In the past two decades, spectral imaging technologies have expanded the capacity of fluorescence microscopy for accurate detection of multiple labels, separation of labels from cellular and tissue autofluorescence, and analysis of autofluorescence signatures. These technologies have been implemented using a range of optical techniques, such as tunable filters, diffraction gratings, prisms, interferometry, and custom Bayer filters. Each of these techniques has associated strengths and weaknesses with regard to spectral resolution, spatial resolution, temporal resolution, and signal-to-noise characteristics. We have previously shown that spectral scanning of the fluorescence excitation spectrum can provide greatly increased signal strength compared to traditional emission-scanning approaches. Here, we present results from utilizing a Hyperspectral Imaging Fluorescence Excitation Scanning (HIFEX) microscope system for live cell imaging. Live cell signaling studies were performed using HEK 293 and rat pulmonary microvascular endothelial cells (PMVECs), transfected with either a cAMP FRET reporter or a Ca2+ reporter. Cells were further labeled to visualize subcellular structures (nuclei, membrane, mitochondria, etc.). Spectral images were acquired using a custom inverted microscope (TE2000, Nikon Instruments) equipped with a 300W Xe arc lamp and tunable excitation filter (VF- 5, Sutter Instrument Co., equipped with VersaChrome filters, Semrock), and run through MicroManager. Timelapse spectral images were acquired from 350-550 nm, in 5 nm increments. Spectral image data were linearly unmixed using custom MATLAB scripts. Results indicate that the HIFEX microscope system can acquire live cell image data at acquisition speeds of 8 ms/wavelength band with minimal photobleaching, sufficient for studying moderate speed cAMP and Ca2+ events.
more »
« less
Hyperspectral imaging fluorescence excitation scanning spectral characteristics of remodeled mouse arteries
Coronary artery disease (CAD), or atherosclerosis, is responsible for nearly a third of all American deaths annually. Detection of plaques and differentiation of plaque stage remains a complicating factor for treatment. Classification of plaque before significant blockage or rupture could inform clinical decisions and prevent mortality. Current detection methods are either nonspecific, slow, or require the use of potentially harmful contrast agents. Recent advances in hyperspectral imaging could be used to detect changes in the autofluorescence of arteries associated with vessel remodeling and subsequent plaque formation and could detect and classify existing lesions. Here, we present data comparing spectral image characteristics of a mouse model designed to undergo vessel remodeling.
C57Bl/6 mice underwent ligation of three of four caudal branches of the left common carotid artery (left external carotid, internal carotid, and occipital artery) with the superior thyroid artery left intact under IACUC approved protocol. Vessels were harvested at a variety of timepoints to compare degrees of remodeling, including 4 weeks and 5 months post-surgery. Immediately following harvest, vessels were prepared by longitudinal opening to expose the luminal surface to a 20X objective. A custom inverted microscope (TE-2000, Nikon Instruments) with a Xe arc lamp and thin film tunable filter arrary (Versachrome, Semrock, Inc.) were used to achieve spectral imaging. Excitation scans utilized wavelengths between 340 nm and 550 nm in 5 nm increments. Hyperspectral data were generated and analyzed with custom Matlab scripts and visualized in ENVI. Preliminary data suggest consistent spectral features associated with control and remodeled vessels.
© (2019) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.
more »
« less
- Award ID(s):
- 1725937
- NSF-PAR ID:
- 10107072
- Date Published:
- Journal Name:
- Proc. SPIE 10890, Label-free Biomedical Imaging and Sensing (LBIS) 2019, 108902M
- Volume:
- 10890
- Page Range / eLocation ID:
- 94
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Background: Recent reports have raised concern about the risk of vessel wall injury (VWI) when pulling out current laser-cut stent retrievers with active strut apposition to the vessel walls. Development of braided stroke thrombectomy-assist devices for use in conjunction with aspiration systems may be gentler in the internal carotid (ICA) and basilar vessels (with regards to radial force) compared to existing laser-cut stent retrievers. Methods: Radial force (RF) bench testing was performed using a radial compression station (Blockwise Engineering, Phoenix, AZ). The average total radial force (RF) in Newtons (N) generated (average of 3 readings) in vessel diameters (d) (Range 3.25 to 4.00mm) seen in proximal LVOs of the anterior circulation (such as in the internal carotid artery - ICA), and vessel diameters (d) (Range 2.50 to 3.25mm) seen in the posterior circulation (such as in the basilar artery - BA) was measured. The Solitaire Platinum Revascularization Device (Medtronic, Irvine, CA) was used as the predicate device. All thrombectomy and thrombectomy-assist devices were compared in terms of the RF being higher or lower (%) to the predicate device. Results: The results of the radial force testing are shown in the table below. The total radial force (RF) of the SHELTER® Retriever (part of Insera System, Insera Therapeutics, Inc., Dallas, TX), a braided thrombectomy-assist device is significantly lower (@ d=2.5mm: 58%) than the predicate device (@ d=2.5mm: 100%) and other laser-cut stent retrievers (@ d=2.5mm: 103% to 152%). Thrombectomy devices with lower OD had higher radial forces than larger devices. Conclusion: Novel braided stroke thrombectomy-assist devices for use in conjunction with aspiration systems have lower radial force compared to existing laser-cut stent retrievers in the ICA and BA vessel diameters. Further studies in-vivo need to assess the impact of lower radial force on minimizing VWI. Funding Source: This study was funded in part by a research grant (NSF Award: 1819491; PI: Vallabh Janardhan, MD) from the National Science Foundation (NSF). Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation. Conference Proceeding: This paper was presented in part at the 2018 Annual Meeting of the Society of Vascular & Interventional Neurology (SVIN), November 14-17, 2018 in San Diego, CAmore » « less
-
Hyperspectral imaging technologies have shown great promise for biomedical applications. These techniques have been especially useful for detection of molecular events and characterization of cell, tissue, and biomaterial composition. Unfortunately, hyperspectral imaging technologies have been slow to translate to clinical devices – likely due to increased cost and complexity of the technology as well as long acquisition times often required to sample a spectral image. We have demonstrated that hyperspectral imaging approaches which scan the fluorescence excitation spectrum can provide increased signal strength and faster imaging, compared to traditional emission-scanning approaches. We have also demonstrated that excitation-scanning approaches may be able to detect spectral differences between colonic adenomas and adenocarcinomas and normal mucosa in flash-frozen tissues. Here, we report feasibility results from using excitation-scanning hyperspectral imaging to screen pairs of fresh tumoral and nontumoral colorectal tissues. Tissues were imaged using a novel hyperspectral imaging fluorescence excitation scanning microscope, sampling a wavelength range of 360-550 nm, at 5 nm increments. Image data were corrected to achieve a NIST-traceable flat spectral response. Image data were then analyzed using a range of supervised and unsupervised classification approaches within ENVI software (Harris Geospatial Solutions). Supervised classification resulted in >99% accuracy for single-patient image data, but only 64% accuracy for multi-patient classification (n=9 to date), with the drop in accuracy due to increased false-positive detection rates. Hence, initial data indicate that this approach may be a viable detection approach, but that larger patient sample sizes need to be evaluated and the effects of inter-patient variability studied.more » « less
-
more » « less
Abstract Background Four-dimensional flow cardiovascular magnetic resonance imaging (4D flow CMR) allows comprehensive assessment of pulmonary artery (PA) flow dynamics. Few studies have characterized longitudinal changes in pulmonary flow dynamics and right ventricular (RV) recovery following a pulmonary endarterectomy (PEA) for patients with chronic thromboembolic pulmonary hypertension (CTEPH). This can provide novel insights of RV and PA dynamics during recovery. We investigated the longitudinal trajectory of 4D flow metrics following a PEA including velocity, vorticity, helicity, and PA vessel wall stiffness.
Methods Twenty patients with CTEPH underwent pre-PEA and > 6 months post-PEA CMR imaging including 4D flow CMR; right heart catheter measurements were performed in 18 of these patients. We developed a semi-automated pipeline to extract integrated 4D flow-derived main, left, and right PA (MPA, LPA, RPA) volumes, velocity flow profiles, and secondary flow profiles. We focused on secondary flow metrics of vorticity, volume fraction of positive helicity (clockwise rotation), and the helical flow index (HFI) that measures helicity intensity.
Results Mean PA pressures (mPAP), total pulmonary resistance (TPR), and normalized RV end-systolic volume (RVESV) decreased significantly post-PEA (P < 0.002). 4D flow-derived PA volumes decreased (P < 0.001) and stiffness, velocity, and vorticity increased (P < 0.01) post-PEA. Longitudinal improvements from pre- to post-PEA in mPAP were associated with longitudinal decreases in MPA area (r = 0.68, P = 0.002). Longitudinal improvements in TPR were associated with longitudinal increases in the maximum RPA HFI (r=-0.85, P < 0.001). Longitudinal improvements in RVESV were associated with longitudinal decreases in MPA fraction of positive helicity (r = 0.75, P = 0.003) and minimum MPA HFI (r=-0.72, P = 0.005).
Conclusion We developed a semi-automated pipeline for analyzing 4D flow metrics of vessel stiffness and flow profiles. PEA was associated with changes in 4D flow metrics of PA flow profiles and vessel stiffness. Longitudinal analysis revealed that PA helicity was associated with pulmonary remodeling and RV reverse remodeling following a PEA.
-
Autofluorescence has historically been considered a nuisance in medical imaging. Many endogenous fluorophores, specifically, collagen, elastin, NADH, and FAD, are found throughout the human body. Diagnostically, these signals can be prohibitive since they can outcompete signals introduced for diagnostic purposes. Recent advances in hyperspectral imaging have allowed the acquisition of significantly more data in a shorter time period by scanning the excitation spectra of fluorophores. The reduced acquisition time and increased signal-to-noise ratio allow for separation of significantly more fluorophores than previously possible. Here, we propose to utilize excitation-scanning of autofluorescence to examine tissues and diagnose pathologies. Spectra of autofluorescent molecules were obtained using a custom inverted microscope (TE-2000, Nikon Instruments) with a Xe arc lamp and thin film tunable filter array (VersaChrome, Semrock, Inc.) Scans utilized excitation wavelengths from 360 nm to 550 nm in 5 nm increments. The resultant spectra were used to examine hyperspectral image stacks from various collaborative studies, including an atherosclerotic rat model and a colon cancer study. Hyperspectral images were analyzed with ENVI and custom Matlab scripts including linear spectral unmixing (LSU) and principal component analysis (PCA). Initial results suggest the ability to separate the signals of endogenous fluorophores and measure the relative concentrations of fluorophores among healthy and diseased states of similar tissues. These results suggest pathology-specific changes to endogenous fluorophores can be detected using excitationscanning hyperspectral imaging. Future work will expand the library of pure molecules and will examine more defined disease states.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Conference Paper:
- https://doi.org/10.1117/12.2510770
