Hyperspectral imaging in highly scattering media by the spectral phasor approach using two filters.

Hyperspectral imaging is a common technique in fluorescence microscopy to obtain the emission spectrum at each pixel of an image. However, methods to obtain spectral resolution based on diffraction gratings or integrated prisms work poorly when the sample is strongly scattering. We developed a microscope named the DIVER that collects the fluorescence emission over a very large angle. Since the fluorescence light after passing through the multiple scattering sample is not collimated, the use of grating or prisms strongly limits the amount of light that can be used with available hyperspectral devices. Here we show that 2 filters that accept uncollimated light over a large aperture are sufficient to calculate the spectral phasor rather than displaying the entire spectrum. Using the properties of the spectral phasors, we can resolve spectral components and perform the type of data analyses that are usually performed in hyperspectral image analysis.

Fiber pattern removal and image reconstruction method for snapshot mosaic hyperspectral endoscopic images.

Hyperspectral endoscopic imaging has the potential to enhance clinical diagnostics and outcome. Most commercial endoscopes utilize imaging fiber bundles to transmit the collected signal from the patient to the medical operator. These bundles consist of several fiber cores surrounded by a cladding layer creating comb structure-like artifacts, which complicate further analysis, both spatially and spectrally. Here we present an optical fiber pattern removal algorithm which we applied to hyperspectral bronchoscopic images robustly and quantitatively without the need for specific optical or electrical hardware. We validate the performance of the pattern removal by using a novel hyperspectral phasor approach. This algorithm can be generalized to all forms of fiber bundle hyperspectral endoscopy.

Blood vessel detection, localization and estimation using a smart laparoscopic grasper: a Monte Carlo study.

For centuries, surgeons have relied on their sense of touch to identify vital structures such as blood vessels in traditional open surgery. Over the past two decades, surgeons have shifted to minimally invasive surgical (MIS) approaches, including laparoscopic surgery, which include benefits such as less scarring, less risk for infection, and quicker recovery times. In fact, some surgeries such as cholecystectomies have seen more than an 80% adoption of this technique because of those benefits. However, due to the fundamental challenges associated with using laparoscopic surgery, there has been a lower adoption in more complex specialties, such as colorectal and thoracic surgery, where the field of surgery has bleeding, fat, scar tissue, and adhesions. These problems are exacerbated by complicating factors such as inflammation, cancer, chronic disease, obesity, and re-operations. Importantly, surgeons will often convert from laparoscopy to open surgery if they can no longer proceed using the minimally invasive approach because of issues described with these complicating factors, thereby negating the benefits that the patient would have seen. When the surgeon does attempt these procedures with those issues, the surgery takes on average 30 min - 1 hour longer. A new method by which surgeons can visualize structures like blood vessels could reduce the conversion rates and operating time, thereby driving a greater adoption of laparoscopic surgery in these complex procedures. Here, we show that by adding near infrared (NIR) LEDs and a linear image sensor onto the opposing jaws of the laparoscopic graspers, blood vessels that are embedded within tissues can be detected and localized efficiently, even those not visible using current imaging techniques. We show the results of Monte Carlo simulations to support our claim, including that blood vessels ranging from 2 to 6 mm and buried under up to 1 cm of tissue can be detected. We also report developing a smart grasper handheld prototype to run ex vivo experiments. The results of these experiments matched with those of the Monte Carlo simulations and the estimated blood vessel size showed a strong correlation with the actual size. This technology will be incorporated into already existing laparoscopic tools to assist surgeons during MIS procedures.

On-chip light-sheet fluorescence imaging flow cytometry at a high flow speed of 1 m/s.

We present on-chip fluorescence imaging flow cytometry by light-sheet excitation on a mirror-embedded microfluidic chip. The method allows us to obtain microscopy-grade fluorescence images of cells flowing at a high speed of 1 m/s, which is comparable to the flow speed of conventional non-imaging flow cytometers. To implement the light-sheet excitation of flowing cells in a microchannel, we designed and fabricated a mirror-embedded PDMS-based microfluidic chip. To show its broad utility, we used the method to classify large populations of microalgal cells (Euglena gracilis) and human cancer cells (human adenocarcinoma cells). Our method holds promise for large-scale single-cell analysis.

High speed functional imaging with source localized multifocal two-photon microscopy.

Multifocal two-photon microscopy (MTPM) increases imaging speed over single-focus scanning by parallelizing fluorescence excitation. The imaged fluorescence's susceptibility to crosstalk, however, severely degrades contrast in scattering tissue. Here we present a source-localized MTPM scheme optimized for high speed functional fluorescence imaging in scattering mammalian brain tissue. A rastered line array of beamlets excites fluorescence imaged with a complementary metal-oxide-semiconductor (CMOS) camera. We mitigate scattering-induced crosstalk by temporally oversampling the rastered image, generating grouped images with structured illumination, and applying Richardson-Lucy deconvolution to reassign scattered photons. Single images are then retrieved with a maximum intensity projection through the deconvolved image groups. This method increased image contrast at depths up to 112 µm in scattering brain tissue and reduced functional crosstalk between pixels during neuronal calcium imaging. Source-localization did not affect signal-to-noise ratio (SNR) in densely labeled tissue under our experimental conditions. SNR decreased at low frame rates in sparsely labeled tissue, with no effect at frame rates above 50 Hz. Our non-descanned source-localized MTPM system enables high SNR, 100 Hz capture of fluorescence transients in scattering brain, increasing the scope of MTPM to faster and smaller functional signals.

Simultaneous acquisition of neuronal morphology and cytoarchitecture in the same Golgi-stained brain.

Acquiring an accurate orientation reference is a prerequisite for precisely analysing the morphological features of Golgi-stained neurons in the whole brain. However, the same reflective imaging contrast of Golgi staining for morphology and Nissl staining for cytoarchitecture leads to the failure of distinguishing soma morphology and simultaneously co-locate cytoarchitecture. Here, we developed the dual-mode micro-optical sectioning tomography (dMOST) method to simultaneously image the reflective and fluorescent signals in three dimensions. We evaluated the feasibility of real-time fluorescent counterstaining on Golgi-stained brain tissue. With our system, we acquired whole-brain data sets of physiological and pathological Golgi-stained mouse model brains with fluorescence-labelled anatomical annotation at single-neuron resolution. We also obtained the neuronal morphology of macaque monkey brain tissue using this method. The results show that real-time acquisition of the co-located cytoarchitecture reference in the same brain greatly facilitates the precise morphological analysis of Golgi-stained neurons.

50 Hz volumetric functional imaging with continuously adjustable depth of focus.

Understanding how neural circuits control behavior requires monitoring a large population of neurons with high spatial resolution and volume rate. Here we report an axicon-based Bessel beam module with continuously adjustable depth of focus (CADoF), that turns frame rate into volume rate by extending the excitation focus in the axial direction while maintaining high lateral resolutions. Cost-effective and compact, this CADoF Bessel module can be easily integrated into existing two-photon fluorescence microscopes. Simply translating one of the relay lenses along its optical axis enabled continuous adjustment of the axial length of the Bessel focus. We used this module to simultaneously monitor activity of spinal projection neurons extending over 60 µm depth in larval zebrafish at 50 Hz volume rate with adjustable axial extent of the imaged volume.

Complex differential variance angiography with noise-bias correction for optical coherence tomography of the retina.

Complex differential variance (CDV) provides phase-sensitive angiographic imaging for optical coherence tomography (OCT) with immunity to phase-instabilities of the imaging system and small-scale axial bulk motion. However, like all angiographic methods, measurement noise can result in erroneous indications of blood flow that confuse the interpretation of angiographic images. In this paper, a modified CDV algorithm that corrects for this noise-bias is presented. This is achieved by normalizing the CDV signal by analytically derived upper and lower limits. The noise-bias corrected CDV algorithm was implemented into an experimental 1 µm wavelength OCT system for retinal imaging that used an eye tracking scanner laser ophthalmoscope at 815 nm for compensation of lateral eye motions. The noise-bias correction improved the CDV imaging of the blood flow in tissue layers with a low signal-to-noise ratio and suppressed false indications of blood flow outside the tissue. In addition, the CDV signal normalization suppressed noise induced by galvanometer scanning errors and small-scale lateral motion. High quality cross-section and motion-corrected en face angiograms of the retina and choroid are presented.

Novel endoscope with increased depth of field for imaging human nasal tissue by microscopic optical coherence tomography.

Intravital microscopy (IVM) offers the opportunity to visualize static and dynamic changes of tissue on a cellular level. It is a valuable tool in research and may considerably improve clinical diagnosis. In contrast to confocal and non-linear microscopy, optical coherence tomography (OCT) with microscopic resolution (mOCT) provides intrinsically cross-sectional imaging. Changing focus position is not needed, which simplifies especially endoscopic imaging. For in-vivo imaging, here we are presenting endo-microscopic OCT (emOCT). A graded-index-lens (GRIN) based 2.75 mm outer diameter rigid endoscope is providing 1.5 - 2 µm nearly isotropic resolution over an extended field of depth. Spherical and chromatic aberrations are used to elongate the focus length. Simulation of the OCT image formation, suggests a better overall image quality in this range compared to a focused Gaussian beam. Total imaging depth at a reduced sensitivity and lateral resolution is more than 200 µm. Using a frame rate of 80 Hz cross-sectional images of concha nasalis were demonstrated in humans, which could resolve cilial motion, cellular structures of the epithelium, vessels and blood cells. Mucus transport velocity was successfully determined. The endoscope may be used for diagnosis and treatment control of different lung diseases like cystic fibrosis or primary ciliary dyskinesia, which manifest already at the nasal mucosa.

Ultrasound-mediation of self-illuminating reporters improves imaging resolution in optically scattering media.

In vivo imaging of self-illuminating bio-and chemiluminescent reporters is used to observe the physiology of small animals. However, strong light scattering by biological tissues results in poor spatial resolution of the optical imaging, which also degrades the quantitative accuracy. To overcome this challenging problem, focused ultrasound is used to modulate the light from the reporter at the ultrasound frequency. This produces an ultrasound switchable light 'beacon' that reduces the influence of light scattering in order to improve spatial resolution. The experimental results demonstrate that apart from light modulation at the ultrasound frequency (AC signal at 3.5 MHz), ultrasound also increases the DC intensity of the reporters. This is shown to be due to a temperature rise caused by insonification that was minimized to be within acceptable mammalian tissue safety thresholds by adjusting the duty cycle of the ultrasound. Line scans of bio-and chemiluminescent objects embedded within a scattering medium were obtained using ultrasound modulated (AC) and ultrasound enhanced (DC) signals. Lateral resolution is improved by a factor of 12 and 7 respectively, as compared to conventional CCD imaging. Two chemiluminescent sources separated by ~10 mm at ~20 mm deep inside a 50 mm thick chicken breast have been successfully resolved with an average signal-to-noise ratio of approximately 8-10 dB.

Erratum: Methods for detecting host genetic modifiers of tumor vascular function using dynamic near-infrared fluorescence imaging: errata.

[This corrects the article on p. 543 in vol. 9, PMID: 29552392.].

Methods for detecting host genetic modifiers of tumor vascular function using dynamic near-infrared fluorescence imaging.

Vascular supply is a critical component of the tumor microenvironment (TME) and is essential for tumor growth and metastasis, yet the endogenous genetic modifiers that impact vascular function in the TME are largely unknown. To identify the host TME modifiers of tumor vascular function, we combined a novel genetic mapping strategy [Consomic Xenograft Model] with near-infrared (NIR) fluorescence imaging and multiparametric analysis of pharmacokinetic modeling. To detect vascular flow, an intensified cooled camera based dynamic NIR imaging system with 785 nm laser diode based excitation was used to image the whole-body fluorescence emission of intravenously injected indocyanine green dye. Principal component analysis was used to extract the spatial segmentation information for the lungs, liver, and tumor regions-of-interest. Vascular function was then quantified by pK modeling of the imaging data, which revealed significantly altered tissue perfusion and vascular permeability that were caused by host genetic modifiers in the TME. Collectively, these data demonstrate that NIR fluorescent imaging can be used as a non-invasive means for characterizing host TME modifiers of vascular function that have been linked with tumor risk, progression, and response to therapy.

In vivo photoacoustic lipid imaging in mice using the second near-infrared window.

Photoacoustic imaging has emerged as a promising technique to improve preclinical and clinical imaging by providing users with label-free optical contrast of tissue. Here, we present a proof-of-concept study for noninvasive in vivo murine lipid imaging using 1210 nm light to investigate differences in periaortic fat among mice of different gender, genotypes, and maturation. Acquired lipid signals suggest that adult male apoE-/- mice have greater periaortic fat accumulation compared to adolescent males, apoE-/- females, and wild-type mice. These results demonstrate the potential of photoacoustic tomography for studying vascular pathophysiology and improving the diagnosis of lipid-based diseases.

Real-time volumetric lipid imaging in vivo by intravascular photoacoustics at 20 frames per second.

Lipid deposition can be assessed with combined intravascular photoacoustic/ultrasound (IVPA/US) imaging. To date, the clinical translation of IVPA/US imaging has been stalled by a low imaging speed and catheter complexity. In this paper, we demonstrate imaging of lipid targets in swine coronary arteries in vivo, at a clinically useful frame rate of 20 s-1. We confirmed image contrast for atherosclerotic plaque in human samples ex vivo. The system is on a mobile platform and provides real-time data visualization during acquisition. We achieved an IVPA signal-to-noise ratio of 20 dB. These data show that clinical translation of IVPA is possible in principle.

Fluorescence depth estimation from wide-field optical imaging data for guiding brain tumor resection: a multi-inclusion phantom study.

Studies have shown that fluorescent agents demarcate tumor from surrounding brain tissue and offer intraoperative guidance during resection. However, visualization of fluorescence signal from tumor below the surgical surface or through the appearance of blood in the surgical field is challenging. We have previously described red light imaging techniques for estimating fluorescent depths in turbid media. In this study, we evaluate these methods over a broader range of fluorophore concentrations, and investigate the ability to resolve multiple fluorescent emissions in the same plane or at different depths along the axis of imaging. A tungsten halogen lamp is used as a broadband white light source for reflectance imaging. Fluorescence from Alexa Fluor 647 is excited with a 635 nm diode laser. Reflectance and fluorescence spectral data are gathered between 670 and 720 nm with the use of a liquid crystal tunable filter and recorded on a sCMOS camera. Results show that two fluorescent emissions can be resolved within 2 mm if they are in the same plane or within 3 mm if they are at different depths along the axis of imaging up to 6 mm below the surface.

Design and evaluation of a compound acoustic lens for photoacoustic computed tomography.

In photoacoustic computed tomography, the limited directivity of the detectors may cause deformation of off-center targets and lead to an imbalanced resolution in the imaging area. To improve the directivity of the acoustic detectors, several negative acoustic lenses have been proposed. In this study, we develop a new compound acoustic lens fabricated by integrating a concave polydimethylsiloxane (PDMS) lens and a convex epoxy lens. Both theoretical simulations and experimental evaluations demonstrate that the compound lens provides a larger directivity compared to single lenses made of PDMS, epoxy, and liquid. The measured acceptance angles of a 6-mm piezoelectric acoustic transducer equipped with the compound, epoxy, liquid, and PDMS lenses are 55°, 36°, 25°, and 20°, respectively. No deformation is observed in the off-center targets by using compound lens. However, serious deformation appears in the cases using single lenses.

Deep tissue photoacoustic computed tomography with a fast and compact laser system.

Photoacoustic computed tomography (PACT) holds great promise for biomedical imaging, but wide-spread implementation is impeded by the bulkiness of flash-lamp-pumped laser systems, which typically weigh between 50 - 200 kg, require continuous water cooling, and operate at a low repetition rate. Here, we demonstrate that compact lasers based on emerging diode technologies are well-suited for preclinical and clinical PACT. The diode-pumped laser used in this study had a miniature footprint (13 × 14 × 7 cm3), weighed only 1.6 kg, and outputted up to 80 mJ per pulse at 1064 nm. In vitro, the laser system readily provided over 4 cm PACT depth in chicken breast tissue. In vivo, in addition to high resolution, non-invasive brain imaging in living mice, the system can operate at 50 Hz, which enabled high-speed cross-sectional imaging of murine cardiac and respiratory function. The system also provided high quality, high-frame rate, and non-invasive three-dimensional mapping of arm, palm, and breast vasculature at multi centimeter depths in living human subjects, demonstrating the clinical viability of compact lasers for PACT.

Non-invasive terahertz imaging of tissue water content for flap viability assessment.

Accurate and early prediction of tissue viability is the most significant determinant of tissue flap survival in reconstructive surgery. Perturbation in tissue water content (TWC) is a generic component of the tissue response to such surgeries, and, therefore, may be an important diagnostic target for assessing the extent of flap viability in vivo. We have previously shown that reflective terahertz (THz) imaging, a non-ionizing technique, can generate spatially resolved maps of TWC in superficial soft tissues, such as cornea and wounds, on the order of minutes. Herein, we report the first in vivo pilot study to investigate the utility of reflective THz TWC imaging for early assessment of skin flap viability. We obtained longitudinal visible and reflective THz imagery comparing 3 bipedicled flaps (i.e. survival model) and 3 fully excised flaps (i.e. failure model) in the dorsal skin of rats over a postoperative period of 7 days. While visual differences between both models manifested 48 hr after surgery, statistically significant (p < 0.05, independent t-test) local differences in TWC contrast were evident in THz flap image sets as early as 24 hr. Excised flaps, histologically confirmed as necrotic, demonstrated a significant, yet localized, reduction in TWC in the flap region compared to non-traumatized skin. In contrast, bipedicled flaps, histologically verified as viable, displayed mostly uniform, unperturbed TWC across the flap tissue. These results indicate the practical potential of THz TWC sensing to accurately predict flap failure 24 hours earlier than clinical examination.

Time-stretch microscopy on a DVD for high-throughput imaging cell-based assay.

Cell-based assay based on time-stretch imaging is recognized to be well-suited for high-throughput phenotypic screening. However, this ultrafast imaging technique has primarily been limited to suspension-cell assay, leaving a wide range of solid-substrate assay formats uncharted. Moreover, time-stretch imaging is generally restricted to intrinsic biophysical phenotyping, but lacks the biomolecular signatures of the cells. To address these challenges, we develop a spinning time-stretch imaging assay platform based on the functionalized digital versatile disc (DVD). We demonstrate that adherent cell culture and biochemically-specific cell-capture can now be assayed with time-stretch microscopy, thanks to the high-speed DVD spinning motion that naturally enables on-the-fly cellular imaging at an ultrafast line-scan rate of >10MHz. As scanning the whole DVD at such a high speed enables ultra-large field-of-view imaging, it could be favorable for scaling both the assay throughput and content as demanded in many applications, e.g. drug discovery, and rare cancer cell screening.

Clinically compatible flexible wide-field multi-color fluorescence endoscopy with a porcine colon model.

Early detection of structural or molecular changes in dysplastic epithelial tissues is crucial for cancer screening and surveillance. Multi-targeting molecular endoscopic fluorescence imaging may improve noninvasive detection of precancerous lesions in the colon. Here, we report the first clinically compatible, wide-field-of-view, multi-color fluorescence endoscopy with a leached fiber bundle scope using a porcine model. A porcine colon model that resembles the human colon is used for the detection of surrogate tumors composed of multiple biocompatible fluorophores (FITC, ICG, and heavy metal-free quantum dots (hfQDs)). With an ex vivo porcine colon tumor model, molecular imaging with hfQDs conjugated with MMP14 antibody was achieved by spraying molecular probes on a mucosa layer that contains xenograft tumors. With an in vivo porcine colon embedded with surrogate tumors, target-to-background ratios of 3.36 ± 0.43, 2.70 ± 0.72, and 2.10 ± 0.13 were achieved for FITC, ICG, and hfQD probes, respectively. This promising endoscopic technology with molecular contrast shows the capacity to reveal hidden tumors and guide treatment strategy decisions.