Standard algorithms for phase unwrapping often fail for interferometric quantitative phase imaging (QPI) of biological samples due to the variable morphology of these samples and the requirement to image at low light intensities to avoid phototoxicity. We describe a new algorithm combining random walk-based image segmentation with linear discriminant analysis (LDA)-based feature detection, using assumptions about the morphology of biological samples to account for phase ambiguities when standard methods have failed. We present three versions of our method: first, a method for LDA image segmentation based on a manually compiled training dataset; second, a method using a random walker (RW) algorithm informed by the assumed properties of a biological phase image; and third, an algorithm which combines LDA-based edge detection with an efficient RW algorithm. We show that the combination of LDA plus the RW algorithm gives the best overall performance with little speed penalty compared to LDA alone, and that this algorithm can be further optimized using a genetic algorithm to yield superior performance for phase unwrapping of QPI data from biological samples.

Extra-ocular tendons (EOTs) transmit the oculorotary force of the muscles to the eyeball to generate dynamic eye movements and align the eyes, yet the mechanical properties of the EOTs remain undefined. The EOTs are known to be composed of parallel bundles of small fibers whose mechanical properties must be determined in order to characterize the overall behavior of EOTs. The current study aimed to investigate the transverse Young׳s modulus of EOT fiber bundles using atomic force microscopy (AFM). Fresh bovine EOT fiber bundle specimens were maintained under temperature and humidity control, and indented 100nm by the inverted pyramid tip of an AFM (Veeco Digital Instruments, NY). Ten indentations were conducted for each of 3 different locations of 10 different specimens from each of 6 EOTs, comprising a total of 1800 indentations. Young׳s modulus for each EOT was determined using a Hertzian contact model. Young׳s moduli for fiber bundles from all six EOTs were determined. Mean Young׳s moduli for fiber bundles were similar for the six anatomical EOTs: lateral rectus 60.12±2.69 (±SD)MPa, inferior rectus 59.69±5.34MPa, medial rectus 56.92±1.91MPa, superior rectus 59.66±2.64MPa, inferior oblique 57.7±1.36MPa, and superior oblique 59.15±2.03. Variation in Young׳s moduli among the six EOTs was not significant (P>0.25). The Young׳s modulus of bovine EOT fibers is highly uniform among the six extraocular muscles, suggesting that each EOT is assembled from fiber bundles representing the same biomechanical elements. This uniformity will simplify overall modeling.

Auditory signals (A) are perceived as lasting longer than visual signals (V) of the same physical duration when they are compared together. Despite considerable debate about how this illusion arises psychologically, the neural underpinnings have not been studied. We used functional magnetic resonance imaging (fMRI) to investigate the neural bases of audiovisual temporal distortions and more generally, intersensory timing. Adults underwent fMRI while judging the relative duration of successively presented standard interval-comparison interval (CI) pairs, which were unimodal (A-A, V-V) or crossmodal (V-A, A-V). Mechanisms of time dilation and compression were identified by comparing the two crossmodal pairs. Mechanisms of intersensory timing were identified by comparing the unimodal and crossmodal conditions. The behavioral results showed that auditory CIs were perceived as lasting longer than visual CIs. There were three novel fMRI results. First, time dilation and compression were distinguished by differential activation of higher-sensory areas (superior temporal, posterior insula, middle occipital), which typically showed stronger effective connectivity when time was dilated (V-A). Second, when time was compressed (A-V) activation was greater in frontal cognitive-control centers, which guide decision making. These areas did not exhibit effective connectivity. Third, intrasensory timing was distinguished from intersensory timing partly by decreased striatal and increased superior parietal activation. These regions showed stronger connectivity with visual, memory, and cognitive-control centers during intersensory timing. Altogether, the results indicate that time dilation and compression arise from the connectivity strength of higher-sensory systems with other areas. Conversely, more extensive network interactions are needed with core timing (striatum) and attention (superior parietal) centers to integrate time codes for intersensory signals.

Quantitative phase imaging (QPI) is a label-free, wide-field microscopy approach with significant opportunities for biomedical applications. QPI uses the natural phase shift of light as it passes through a transparent object, such as a mammalian cell, to quantify biomass distribution and spatial and temporal changes in biomass. Reported in cell studies more than 60 years ago, ongoing advances in QPI hardware and software are leading to numerous applications in biology, with a dramatic expansion in utility over the past two decades. Today, investigations of cell size, morphology, behavior, cellular viscoelasticity, drug efficacy, biomass accumulation and turnover, and transport mechanics are supporting studies of development, physiology, neural activity, cancer, and additional physiological processes and diseases. Here, we review the field of QPI in biology starting with underlying principles, followed by a discussion of technical approaches currently available or being developed, and end with an examination of the breadth of applications in use or under development. We comment on strengths and shortcomings for the deployment of QPI in key biomedical contexts and conclude with emerging challenges and opportunities based on combining QPI with other methodologies that expand the scope and utility of QPI even further.

Cell cycle deregulation is a cancer hallmark that has stimulated the development of mitotic inhibitors with differing mechanisms of action. Quantitative phase imaging (QPI) is an emerging approach for determining cancer cell sensitivities to chemotherapies in vitro. Cancer cell fates in response to mitotic inhibitors are agent- and dose-dependent. Fates that lead to chromosomal instabilities may result in a survival advantage and drug resistance. Conventional techniques for quantifying cell fates are incompatible with growth inhibition assays that produce binary live/dead results. Therefore, we used QPI to quantify post-mitotic fates of G0/G1 synchronized HeLa cervical adenocarcinoma and M202 melanoma cells during 24 h of escalating-dose exposures to mitotic inhibitors, including microtubule inhibitors paclitaxel and colchicine, and an Aurora kinase A inhibitor, VX-680. QPI determined cell fates by measuring changes in cell biomass, morphology, and mean phase-shift. Cell fates fell into three groups: (1) bipolar division from drug failure; (2) cell death or sustained mitotic arrest; and (3) aberrant endocycling or multipolar division. In this proof-of-concept study, colchicine was most effective in producing desirable outcomes of sustained mitotic arrest or death throughout its dosing range, whereas both paclitaxel and VX-680 yielded dose-dependent multipolar divisions or endocycling, respectively. Furthermore, rapid completion of mitosis associated with bipolar divisions whereas prolonged mitosis associated with multipolar divisions or cell death. Overall, QPI measurement of drug-induced cancer cell fates provides a tool to inform the development of candidate agents by quantifying the dosing ranges over which suboptimal inhibitor choices lead to undesirable, aberrant cancer cell fates.

A challenge in Parkinson's disease (PD) is to identify biomarkers of early cognitive change because functioning in some domains may be more prognostic of dementia. Few studies have investigated whether structural magnetic resonance imaging (MRI) correlates in a regionally specific manner with functioning in different cognitive domains. The aim of this study was to identify neuroanatomical correlates of executive functioning, memory, and visual cognition in PD without dementia. 3T MRI was conducted in 51 PD patients and 39 control participants. Brain volumes were measured in structures comprising the frontostriatal cognitive-control system, the medial temporal memory system, the ventral object-based system, and the dorsal spatial-based system. Measures of executive functioning (Stroop Test; Letter Fluency), memory (California Verbal Learning Test), visuospatial cognition (Judgment of Line Orientation), and visuoconstruction (Pentagon Copy) were correlated with volumes comprising each system. Poorer executive functioning largely correlated with decreased frontostriatal volumes. Poorer memory correlated with decreased volumes in all medial temporal regions, but also with frontostriatal volumes. Poorer visuospatial cognition correlated with decreased volumes in the object-based system, whereas poorer visuoconstruction correlated with decreased frontal and object-based system volumes. These relationships were nonsignificant in the control group. This is the first study to demonstrate that subtle changes in multiple cognitive domains in PD without dementia correlate with regional volumes in specific systems implicated in the development of cognitive impairment. The findings suggest that structural MRI holds promise as a marker of early changes in different brain systems, some of which may predict future cognitive deterioration.

A challenge in Parkinson's disease (PD) is to identify biomarkers of early cognitive change because functioning in some domains may be more prognostic of dementia. Few studies have investigated whether structural magnetic resonance imaging (MRI) correlates in a regionally specific manner with functioning in different cognitive domains. The aim of this study was to identify neuroanatomical correlates of executive functioning, memory, and visual cognition in PD without dementia. 3T MRI was conducted in 51 PD patients and 39 control participants. Brain volumes were measured in structures comprising the frontostriatal cognitive-control system, the medial temporal memory system, the ventral object-based system, and the dorsal spatial-based system. Measures of executive functioning (Stroop Test; Letter Fluency), memory (California Verbal Learning Test), visuospatial cognition (Judgment of Line Orientation), and visuoconstruction (Pentagon Copy) were correlated with volumes comprising each system. Poorer executive functioning largely correlated with decreased frontostriatal volumes. Poorer memory correlated with decreased volumes in all medial temporal regions, but also with frontostriatal volumes. Poorer visuospatial cognition correlated with decreased volumes in the object-based system, whereas poorer visuoconstruction correlated with decreased frontal and object-based system volumes. These relationships were nonsignificant in the control group. This is the first study to demonstrate that subtle changes in multiple cognitive domains in PD without dementia correlate with regional volumes in specific systems implicated in the development of cognitive impairment. The findings suggest that structural MRI holds promise as a marker of early changes in different brain systems, some of which may predict future cognitive deterioration.

Quantitative polymerase chain reaction is the current "golden standard" for quantification of nucleic acids; however, its utility is constrained by an inability to easily and reliably detect multiple targets in a single reaction. We have successfully overcome this problem with a novel combination of two widely used approaches: target-specific multiplex amplification with 15 cycles of polymerase chain reaction (PCR), followed by single-molecule detection of amplicons with atomic force microscopy (AFM). In test experiments comparing the relative expression of ten transcripts in two different human total RNA samples, we find good agreement between our single reaction, multiplexed PCR/AFM data, and data from 20 individual singleplex quantitative PCR reactions. This technique can be applied to virtually any analytical problem requiring sensitive measurement concentrations of multiple nucleic acid targets.

This study investigated the ability of individuals with Parkinson's disease (PD) to synthesize temporal information across the senses, namely audition and vision. Auditory signals (A) are perceived as lasting longer than visual signals (V) when they are compared together, since attentsion is captured and sustained more easily than for visual information. We used the audiovisual illusion to probe for disturbances in brain networks that govern the resolution of time in two intersensory conditions that putatively differ in their attention demands. PD patients and controls judged the relative duration of successively presented pairs of unimodal (AA, VV) and crossmodal (VA, AV) signals whilst undergoing fMRI. There were four main findings. First, underestimation of time was exaggerated in PD when timing depended on controlled attention (AV), whereas subtle deficits were found when audition dominated and attention was more easily sustained (VA). Second, group differences in regional activation were observed only for the AV-unimodal comparison, where the PD group failed to modulate basal ganglia, anterior insula, and inferior cerebellum activity in accord with the timing condition. Third, the intersensory timing conditions were dissociated by patterns of abnormal functional connectivity. When intersensory timing emphasized controlled attention, patients showed weakened connectivity of the cortico-thalamus-basal ganglia (CTBG) circuit and the anterior insula with widespread cortical regions, yet enhanced cerebellar connectivity. When audition dominated intersensory timing, patients showed enhanced connectivity of CTBG elements, the anterior insula, and the cerebellum with the caudate tail and frontal cortex. Fourth, abnormal connectivity measures showed excellent sensitivity and specificity in accurately classifying subjects. The results demonstrate that intersensory timing deficits in PD were well characterized by context-dependent patterns of functional connectivity within a presumed core timing system (CTBG) and a ventral attention hub (anterior insula), and enhanced cerebellar connectivity irrespective of the hypothesized attention demands of timing.

While adult heart muscle is the least regenerative of tissues, embryonic cardiomyocytes are proliferative, with embryonic stem (ES) cells providing an endless reservoir. In addition to secreted factors and cell-cell interactions, the extracellular microenvironment has been shown to play an important role in stem cell lineage specification, and understanding how scaffold elasticity influences cardiac differentiation is crucial to cardiac tissue engineering. Though previous studies have analyzed the role of the matrix elasticity on the function of differentiated cardiomyocytes, whether it affects the induction of cardiomyocytes from pluripotent stem cells is poorly understood. Here, we examined the role of matrix rigidity on the cardiac differentiation using mouse and human ES cells. Culture on polydimethylsiloxane (PDMS) substrates of varied monomer-to-crosslinker ratios revealed that rigid extracellular matrices promote a higher yield of de novo cardiomyocytes from undifferentiated ES cells. Using an genetically modified ES system that allows us to purify differentiated cardiomyocytes by drug selection, we demonstrate that rigid environments induce higher cardiac troponin T expression, beating rate of foci, and expression ratio of adult α- to fetal β- myosin heavy chain in a purified cardiac population. M-mode and mechanical interferometry image analyses demonstrate that these ES-derived cardiomyocytes display functional maturity and synchronization of beating when co-cultured with neonatal cardiomyocytes harvested from a developing embryo. Together, these data identify matrix stiffness as an independent factor that instructs not only the maturation of the already differentiated cardiomyocytes but also the induction and proliferation of cardiomyocytes from undifferentiated progenitors. Manipulation of the stiffness will help direct the production of functional cardiomyocytes en masse from stem cells for regenerative medicine purposes.