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Quantitative diffuse reflectance spectroscopy (DRS) was developed for label-free, noninvasive, and real-time assessment of implanted tissue-engineered devices manufactured from primary human oral keratinocytes (six batches in two 5-patient cohorts). Constructs were implanted in a murine model for 1 and 3 weeks. DRS evaluated construct success in situ using optical absorption (hemoglobin concentration and oxygenation, attributed to revascularization) and optical scattering (attributed to cellular density and layer thickness). Destructive pre- and postimplantation histology distinguished experimental control from stressed constructs, whereas noninvasive preimplantation measures of keratinocyte glucose consumption and residual glucose in spent culture media did not. In constructs implanted for 1 week, DRS distinguished control due to stressed and compromised from healthy constructs. In constructs implanted for 3 weeks, DRS identified constructs with higher postimplantation success. These results suggest that quantitative DRS is a promising, clinically compatible technology for rapid, noninvasive, and localized tissue assessment to characterize tissue-engineered construct success in vivo. IMPACT STATEMENT: Despite the recent advance in tissue engineering and regenerative medicine, there is still a lack of nondestructive tools to longitudinally monitor the implanted tissue-engineered devices. In this study, we demonstrate the potential of quantitative diffuse reflectance spectroscopy as a clinically viable technique for noninvasive, label-free, and rapid characterization of graft success in situ.

Many conventional methods to assess engineered tissue morphology and viability are destructive techniques with limited utility for tissue constructs intended for implantation in patients. Sterile label-free optical molecular imaging methods analyzed tissue endogenous fluorophores without staining, noninvasively and quantitatively assessing engineered tissue, in lieu of destructive assessment methods. The objective of this study is to further investigate label-free optical metrics and their correlation with destructive methods. Tissue-engineered constructs (n = 33 constructs) fabricated with primary human oral keratinocytes (n = 10 patients) under control, thermal stress, and rapamycin treatment manufacturing conditions exhibited a range of tissue viability states, as evaluated by quantitative histology scoring, WST-1 assay, Ki-67 immunostaining imaging, and label-free optical molecular imaging methods. Both histology sections of fixed tissues and cross-sectioned label-free optical images of living tissues provided quantitative spatially selective information on local tissue morphology, but optical methods noninvasively characterized both local tissue morphology and cellular viability at the same living tissue site. Furthermore, optical metrics noninvasively assessed living tissue viability with a statistical significance consistent with the destructive tissue assays WST-1 and histology. Over the range of cell viability states created experimentally, optical metrics noninvasively and quantitatively characterized living tissue viability and correlated with the destructive WST-1 tissue assay. By providing, under sterile conditions, noninvasive metrics that were comparable with conventional destructive tissue assays, label-free optical molecular imaging has the potential to monitor and assess engineered tissue construct viability before surgical implantation. IMPACT STATEMENT: The U.S. Food and Drug Administration has identified the lack of noninvasive evaluation method as an essential missing experimental release criterion strongly desired for clinical translation of tissue-engineered constructs. This study addresses the need by investigating label-free optical metrics for monitoring engineered tissues with a range of viability states. The noninvasive optical method was compared with multiple traditional assays and was found to correlate with a destructive tissue assay. The study would draw interest from many fields, including academics and professionals working in biomedical optics, tissue engineering, regenerative medicine, and the broader medical audience due to its potential applications to any cell-based construct.

Incidences of low-trauma fractures among osteopenic women may be related to changes in bone quality. In this blinded, prospective-controlled study, compositional and heterogeneity contributors of bone quality to fracture risk were examined. We hypothesize that Raman spectroscopy can differentiate between osteopenic women with one or more fractures (cases) from women without fractures (controls). This study involved the Raman spectroscopic analysis of cortical and cancellous bone composition using iliac crest biopsies obtained from 59-cases and 59-controls, matched for age (62.0 ± 7.5 and 61.7 ± 7.3 years, respectively, p = 0.38) and hip bone mineral density (BMD, 0.827 ± 0.083 and 0.823 ± 0.072 g/cm3, respectively, p = 0.57). Based on aggregate univariate case-control and odds ratio based logistic regression analyses, we discovered two Raman ratiometric parameters that were predictive of past fracture risk. Specifically, 1244/1268 and 1044/959 cm−1 ratios, were identified as the most differential aspects of bone quality in cortical cases with odds ratios of 0.617 (0.406–0.938 95% CI, p = 0.024) and 1.656 (1.083–2.534 95% CI, p = 0.020), respectively. Both 1244/1268 and 1044/959 cm−1 ratios exhibited moderate sensitivity (59.3–64.4%) but low specificity (49.2–52.5%). These results suggest that the organization of mineralized collagen fibrils were significantly altered in cortical cases compared to controls. In contrast, compositional and heterogeneity parameters related to mineral/matrix ratios, B-type carbonate substitutions, and mineral crystallinity, were not significantly different between cases and controls. In conclusion, a key outcome of this study is the significant odds ratios obtained for two Raman parameters (1244/1268 and 1044/959 cm−1 ratios), which from a diagnostic perspective, may assist in the screening of osteopenic women with suspected low-trauma fractures. One important implication of these findings includes considering the possibility that changes in the organization of collagen compositional structure plays a far greater role in postmenopausal women with osteopenic fractures.

Fluorescence lifetime sensing has been shown to noninvasively characterize the preimplantation health and viability of engineered tissue constructs. However, current practices to monitor postimplantation construct integration are either qualitative (visual assessment) or destructive (tissue histology). We employed label-free fluorescence lifetime spectroscopy for quantitative, noninvasive optical assessment of engineered tissue constructs that were implanted into a murine model. The portable system was designed to be suitable for intravital measurements and included a handheld probe to precisely and rapidly acquire data at multiple sites per construct. Our model tissue constructs were manufactured from primary human cells to simulate patient variability based on a standard protocol, and half of the manufactured constructs were stressed to create a range of health states. Secreted amounts of three cytokines that relate to cellular viability were measured in vitro to assess preimplantation construct health: interleukin-8 (IL-8), human β-defensin 1 (hBD-1), and vascular endothelial growth factor (VEGF). Preimplantation cytokine secretion ranged from 1.5 to 33.5 pg/mL for IL-8, from 3.4 to 195.0 pg/mL for hBD-1, and from 0.1 to 154.3 pg/mL for VEGF. In vivo optical sensing assessed constructs at 1 and 3 weeks postimplantation. We found that at 1 week postimplantation, in vivo optical parameters correlated with in vitro preimplantation secretion levels of all three cytokines (p

Background Angiogenesis, the formation of blood vessels, is a critical aspect of wound healing. Disorders of wound healing are often characterized by lack of angiogenesis, a condition frequently observed in aging and diabetic patients. Current techniques for assessing blood at injury sites are limited to contrast-imaging, including angiography. However, these techniques do not directly observe oxygenation of blood and are not amenable to serial evaluation. A multimodal noninvasive reflectance and Raman spectrometer have been proposed to help clinicians as a point-of-care tool to interrogate local angiogenesis and tissue architecture, respectively. The spectrometer system is a rapid, noninvasive, and label-free technology well-suited for the clinical environment. Materials and methods To demonstrate feasibility, the spectrometer system was used to interrogate angiogenesis serially over 9 wk as a result of heterotopic ossification (HO) development in a validated murine model. End-stage HO was confirmed by micro-computed tomography. Results Our preliminary results suggest that reflectance spectroscopy can be used to delineate vessel formation and that pathologic wounds may be characterized by unique spectra. In our model, HO formed at sites 1-3, whereas sites 4 and 5 did not have radiographic evidence of HO. Conclusions A point-of-care system like that demonstrated here shows potential as a noninvasive tool to assess local angiogenesis and tissue architecture that may allow for timely intervention in a clinical setting.

Bone composition and biomechanics at the tissue-level are important contributors to whole bone strength. Sclerostin antibody (Scl-Ab) is a candidate anabolic therapy for the treatment of osteoporosis that increases bone formation, bone mass, and bone strength in animal studies, but its effect on bone quality at the tissue-level has received little attention. Pre-clinical studies of Scl-Ab have recently expanded to include diseases with altered collagen and material properties such as Osteogenesis Imperfecta (OI). The purpose of this study was to investigate the role of Scl-Ab on bone quality by determining bone material composition and tissue-level mechanical properties in normal wild type (WT) tissue, as well as mice with a typical OI Gly→Cys mutation (Brtl/+) in type I collagen. Rapidly growing (3-week-old) and adult (6-month-old) WT and Brtl/+ mice were treated for 5 weeks with Scl-Ab. Fluorescent guided tissue-level bone composition analysis (Raman spectroscopy) and biomechanical testing (nanoindentation) were performed at multiple tissue ages. Scl-Ab increased mineral to matrix in adult WT and Brtl/+ at tissue ages of 2–4wks. However, no treatment related changes were observed in mineral to matrix levels at mid-cortex, and elastic modulus was not altered by Scl-Ab at any tissue age. Increased mineral-to-matrix was phenotypically observed in adult Brtl/+ OI mice (at tissue ages >3wk) and rapidly growing Brtl/+ (at tissue ages > 4wk) mice compared to WT. At identical tissue ages defined by fluorescent labels adult mice had generally lower mineral to matrix ratios and a greater elastic modulus than rapidly growing mice, demonstrating that bone matrix quality can be influenced by animal age and tissue age alike. In summary, these data suggest that Scl-Ab alters the matrix chemistry of newly formed bone while not affecting the elastic modulus, induces similar changes between Brtl/+ and WT mice, and provides new insight into the interaction between tissue age and animal age on bone quality.

Clinical translation of engineered tissue constructs requires noninvasive methods to assess construct health and viability after implantation in patients. However, current practices to monitor post-implantation construct integration are either qualitative (visual assessment) or destructive (tissue histology). As label-free fluorescence lifetime sensing can noninvasively characterize pre-implantation construct viability, we employed a handheld fluorescence lifetime spectroscopy probe to quantitatively and noninvasively assess tissue constructs that were implanted in a murine model. We designed the system to be suitable for intravital measurements: portability, localization with precise maneuverability, and rapid data acquisition. Our model tissue constructs were manufactured from primary human cells to simulate patient variability and were stressed to create a range of health states. Secreted amounts of three cytokines that relate to cellular viability were measured in vitro to assess pre-implantation construct health. In vivo optical sensing assessed tissue integration of constructs at one-week and three-weeks post-implantation. At one-week post-implantation, optical parameters correlated with in vitro pre-implantation secretion levels of all three cytokines (p < 0.05). This relationship was no longer seen at three-weeks post-implantation, suggesting comparable tissue integration independent of preimplantation health. Histology confirmed re-epithelialization of these constructs independent of pre-implantation health state, supporting the lack of a correlation. These results suggest that clinical optical diagnostic tools based on label-free fluorescence lifetime sensing of endogenous tissue fluorophores could noninvasively monitor post-implantation integration of engineered tissues.

Nonlinear optical molecular imaging and quantitative analytic methods were developed to non-invasively assess the viability of tissue-engineered constructs manufactured from primary human cells. Label-free optical measures of local tissue structure and biochemistry characterized morphologic and functional differences between controls and stressed constructs. Rigorous statistical analysis accounted for variability between human patients. Fluorescence intensity-based spatial assessment and metabolic sensing differentiated controls from thermally-stressed and from metabolically-stressed constructs. Fluorescence lifetime-based sensing differentiated controls from thermally-stressed constructs. Unlike traditional histological (found to be generally reliable, but destructive) and biochemical (non-invasive, but found to be unreliable) tissue analyses, label-free optical assessments had the advantages of being both non-invasive and reliable. Thus, such optical measures could serve as reliable manufacturing release criteria for cell-based tissue-engineered constructs prior to human implantation, thereby addressing a critical regulatory need in regenerative medicine.

Calciphylaxis is a painful, debilitating, and premorbid condition, which presents as calcified vasculature and soft tissues. Traditional diagnosis of calciphylaxis lesions requires an invasive biopsy, which is destructive, time consuming, and often leads to exacerbation of the condition and infection. Furthermore, it is difficult to find small calcifications within a large wound bed. To address this need, a noninvasive diagnostic tool may help clinicians identify ectopic calcified mineral and determine the disease margin. We propose Raman spectroscopy as a rapid, point-of-care, noninvasive, and label-free technology to detect calciphylaxis mineral. Debrided calciphylactic tissue was collected from six patients and assessed by microcomputed tomography (micro-CT). Micro-CT confirmed extensive deposits in three specimens, which were subsequently examined with Raman spectroscopy. Raman spectra confirmed that deposits were consistent with carbonated apatite, consistent with the literature. Raman spectroscopy shows potential as a noninvasive technique to detect calciphylaxis in a clinical environment.

Osteogenesis imperfecta (OI) is a genetic disorder resulting in defective collagen or collagen-associated proteins and fragile, brittle bones. To date, therapies to improve OI bone mass, such as bisphosphonates, have increased bone mass in the axial skeleton of OI patients, but have shown limited effects at reducing long bone fragility. Sclerostin antibody (Scl- Ab), currently in clinical trials for osteoporosis, stimulates bone formation and may have the potential to reduce long bone fracture rates in OI patients. Scl-Ab has been investigated as an anabolic therapy for OI in the Brtl/+ mouse model of moderately severe Type IV OI. While Scl-Ab increases long bone mass in the Brtl/+ mouse, it is not known whether material properties and composition changes also occur. Here, we report on the effects of Scl-Ab on wild type and Brtl/+ young (3 week) and adult (6 month) male mice. Scl-Ab was administered over 5 weeks (25mg/kg, 2x/week). Raman microspectroscopy and nanoindentation are used for bone composition and biomechanical bone property measurements in excised bone. Fluorescent labels (calcein and alizarin) at 4 time points over the entire treatment period are used to enable measurements at specific tissue age. Differences between wild type and Brtl/+ groups included variations in the mineral and matrix lattices, particularly the phosphate v1, carbonate v1, and the v(CC) proline and hydroxyproline stretch vibrations. Results of Raman spectroscopy corresponded to nanoindentation findings which indicated that old bone (near midcortex) is stiffer (higher elastic modulus) than new bone. We compare and contrast mineral to matrix and carbonate to phosphate ratios in young and adult mice with and without treatment.

Nonlinear optical microscopy methods were developed for quantitative, noninvasive, and label-free assessment of living engineered tissues fabricated from primary human cells. Clinical applications in tissue engineering and regenerative medicine will be discussed.

In this study, we describe a direct fit photon-tissue interaction model to quantitatively analyze reflectance spectra of biological tissue samples. The model rapidly extracts biologically-relevant parameters associated with tissue optical scattering and absorption. This model was employed to analyze reflectance spectra acquired from freshly excised human pancreatic pre-cancerous tissues (intraductal papillary mucinous neoplasm (IPMN), a common precursor lesion to pancreatic cancer). Compared to previously reported models, the direct fit model improved fit accuracy and speed. Thus, these results suggest that such models could serve as real-time, quantitative tools to characterize biological tissues assessed with reflectance spectroscopy.

In a pilot study, multimodal optical spectroscopy coupled with quantitative tissue-optics models distinguished intraductal papillary mucinous neoplasm (IPMN), a common precursor to pancreatic cancer, from normal tissues in freshly excised human pancreas. A photon-tissue interaction (PTI) model extracted parameters associated with cellular nuclear size and refractive index (from reflectance spectra) and extracellular collagen content (from fluorescence spectra). The results suggest that tissue optical spectroscopy has the potential to characterize pre-cancerous neoplasms in human pancreatic tissues.

Fluorescence lifetime imaging microscopy (FLIM) is a method for measuring fluorophore lifetimes with microscopic spatial resolution, providing a useful tool for cell biologists to detect, visualize, and investigate structure and function of biological systems. In this chapter, we begin by introducing the basic theory of fluorescence lifetime, including the characteristics of fluorophore decay, followed by a discussion of factors affecting fluorescence lifetimes and the potential advantages of fluorescence lifetime as a source of image contrast. Experimental methods for creating lifetime maps, including both time- and frequency-domain experimental approaches, are then introduced. Then, FLIM data analysis methods are discussed, including rapid lifetime determination, multiexponential fitting, Laguerre polynomial fitting, and phasor plot analysis. After, data analysis methods are introduced that improve lifetime precision of FLIM maps based upon optimal virtual gating and total variation denoising. The chapter concludes by highlighting several recent FLIM applications for quantitative biological imaging, including Förster resonance energy transfer-FLIM, fluorescence correlation spectroscopy-FLIM, multispectral-FLIM, and multiphoton-FLIM.

Pancreatic adenocarcinoma has a five-year survival rate of less than 6%. This low survival rate is attributed to the lack of accurate detection methods, which limits diagnosis to late-stage disease. Here, an in vivo pilot study assesses the feasibility of optical spectroscopy to improve clinical detection of pancreatic adenocarcinoma. During surgery on 6 patients, we collected spectrally-resolved reflectance and fluorescence in vivo. Site-matched in vivo and ex vivo data agreed qualitatively and quantitatively. Quantified differences between adenocarcinoma and normal tissues in vivo were consistent with previous results from a large ex vivo data set. Thus, optical spectroscopy is a promising method for the improved diagnosis of pancreatic cancer in vivo.

Fluorescence lifetime imaging microscopy (FLIM) is a method for measuring fluorophore lifetimes with microscopic spatial resolution, providing a useful tool for cell biologists to detect, visualize, and investigate structure and function of biological systems. In this chapter, we begin by introducing the basic theory of fluorescence lifetime, including the characteristics of fluorophore decay, followed by a discussion of factors affecting fluorescence lifetimes and the potential advantages of fluorescence lifetime as a source of image contrast. Experimental methods for creating lifetime maps, including both time- and frequency-domain experimental approaches, are then introduced. Then, FLIM data analysis methods are discussed, including rapid lifetime determination, multiexponential fitting, Laguerre polynomial fitting, and phasor plot analysis. After, data analysis methods are introduced that improve lifetime precision of FLIM maps based upon optimal virtual gating and total variation denoising. The chapter concludes by highlighting several recent FLIM applications for quantitative biological imaging, including Forster resonance energy transfer-FLIM, fluorescence correlation spectroscopy-FLIM, multispectral-FLIM, and multiphoton-FLIM.

Label-free nonlinear optical microscopy was employed to monitor primary human cells in tissue-engineered constructs. Novel quantitative analysis techniques were developed that successfully distinguished viable from non-viable engineered tissues.

Fluorescence lifetime imaging microscopy (FLIM) was employed to noninvasively characterize metabolic function in primary human oral keratinocytes used to develop functional engineered tissues. Living cells were compared under control culture conditions and systematic variations to investigate cellular function and viability. Nonlinear optical microscopy via two-photon excitation was employed to image cellular metabolic biomarkers NAD(P)H and FAD with thin optical sectioning and minimal out-of-focus fluorophore photobleaching. Novel post-processing FLIM algorithms were developed and tested. Results suggest that FLIM may provide useful information about live cell function and viability.

Fluorescence redox imaging was employed to monitor the metabolic activity of primary human oral keratinocytes prior to the development of tissue-engineered constructs. Keratinocytes with controlled culture conditions were treated with varying levels of chemical stimuli, resulting in differing cellular morphology, growth rate, and metabolic activity. Fluorescence images of keratinocytes were noninvasively acquired from endogenous intracellular metabolic fluorophores NAD(P)H and FAD. A redox ratio quantitatively analyzed each pair of images, showing that fluorescence redox imaging may be a novel technique to characterize live cell viability

There is a growing need for the development of computational models that can account for complex tissue morphology in simulations of photon propagation. We describe the development and validation of a user-friendly, MATLAB-based Monte Carlo code that uses analytically-defined surface meshes to model heterogeneous tissue geometry. The code can use information from non-linear optical microscopy images to discriminate the fluorescence photons (from endogenous or exogenous fluorophores) detected from different layers of complex turbid media. We present a specific application of modeling a layered human tissue-engineered construct (Ex Vivo Produced Oral Mucosa Equivalent, EVPOME) designed for use in repair of oral tissue following surgery. Second-harmonic generation microscopic imaging of an EVPOME construct (oral keratinocytes atop a scaffold coated with human type IV collagen) was employed to determine an approximate analytical expression for the complex shape of the interface between the two layers. This expression can then be inserted into the code to correct the simulated fluorescence for the effect of the irregular tissue geometry.

Pancreatic adenocarcinoma has a five-year survival rate of only 6%, largely because current diagnostic methods cannot reliably detect the disease in its early stages. Reflectance and fluorescence spectroscopies have the potential to provide quantitative, minimally-invasive means of distinguishing pancreatic adenocarcinoma from normal pancreatic tissue and chronic pancreatitis. The first collection of wavelength-resolved reflectance and fluorescence spectra and time-resolved fluorescence decay curves from human pancreatic tissues was acquired with clinically-compatible instrumentation. Mathematical models of reflectance and fluorescence extracted parameters related to tissue morphology and biochemistry that were statistically significant for distinguishing between pancreatic tissue types. These results suggest that optical spectroscopy has the potential to detect pancreatic disease in a clinical setting.

Tissue-engineered constructs require noninvasive monitoring of cellular viability prior to implantation. In a preclinical study on human Ex Vivo Produced Oral Mucosa Equivalent (EVPOME) constructs, nonlinear optical molecular imaging was employed to extract morphological and functional information from intact constructs. Multiphoton excitation fluorescence images were acquired using endogenous fluorescence from cellular nicotinamide adenine dinucleotide phosphate [NAD(P)H] and flavin adenine dinucleotide (FAD). The images were analyzed to report quantitatively on tissue structure and metabolism (redox ratio). Both thickness variations over time and cell distribution variations with depth were identified, while changes in redox were quantified. Our results show that nonlinear optical molecular imaging has the potential to visualize and quantitatively monitor the growth and viability of a tissue-engineered construct over time. (110 words)

Endogenous fluorescence redox imaging was developed to noninvasively assess cell viability in 3-dimensional tissue-engineered constructs prior to implantation. A lower redox ratio was observed from samples with higher proliferation.

A specialized transient digitizer system was developed for spectroscopic collection of fluorescence wavelength-time matrices (WTMs) from biological tissues. The system is compact, utilizes fiber optic probes for clinical compatibility, and offers rapid collection of high signal-to-noise ratio (>100) time- and wavelength- resolved fluorescence. The system is compatible with excitation sources operating in excess of 25 kHz. Wavelength-resolved measurement range is 300?800 nm with ≥ 0.01 nm steps. Time-resolved measurement depth is 128 ns with fixed 0.2 ns steps. The information-rich WTM data provides comprehensive fluorescence sensing capabilities, as demonstrated on tissue simulating phantoms. Extracting wavelength-resolved fluorophore lifetimes illustrates the potential of using the technology to resolve exogenous or endogenous fluorophore contributions in tissue samples in a clinical setting for tissue diagnostics and monitoring.

We report data collected with a specialized transient digitizer, high repetition rate microchip laser sources, and fiber optic light delivery and collection for rapid remote sensing in tissue-simulating phantoms. The instrumentation is highly suitable for eventual translation to a clinical setting owing to the speed of data acquisition and small footprint. Ranges for data acquisition time and instrument sensitivity were determined by measuring wavelength time matrices (WTMs) from tissue-simulating phantoms. Accuracy of WTM data was validated by comparison with Monte-Carlo simulations of fluorescent light propagation in turbid media.

Clinically-compatible technology was developed to measure wavelength- and time-resolved fluorescence intensities from biological tissues. Validation studies were conducted on tissue-simulating phantoms and the results were consistent with theoretical predictions with < 4% deviation.

We report novel optical diagnostic algorithms for clinical pancreatic tissue classification, including a photon-tissue interaction model developed to extract biophysical parameters from reflectance and fluorescence spectra to distinguish pancreatic adenocarcinoma from normal tissue and pancreatitis.

A photon-tissue interaction (PTI) model was developed and employed to analyze 96 pairs of reflectance and fluorescence spectra from freshly excised human pancreatic tissues. For each pair of spectra, the PTI model extracted a cellular nuclear size parameter from the measured reflectance, and the relative contributions of extracellular and intracellular fluorophores to the intrinsic fluorescence. The results suggest that reflectance and fluorescence spectroscopies have the potential to quantitatively distinguish among pancreatic tissue types, including normal pancreatic tissue, pancreatitis, and pancreatic adenocarcinoma.

A fiber-optic system was developed to rapidly acquire tissue fluorescence wavelength-time matrices (WTMs) with high signal-to-noise ratio (SNR). The essential system components (473 nm microchip laser operating at 3 kHz repetition frequency, fiber-probe assemblies, emission monochromator, photomultiplier tube, and digitizer) were assembled into a compact and clinically-compatible unit. Data were acquired from fluorescence standards and tissue-simulating phantoms to test system performance. Fluorescence decay waveforms with SNR > 100 at the decay curve peak were obtained in less than 30 ms. With optimized data transfer and monochromator stepping functions, it should be feasible to acquire a full WTM at 5 nm emission wavelength intervals over a 200 nm range in under 2 seconds.