Optical measurement of microvascular oxygenation and blood flow responses in awake mouse cortex during functional activation.

The cerebral cortex has a number of conserved morphological and functional characteristics across brain regions and species. Among them, the laminar differences in microvascular density and mitochondrial cytochrome c oxidase staining suggest potential laminar variability in the baseline O2 metabolism and/or laminar variability in both O2 demand and hemodynamic response. Here, we investigate the laminar profile of stimulus-induced intravascular partial pressure of O2 (pO2) transients to stimulus-induced neuronal activation in fully awake mice using two-photon phosphorescence lifetime microscopy. Our results demonstrate that stimulus-induced changes in intravascular pO2 are conserved across cortical layers I-IV, suggesting a tightly controlled neurovascular response to provide adequate O2 supply across cortical depth. In addition, we observed a larger change in venular O2 saturation (ΔsO2) compared to arterioles, a gradual increase in venular ΔsO2 response towards the cortical surface, and absence of the intravascular "initial dip" previously reported under anesthesia. This study paves the way for quantification of layer-specific cerebral O2 metabolic responses, facilitating investigation of brain energetics in health and disease and informed interpretation of laminar blood oxygen level dependent functional magnetic resonance imaging signals.

Quantitation of cerebral oxygen tension using phasor analysis and phosphorescence lifetime imaging microscopy (PLIM).

Time-domain measurements for fluorescence lifetime imaging microscopy (FLIM) and phosphorescence lifetime imaging microscopy (PLIM) are conventionally computed by nonlinear curve fitting techniques to model the time-resolved profiles as mono- or multi-exponential decays. However, these techniques are computationally intensive and prone to fitting errors. The phasor or "polar plot" analysis method has recently gained attention as a simple method to characterize fluorescence lifetime. Here, we adapted the phasor analysis method for absolute quantitation of phosphorescence lifetimes of oxygen-sensitive phosphors and used the phasor-derived lifetime values to quantify oxygen partial pressure (pO2) in cortical microvessels of awake mice. Our results, both experimental and simulated, demonstrate that oxygen measurements obtained from computationally simpler phasor analysis agree well with traditional curve fitting calculations. To our knowledge, the current study constitutes the first application of the technique for characterizing microsecond-length, time-domain phosphorescence measurements and absolute, in vivo quantitation of a vital physiological parameter. The method shows promise for monitoring cerebral metabolism and pathological changes in preclinical rodent models.

A Suite of Neurophotonic Tools to Underpin the Contribution of Internal Brain States in fMRI.

Recent developments in optical microscopy, applicable for large-scale and longitudinal imaging of cortical activity in behaving animals, open unprecedented opportunities to gain a deeper understanding of neurovascular and neurometabolic coupling during different brain states. Future studies will leverage these tools to deliver foundational knowledge about brain state-dependent regulation of cerebral blood flow and metabolism as well as regulation as a function of brain maturation and aging. This knowledge is of critical importance to interpret hemodynamic signals observed with functional magnetic resonance imaging (fMRI).

Author Correction: Potential circadian effects on translational failure for neuroprotection.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Potential circadian effects on translational failure for neuroprotection.

Neuroprotectant strategies that have worked in rodent models of stroke have failed to provide protection in clinical trials. Here we show that the opposite circadian cycles in nocturnal rodents versus diurnal humans1,2 may contribute to this failure in translation. We tested three independent neuroprotective approaches-normobaric hyperoxia, the free radical scavenger α-phenyl-butyl-tert-nitrone (αPBN), and the N-methyl-D-aspartic acid (NMDA) antagonist MK801-in mouse and rat models of focal cerebral ischaemia. All three treatments reduced infarction in day-time (inactive phase) rodent models of stroke, but not in night-time (active phase) rodent models of stroke, which match the phase (active, day-time) during which most strokes occur in clinical trials. Laser-speckle imaging showed that the penumbra of cerebral ischaemia was narrower in the active-phase mouse model than in the inactive-phase model. The smaller penumbra was associated with a lower density of terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)-positive dying cells and reduced infarct growth from 12 to 72 h. When we induced circadian-like cycles in primary mouse neurons, deprivation of oxygen and glucose triggered a smaller release of glutamate and reactive oxygen species, as well as lower activation of apoptotic and necroptotic mediators, in 'active-phase' than in 'inactive-phase' rodent neurons. αPBN and MK801 reduced neuronal death only in 'inactive-phase' neurons. These findings suggest that the influence of circadian rhythm on neuroprotection must be considered for translational studies in stroke and central nervous system diseases.

In vivo deep-tissue microscopy with UCNP/Janus-dendrimers as imaging probes: resolution at depth and feasibility of ratiometric sensing.

Lanthanide-based upconverting nanoparticles (UCNPs) are known for their remarkable ability to convert near-infrared energy into higher energy light, offering an attractive platform for construction of biological imaging probes. Here we focus on in vivo high-resolution microscopy - an application for which the opportunity to carry out excitation at low photon fluxes in non-linear regime makes UCNPs stand out among all multiphoton probes. To create biocompatible nanoparticles we employed Janus-type dendrimers as surface ligands, featuring multiple carboxylates on one 'face' of the molecule, polyethylene glycol (PEG) residues on another and Eriochrome Cyanine R dye as the core. The UCNP/Janus-dendrimers showed outstanding performance as vascular markers, allowing for depth-resolved mapping of individual capillaries in the mouse brain down to a remarkable depth of ∼1000 µm under continuous wave (CW) excitation with powers not exceeding 20 mW. Using a posteriori deconvolution, high-resolution images could be obtained even at high scanning speeds in spite of the blurring caused by the long luminescence lifetimes of the lanthanide ions. Secondly, the new UCNP/dendrimers allowed us to evaluate the feasibility of quantitative analyte imaging in vivo using a popular ratiometric UCNP-to-ligand excitation energy transfer (EET) scheme. Our results show that the ratio of UCNP emission bands, which for quantitative sensing should respond selectively to the analyte of interest, is also strongly affected by optical heterogeneities of the medium. On the other hand, the luminescence decay times of UCNPs, which are independent of the medium properties, are modulated via EET only insignificantly. As such, quantitative analyte sensing in biological tissues with UCNP-based probes still remains a challenge.

cGMP-dependent protein kinase I in vascular smooth muscle cells improves ischemic stroke outcome in mice.

Recent works highlight the therapeutic potential of targeting cyclic guanosine monophosphate (cGMP)-dependent pathways in the context of brain ischemia/reperfusion injury (IRI). Although cGMP-dependent protein kinase I (cGKI) has emerged as a key mediator of the protective effects of nitric oxide (NO) and cGMP, the mechanisms by which cGKI attenuates IRI remain poorly understood. We used a novel, conditional cGKI knockout mouse model to study its role in cerebral IRI. We assessed neurological deficit, infarct volume, and cerebral perfusion in tamoxifen-inducible vascular smooth muscle cell-specific cGKI knockout mice and control animals. Stroke experiments revealed greater cerebral infarct volume in smooth muscle cell specific cGKI knockout mice (males: 96 ± 16 mm3; females: 93 ± 12 mm3, mean±SD) than in all control groups: wild type (males: 66 ± 19; females: 64 ± 14), cGKI control (males: 65 ± 18; females: 62 ± 14), cGKI control with tamoxifen (males: 70 ± 8; females: 68 ± 10). Our results identify, for the first time, a protective role of cGKI in vascular smooth muscle cells during ischemic stroke injury. Moreover, this protective effect of cGKI was found to be independent of gender and was mediated via improved reperfusion. These results suggest that cGKI in vascular smooth muscle cells should be targeted by therapies designed to protect brain tissue against ischemic stroke.

More homogeneous capillary flow and oxygenation in deeper cortical layers correlate with increased oxygen extraction.

Our understanding of how capillary blood flow and oxygen distribute across cortical layers to meet the local metabolic demand is incomplete. We addressed this question by using two-photon imaging of resting-state microvascular oxygen partial pressure (PO2) and flow in the whisker barrel cortex in awake mice. Our measurements in layers I-V show that the capillary red-blood-cell flux and oxygenation heterogeneity, and the intracapillary resistance to oxygen delivery, all decrease with depth, reaching a minimum around layer IV, while the depth-dependent oxygen extraction fraction is increased in layer IV, where oxygen demand is presumably the highest. Our findings suggest that more homogeneous distribution of the physiological observables relevant to oxygen transport to tissue is an important part of the microvascular network adaptation to local brain metabolism. These results will inform the biophysical models of layer-specific cerebral oxygen delivery and consumption and improve our understanding of the diseases that affect cerebral microcirculation.

Two-photon phosphorescence lifetime microscopy of retinal capillary plexus oxygenation in mice.

Impaired oxygen delivery and/or consumption in the retinal tissue underlies the pathophysiology of many retinal diseases. However, the essential tools for measuring oxygen concentration in retinal capillaries and studying oxygen transport to retinal tissue are still lacking. We show that two-photon phosphorescence lifetime microscopy can be used to map absolute partial pressures of oxygen (pO2) in the retinal capillary plexus. Measurements were performed at various retinal depths in anesthetized mice under systemic normoxic and hyperoxic conditions. We used a newly developed two-photon phosphorescent oxygen probe, based on a two-photon absorbing platinum tetraphthalimidoporphyrin, and commercially available optics without correction for optical aberrations of the eye. The transverse and axial distances within the tissue volume were calibrated using a model of the eye's optical system. We believe this is the first demonstration of in vivo depth-resolved imaging of pO2 in retinal capillaries. Application of this method has the potential to advance our understanding of oxygen delivery on the microvascular scale and help elucidate mechanisms underlying various retinal diseases.

Ultrahigh-speed, phase-sensitive full-field interferometric confocal microscopy for quantitative microscale physiology.

We developed ultra-high-speed, phase-sensitive, full-field reflection interferometric confocal microscopy (FFICM) for the quantitative characterization of in vivo microscale biological motions and flows. We demonstrated 2D frame rates in excess of 1 kHz and pixel throughput rates up to 125 MHz. These fast FFICM frame rates were enabled by the use of a low spatial coherence, high-power laser source. Specifically, we used a dense vertical cavity surface emitting laser (VCSEL) array that synthesized low spatial coherence light through a large number of narrowband, mutually-incoherent emitters. Off-axis interferometry enabled single-shot acquisition of the complex-valued interferometric signal. We characterized the system performance (~2 µm lateral resolution, ~8 µm axial gating depth) with a well-known target. We also demonstrated the use of this highly parallelized confocal microscopy platform for visualization and quantification of cilia-driven surface flows and cilia beat frequency in an important animal model (Xenopus embryos) with >1 kHz frame rate. Such frame rates are needed to see large changes in local flow velocity over small distance (high shear flow), in this case, local flow around a single ciliated cell. More generally, our results are an important demonstration of low-spatial coherence, high-power lasers in high-performance, quantitative biomedical imaging.

High-speed line-field confocal holographic microscope for quantitative phase imaging.

We present a high-speed and phase-sensitive reflectance line-scanning confocal holographic microscope (LCHM). We achieved rapid confocal imaging using a fast line-scan CCD camera and quantitative phase imaging using off-axis digital holography (DH) on a 1D, line-by-line basis in our prototype experiment. Using a 20 kHz line scan rate, we achieved a frame rate of 20 Hz for 512x512 pixels en-face confocal images. We realized coherent holographic detection two different ways. We first present a LCHM using off-axis configuration. By using a microscope objective of a NA 0.65, we achieved axial and lateral resolution of ~3.5 micrometers and ~0.8 micrometers, respectively. We demonstrated surface profile measurement of a phase target at nanometer precision and the digital refocusing of a defocused confocal en-face image. Ultrahigh temporal resolution M mode is demonstrated by measuring the vibration of a PZT-actuated mirror driven by a sine wave at 1 kHz. We then report our experimental work on a LCHM using an in-line configuration. In this in-line LCHM, the coherent detection is enabled by moving the reference arm at a constant speed, thereby introducing a Doppler frequency shift that leads to spatial interference fringes along the scanning direction. Lastly, we present a unified formulation that treats off-axis and in-line LCHM in a unified joint spatiotemporal modulation framework and provide a connection between LCHM and the traditional off-axis DH. The presented high-speed LCHM may find applications in optical metrology and biomedical imaging.

Spectral demultiplexing in holographic and fluorescent on-chip microscopy.

Lensfree on-chip imaging and sensing platforms provide compact and cost-effective designs for various telemedicine and lab-on-a-chip applications. In this work, we demonstrate computational solutions for some of the challenges associated with (i) the use of broadband, partially-coherent illumination sources for on-chip holographic imaging, and (ii) multicolor detection for lensfree fluorescent on-chip microscopy. Specifically, we introduce spectral demultiplexing approaches that aim to digitally narrow the spectral content of broadband illumination sources (such as wide-band light emitting diodes or even sunlight) to improve spatial resolution in holographic on-chip microscopy. We also demonstrate the application of such spectral demultiplexing approaches for wide-field imaging of multicolor fluorescent objects on a chip. These computational approaches can be used to replace e.g., thin-film interference filters, gratings or other optical components used for spectral multiplexing/demultiplexing, which can form a desirable solution for cost-effective and compact wide-field microscopy and sensing needs on a chip.

Cost-effective and rapid blood analysis on a cell-phone.

We demonstrate a compact and cost-effective imaging cytometry platform installed on a cell-phone for the measurement of the density of red and white blood cells as well as hemoglobin concentration in human blood samples. Fluorescent and bright-field images of blood samples are captured using separate optical attachments to the cell-phone and are rapidly processed through a custom-developed smart application running on the phone for counting of blood cells and determining hemoglobin density. We evaluated the performance of this cell-phone based blood analysis platform using anonymous human blood samples and achieved comparable results to a standard bench-top hematology analyser. Test results can either be stored on the cell-phone memory or be transmitted to a central server, providing remote diagnosis opportunities even in field settings.

Lensless fluorescent microscopy on a chip.

On-chip lensless imaging in general aims to replace bulky lens-based optical microscopes with simpler and more compact designs, especially for high-throughput screening applications. This emerging technology platform has the potential to eliminate the need for bulky and/or costly optical components through the help of novel theories and digital reconstruction algorithms. Along the same lines, here we demonstrate an on-chip fluorescent microscopy modality that can achieve e.g., <4 µm spatial resolution over an ultra-wide field-of-view (FOV) of >0.6-8 cm(2) without the use of any lenses, mechanical-scanning or thin-film based interference filters. In this technique, fluorescent excitation is achieved through a prism or hemispherical-glass interface illuminated by an incoherent source. After interacting with the entire object volume, this excitation light is rejected by total-internal-reflection (TIR) process that is occurring at the bottom of the sample micro-fluidic chip. The fluorescent emission from the excited objects is then collected by a fiber-optic faceplate or a taper and is delivered to an optoelectronic sensor array such as a charge-coupled-device (CCD). By using a compressive-sampling based decoding algorithm, the acquired lensfree raw fluorescent images of the sample can be rapidly processed to yield e.g., <4 µm resolution over an FOV of >0.6-8 cm(2). Moreover, vertically stacked micro-channels that are separated by e.g., 50-100 µm can also be successfully imaged using the same lensfree on-chip microscopy platform, which further increases the overall throughput of this modality. This compact on-chip fluorescent imaging platform, with a rapid compressive decoder behind it, could be rather valuable for high-throughput cytometry, rare-cell research and microarray-analysis.

Wide-field lensless fluorescent microscopy using a tapered fiber-optic faceplate on a chip.

We demonstrate lensless fluorescent microscopy over a large field-of-view of ~60 mm(2) with a spatial resolution of <4 µm. In this on-chip fluorescent imaging modality, the samples are placed on a fiber-optic faceplate that is tapered such that the density of the fiber-optic waveguides on the top facet is >5 fold larger than the bottom one. Placed on this tapered faceplate, the fluorescent samples are pumped from the side through a glass hemisphere interface. After excitation of the samples, the pump light is rejected through total internal reflection that occurs at the bottom facet of the sample substrate. The fluorescent emission from the sample is then collected by the smaller end of the tapered faceplate and is delivered to an opto-electronic sensor-array to be digitally sampled. Using a compressive sampling algorithm, we decode these raw lensfree images to validate the resolution (<4 µm) of this on-chip fluorescent imaging platform using microparticles as well as labeled Giardia muris cysts. This wide-field lensfree fluorescent microscopy platform, being compact and high-throughput, might provide a valuable tool especially for cytometry, rare cell analysis (involving large area microfluidic systems) as well as for microarray imaging applications.

Lensfree fluorescent on-chip imaging of transgenic Caenorhabditis elegans over an ultra-wide field-of-view.

We demonstrate lensfree on-chip fluorescent imaging of transgenic Caenorhabditis elegans (C. elegans) over an ultra-wide field-of-view (FOV) of e.g., >2-8 cm(2) with a spatial resolution of ∼10 µm. This is the first time that a lensfree on-chip platform has successfully imaged fluorescent C. elegans samples. In our wide-field lensfree imaging platform, the transgenic samples are excited using a prism interface from the side, where the pump light is rejected through total internal reflection occurring at the bottom facet of the substrate. The emitted fluorescent signal from C. elegans samples is then recorded on a large area opto-electronic sensor-array over an FOV of e.g., >2-8 cm(2), without the use of any lenses, thin-film interference filters or mechanical scanners. Because fluorescent emission rapidly diverges, such lensfree fluorescent images recorded on a chip look blurred due to broad point-spread-function of our platform. To combat this resolution challenge, we use a compressive sampling algorithm to uniquely decode the recorded lensfree fluorescent patterns into higher resolution images, demonstrating ∼10 µm resolution. We tested the efficacy of this compressive decoding approach with different types of opto-electronic sensors to achieve a similar resolution level, independent of the imaging chip. We further demonstrate that this wide FOV lensfree fluorescent imaging platform can also perform sequential bright-field imaging of the same samples using partially-coherent lensfree digital in-line holography that is coupled from the top facet of the same prism used in fluorescent excitation. This unique combination permits ultra-wide field dual-mode imaging of C. elegans on a chip which could especially provide a useful tool for high-throughput screening applications in biomedical research.

Lensfree On-Chip Microscopy and Tomography for Bio-Medical Applications.

Lensfree on-chip holographic microscopy is an emerging technique that offers imaging of biological specimens over a large field-of-view without using any lenses or bulky optical components. Lending itself to a compact, cost-effective and mechanically robust architecture, lensfree on-chip holographic microscopy can offer an alternative toolset addressing some of the emerging needs of microscopic analysis and diagnostics in low-resource settings, especially for telemedicine applications. In this review, we summarize the latest achievements in lensfree optical microscopy based on partially coherent on-chip holography, including portable telemedicine microscopy, cell-phone based microscopy and field-portable optical tomographic microscopy. We also discuss some of the future directions for telemedicine microscopy and its prospects to help combat various global health challenges.

Lensless fluorescent on-chip microscopy using a fiber-optic taper.

We demonstrate a lensfree on-chip fluorescent microscopy platform that can image fluorescently labeled cells over ~60 mm(2) field-of-view with <4 urn spatial resolution. In this lensfree imaging system, micro-objects of interest are directly located on a tapered fiber-optic faceplate which has > 5-fold higher density of fiber-optic waveguides in its top facet compared to the bottom facet. For excitation, an incoherent light source (e.g., a simple light emitting diode--LED) is used to pump fluorescent objects through a glass hemi-sphere interface. Upon interacting with the entire sample volume, the excitation light is rejected by total internal reflection process occurring at the bottom of the sample substrate. Fluorescent emission from the objects is then collected by the smaller facet of the tapered faceplate and is delivered to a detector-array with an image magnification of ~2.4X. A compressive sampling based decoding algorithm is used for sparse signal recovery, which further increases the space-bandwidth-product of our lensfree on-chip fluorescent imager. We validated the performance of this lensfree imaging platform using fluorescent micro-particles as well as labeled water-borne parasites (e.g., Giardia Muris cysts). Such a compact and wide-field fluorescent microscopy platform could be valuable for cytometry and rare cell imaging applications as well as for micro array research.

Lensfree color imaging on a nanostructured chip using compressive decoding.

We demonstrate subpixel level color imaging capability on a lensfree incoherent on-chip microscopy platform. By using a nanostructured substrate, the incoherent emission from the object plane is modulated to create a unique far-field diffraction pattern corresponding to each point at the object plane. These lensfree diffraction patterns are then sampled in the far-field using a color sensor-array, where the pixels have three different types of color filters at red, green, and blue (RGB) wavelengths. The recorded RGB diffraction patterns (for each point on the structured substrate) form a basis that can be used to rapidly reconstruct any arbitrary multicolor incoherent object distribution at subpixel resolution, using a compressive sampling algorithm. This lensfree computational imaging platform could be quite useful to create a compact fluorescent on-chip microscope that has color imaging capability.

Lensless wide-field fluorescent imaging on a chip using compressive decoding of sparse objects.

We demonstrate the use of a compressive sampling algorithm for on-chip fluorescent imaging of sparse objects over an ultra-large field-of-view (>8 cm(2)) without the need for any lenses or mechanical scanning. In this lensfree imaging technique, fluorescent samples placed on a chip are excited through a prism interface, where the pump light is filtered out by total internal reflection after exciting the entire sample volume. The emitted fluorescent light from the specimen is collected through an on-chip fiber-optic faceplate and is delivered to a wide field-of-view opto-electronic sensor array for lensless recording of fluorescent spots corresponding to the samples. A compressive sampling based optimization algorithm is then used to rapidly reconstruct the sparse distribution of fluorescent sources to achieve approximately 10 microm spatial resolution over the entire active region of the sensor-array, i.e., over an imaging field-of-view of >8 cm(2). Such a wide-field lensless fluorescent imaging platform could especially be significant for high-throughput imaging cytometry, rare cell analysis, as well as for micro-array research.