Targeted SERS nanosensors measure physicochemical gradients and free energy changes in live 3D tumor spheroids.

Use of multicellular tumor spheroids (MTS) to investigate therapies has gained impetus because they have potential to mimic factors including zonation, hypoxia and drug-resistance. However, analysis remains difficult and often destroys 3D integrity. Here we report an optical technique using targeted nanosensors that allows in situ 3D mapping of redox potential gradients whilst retaining MTS morphology and function. The magnitude of the redox potential gradient can be quantified as a free energy difference (ΔG) and used as a measurement of MTS viability. We found that by delivering different doses of radiotherapy to MTS we could correlate loss of ΔG with increasing therapeutic dose. In addition, we found that resistance to drug therapy was indicated by an increase in ΔG. This robust and reproducible technique allows interrogation of an in vitro tumor-model's bioenergetic response to therapy, indicating its potential as a tool for therapy development.Leverhulme Trust (Grant ID: RPG-2012-680), Jamie King Cancer Research FundThis is the final version of the article. It first appeared from the Royal Society of Chemistry via http://dx.doi.org/10.1039/C6NR06031

Debiased ambient vibrations optical coherence elastography to profile cell, organoid and tissue mechanical properties

The role of the mechanical environment in defining tissue function, development and growth has been shown to be fundamental. Assessment of the changes in stiffness of tissue matrices at multiple scales has relied mostly on invasive and often specialist equipment such as AFM or mechanical testing devices poorly suited to the cell culture workflow.In this paper, we have developed a unbiased passive optical coherence elastography method, exploiting ambient vibrations in the sample that enables real-time noninvasive quantitative profiling of cells and tissues. We demonstrate a robust method that decouples optical scattering and mechanical properties by actively compensating for scattering associated noise bias and reducing variance. The efficiency for the method to retrieve ground truth is validated in silico and in vitro, and exemplified for key applications such as time course mechanical profiling of bone and cartilage spheroids, tissue engineering cancer models, tissue repair models and single cell. Our method is readily implementable with any commercial optical coherence tomography system without any hardware modifications, and thus offers a breakthrough in on-line tissue mechanical assessment of spatial mechanical properties for organoids, soft tissues and tissue engineering

Doppler optical coherence tomography imaging of local fluid flow and shear stress within microporous scaffolds

Establishing a relationship between perfusion rate and fluid shear stress in a 3-dimensional cell culture environment is an ongoing and challenging task faced by tissue engineers. In this study, we explore Doppler optical coherence tomography (DOCT) as a potential imaging tool for in situ monitoring of local fluid flow profiles inside porous chitosan scaffolds. From the measured fluid flow profiles, the fluid shear stresses are evaluated. We examine the localized fluid flow and shear stress within low and high porosity chitosan scaffolds, which are subjected to a constant input flow rate of 0.5 ml·min(-1). The DOCT results show that the behaviour of the fluid flow and shear stress in micropores is strongly dependent on the micropore interconnectivity, porosity, and size of pores within the scaffold. For low porosity and high porosity chitosan scaffolds examined, the measured local fluid flow and shear stress varied from micropore to micropore with a mean shear stress of 0.49±0.3 dyn·cm(-2) and 0.38±0.2 dyn·cm(-2), respectively. In addition, we show that the scaffold’s porosity and interconnectivity can be quantified by combining analyses of the 3-dimensional structural and flow images obtained from DOCT

Label-free assessment of adipose-derived stem cell differentiation using coherent anti-Stokes Raman scattering and multiphoton microscopy

©2012 IEEE. Personal use of this material is permitted. Permission from IEEE must be obtained for all other users, including reprinting/ republishing this material for advertising or promotional purposes, creating new collective works for resale or redistribution to servers or lists, or reuse of any copyrighted components of this work in other works.Presented at the 2012 IEEE/RSJ International Conference on Intelligent Robots and Systems (IROS), 7-12 October 2012, Vilamoura, Portugal.We propose a novel method for a robot to separate and segment objects in a cluttered tabletop environment. The method leverages the fact that external object boundaries produce visible edges within an object cluster. We achieve this singulation of objects by using the robot arm to perform pushing actions specifically selected to test whether particular visible edges correspond to object boundaries. We verify the separation of objects after a push by examining the clusters formed by geometric segmentation of regions residing on the table surface. To avoid explicitly representing and tracking edges across push behaviors we aggregate over all edges in a given orientation by representing the push-history as an orientation histogram. By tracking the history of directions pushed for each object cluster we can build evidence that a cluster cannot be further separated. We present quantitative and qualitative experimental results performed in a real home environment by a mobile manipulator using input from an RGB-D camera mounted on the robot’s head. We show that our pushing strategy can more reliably obtain singulation in fewer pushes than an approach, that does not explicitly reason about boundary information

Molecular photo-thermal optical coherence phase microscopy using gold nanorods

Optical coherence tomography (OCT) is a non-invasive interferometry imaging technique with micrometre scale resolution at millimetre scale penetration depths in highly scattering tissues. This study describes a new evolution of OCT, termed molecular optical coherence phase microscopy (molecular OCPM), which is capable of imaging the expression of molecular markers at the cellular level using gold nanorods as photothermal imaging agents. Gold nanorods were selected as the imaging agents due to their excellent photothermal energy conversion efficiency and tuneable plasmon bands. The gold nanorods were surface functionalized to achieve efficient and specific targeting of the tyrosine kinase human epidermal growth factor receptor HER2 molecular markers used as a model tumor biomarker. Phase modulation retrieval was used to generate photothermal maps which were overlayed on intensity images. Phase modulation within the filter corresponding to the laser excitation modulation frequency was clearly observed for cells targeted with the molecular photothermal imaging agents. These results confirm the ability of photothermal optical coherence phase microscopy to image accurately at the cellular level gold nanorods molecularly targeted to a biomarker expressed on cancer cell membranes, paving the way for its application to novel bioimaging procedures

A novel optical coherence tomography-based micro-indentation technique for mechanical characterization of hydrogels

Depth-sensing micro-indentation has been well recognized as a powerful tool for characterizing mechanical properties of solid materials due to its non-destructive approach. Based on the depth-sensing principle, we have developed a new indentation method combined with a high-resolution imaging technique, optical coherence tomography, which can accurately measure the deformation of hydrogels under a spherical indenter at constant force. The Hertz contact theory has been applied for quantitatively correlating the indentation force and the deformation with the mechanical properties of the materials. Young's moduli of hydrogels estimated by the new method are comparable with those measured by conventional depth-sensing micro-indentation. The advantages of this new method include its capability to characterize mechanical properties of bulk soft materials and amenability to perform creeping tests. More importantly, the measurement can be performed under sterile conditions allowing non-destructive, in situ and real-time investigations on the changes in mechanical properties of soft materials (e.g. hydrogel). This unique character can be applied for various biomechanical investigations such as monitoring reconstruction of engineered tissues

Cellular Micromotion Monitored by Long-Range Surface Plasmon Resonance with Optical Fluctuation Analysis

Long-range surface plasmon resonance (LRSPR) is a powerful biosensing technology due to a substantially larger probing depth into the medium and sensitivity, compared with conventional SPR. We demonstrate here that LRSPR can provide sensitive noninvasive measurement of the dynamic fluctuation of adherent cells, often referred to as the cellular micromotion. Proof of concept was achieved using confluent layers of 3T3 fibroblast cells and MDA-MB-231 cancer cells. The slope of the power spectral density (PSD) of the optical fluctuations was calculated to determine the micromotion index, and significant differences were measured between live and fixed cell layers. Furthermore, the performances of LRSPR and conventional surface plasmon resonance (cSPR) were compared with respect to micromotion monitoring. Our study showed that the micromotion index of cells measured by LRSPR sensors was higher than when measured with cSPR, suggesting a higher sensitivity of LRSPR to the micromotion of cells. To investigate further this finding, simulations were conducted to establish the relative sensitivities of LRSPR and cSPR to membrane fluctuations. Increased signal intensity was predicted for LRSPR in comparison to cSPR, suggesting that membrane fluctuations play a significant role in the optical micromotion measured in LRSPR. Analogous to cellular micromotion measured using impedance techniques, LRSPR micromotion has the potential to provide important biological information on the metabolic activity and viability of adherent cells