Primary blast injury is caused by the direct impact of an overpressurization wave on the body. Due to limitations of current models, we have developed a novel approach to study primary blast‐induced ...traumatic brain injury. Specifically, we employ a bioengineered 3D brain‐like human tissue culture system composed of collagen‐infused silk protein donut‐like hydrogels embedded with human IPSC‐derived neurons, human astrocytes, and a human microglial cell line. We have utilized this system within an advanced blast simulator (ABS) to expose the 3D brain cultures to a blast wave that can be precisely controlled. These 3D cultures are enclosed in a 3D‐printed surrogate skull‐like material containing media which are then placed in a holder apparatus inside the ABS. This allows for exposure to the blast wave alone without any secondary injury occurring. We show that blast induces an increase in lactate dehydrogenase activity and glutamate release from the cultures, indicating cellular injury. Additionally, we observe a significant increase in axonal varicosities after blast. These varicosities can be stained with antibodies recognizing amyloid precursor protein. The presence of amyloid precursor protein deposits may indicate a blast‐induced axonal transport deficit. After blast injury, we find a transient release of the known TBI biomarkers, UCHL1 and NF‐H at 6 h and a delayed increase in S100B at 24 and 48 h. This in vitro model will enable us to gain a better understanding of clinically relevant pathological changes that occur following primary blast and can also be utilized for discovery and characterization of biomarkers.
In vitro tissue engineering provides a novel approach to investigate the cellular and molecular effects of traumatic brain injury. Here, we describe a bioengineered 3D brain‐like human tissue culture system to study primary blast‐induced traumatic brain injury.
Alterations in the balance between different metabolic pathways used to meet cellular bioenergetic and biosynthetic demands are considered hallmarks of cancer. Optical imaging relying on endogenous ...fluorescence has been used as a noninvasive approach to assess tissue metabolic changes during cancer development. However, quantitative correlations of optical assessments with variations in the concentration of relevant metabolites or in the specific metabolic pathways that are involved have been lacking. In this study, we use high-resolution, depth-resolved imaging, relying entirely on endogenous two-photon excited fluorescence in combination with invasive biochemical assays and mass spectrometry to demonstrate the sensitivity and quantitative nature of optical redox ratio tissue assessments. We identify significant differences in the optical redox ratio of live, engineered normal and precancerous squamous epithelial tissues. We establish that while decreases in the optical redox ratio are associated with enhanced levels of glycolysis relative to oxidative phosphorylation, increases in glutamine consumption to support energy production are associated with increased optical redox ratio values. Such mechanistic insights in the origins of optical metabolic assessments are critical for exploiting fully the potential of such noninvasive approaches to monitor and understand important metabolic changes that occur in live tissues at the onset of cancer or in response to treatment.
Active changes in mitochondrial structure and organization facilitate cellular homeostasis. Because aberrant mitochondrial dynamics are implicated in a variety of human diseases, their assessment is ...potentially useful for diagnosis, therapy, and disease monitoring. Because current techniques for evaluating mitochondrial morphology are invasive or necessitate mitochondria-specific dyes, their clinical translation is limited. We report that mitochondrial dynamics can be monitored in vivo, within intact human skin by relying entirely on endogenous two-photon-excited fluorescence from the reduced metabolic coenzyme nicotinamide adenine dinucleotide (NADH). We established the sensitivity of this approach with in vivo, fast temporal studies of arterial occlusion-reperfusion, which revealed acute changes in the mitochondrial metabolism and dynamics of the lower human epidermal layers. In vitro hypoxic-reperfusion studies validated that the in vivo outcomes were a result of NADH fluorescence changes. To demonstrate the diagnostic potential of this approach, we evaluated healthy and cancerous human skin epithelia. Healthy tissues displayed consistent, depth-dependent morphological and mitochondrial organization patterns that varied with histological stratification and intraepithelial mitochondrial protein expression. In contrast, these consistent patterns were absent in cancerous skin lesions. We exploited these differences to successfully differentiate healthy from cancerous tissues using a predictive classification approach. Collectively, these results demonstrate that our label-free, automated, near real-time assessments of mitochondrial organization-relying solely on endogenous contrast-could be useful for accurate, noninvasive in vivo diagnosis.
In this paper, we present a 2-photon imaging probe system featuring a novel fluorescence collection method with improved and reliable efficiency. The system aims to miniaturize the potential of ...2-photon imaging in the metabolic and morphological characterization of cervical tissue at sub-micron resolution over large imaging depths into a flexible and clinically viable platform towards the early detection of cancers. Clinical implementation of such a probe system is challenging due to inherently low levels of autofluorescence, particularly when imaging deep in highly scattering tissues. For an efficient collection of fluorescence signals, our probe employs 12 0.5 NA collection fibers arranged around a miniaturized excitation objective. By bending and terminating a multitude of collection fibers at a specific angle, we increase collection area and directivity significantly. Positioning of these fibers allows the collection of fluorescence photons scattered away from their ballistic trajectory multiple times, which offers a system collection efficiency of 4%, which is 55% of what our bench-top microscope with 0.75 NA objective achieves. We demonstrate that the collection efficiency is largely maintained even at high scattering conditions and high imaging depths. Radial symmetry of arrangement maintains uniformity of collection efficiency across the whole FOV. Additionally, our probe can image at different tissue depths via axial actuation by a dc servo motor, allowing depth dependent tissue characterization. We designed our probe to perform imaging at 775 nm, targeting 2-photon autofluorescence from NAD(P)H and FAD molecules, which are often used in metabolic tissue characterization. An air core photonic bandgap fiber delivers laser pulses of 100 fs duration to the sample. A miniaturized objective designed with commercially available lenses of 3 mm diameter focuses the laser beam on tissue, attaining lateral and axial imaging resolutions of 0.66 µm and 4.65 µm, respectively. Characterization results verify that our probe achieves collection efficiency comparable to our optimized bench-top 2-photon imaging microscope, minimally affected by imaging depth and radial positioning. We validate autofluorescence imaging capability with excised porcine vocal fold tissue samples. Images with 120 µm FOV and 0.33 µm pixel sizes collected at 2 fps confirm that the 300 µm imaging depth was achieved.
Silk-based biomaterial systems have been previously explored for a variety of medical and nonmedical materials needs. The unique biophysical features of silks provide options to generate highly ...tailored structures and morphologies with this unique family of fibrous proteins. To exploit these features, we have optimized the all aqueous processing of silk fibroin into novel surface nanopatterned protein materials. We have exploited control of this nanomorphology to optimize the optical features of these silk protein systems. We demonstrate control of surface morphology down to 125 nm, with fidelity over large length scales. This surface nanopatterning allows the silk protein to be formed into diffractive optics such as diffraction gratings, pattern generators, and lenses due to novel aqueous processing into optically clear materials via control of β sheet crystallinity. Further, we incorporate biological components, such as hemoglobin and the enzyme peroxidase, during the process of forming the silk diffraction gratings. The ambient processing of the silk protein in water, in combination with these bioactive components, allows these entrained molecules to retain activity and provide added functions and selectivity to the optically active silk films. Thus, combinations of biochemical and optical readout is feasible and provides in a single, disposable/all degradable element with both spectral discrimination and biological function. These new surface nanopatterned, bioactive silk protein-based material systems offer a unique combination of features potentially useful for a range of biosensor needs, particularly when considered in concert with the remarkable mechanical properties of these proteins, their biocompatibility, and controllable biodegradation.
Foam cells perform critical functions in atherosclerosis. We hypothesize that coronary segments with superficial foam cells (SFCs) situated in a region of interest with a depth of 200 mum can be ...identified using intrinsic fluorescence spectroscopy (IFS) and diffuse reflectance spectroscopy (DRS). This is a key step in our ongoing program to develop a spectroscopic technique for real-time in vivo diagnosis of vulnerable atherosclerotic plaque.
We subjected 132 human coronary segments to in vitro IFS and DRS. We detected SFCs in 13 thick fibrous cap atheromas and 8 pathologic intimal thickening (PIT) lesions. SFCs colocalized with accumulations of smooth muscle cells and proteoglycans, including hyaluronan (P<0.001). Two spectroscopic parameters were generated from analysis of IFS at 480 nm excitation and DRS. A discriminatory algorithm using these parameters identified specimens with SFC area >40%, 20%, 10%, 5%, 2.5%, and 0% of the region of interest with 98%, 98%, 93%, 94%, 93%, and 90% accuracy, respectively.
Our combined IFS and DRS technique accurately detects SFCs in thick fibrous cap atheromas and PIT lesions. Because SFCs are associated with histological markers of plaque erosion, our spectroscopic technique could prove useful in identifying vulnerable plaques.
LSS and DRS are techniques that provide diagnostically useful information based on changes in the intensity of backscattered light that has experienced either a single or multiple scattering events. ...These changes can be detected effectively using statistical and model-based algorithms. In addition to providing a means to detect the presence of dysplasia noninvasively, the latter approach yields potentially significant insights into the processes that are involved in the development of dysplasia in BE. Because DRS and LSS provide complementary information to each other and to other spectroscopic modalities, such as fluorescence, it is possible that a combination of all these techniques will ultimately provide a highly accurate real-time method for detecting BE dysplasia. Extending point spectroscopic measurements to imaging of the esophagus will be challenging, but continuous progess in the development of powerful light sources, sensitive detectors, and micro-optical elements should allow this method to become a reality in the near future.
Abstract Non-invasive approaches to assess tissue function could improve significantly current methods to diagnose diseases and optimize engineered tissues. In this study, we describe a two-photon ...excited fluorescence microscopy approach that relies entirely on endogenous fluorophores to dynamically quantify functional metabolic readouts from individual cells within three-dimensional engineered tissues undergoing adipogenic differentiation over six months. Specifically, we employ an automated approach to analyze 3D image volumes and extract a redox ratio of metabolic cofactors. We identify a decrease in redox ratio over the first two months of culture that is associated with stem cell differentiation and lipogenesis. In addition, we demonstrate that the presence of endothelial cells facilitate greater cell numbers deeper within the engineered tissues. Since traditional assessments of engineered tissue structure and function are destructive and logistically intensive, this non-destructive, label-free approach offers a potentially powerful high-content characterization tool for optimizing tissue engineering protocols and assessing engineered tissue implants.
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