The reliable diagnosis of a mild traumatic brain injury (mTBI) is a pervasive problem in sports and in the military. The frequency and severity of each occurrence, while difficult to quantify, may impact long term cognitive function and quality of life. Despite the new revelations concerning brain disfunction from head injuries, individuals still feel pressure to remain on the field despite a debilitating injury. In this study, we evaluated the accuracy of a system that could be employed on the sidelines or in the locker room to provide an immediate objective mTBI assessment. Participants consisted of 38 individuals with a recent mTBI and 47 controls with no history of mTBI within the last 5 years. Participants were administered a simple symptom questionnaire, behavioral tests, and resting state EEG was measured using three frontopolar electrodes. An advanced machine learning algorithm called boosting was utilized to classify subjects into either injured or controls using power spectral densities on 1-min of resting EEG and the symptom questionnaire. Results based on leave-one-out cross-validation revealed that the addition of EEG measurements boosted the accuracy to approximately 91 ± 2% compared to 82 ± 4% from the symptom questionnaire alone. This study demonstrated the potential benefit of including EEG measurements to diagnose suspected brain injury patients. This is a step toward accurate and objective classification measurements that can be implemented on the field as a future injury assessment tool.

Functional near infrared spectroscopy (fNIRS) is a non-invasive functional neuroimaging modality. Although it is amenable to use in infants and young children, there is a lack of fNIRS research within the toddler age range. In this study, we used fNIRS to measure cerebral hemodynamics in the prefrontal cortex (PFC) in 18-36 months old toddlers (n=29) as part of a longitudinal study that enrolled typically-developing toddlers as well as those "at risk" for language and other delays based on presence of early language delays. In these toddlers, we explored two hemodynamic response indices during periods of rest during which time audiovisual children's programming was presented. First, we investigate Lateralization Index, based on differences in oxy-hemoglobin saturation from left and right prefrontal cortex. Then, we measure oxygenation variability (OV) index, based on variability in oxygen saturation at frequencies attributed to cerebral autoregulation. Preliminary findings show that lower cognitive (including language) abilities are associated with fNIRS measures of both lower OV index and more extreme Lateralization index values. These preliminary findings show the feasibility of using fNIRS in toddlers, including those at risk for developmental delay, and lay the groundwork for future studies.

We first discuss the main obstacles for clinical translation of biophotonics technologies, in particular, the different valleys of death, after which we present different examples of ways to bridge these gaps, remove the obstacles, and promote clinical translation.

We report an accurate method to count red blood cells, platelets, and white blood cells, as well as to determine hemoglobin in the blood of humans, horses, dogs, cats, and cows. Red and white blood cell counts can also be performed on human body fluids such as cerebrospinal fluid, synovial fluid, and peritoneal fluid. The approach consists of using a compact, custom-built microscope to record large field-of-view, bright-field, and fluorescence images of samples that are stained with a single dye and using automatic algorithms to count blood cells and detect hemoglobin. The total process takes about 15 min, including 5 min for sample preparation, and 10 min for data collection and analysis. The minimum volume of blood needed for the test is 0.5 μL, which allows for minimally invasive sample collection such as using a finger prick rather than a venous draw. Blood counts were compared to gold-standard automated clinical instruments, with excellent agreement between the two methods as determined by a Bland-Altman analysis. Accuracy of counts on body fluids was consistent with hand counting by a trained clinical lab scientist, where our instrument demonstrated an approximately 100-fold lower limit of detection compared to current automated methods. The combination of a compact, custom-built instrument, simple sample collection and preparation, and automated analysis demonstrates that this approach could benefit global health through use in low-resource settings where central hematology laboratories are not accessible.

The objective of this study was to develop a method for simultaneously measuring the calcium and contraction dynamics of single, live cardiomyocytes at high spatial resolutions. Such measurements are important to investigate local calcium release and the mechanical response at the sarcomere level (i.e. the basic unit of contraction), which have important implications in cardiac dysfunction and arrhythmias in conditions such as hypertension, atrial fibrillation, and myocardial infarction. Here, we describe a multimodal second harmonic generation (SHG) and two photon fluorescence (2PF) microscopy technique that is used to simultaneously measure subsarcomere calcium and contraction events at high spatial and temporal resolutions. The method takes advantage of the label-free nature of SHG for imaging the sarcomeres and the high spatial colocalization of the SHG signal and the fluorescence signal excited from calcium indicators. This microscope was used to measure calcium sparks and waves and associated contractions in subcellular microdomains, leading to the generation of subcellular strain. We anticipate this new imaging tool will play an important role in studying mechanical stress-induced heart disease.

RATIONALE:Cardiac myocyte contraction is caused by Ca(2+) binding to troponin C, which triggers the cross-bridge power stroke and myofilament sliding in sarcomeres. Synchronized Ca(2+) release causes whole cell contraction and is readily observable with current microscopy techniques. However, it is unknown whether localized Ca(2+) release, such as Ca(2+) sparks and waves, can cause local sarcomere contraction. Contemporary imaging methods fall short of measuring microdomain Ca(2+)-contraction coupling in live cardiac myocytes.OBJECTIVE:To develop a method for imaging sarcomere level Ca(2+)-contraction coupling in healthy and disease model cardiac myocytes.METHODS AND RESULTS:Freshly isolated cardiac myocytes were loaded with the Ca(2+)-indicator fluo-4. A confocal microscope equipped with a femtosecond-pulsed near-infrared laser was used to simultaneously excite second harmonic generation from A-bands of myofibrils and 2-photon fluorescence from fluo-4. Ca(2+) signals and sarcomere strain correlated in space and time with short delays. Furthermore, Ca(2+) sparks and waves caused contractions in subcellular microdomains, revealing a previously underappreciated role for these events in generating subcellular strain during diastole. Ca(2+) activity and sarcomere strain were also imaged in paced cardiac myocytes under mechanical load, revealing spontaneous Ca(2+) waves and correlated local contraction in pressure-overload-induced cardiomyopathy.CONCLUSIONS:Multimodal second harmonic generation 2-photon fluorescence microscopy enables the simultaneous observation of Ca(2+) release and mechanical strain at the subsarcomere level in living cardiac myocytes. The method benefits from the label-free nature of second harmonic generation, which allows A-bands to be imaged independently of T-tubule morphology and simultaneously with Ca(2+) indicators. Second harmonic generation 2-photon fluorescence imaging is widely applicable to the study of Ca(2+)-contraction coupling and mechanochemotransduction in both health and disease.

Tahoe Forest Hospital is a rural health care facility and designated Critical Access Hospital located in Truckee, California. The North Lake Tahoe region is a recreational destination for people from San Francisco, Silicon Valley, and the Sacramento areas of Northern California. It also is the home of successful entrepreneurs and world-renowned scientists and engineers who came to the area to pursue their careers or retire in the community they had come to love.

Current flow-based blood counting devices require significant medical infrastructure and are not appropriate for field use. In this article we report on the development of a sample preparation, measurement, and analysis method that permits automated and accurate counting of red blood cells (RBCs), white blood cells (WBCs), and platelets, as well as allowing a 3-part differential of the WBCs to be performed on extremely small volumes of whole blood. This method is compatible with portable instrumentation that can be deployed in the field. The method consists of serially diluting blood samples first with sodium dodecyl sulfate dissolved in phosphate buffered saline, then in acridine orange dissolved in phosphate buffered saline, followed by fluorescence and dark field imaging with low magnification objectives. Image analysis is performed to extract cell counts and differentials. We performed a paired analysis of 20 volunteers with complete blood count values both within and beyond the normal reference range using a commercial automated hematology analyzer and the image-based method, with the new method achieving accuracies comparable to that of the commercial system. Because the sample preparation and imaging are simple and inexpensive to implement, this method has applications for pediatrics, clinician offices, and global health in regions that do not have access to central hematology laboratories.

Laser tweezers Raman spectroscopy was used to characterize the oxygenation response of single normal adult, sickle, and cord blood red blood cells (RBCs) to an applied mechanical force. Individual cells were subjected to different forces by varying the laser power of a single-beam optical trap, and the intensities of several oxygenation-specific Raman spectral peaks were monitored to determine the oxygenation state of the cells. For all three cell types, an increase in laser power (or mechanical force) induced a greater deoxygenation of the cell. However, sickle RBCs deoxygenated more readily than normal RBCs when subjected to the same optical forces. Conversely, cord blood RBCs were able to maintain their oxygenation better than normal RBCs. These results suggest that differences in the chemical or mechanical properties of fetal, normal, and sickle cells affect the degree to which applied mechanical forces can deoxygenate the cell. Populations of normal, sickle, and cord RBCs were identified and discriminated based on this mechanochemical phenomenon. This study demonstrates the potential application of laser tweezers Raman spectroscopy as a single-cell, label-free analytical tool to characterize the functional (e.g., mechanical deformability, oxygen binding) properties of normal and diseased RBCs.

Laser tweezers Raman spectroscopy (LTRS) combines optical trapping with micro-Raman spectroscopy to enable label-free biochemical analysis of individual cells and small biological particles in suspension. The integration of the two technologies greatly simplifies the sample preparation and handling of suspension cells for spectroscopic analysis in physiologically meaningful conditions. In our group, LTRS has been used to study the effects of external perturbations, both chemical and mechanical, on the biochemistry of the cell. Single cell dynamics can be studied by performing longitudinal studies to continuously monitor the response of the cell as it interacts with its environment. The ability to carry out these measurements in-vitro makes LTRS an attractive tool for many biomedical applications. Here, we discuss the use of LTRS to study the response of cancer cells to chemotherapeutics and bacteria cells to antibiotics and show that the life cycle and apoptosis of the cells can be detected. These results show the promise of LTRS for drug discovery/screening, antibiotic susceptibility testing, and chemotherapy response monitoring applications. In separate experiments, we study the response of red blood cells to the mechanical forces imposed on the cell by the optical tweezers. A laser power dependent deoxygenation of the red blood cell in the single beam trap is reported. Normal, sickle cell, and fetal red blood cells have a different behavior that enables the discrimination of the cell types based on this mechanochemical response. These results show the potential utility of LTRS for diagnosing and studying red blood cell diseases.

The diagnostic potential of autofluorescence (AF) microscopy under ultraviolet (UV) excitation is explored using ex vivo human specimens. The aim is to establish optical patterns (the rules for interpretation) that correspond to normal and abnormal histologies of the esophagus, spanning from early benign modifications (Barrett's esophagus) to subsequent dysplastic change and progression toward carcinoma. This was achieved by developing an image library categorized by disease progression. We considered morphological changes of disease as they are compared with histological diagnosis of the pathological specimen, as well as control samples of normal esophagus, proximal stomach, and small intestine tissue. Our experimental results indicate that UV AF microscopy could provide real-time histological information for visualizing changes in tissue microstructure that are currently undetectable using conventional endoscopic methods.

The 53 papers in this special issue cover a broad range of areas, including next generation ultrafast sources and technologies, ultrafast spectroscopy and microscopy, and ultrafast x-ray and strong field science and applications.

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) are a potentially unlimited source of cardiomyocytes (CMs) for cardiac transplantation therapies. The establishment of pure PSC-CM populations is important for this application, but is hampered by a lack of CM-specific surface markers suitable for their identification and sorting. Contemporary purification techniques are either non-specific or require genetic modification. We report a second harmonic generation (SHG) signal detectable in PSC-CMs that is attributable to sarcomeric myosin, dependent on PSC-CM maturity, and retained while PSC-CMs are in suspension. Our study demonstrates the feasibility of developing a SHG-activated flow cytometer for the non-invasive purification of PSC-CMs.

In this paper we report the development of two attachments to a commercial cell phone that transform the phone's integrated lens and image sensor into a 350x microscope and visible-light spectrometer. The microscope is capable of transmission and polarized microscopy modes and is shown to have 1.5 micron resolution and a usable field-of-view of 150 x 50 with no image processing, and approximately 350 x 350 when post-processing is applied. The spectrometer has a 300 nm bandwidth with a limiting spectral resolution of close to 5 nm. We show applications of the devices to medically relevant problems. In the case of the microscope, we image both stained and unstained blood-smears showing the ability to acquire images of similar quality to commercial microscope platforms, thus allowing diagnosis of clinical pathologies. With the spectrometer we demonstrate acquisition of a white-light transmission spectrum through diffuse tissue as well as the acquisition of a fluorescence spectrum. We also envision the devices to have immediate relevance in the educational field.

The non-invasiveness of autofluorescence technology may reduce sampling error and time delay for histopathology diagnosis. We establish biophotonic methods and guidelines to visualize and interpret early epithelial tissue changes that signify disease. Flexible and rigid fiber endomicroscopy instrumentation design parameters feasible for translation towards clinical use are in development.

Laser tweezers Raman spectroscopy (LTRS) was used to demonstrate that a red blood cell (RBC) in a single beam optical trap transitions from an oxygenated to a partially deoxygenated state with increasing trapping power. Continuous switching between the two states is possible by repeatedly cycling between low and high trapping powers. Alterations in the hemoglobin conformation and interactions due to cell folding in the trap are proposed to be responsible for the transition. This study demonstrates that mechanically induced biochemical changes by optical forces need to be considered when applying single beam optical tweezers for cell analysis. LTRS holds promise as a functional assay to characterize normal and diseased RBCs based on their biochemical response to the forces of a single beam optical trap.

Abstract Abstract 4847 The number of individuals with sickle cell disease (SCD) in the U.S. is estimated to be over 70,000, and the average life span of a SCD patient is 39 years. Despite the high incidence of SCD in the U.S. and its significant negative effects on patient health, there are currently only limited treatment options for this disease. Also, although a single mutation in the globin gene is known to cause SCD, there is significant clinical diversity. As technology advances, many studies, including genome-wide analyses and proteomics, have been conducted to understand the disease mechanism and find better treatments. However, none has yet led to the development of novel therapies. We need a practical approach to study red blood cells (RBCs) to increase our knowledge of this heterogeneous disease. Laser tweezers Raman spectroscopy (LTRS) is a label-free, laser-based method for the biochemical analysis of single living cells. Based on the phenomenon of inelastic light scattering of photons by molecular bonds, Raman spectroscopy can provide detailed information, in the form of a Raman spectrum, about the molecular structure and conformations inside single living cells. Moreover, the integration of laser tweezers enables 1) simple, convenient analysis of single suspension cells such as RBCs and 2) a method to impart a mechanical force on a cell (via the optical forces) and simultaneously monitor the biochemical response of that cell to the applied force. Previous studies using optical tweezers have shown different RBC elasticity between samples from SCD patients treated with hydroxyurea and those that were not treated with hydroxyurea. Raman spectroscopy has been applied to both normal and pathological RBCs in previous studies, but the results were limited to the identification of characteristic signatures. Our integration of the two methods, Raman spectroscopy and laser tweezers, is a new biophotonic approach for studying the intricate relationship between the biochemical and mechanical properties of a single cell that was not previously possible and is not easily carried out with existing methods. As such, we believe that this recent emerging single cell-based technology can be developed and used as an RBC functional assay. We have previously shown that LTRS can detect a force dependent oxygenation state transition in individual RBCs trapped at different laser powers, presumably due to the optical forces stretching the cell and inducing changes in the hemoglobin-hemoglobin and hemoglobin-membrane interactions that change the oxygen content of the cell. We have also shown characteristic Raman signatures indicating different oxygen content in different RBC samples at specific applied forces. Signatures were different among RBCs from cord, normal adult, and SCD patient blood, suggesting laser tweezers Raman spectroscopy can be used to measure the different force dependent oxygen content in a single RBC. We are currently validating our system to determine whether Raman spectroscopy can be used to identify and distinguish gene-corrected RBCs from normal and sickle RBCs. Our preliminary results showed distinct Raman fingerprints associated with the membrane structure of RBCs. Spectral fingerprints were observed to be different between normal and sickle RBCs, and gene-corrected RBCs were observed to have Raman profiles similar to those of normal RBCs. We speculate that the signature overlap between normal and gene-corrected samples is due to partial gene correction. These results are encouraging and strongly suggest that Raman spectroscopy can not only identify molecular structures but also be used as a functional assay. Disclosures: No relevant conflicts of interest to declare.

We explore autofluorescence endomicroscopy as a potential tool for real-time visualization of epithelial tissue microstructure and organization in a clinical setting. The design parameters are explored using two experimental systems--an Olympus Medical Systems Corp. stand-alone clinical prototype probe, and a custom built bench-top rigid fiber conduit prototype. Both systems entail ultraviolet excitation at 266 nm and/or 325 nm using compact laser sources. Preliminary results using ex vivo animal and human tissue specimens suggest that this technology can be translated toward in vivo application to address the need for real-time histology.