Label-free visualization and characterization of extracellular vesicles in breast cancer.

Despite extensive interest, extracellular vesicle (EV) research remains technically challenging. One of the unexplored gaps in EV research has been the inability to characterize the spatially and functionally heterogeneous populations of EVs based on their metabolic profile. In this paper, we utilize the intrinsic optical metabolic and structural contrast of EVs and demonstrate in vivo/in situ characterization of EVs in a variety of unprocessed (pre)clinical samples. With a pixel-level segmentation mask provided by the deep neural network, individual EVs can be analyzed in terms of their optical signature in the context of their spatial distribution. Quantitative analysis of living tumor-bearing animals and fresh excised human breast tissue revealed abundance of NAD(P)H-rich EVs within the tumor, near the tumor boundary, and around vessel structures. Furthermore, the percentage of NAD(P)H-rich EVs is highly correlated with human breast cancer diagnosis, which emphasizes the important role of metabolic imaging for EV characterization as well as its potential for clinical applications. In addition to the characterization of EV properties, we also demonstrate label-free monitoring of EV dynamics (uptake, release, and movement) in live cells and animals. The in situ metabolic profiling capacity of the proposed method together with the finding of increasing NAD(P)H-rich EV subpopulations in breast cancer have the potential for empowering applications in basic science and enhancing our understanding of the active metabolic roles that EVs play in cancer progression.

Assessing the Effect of Middle Ear Effusions on Wideband Acoustic Immittance Using Optical Coherence Tomography.

OBJECTIVES: Wideband acoustic immittance (WAI) noninvasively assesses middle ear function by measuring the sound conduction over a range of audible frequencies. Although several studies have shown the potential of WAI for detecting the presence of middle ear effusions (MEEs), determining the effects of MEE type and amount on WAI in vivo has been challenging due to the anatomical location of middle ear cavity. The purpose of this study is to correlate WAI measurements with physical characteristics of the middle ear and MEEs determined by optical coherence tomography (OCT), a noninvasive optical imaging technique. DESIGN: Sixteen pediatric subjects (average age of 7 ± 4 years) were recruited from the primary care clinic at Carle Foundation Hospital (Urbana, IL). A total of 22 ears (normal: 15 ears, otitis media with effusion: 6 ears, and acute otitis media: 1 ear, based on physician's diagnosis) were examined via standard otoscopy, tympanometry, OCT imaging, and WAI measurements in a busy, community-based clinical setting. Cross-sectional OCT images were analyzed to quantitatively assess the presence, type (relative turbidity based on the amount of scattering), and amount (relative fluid level) of MEEs. These OCT metrics were utilized to categorize subject ears into no MEE (control), biofilm without a MEE, serous-scant, serous-severe, mucoid-scant, and mucoid-severe MEE groups. The absorbance levels in each group were statistically evaluated at α = 0.05. RESULTS: The absorbance of the control group showed a similar trend when compared with a pediatric normative dataset, and the presence of an MEE generally decreased the power absorbance. The mucoid MEE group showed significantly less power absorbance from 2.74 to 4.73 kHz (p < 0.05) when compared with the serous MEE group, possibly due to the greater mass impeding the middle ear system. Similarly, the greater amount of middle ear fluid contributed to the lower power absorbance from 1.92 to 2.37 kHz (p< 0.05), when compared with smaller amounts of fluid. As expected, the MEEs with scant fluid only significantly affected the power absorbance at frequencies greater than 4.85 kHz. A large variance in the power absorbance was observed between 2 and 5 kHz, suggesting the dependence on both the type and amount of MEE. CONCLUSIONS: Physical characteristics of the middle ear and MEEs quantified from noninvasive OCT images can be helpful to understand abnormal WAI measurements. Mucoid MEEs decrease the power absorbance more than serous MEEs, and the greater amounts of MEE decreases the power absorbance, especially at higher (>2 kHz) frequencies. As both the type and amount of MEE can significantly affect WAI measurements, further investigations to correlate acoustic measurements with physical characteristics of middle ear conditions in vivo is needed.

In Vivo Optical Characterization of Middle Ear Effusions and Biofilms During Otitis Media.

Otitis media (OM), a common ear infection, is characterized by the presence of an accumulated middle ear effusion (MEE) in a normally air-filled middle ear cavity. While assessing the MEE plays a critical role in the overall management of OM, identifying and examining the MEE is challenging with the current diagnostic tools since the MEE is located behind the semi-opaque eardrum. The objective of this cross-sectional, observational study is to non-invasively visualize and characterize MEEs and bacterial biofilms in the middle ear. A portable, handheld, otoscope-integrated optical coherence tomography (OCT) system combined with novel analytical methods has been developed. In vivo middle ear OCT images were acquired from 53 pediatric subjects (average age of 3.9 years; all awake during OCT imaging) diagnosed with OM and undergoing a surgical procedure (ear tube surgery) to aspirate the MEE and aerate the middle ear. In vivo middle ear OCT acquired prior to the surgery was compared with OCT of the freshly extracted MEEs, clinical diagnosis, and post-operative evaluations. Among the subjects who were identified with the presence of MEEs, 89.6% showed the presence of the TM-adherent biofilm in in vivo OCT. This study provides an atlas of middle ear OCT images exhibiting a range of depth-resolved MEE features, which can only be visualized and assessed non-invasively through OCT. Quantitative metrics of OCT images acquired prior to the surgery were statistically correlated with surgical evaluations of MEEs. Measurements of MEE characteristics will provide new readily available information that can lead to improved diagnosis and management strategies for the highly prevalent OM in children.

Label-free optical redox ratio from urinary extracellular vesicles as a screening biomarker for bladder cancer.

Extracellular vesicles (EVs) have been studied for their potential applications in cancer screening, diagnosis, and treatment monitoring. Most studies have focused on the bulk content of EVs; however, it is also informative to investigate their metabolic status, and changes under different physiological and environmental conditions. In this study, noninvasive, multimodal, label-free nonlinear optical microscopy was used to evaluate the optical redox ratio of large EVs (microvesicles) isolated from the urine of 11 dogs in three cohorts (4 healthy, 4 transitional cell carcinoma (TCC) of the bladder, and 3 prostate cancer). The optical redox ratio is a common metric comparing the autofluorescence intensities of metabolic cofactors FAD and NAD(P)H to characterize the metabolic profile of cells and tissues, and has recently been applied to EVs. The optical redox ratio revealed that dogs with TCC of the bladder had a more than 2-fold increase in NAD(P)H-rich urinary EVs (uEVs) when compared to healthy dogs, whereas dogs with prostate cancer had no significant difference. The optical redox ratio values of uEVs kept at -20°C for 48 hours were significantly different from those of freshly isolated uEVs, indicating that this parameter is more reliable when assessing freshly isolated uEVs. These results suggest that the label-free optical redox ratio of uEVs, indicating relative rates of glycolysis and oxidative phosphorylation of parent cells and tissues, may act as a potential screening biomarker for bladder cancer.

High-speed label-free two-photon fluorescence microscopy of metabolic transients during neuronal activity.

The brain is an especially active metabolic system, requiring a large supply of energy following neuronal activation. However, direct observation of cellular metabolic dynamics associated with neuronal activation is challenging with currently available imaging tools. In this study, an optical imaging approach combining imaging of calcium transients and the metabolic co-enzyme nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) is utilized to track the metabolic dynamics in hippocampal neuron cultures. Results show distinct cellular components for the NAD(P)H response following neuronal activity, where notable differences in the NAD(P)H dynamics between neurons and astrocytes can be directly observed. Additionally, tracking of these responses across a large field of view is demonstrated for metabolic profiling of neuronal activation. Observation of neuronal dynamics using these methods allows for closer examination of the complex metabolic machinery of the brain, and may lead to a better understanding of the cellular metabolism of neuronal activation.

Biomechanical sensing of in vivo magnetic nanoparticle hyperthermia-treated melanoma using magnetomotive optical coherence elastography.

Rationale: Magnetic nanoparticle hyperthermia (MH) therapy is capable of thermally damaging tumor cells, yet a biomechanically-sensitive monitoring method for the applied thermal dosage has not been established. Biomechanical changes to tissue are known indicators for tumor diagnosis due to its association with the structural organization and composition of tissues at the cellular and molecular level. Here, by exploiting the theranostic functionality of magnetic nanoparticles (MNPs), we aim to explore the potential of using stiffness-based metrics that reveal the intrinsic biophysical changes of in vivo melanoma tumors after MH therapy. Methods: A total of 14 melanoma-bearing mice were intratumorally injected with dextran-coated MNPs, enabling MH treatment upon the application of an alternating magnetic field (AMF) at 64.7 kHz. The presence of the MNP heating sources was detected by magnetomotive optical coherence tomography (MM-OCT). For the first time, the elasticity alterations of the hyperthermia-treated, MNP-laden, in vivo tumors were also measured with magnetomotive optical coherence elastography (MM-OCE), based on the mechanical resonant frequency detected. To investigate the correlation between stiffness changes and the intrinsic biological changes, histopathology was performed on the excised tumor after the in vivo measurements. Results: Distinct shifts in mechanical resonant frequency were observed only in the MH-treated group, suggesting a heat-induced stiffness change in the melanoma tumor. Moreover, tumor cellularity, protein conformation, and temperature rise all play a role in tumor stiffness changes after MH treatment. With low cellularity, tumor softens after MH even with low temperature elevation. In contrast, with high cellularity, tumor softening occurs only with a low temperature rise, which is potentially due to protein unfolding, whereas tumor stiffening was seen with a higher temperature rise, likely due to protein denaturation. Conclusions: This study exploits the theranostic functionality of MNPs and investigates the MH-induced stiffness change on in vivo melanoma-bearing mice with MM-OCT and MM-OCE for the first time. It was discovered that the elasticity alteration of the melanoma tumor after MH treatment depends on both thermal dosage and the morphological features of the tumor. In summary, changes in tissue-level elasticity can potentially be a physically and physiologically meaningful metric and integrative therapeutic marker for MH treatment, while MM-OCE can be a suitable dosimetry technique.

Efficacy of endotracheal tube suctioning in intubated intensive care unit patients determined by in vivo catheter-based optical coherence tomography-a pilot study.

BACKGROUND: Mechanical ventilation using an endotracheal tube (ETT) is one of the critical interventions given to patients in the intensive care unit (ICU). ETTs are associated with the formation of biofilms, placing patients at increased risk for developing ventilator-associated pneumonia (VAP). ETT suctioning is used to remove secretions, reduce bacterial colonization, and reduce the rate of biofilm formation. However, current standard-of-care suctioning procedures do not adequately eliminate all secretions from the ETT. METHODS: This observational study was conducted in a cohort of 4 subjects admitted to the ICU and intubated with an ETT, irrespective of ethnicity, gender, or race. A total of 23 suctioning procedures were evaluated with in vivo three-dimensional (3D) optical coherence tomography (OCT) imaging, before and after suctioning. A secretion density metric was derived from the OCT data to quantify the amount of secretions present within the ETT, and an attenuation coefficient metric was derived to detect and quantify the presence of biofilms. Analyzed OCT images were correlated with clinical and microscopy data. RESULTS: Data obtained suggests that the current standard-of-care suctioning procedure is inefficient at clearing secretions or preventing the formation of biofilms. The presence of biofilms was corroborated with both post-intubation microscopy of the ETTs, as well as with clinical data. CONCLUSIONS: We conclude that the standard-of-care suctioning method does not eliminate secretions nor reduce the formation of biofilm in ETTs. Our in situ imaging method was sensitive to the presence of secretions, biofilms, and quantitative, and can be used for investigating different suctioning protocols in the future.

In vivo dynamic characterization of the human tympanic membrane using pneumatic optical coherence tomography.

Decreased mobility of the human eardrum, the tympanic membrane (TM), is an essential indicator of a prevalent middle ear infection. The current diagnostic method to assess TM mobility is via pneumatic otoscopy, which provides subjective and qualitative information of subtle motion. In this study, a handheld spectral-domain pneumatic optical coherence tomography system was developed to simultaneously measure the displacement of the TM, air pressure inputs applied to a sealed ear canal, and to perform digital pneumatic otoscopy. A novel approach based on quantitative parameters is presented to characterize spatial and temporal variations of the dynamic TM motion. Furthermore, the TM motions of normal middle ears are compared with those of ears with middle ear infections. The capability of noninvasively measuring the rapid motion of the TM is beneficial to understand the complex dynamics of the human TM, and can ultimately lead to improved diagnosis and management of middle ear infections.

Longitudinal monitoring of cell metabolism in biopharmaceutical production using label-free fluorescence lifetime imaging microscopy.

Chinese hamster ovary (CHO) cells are routinely used in the biopharmaceutical industry for production of therapeutic monoclonal antibodies (mAbs). Although multiple offline and time-consuming measurements of spent media composition and cell viability assays are used to monitor the status of culture in biopharmaceutical manufacturing, the day-to-day changes in the cellular microenvironment need further in-depth characterization. In this study, two-photon fluorescence lifetime imaging microscopy (2P-FLIM) was used as a tool to directly probe into the health of CHO cells from a bioreactor, exploiting the autofluorescence of intracellular nicotinamide adenine dinucleotide phosphate (NAD(P)H), an enzymatic cofactor that determines the redox state of the cells. A custom-built multimodal microscope with two-photon FLIM capability was utilized to monitor changes in NAD(P)H fluorescence for longitudinal characterization of a changing environment during cell culture processes. Three different cell lines were cultured in 0.5 L shake flasks and 3 L bioreactors. The resulting FLIM data revealed differences in the fluorescence lifetime parameters, which were an indicator of alterations in metabolic activity. In addition, a simple principal component analysis (PCA) of these optical parameters was able to identify differences in metabolic progression of two cell lines cultured in bioreactors. Improved understanding of cell health during antibody production processes can result in better streamlining of process development, thereby improving product titer and verification of scale-up. To our knowledge, this is the first study to use FLIM as a label-free measure of cellular metabolism in a biopharmaceutically relevant and clinically important CHO cell line.

Sex-specific genetic dissection of diabetes in a rodent model identifies Ica1 and Ndufa4 as major candidate genes.

The aim of the study was to initiate a sex-specific investigation of the molecular basis of diabetes, using a genomic approach in the Cohen Diabetic rat model of diet-induced Type 2 diabetes. We used an F2 population resulting from a cross between Cohen Diabetic susceptible (CDs) and resistant (CDr) and consisting of 132 males and 159 females to detect relevant QTLs by linkage and cosegregation analyses. To confirm the functional relevance of the QTL, we applied the "chromosome substitution" strategy. We identified candidate genes within the quantitative trait locus (QTL) and studied their differential expression. We sequenced the differentially expressed candidate genes to account for differences in their expression. We confirmed in this new cross in males a previously detected major QTL on rat chromosome 4 (RNO4); we identified in females this major QTL as well. We found three additional diabetes-related QTLs on RNO11, 13, and 20 in females only. We pursued the investigation of the QTL on RNO4 and generated a CDs.4(CDr) consomic strain, which provided us with functional confirmation for the contribution of the QTL to the diabetic phenotype in both sexes. We successfully narrowed the QTL span to 2.6 cM and identified within it six candidate genes, but only two of which, Ica1 (islet cell autoantigen 1) and Ndufa4 (NADH dehydrogenase ubiquinone) were differentially expressed between CDs and CDr. We sequenced the exons and promoter regions of Ica1 and Ndufa4 but did not identify sequence variations between the strains. The detection of the QTL on RNO4 in both sexes suggests involvement of Ica1, Ndufa4, the Golgi apparatus, the mitochondria and genetic susceptibility to dietary-environmental factors in the pathophysiology of diabetes in our model. The additional sex-specific QTLs are likely to account for differences in the diabetic phenotype between the sexes.

Geno-transcriptomic dissection of proteinuria in the uninephrectomized rat uncovers a molecular complexity with sexual dimorphism.

Investigation of proteinuria, whose pathophysiology remains incompletely understood, is confounded by differences in the phenotype between males and females. We initiated a sex-specific geno-transcriptomic dissection of proteinuria in uninephrectomized male and female Sabra rats that spontaneously develop focal and segmental glomerulosclerosis, testing the hypothesis that different mechanisms might underlie the pathophysiology of proteinuria between the sexes. In the genomic arm, we scanned the genome of 136 male and 111 female uninephrectomized F2 populations derived from crosses between SBH/y and SBN/y. In males, we identified proteinuria-related quantitative trait loci (QTLs) on RNO2 and 20 and protective QTLs on RNO6 and 9. In females, we detected proteinuria-related QTLs on RNO11, 13, and 20. The only QTL overlap between the sexes was on RNO20. Using consomic strains, we confirmed the functional significance of this QTL in both sexes. In the transcriptomic arm, we searched on a genomewide scale for genes that were differentially expressed in kidneys of SBH/y and SBN/y with and without uninephrectomy. These studies identified within each sex differentially expressed genes of relevance to proteinuria. Integrating genomics with transcriptomics, we identified differentially expressed genes that mapped within the boundaries of the proteinuria-related QTLs, singling out 24 transcripts in males and 30 in females, only 4 of which (Tubb5, Ubd, Psmb8, and C2) were common to both sexes. Data mining revealed that these transcripts are involved in multiple molecular mechanisms, including immunity, inflammation, apoptosis, matrix deposition, and protease activity, with no single molecular pathway predominating in either sex. These results suggest that the pathophysiology of proteinuria is highly complex and that some of the underlying mechanisms are shared between the sexes, while others are sex specific and may account for the difference in the proteinuric phenotype between males and females.

Video-rate multimodal multiphoton imaging and three-dimensional characterization of cellular dynamics in wounded skin.

To date, numerous studies have been performed to elucidate the complex cellular dynamics in skin diseases, but few have attempted to characterize these cellular events under conditions similar to the native environment. To address this challenge, a three-dimensional (3D) multimodal analysis platform was developed for characterizing in vivo cellular dynamics in skin, which was then utilized to process in vivo wound healing data to demonstrate its applicability. Special attention is focused on in vivo biological parameters that are difficult to study with ex vivo analysis, including 3D cell tracking and techniques to connect biological information obtained from different imaging modalities. These results here open new possibilities for evaluating 3D cellular dynamics in vivo, and can potentially provide new tools for characterizing the skin microenvironment and pathologies in the future.

Handheld optical coherence tomography for clinical assessment of dental plaque and gingiva.

SIGNIFICANCE: Optical coherence tomography (OCT) offers high spatial resolution and contrast for imaging intraoral structures, yet few studies have investigated its clinical feasibility for dental plaque and gingiva imaging in vivo. Furthermore, the accessibility is often limited to anterior teeth due to bulky imaging systems and probes. AIM: A custom-designed, handheld probe-based, spectral-domain OCT system with an interchangeable attachment was developed to assess dental plaque and gingival health in a clinical setting. APPROACH: Healthy volunteers and subjects with gingivitis and sufficient plaque were recruited. The handheld OCT system was operated by trained dental hygienists to acquire images of dental plaque and gingiva at various locations and after one-week use of oral hygiene products. RESULTS: The handheld OCT can access premolars, first molars, and lingual sides of teeth to visualize the plaque distribution. OCT intensity-based texture analysis revealed lower intensity from selected sites in subjects with gingivitis. The distribution of the dental plaque after one-week use of the oral hygiene products was compared, showing the capability of OCT as a longitudinal tracking tool. CONCLUSIONS: OCT has a strong potential to display and assess dental plaque and gingiva in a clinical setting. Meanwhile, technological challenges remain to perform systematic longitudinal tracking and comparative analyses.

Dynamic Tracking Algorithm for Time-Varying Neuronal Network Connectivity using Wide-Field Optical Image Video Sequences.

Propagation of signals between neurons and brain regions provides information about the functional properties of neural networks, and thus information transfer. Advances in optical imaging and statistical analyses of acquired optical signals have yielded various metrics for inferring neural connectivity, and hence for mapping signal intercorrelation. However, a single coefficient is traditionally derived to classify the connection strength between two cells, ignoring the fact that neural systems are inherently time-variant systems. To overcome these limitations, we utilized a time-varying Pearson's correlation coefficient, spike-sorting, wavelet transform, and wavelet coherence of calcium transients from DIV 12-15 hippocampal neurons from GCaMP6s mice after applying various concentrations of glutamate. Results provide a comprehensive overview of resulting firing patterns, network connectivity, signal directionality, and network properties. Together, these metrics provide a more comprehensive and robust method of analyzing transient neural signals, and enable future investigations for tracking the effects of different stimuli on network properties.

Non-invasive monitoring of pharmacodynamics during the skin wound healing process using multimodal optical microscopy.

OBJECTIVE: Impaired diabetic wound healing is one of the serious complications associated with diabetes. In patients with diabetes, this impairment is characterized by several physiological abnormalities such as metabolic changes, reduced collagen production, and diminished angiogenesis. We designed and developed a multimodal optical imaging system that can longitudinally monitor formation of new blood vessels, metabolic changes, and collagen deposition in a non-invasive, label-free manner. RESEARCH DESIGN AND METHODS: The closure of a skin wound in (db/db) mice, which presents delayed wound healing pathologically similar to conditions in human type 2 diabetes mellitus, was non-invasively followed using the custom-built multimodal microscope. In this microscope, optical coherence tomography angiography was used for studying neovascularization, fluorescence lifetime imaging microscopy for nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) assessment, fluorescence intensity changes of NAD(P)H and flavin adenine dinucleotide (FAD) cofactors for evaluating metabolic changes, and second harmonic generation microscopy for analyzing collagen deposition and organization. The animals were separated into four groups: control, placebo, low concentration (LC), and high concentration (HC) treatment. Images of the wound and surrounding areas were acquired at different time points during a 28-day period. RESULTS: Various physiological changes measured using the optical imaging modalities at different phases of wound healing were compared. A statistically significant improvement in the functional relationship between angiogenesis, metabolism, and structural integrity was observed in the HC group. CONCLUSIONS: This study demonstrated the capability of multimodal optical imaging to non-invasively monitor various physiological aspects of the wound healing process, and thus become a promising tool in the development of better diagnostic, treatment, and monitoring strategies for diabetic wound care.

Assessing the severity of psoriasis through multivariate analysis of optical images from non-lesional skin.

Patients with psoriasis represent a heterogeneous population with individualized disease expression. Psoriasis can be monitored through gold standard histopathology of biopsy specimens that are painful and permanently scar. A common associated measure is the use of non-invasive assessment of the Psoriasis Area and Severity Index (PASI) or similarly derived clinical assessment based scores. However, heterogeneous manifestations of the disease lead to specific PASI scores being poorly reproducible and not easily associated with clinical severity, complicating the efforts to monitor the disease. To address this issue, we developed a methodology for non-invasive automated assessment of the severity of psoriasis using optical imaging. Our analysis shows that two-photon fluorescence lifetime imaging permits the identification of biomarkers present in both lesional and non-lesional skin that correlate with psoriasis severity. This ability to measure changes in lesional and healthy-appearing skin provides a new pathway for independent monitoring of both the localized and systemic effects of the disease. Non-invasive optical imaging was conducted on lesions and non-lesional (pseudo-control) skin of 33 subjects diagnosed with psoriasis, lesional skin of 7 subjects diagnosed with eczema, and healthy skin of 18 control subjects. Statistical feature extraction was combined with principal component analysis to analyze pairs of two-photon fluorescence lifetime images of stratum basale and stratum granulosum layers of skin. We found that psoriasis is associated with biochemical and structural changes in non-lesional skin that can be assessed using clinically available two-photon fluorescence lifetime microscopy systems.

Simultaneous two-photon activation and imaging of neural activity based on spectral-temporal modulation of supercontinuum light.

SIGNIFICANCE: Recent advances in nonlinear optics in neuroscience have focused on using two ultrafast lasers for activity imaging and optogenetic stimulation. Broadband femtosecond light sources can obviate the need for multiple lasers by spectral separation for chromatically targeted excitation. AIM: We present a photonic crystal fiber (PCF)-based supercontinuum source for spectrally resolved two-photon (2P) imaging and excitation of GCaMP6s and C1V1-mCherry, respectively. APPROACH: A PCF is pumped using a 20-MHz repetition rate femtosecond laser to generate a supercontinuum of light, which is spectrally separated, compressed, and recombined to image GCaMP6s (930 nm excitation) and stimulate the optogenetic protein, C1V1-mCherry (1060 nm excitation). Galvanometric spiral scanning is employed on a single-cell level for multiphoton excitation and high-speed resonant scanning is employed for imaging of calcium activity. RESULTS: Continuous wave lasers were used to verify functionality of optogenetic activation followed by directed 2P excitation. Results from these experiments demonstrate the utility of a supercontinuum light source for simultaneous, single-cell excitation and calcium imaging. CONCLUSIONS: A PCF-based supercontinuum light source was employed for simultaneous imaging and excitation of calcium dynamics in brain tissue. Pumped PCFs can serve as powerful light sources for imaging and activation of neural activity, and overcome the limited spectra and space associated with multilaser approaches.

In vivo detection of endotracheal tube biofilms in intubated critical care patients using catheter-based optical coherence tomography.

The formation of biofilms in the endotracheal tubes (ETTs) of intubated patients on mechanical ventilation is associated with a greater risk of ventilator-associated pneumonia and death. New technologies are needed to detect and monitor ETTs in vivo for the presence of these biofilms. Longitudinal OCT imaging was performed in mechanically ventilated subjects at 24-hour intervals until extubation to detect the formation and temporal changes of in vivo ETT biofilms. OCT-derived attenuation coefficient images were used to differentiate between mucus and biofilm. Extubated ETTs were examined with optical and electron microscopy, and all imaging results were correlated with standard-of-care clinical test reports. OCT and attenuation coefficient images from four subjects were positive for ETT biofilms and were negative for two subjects. The processed and stained extubated ETTs and clinical reports confirmed the presence/absence of biofilms in all subjects. Our findings confirm that OCT can detect and differentiate between biofilm-positive and biofilm-negative groups (P < 10-5 ). OCT image-based features may serve as biomarkers for direct in vivo detection of ETT biofilms and help drive investigation of new management strategies to reduce the incidence of VAP.

Tracking metabolic dynamics of apoptosis with high-speed two-photon fluorescence lifetime imaging microscopy.

Programmed cell death, or apoptosis, is an essential process in development and homeostasis, and disruptions in associated pathways are responsible for a wide variety of diseases such as cancer, developmental abnormalities, and Alzheimer's disease. On the other hand, cell death, in many cases, is the desired outcome of therapeutic treatments targeting diseases such as cancer. Recently, metabolic imaging based on two-photon fluorescence microscopy has been developed and shown to be highly sensitive to certain cell death processes, most notably apoptosis, thus having the potential as an advanced label-free screening tool. However, the typically low acquisition rates of this imaging technique have resulted in a limited throughput approach, allowing only a small population of cells to be tracked at well-separated time points. To address this limitation, a high-speed two-photon fluorescence lifetime imaging microscopy (2P-FLIM) platform capable of video-rate imaging is applied to study and further characterize the metabolic dynamics associated with cell death. Building upon previous work demonstrating the capabilities of this system, this microscope is utilized to study rapid metabolic changes during cell death induction, such as dose-dependency of metabolic response, response in invasive vs. noninvasive cancer cells, and response in an apoptosis-resistant cell line, which is further shown to undergo autophagy in response to toxic stimuli. Results from these experiments show that the early apoptosis-related metabolic dynamics are strongly correlated with important cellular parameters including responsiveness to apoptosis-inducing stimuli. The high speed and sensitivity of the presented imaging approach enables new investigations into this highly dynamic and complex process.

Simultaneous label-free autofluorescence and multi-harmonic imaging reveals in vivo structural and metabolic changes in murine skin.

Simultaneous quantification of multifarious cellular metabolites and the extracellular matrix in vivo has been long sought. Simultaneous label-free autofluorescence and multi-harmonic (SLAM) microscopy has achieved simultaneous four-channel nonlinear imaging to study tissue structure and metabolism. In this study, we implemented two laser systems and directly compared SLAM microscopy with conventional two-photon microscopy for in vivo imaging. We found that three-photon imaging of adenine dinucleotide (phosphate) (NAD(P)H) in SLAM microscopy using our tailored laser source provided better resolution, contrast, and background suppression than conventional two-photon imaging of NAD(P)H. We also integrated fluorescence lifetime imaging with SLAM microscopy, and enabled differentiation of free and bound NAD(P)H. We imaged murine skin in vivo and showed that changes in tissue structure, cell dynamics, and metabolism can be monitored simultaneously in real-time. We also discovered an increase in metabolism and protein-bound NAD(P)H in skin cells during the early stages of wound healing.