of signaling specificity. However, to date, it has been difficult to track second messenger signals at multiple locations within a single cell. This difficulty is largely due to the inability to measure multiplexed florescence signals in real time. To overcome this limitation, we have utilized both emission scan- and excitation scan-based hyperspectral imaging approaches to track second messenger signals as well as labeled cellular structures and/or proteins in the same cell. We have previously reported that hyperspectral imaging techniques improve the signal-to-noise ratios of both fluorescence and FRET measurements, and are thus well suited for the measurement of localized second messenger signals. Using these approaches, we have measured near plasma membrane and near nuclear membrane cAMP signals, as well as distributed signals within the cytosol, in several cell types including airway smooth muscle, pulmonary endothelial, and HEK-293 cells. We have also measured cAMP and Ca2+ signals near autofluorescent structures that appear to be golgi. Our data demonstrate that hyperspectral imaging approaches provide unique insight into the spatial and kinetic distributions of cAMP and Ca2+ signals in single cells.
... We have previously shown that automated subcellular analysis of second messenger signals for multilabel FRET studies can be performed using hyperspectral microscopic. [13][14][15] We expect that this excitation-scanning hyperspectral imaging implementation will allow these same cell signaling studies with greatly increased temporal sampling. ...
The majority of microscopic and endoscopic technologies utilize white light illumination. For a number of applications, hyper-spectral imaging can be shown to have significant improvements over standard white-light imaging techniques. This is true for both microscopy and in vivo imaging. However, hyperspectral imaging methods have suffered from slow application times. Often, minutes are required to gather a full imaging stack. Here we will describe and evaluate a novel excitation-scanning hyperspectral imaging system and discuss some applications. We have developed and are optimizing a novel approach called excitation-scanning hyperspectral imaging that provides an order of magnitude increased signal strength. This excitation scanning technique has enabled us to produce a microscopy system capable of high speed hyperspectral imaging with the potential for live video acquisition. The excitation-scanning hyperspectral imaging technology we developed may impact a range of applications. The current design uses digital strobing to illuminate at 16 wavelengths with millisecond image acquisition time. Analog intensity control enables a fully customizable excitation profile. A significant advantage of excitation-scanning hyperspectral imaging is can identify multiple targets simultaneously in real time. Finally, we are exploring utilizing this technology for a variety of applications ranging from measuring cAMP distribution in three dimensions within a cell to electrophysiology.
... We have previously shown that automated subcellular analysis of second messenger signals for multilabel FRET studies can be performed using hyperspectral microscopic. [13][14][15] ...
Currently, the majority of microscopic and endoscopic technologies utilize white light illumination. For a number of applications, hyper-spectral imaging can be shown to have significant improvements over standard white-light imaging techniques. This is true for both microscopy and in vivo imaging. However, hyperspectral imaging methods have suffered from slow application times. Often, minutes are required to gather a full imaging stack. Here we will describe the system and evaluate optimizations and applications of a novel excitation-scanning hyperspectral imaging system. We have developed and are optimizing a novel approach called excitation-scanning hyperspectral imaging that provides an order of magnitude increased signal strength. Optimization of the light path, optical components and illumination sources have allowed us to achieve high speed image acquisition. This high speed allows for potential live video acquisition. This excitation-scanning hyperspectral imaging technology has potential to impact a range of applications. The current system allows triggering of up to 16 wavelengths at less than 1 millisecond per image using digital strobing. Analog intensity control is also provided for a fully customizable excitation profile. A significant advantage of excitation-scanning hyperspectral imaging is can identify multiple targets simultaneously in real time. We are optimizing the system to compare sensitivity and specificity of excitation-scanning hyperspectral imaging with pathology techniques. Finally, we are exploring utilizing this technology to measure cAMP distribution in three dimensions within a cell.
... Hyperspectral image stacks were unmixed using a linear unmixing algorithm and the reference library ( Figure 1) implemented in custom MATLAB (Mathworks) scripts. FRET levels were calculated as described elsewhere [16][17][18] . cAMP levels were calculated from measured FRET values based upon the standard ligand/receptor binding equation. ...
Cyclic AMP (cAMP) is a ubiquitous second messenger known to differentially regulate many cellular functions. Several lines of evidence suggest that the distribution of cAMP within cells is not uniform. However, to date, no studies have measured the kinetics of 3D cAMP distributions within cells. This is largely due to the low signal-to-noise ratio of FRET-based probes. We previously reported that hyperspectral imaging improves the signal-to-noise ratio of FRET measurements. Here we utilized hyperspectral imaging approaches to measure FRET signals in five dimensions (5D) - three spatial (x, y, z), wavelength (λ), and time (t) - allowing us to visualize cAMP gradients in pulmonary endothelial cells. cAMP levels were measured using a FRET-based sensor (H188) comprised of a cAMP binding domain sandwiched between FRET donor and acceptor - Turquoise and Venus fluorescent proteins. We observed cAMP gradients in response to 0.1 or 1 μM isoproterenol, 0.1 or 1 μM PGE1, or 50 μM forskolin. Forskolin- and isoproterenol-induced cAMP gradients formed from the apical (high cAMP) to basolateral (low cAMP) face of cells. In contrast, PGE1-induced cAMP gradients originated from both the basolateral and apical faces of cells. Data suggest that 2D (x,y) studies of cAMP compartmentalization may lead to erroneous conclusions about the existence of cAMP gradients, and that 3D (x,y,z) studies are required to assess mechanisms of signaling specificity. Results demonstrate that 5D imaging technologies are powerful tools for measuring biochemical processes in discrete subcellular domains. This work was supported by NIH P01HL066299, R01HL058506, S10RR027535, AHA 16PRE27130004 and the Abraham Mitchell Cancer Research Fund.
... Hyperspectral imaging, a technology originally developed for remote sensing applications 1-3 , has found many applications in the biomedical imaging and microscopy fields [4][5][6][7] . Research applications for hyperspectral imaging have ranged from spectral imaging FRET microscopy measurements to assess molecular properties in single cells [8][9][10][11] to whole-animal in vivo imaging 12 . Clinical applications for hyperspectral imaging have also been proposed, such as for in vivo cancer detection [13][14][15][16][17] . ...
Over the past 2 decades, hyperspectral imaging technologies have been adapted to address the need for molecule-specific identification in the biomedical imaging field. Applications have ranged from single-cell microscopy to whole-animal in vivo imaging and from basic research to clinical systems. Enabling this growth has been the availability of faster, more effective hyperspectral filtering technologies and more sensitive detectors. Hence, the potential for growth of biomedical hyperspectral imaging is high, and many hyperspectral imaging options are already commercially available. However, despite the growth in hyperspectral technologies for biomedical imaging, little work has been done to aid users of hyperspectral imaging instruments in selecting appropriate analysis algorithms. Here, we present an approach for comparing the effectiveness of spectral analysis algorithms by combining experimental image data with a theoretical “what if” scenario. This approach allows us to quantify several key outcomes that characterize a hyperspectral imaging study: linearity of sensitivity, positive detection cut-off slope, dynamic range, and false positive events. We present results of using this approach for comparing the effectiveness of several common spectral analysis algorithms for detecting weak fluorescent protein emission in the midst of strong tissue autofluorescence. Results indicate that this approach should be applicable to a very wide range of applications, allowing a quantitative assessment of the effectiveness of the combined biology, hardware, and computational analysis for detecting a specific molecular signature. © (2017) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.
... Hyperspectral image stacks were unmixed using a linear unmixing algorithm and the reference library ( Figure 1) implemented in custom MATLAB (Mathworks) scripts. FRET levels were calculated as described elsewhere [16][17][18] . cAMP levels were calculated from measured FRET values based upon the standard ligand/receptor binding equation. ...
... We have previously shown that hyperspectral imaging using fluorescence emission scanning can be used to accurately detect discrete molecular signals in cells and tissues. 41,[56][57][58][59][60] However, we have found that fluorescence excitation scanning provides 10-to 30-fold higher signal sensitivity than traditional (emission-based) spectral imaging approaches. 55 This technology allows fluorescence and absorbance image data to be acquired across a range of narrow-wavelength illumination bands, spanning the ultraviolet (UV) through visible spectrum and can easily be adapted for endoscopic use. ...
Optical spectroscopy and hyperspectral imaging have shown the potential to discriminate between cancerous and non-cancerous tissue with high sensitivity and specificity. However, to date, these techniques have not been effectively translated to real-time endoscope platforms. Hyperspectral imaging of the fluorescence excitation spectrum represents new technology that may be well-suited for endoscopic implementation. However, the feasibility of detecting differences between normal and cancerous mucosa using fluorescence excitation-scanning hyperspectral imaging has not been evaluated. The goal of this study was to evaluate the initial feasibility of using fluorescence excitation-scanning hyperspectral imaging for measuring changes in fluorescence excitation spectrum concurrent with colonic adenocarcinoma using a small pre-pilot-scale sample size. Ex vivo analysis was performed using resected pairs of colorectal adenocarcinoma and normal mucosa. Adenocarcinoma was confirmed by histologic evaluation of H&E permanent sections. Specimens were imaged using a custom hyperspectral imaging fluorescence excitation-scanning microscope system. Results demonstrated consistent spectral differences between normal and cancerous tissues over the fluorescence excitation range of 390-450 nm that could be the basis for wavelength-dependent detection of colorectal cancers. Hence, excitation-scanning hyperspectral imaging may offer an alternative approach for discriminating adenocarcinoma from surrounding normal colonic mucosa, but further studies will be required to evaluate the accuracy of this approach using a larger patient cohort.
... In prior work, we have shown that hyperspectral microscopy allows multilabel FRET studies to be performed for automated subcellular analysis of second messenger signals. [11][12][13] Our work indicates that fluorescence excitation-scanning hyperspectral imaging shows promise as an alternative hyperspectral imaging modality for very high-speed imaging. Additionally, our proof of concept and test apparatus shows that hyperspectral imaging has the potential to allow for live video imaging of tissue in vivo and ex vivo. ...
Current microscopic and endoscopic technologies for cancer screening utilize white-light illumination sources. Hyper-spectral imaging has been shown to improve sensitivity while retaining specificity when compared to white-light imaging in both microscopy and in vivo imaging. However, hyperspectral imaging methods have historically suffered from slow acquisition times due to the narrow bandwidth of spectral filters. Often minutes are required to gather a full image stack. We have developed a novel approach called excitation-scanning hyperspectral imaging that provides 2–3 orders of magnitude increased signal strength. This reduces acquisition times significantly, allowing for live video acquisition. Here, we describe a preliminary prototype excitation-scanning hyperspectral imaging system that can be coupled with endoscopes or microscopes for hyperspectral imaging of tissues and cells.
Our system is comprised of three subsystems: illumination, transmission, and imaging. The illumination subsystem employs light-emitting diode arrays to illuminate at different wavelengths. The transmission subsystem utilizes a unique geometry of optics and a liquid light guide. Software controls allow us to interface with and control the subsystems and components. Digital and analog signals are used to coordinate wavelength intensity, cycling and camera triggering. Testing of the system shows it can cycle 16 wavelengths at as fast as 1 ms per cycle. Additionally, more than 18% of the light transmits through the system. Our setup should allow for hyperspectral imaging of tissue and cells in real time.
