Mechanical fluidity of fully suspended biological cells

Mechanical characteristics of single biological cells are used to identify and possibly leverage interesting differences among cells or cell populations. Fluidity---hysteresivity normalized to the extremes of an elastic solid or a viscous liquid---can be extracted from, and compared among, multiple rheological measurements of cells: creep compliance vs. time, complex modulus vs. frequency, and phase lag vs. frequency. With multiple strategies available for acquisition of this nondimensional property, fluidity may serve as a useful and robust parameter for distinguishing cell populations, and for understanding the physical origins of deformability in soft matter. Here, for three disparate eukaryotic cell types deformed in the suspended state via optical stretching, we examine the dependence of fluidity on chemical and environmental influences around a time scale of 1 s. We find that fluidity estimates are consistent in the time and the frequency domains under a structural damping (power-law or fractional derivative)model, but not under an equivalent-complexity lumpedcomponent (spring-dashpot) model; the latter predicts spurious time constants. Although fluidity is suppressed by chemical crosslinking, we find that adenosine triphosphate (ATP) depletion in the cell does not measurably alter the parameter, and thus conclude that active ATP-driven events are not a crucial enabler of fluidity during linear viscoelastic deformation of a suspended cell. Finally, by using the capacity of optical stretching to produce near-instantaneous increases in cell temperature, we establish that fluidity increases with temperature---now measured in a fully suspended, sortable cell without the complicating factor of cell-substratum adhesion

Multimodal optical diagnostics of the microhaemodynamics in upper and lower limbs

The introduction of optical non-invasive diagnostic methods into clinical practice can substantially advance in the detection of early microcirculatory disorders in patients with different diseases. This paper is devoted to the development and application of the optical non-invasive diagnostic approach for the detection and evaluation of the severity of microcirculatory and metabolic disorders in rheumatic diseases and diabetes mellitus. The proposed methods include the joint use of laser Doppler flowmetry, absorption spectroscopy and fluorescence spectroscopy in combination with functional tests. This technique showed the high diagnostic importance for the detection of disturbances in peripheral microhaemodynamics. These methods have been successfully tested as additional diagnostic techniques in the field of rheumatology and endocrinology. The sensitivity and specificity of the proposed diagnostic procedures have been evaluated.<br/

Optical stimulation of the prostate nerves : a potential diagnostic technique

There is wide variability in sexual potency rates (9-86%) after nerve-sparing prostate cancer surgery due to limited knowledge of the location of the cavernous nerves (CN's) on the prostate surface, which are responsible for erectile function. Thus, preservation of the CN's is critical in preserving a man's ability to have spontaneous erections following surgery. Nerve-mapping devices, utilizing conventional Electrical Nerve Stimulation (ENS) techniques, have been used as intra-operative diagnostic tools to assist in preservation of the CN. However, these technologies have proven inconsistent and unreliable in identifying the CN's due to the need for physical contact, the lack of spatial selectivity, and the presence of electrical artifacts in measurements. Optical Nerve Stimulation (ONS), using pulsed infrared laser radiation, is studied as an alternative to ENS. The objective of this study is sevenfold: (1) to develop a laparoscopic laser probe for ONS of the CN's in a rat model, in vivo; (2) to demonstrate faster ONS using continuous-wave infrared laser radiation; (3) to describe and characterize the mechanism of successful ONS using alternative laser wavelengths; (4) to test a compact, inexpensive all-single-mode fiber configuration for optical stimulation of the rat CN studies; (5) to implement fiber optic beam shaping methods for comparison of Gaussian and flat-top spatial beam profiles during ONS; (6) to demonstrate successful ONS of CN's through a thin layer of fascia placed over the nerve and prostate gland; and (7) to verify the experimentally determined therapeutic window for safe and reliable ONS without thermal damage to the CN's by comparison with a computational model for thermal damage. A 5.5-Watt Thulium fiber laser operated at 1870 nm and two pigtailed, single mode, near-IR diode lasers (150-mW, 1455-nm laser and 500-mW, 1550-nm laser) were used for non-contact stimulation of the rat CN's. Successful laser stimulation, as measured by an intracavernous pressure (ICP) response in the penis, was achieved with the laser operating in CW mode. CW optical nerve stimulation provides a significantly faster ICP response time using a lower laser power laser than conventional pulsed stimulation. An all-single-mode fiber design was successfully tested in a rat model. The CN reached a threshold temperature of ~ 42°C, with response times as short as 3 s, and ICP responses in the rat penis of up to 50 mmHg compared to a baseline of 5-10 mmHg. Chemical etching of the distal single-mode-fiber tip produced a concave shape and transformed the Gaussian to a flat-top spatial beam profile, resulting in simplified alignment of the laser beam with the nerve. This novel, all-single-mode-fiber laser nerve stimulation system introduces several advantages including: (1) a less expensive and more compact ONS configuration; (2) elimination of alignment and cleaning bulk optical components; and (3) improved spatial beam profile for simplified alignment. In order to define the upper limit of the therapeutic window for ONS of CN in a rat model, in vivo, identification of the thermal damage threshold for the CN after laser irradiation was investigated by direct comparison of the visible thermal damage data with a theoretical thermal damage calculation utilizing a standard Arrhenius integral model

Microfabricated Sampling Probes for Monitoring Brain Chemistry at High Spatial and Temporal Resolution

Monitoring neurochemical dynamics has played a crucial role in elucidating brain function and related disorders. An essential approach for monitoring neurochemicals is to couple sampling probes to analytical measurements; however, this approach is inherently limited by poor spatial and temporal resolution. In this work, we have developed miniaturized sampling probes and analytical technology to overcome these limitations. Conventional sampling probes were handmade and have several disadvantages, including large sizes (over 220 µm in diameter) and limited design flexibility. To address these disadvantages, we have used microfabrication to manufacture sampling probes. By bulk micromachining of Si, microchannels and small sampling regions can be fabricated within a probe, with an overall dimension of ~100 µm. For development of a dialysis probe, nanoporous anodic aluminum oxide was adapted for monolithically embedding a membrane. Coupling the probe to liquid chromatography-mass spectrometry, multiple neurochemicals were measured at basal conditions, including dopamine and acetylcholine. Comparing to conventional dialysis probes, the microfabricated dialysis probe provided at least 6-fold improvement in spatial resolution and potentially had lower tissue disruption. Furthermore, we have continued the development of a microfabricated push-pull probe. We enhanced functionality of the probe by integrating an additional channel into the probe for chemical delivery. Further, we demonstrated that the probe can feasibly be coupled to droplet microfluidic devices for improved temporal resolution. Nanospray ionization mass spectrometry was used for multiplexed measurements of neurochemicals in nanoliter droplet samples. Utility of the integrated system was demonstrated by monitoring in vivo dynamics during potassium stimulation of 4 neurochemicals, including glutamate and GABA. The probe provided unprecedented spatial resolution and temporal resolution as high as ~5 s. Additionally, we highlighted versatility of the method by coupling the probe to another high-throughput assay, i.e., droplet-based microchip capillary electrophoresis for rapid separation (less than 3 s) and measurement of multiple amino acid neurochemicals. This collection of work illustrates that development of the microfabricated sampling probes and their compatible microfluidic systems are highly beneficial for studying brain chemistry. The integrated miniaturized analytical technology can potentially be useful for solving other problems of biological significance.PHDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/144094/1/nonngern_1.pd

Superpulsed low-level laser therapy protects skeletal muscle of mdx mice against damage, inflammation and morphological changes delaying dystrophy progression.

Aim: To evaluate the effects of preventive treatment with low-level laser therapy (LLLT) on progression of dystrophy in mdx mice. Methods: Ten animals were randomly divided into 2 experimental groups treated with superpulsed LLLT (904 nm, 15 mW, 700 Hz, 1 J) or placebo-LLLT at one point overlying the tibialis anterior muscle (bilaterally) 5 times per week for 14 weeks (from 6th to 20th week of age). Morphological changes, creatine kinase (CK) activity and mRNA gene expression were assessed in animals at 20th week of age. Results: Animals treated with LLLT showed very few morphological changes in skeletal muscle, with less atrophy and fibrosis than animals treated with placebo-LLLT. CK was significantly lower (p = 0.0203) in animals treated with LLLT (864.70 U.l−1, SEM 226.10) than placebo (1708.00 U.l−1, SEM 184.60). mRNA gene expression of inflammatory markers was significantly decreased by treatment with LLLT (p<0.05): TNF-α (placebo-control = 0.51 µg/µl [SEM 0.12], - LLLT = 0.048 µg/µl [SEM 0.01]), IL-1β (placebo-control = 2.292 µg/µl [SEM 0.74], - LLLT = 0.12 µg/µl [SEM 0.03]), IL-6 (placebo-control = 3.946 µg/µl [SEM 0.98], - LLLT = 0.854 µg/µl [SEM 0.33]), IL-10 (placebo-control = 1.116 µg/µl [SEM 0.22], - LLLT = 0.352 µg/µl [SEM 0.15]), and COX-2 (placebo-control = 4.984 µg/µl [SEM 1.18], LLLT = 1.470 µg/µl [SEM 0.73]). Conclusion: Irradiation of superpulsed LLLT on successive days five times per week for 14 weeks decreased morphological changes, skeletal muscle damage and inflammation in mdx mice. This indicates that LLLT has potential to decrease progression of Duchenne muscular dystrophy

NONCONTACT DIFFUSE CORRELATION TOMOGRAPHY OF BREAST TUMOR

Since aggressive cancers are frequently hypermetabolic with angiogenic vessels, quantification of blood flow (BF) can be vital for cancer diagnosis. Our laboratory has developed a noncontact diffuse correlation tomography (ncDCT) system for 3-D imaging of BF distribution in deep tissues (up to centimeters). The ncDCT system employs two sets of optical lenses to project source and detector fibers respectively onto the tissue surface, and applies finite element framework to model light transportation in complex tissue geometries. This thesis reports our first step to adapt the ncDCT system for 3-D imaging of BF contrasts in human breast tumors. A commercial 3-D camera was used to obtain breast surface geometry which was then converted to a solid volume mesh. An ncDCT probe scanned over a region of interest on the breast mesh surface and the measured boundary data were used for 3-D image reconstruction of BF distribution. This technique was tested with computer simulations and in 28 patients with breast tumors. Results from computer simulations suggest that relatively high accuracy can be achieved when the entire tumor was within the sensitive region of diffuse light. Image reconstruction with a priori knowledge of the tumor volume and location can significantly improve the accuracy in recovery of tumor BF contrasts. In vivo ncDCT imaging results from the majority of breast tumors showed higher BF contrasts in the tumor regions compared to the surrounding tissues. Reconstructed tumor depths and dimensions matched ultrasound imaging results when the tumors were within the sensitive region of light propagation. The results demonstrate that ncDCT system has the potential to image BF distributions in soft and vulnerable tissues without distorting tissue hemodynamics. In addition to this primary study, detector fibers with different modes (i.e., single-mode, few-mode, multimode) for photon collection were experimentally explored to improve the signal-to-noise ratio of diffuse correlation spectroscopy flow-oximeter measurements

Next-Generation Raman tomography Instrument for Non-Invasive In Vivo Bone Imaging

Combining diffuse optical tomography methods with Raman spectroscopy of tissue provides the ability for in vivo measurements of chemical and molecular characteristics, which have the potential for being useful in diagnostic imaging. In this study a system for Raman tomography was developed and tested. A third generation microCT coupled system was developed to combine 10 detection fibers and 5 excitation fibers with laser line filtering and a Cytop reference signal. Phantom measurements of hydroxyapatite concentrations from 50 to 300 mg/ml had a linear response. Fiber placement and experiment design was optimized using cadaver animals with live animal measurements acquired to validate the systems capabilities. Promising results from the initial animal experiments presented here, pave the way for a study of longitudinal measurements during fracture healing and the scaling of the Raman tomography system towards human measurements

SAM68 is a physiological regulator of SMN2 splicing in spinal muscular atrophy

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by loss of motor neurons in patients with null mutations in the SMN1 gene. The almost identical SMN2 gene is unable to compensate for this deficiency because of the skipping of exon 7 during pre-messenger RNA (mRNA) processing. Although several splicing factors can modulate SMN2 splicing in vitro, the physiological regulators of this disease-causing event are unknown. We found that knockout of the splicing factor SAM68 partially rescued body weight and viability of SMAΔ7 mice. Ablation of SAM68 function promoted SMN2 splicing and expression in SMAΔ7 mice, correlating with amelioration of SMA-related defects in motor neurons and skeletal muscles. Mechanistically, SAM68 binds to SMN2 pre-mRNA, favoring recruitment of the splicing repressor hnRNP A1 and interfering with that of U2AF65 at the 3' splice site of exon 7. These findings identify SAM68 as the first physiological regulator of SMN2 splicing in an SMA mouse model

Compliant Culture Platforms with Independently Tunable Stiffness and Electrical/Optical Functionality

Interfacing biological cells and solid-state devices is crucial in many applications, ranging from well-established fields, such as electrophisiology, to the newly developed areas of optogenetics and mechanobiology. Most biological cells are anchored to substrates with elastic modulus, E, in the range of ~1 to 100 kPa, the moduli of brain-tissue and osteoid, respectively. On the other hand, bulk semiconductor substrates have ~6 orders of magnitude higher elastic modulus. This large elastic mismatch between devices and cells natural microenvironments is an issue for bio-devices integration, as cells are highly sensitive to mechanical cues. Specifically, cells exert traction forces on their surroundings and adjust their adhesion mechanism, cytoskeleton, locomotion and overall state according to the stiffness of the substrate they are anchored to. This type of behavior makes it a significant challenge to integrate semiconductor devices with biological cells without altering the cell state. I demonstrate a new family of culture platforms to successfully integrate biological cells and electronic/photonic devices from a mechanical perspective. The proposed platforms are referred to as effectively compliant layered substrates (ECLS). ECLS are based on inorganic nanomembranes (NMs) partially suspended v or bonded to compliant substrates. The unique attribute of ECLS is that, the constitutive material of the NM provides the electrical and optical functionality necessary to a device operation, while the NM geometry and the nature of the supporting substrate can be tailored to match the mechanical response of biological tissues. Specifically, I present fabrication and bio-interfacing of ECLS comprising of device-grade, single-crystal Si NMs on a compliant PDMS substrate with tunable elastic modulus from ~kPa to ~MPa. NMs with thickness in the range of ~20-220 nm and ~ 1x1 cm2 lateral areas are used in this study. ECLS are obtained using a two-step process, including synthesis of the compliant supporting substrate and fabrication, release and transfer of the NM onto the compliant host. Characterization of the mechanical properties of the ECLS and of the bare compliant substrate is performed by nanoindentation. Finally, I access a 3T3 fibroblast cell culture on the fabricated ECLS, as well as on bulk silicon and bare soft substrates to investigate cell response to mechanical cues. Specifically, I investigate cytotoxicity of ECLS substrate and conduct a comparative analysis of cell proliferation, morphology, and adhesion mechanisms between bulk Si, and Si-based ECLS with different elastic moduli. Flow-cytometry, bright-field and confocal fluorescence microscopy are used for this study. The proposed ECLS approach has successfully allowed fabrication of device-graded platforms with varying elastic modulus over three orders of magnitude and matching the mechanical properties of a wide range of biological tissues. Fabricated ECLS allowed healthy bioactivity of 3T3 fibroblast with no toxic behavior. 3T3 fibroblast cultured on ECLS with different elastic modulus displayed a drastic change in cytoskeleton (size and shape) and adhesion mechanisms (stress fiber organization and focal adhesions) compared to that of bulk Si

NONINVASIVE MULTIMODAL DIFFUSE OPTICAL IMAGING OF VULNERABLE TISSUE HEMODYNAMICS

Measurement of tissue hemodynamics provides vital information for the assessment of tissue viability. This thesis reports three noninvasive near-infrared diffuse optical systems for spectroscopic measurements and tomographic imaging of tissue hemodynamics in vulnerable tissues with the goal of disease diagnosis and treatment monitoring. A hybrid near-infrared spectroscopy/diffuse correlation spectroscopy (NIRS/DCS) instrument with a contact fiber-optic probe was developed and utilized for simultaneous and continuous monitoring of blood flow (BF), blood oxygenation, and oxidative metabolism in exercising gastrocnemius. Results measured by the hybrid NIRS/DCS instrument in 37 subjects (mean age: 67 ± 6) indicated that vitamin D supplement plus aerobic training improved muscle metabolic function in older population. To reduce the interference and potential infection risk on vulnerable tissues caused by the contact measurement, a noncontact diffuse correlation spectroscopy/tomography (ncDCS/ncDCT) system was then developed. The ncDCS/ncDCT system employed optical lenses to project limited numbers of sources and detectors on the tissue surface. A motor-driven noncontact probe scanned over a region of interest to collect boundary data for three dimensional (3D) tomographic imaging of blood flow distribution. The ncDCS was tested for BF measurements in mastectomy skin flaps. Nineteen (19) patients underwent mastectomy and implant-based breast reconstruction were measured before and immediately after mastectomy. The BF index after mastectomy in each patient was normalized to its baseline value before surgery to get relative BF (rBF). Since rBF values in the patients with necrosis (n = 4) were significantly lower than those without necrosis (n = 15), rBF levels can be used to predict mastectomy skin flap necrosis. The ncDCT was tested for 3D imaging of BF distributions in chronic wounds of 5 patients. Spatial variations in BF contrasts over the wounded tissues were observed, indicating the capability of ncDCT in detecting tissue hemodynamic heterogeneities. To improve temporal/spatial resolution and avoid motion artifacts due to a long mechanical scanning of ncDCT, an electron-multiplying charge-coupled device based noncontact speckle contrast diffuse correlation tomography (scDCT) was developed. Validation of scDCT was done by imaging both high and low BF contrasts in tissue-like phantoms and human forearms. In a wound imaging study using scDCT, significant lower BF values were observed in the burned areas/volumes compared to surrounding normal tissues in two patients with burn. One limitation in this study was the potential influence of other unknown tissue optical properties such as tissue absorption coefficient (µa) on BF measurements. A new algorithm was then developed to extract both µa and BF using light intensities and speckle contrasts measured by scDCT at multiple source-detector distances. The new algorithm was validated using tissue-like liquid phantoms with varied values of µa and BF index. In-vivo validation and application of the innovative scDCT technique with the new algorithm is the subject of future work