An integrated microfluidic chip for immunomagnetic detection and isolation of rare prostate cancer cells from blood.

The quantitative and qualitative analysis of circulating tumor cells (CTCs) has the potential to improve the clinical management of several cancers, including prostate cancer. As such, there is much interest in the isolation of CTCs from the peripheral blood of cancer patients. We report the design, fabrication, and proof-of-principle testing of an integrated permalloy-based microfluidic chip for immunomagnetic isolation of blood-borne prostate cancer cells using an antibody targeting prostate surface membrane antigen (PSMA). The preliminary results using spiked blood samples indicate that the proposed device is consistently capable of isolating prostate cancer cells with high sensitivity (up to 98 %) at clinically relevant low concentrations (down to 20 cells/mL) and an acceptable throughput (100 µL/min).

Doppler ultrasound-based measurement of tendon velocity and displacement for application toward detecting user-intended motion.

The motivation of this research is to non-invasively monitor the wrist tendon's displacement and velocity, for purposes of controlling a prosthetic device. This feasibility study aims to determine if the proposed technique using Doppler ultrasound is able to accurately estimate the tendon's instantaneous velocity and displacement. This study is conducted with a tendon mimicking experiment consisting of two different materials: a commercial ultrasound scanner, and a reference linear motion stage set-up. Audio-based output signals are acquired from the ultrasound scanner, and are processed with our proposed Fourier technique to obtain the tendon's velocity and displacement estimates. We then compare our estimates to an external reference system, and also to the ultrasound scanner's own estimates based on its proprietary software. The proposed tendon motion estimation method has been shown to be repeatable, effective and accurate in comparison to the external reference system, and is generally more accurate than the scanner's own estimates. After establishing this feasibility study, future testing will include cadaver-based studies to test the technique on the human arm tendon anatomy, and later on live human test subjects in order to further refine the proposed method for the novel purpose of detecting user-intended tendon motion for controlling wearable prosthetic devices.

A novel permalloy based magnetic single cell micro array.

Devices capable of automatically aligning cells onto geometrical arrays are of great interest to biomedical researchers. Such devices can facilitate the study of numerous cells while the cells remain physically separated from one another. In this way, cell arrays reduce cell-to-cell interactions while the cells are all subjected to common stimuli, which allows individual cell behaviour to be revealed. The use of arrays allows for the parallel analysis of single cells, facilitates data logging, and opens the door to the use of automated machine-based single cell analysis techniques. A novel permalloy based magnetic single cell micro array (MSCMA) is presented in this paper. The MSCMA creates an array of magnetic traps by generating magnetic flux density peaks at predefined locations. When using cells labelled with immunomagnetic labels, the cells will interact with the magnetic fields, and can be captured at the magnetic trap sites. Prototypes of the MSCMA have been successfully fabricated and tested using both fixed and live Jurkat cells (10 microm average diameter) that were labelled. The prototypes performed as predicted during experimental trials. The experimental results show that the MSCMA can randomly array up to 136 single cells per square mm. The results also show that the number of single cells captured is a function of the trap site density of the MSCMA design and the cell density in the fluid sample.

RoboSCell: an automated single cell arraying and analysis instrument.

Single cell research has the potential to revolutionize experimental methods in biomedical sciences and contribute to clinical practices. Recent studies suggest analysis of single cells reveals novel features of intracellular processes, cell-to-cell interactions and cell structure. The methods of single cell analysis require mechanical resolution and accuracy that is not possible using conventional techniques. Robotic instruments and novel microdevices can achieve higher throughput and repeatability; however, the development of such instrumentation is a formidable task. A void exists in the state-of-the-art for automated analysis of single cells. With the increase in interest in single cell analyses in stem cell and cancer research the ability to facilitate higher throughput and repeatable procedures is necessary. In this paper, a high-throughput, single cell microarray-based robotic instrument, called the RoboSCell, is described. The proposed instrument employs a partially transparent single cell microarray (SCM) integrated with a robotic biomanipulator for in vitro analyses of live single cells trapped at the array sites. Cells, labeled with immunomagnetic particles, are captured at the array sites by channeling magnetic fields through encapsulated permalloy channels in the SCM. The RoboSCell is capable of systematically scanning the captured cells temporarily immobilized at the array sites and using optical methods to repeatedly measure extracellular and intracellular characteristics over time. The instrument's capabilities are demonstrated by arraying human T lymphocytes and measuring the uptake dynamics of calcein acetoxymethylester--all in a fully automated fashion.

In vivo estimation of flexor digitorum superficialis tendon displacement with speckle tracking on 2-D ultrasound images using Laplacian, Gaussian and Rayleigh techniques.

This study applies 2-D speckle tracking using B-scan ultrasound imaging to estimate the instantaneous and total displacement of the middle flexor digitorum superficialis (FDS) tendon proximal to the wrist. This is achieved by performing the study with human patients, during regular carpal tunnel surgeries. B-Scan images were collected with a 12-MHz transducer placed proximal to the wrist, while a video microscope simultaneously imaged the exposed flexor tendons in the palm as a reference for validation. The accuracy of the proposed speckle-based tracking method is compared using log-compressed Rayleigh (Fisher-Tippet)-, Gaussian (sum of squared differences)- and Laplacian (sum of absolute differences)-based statistics as similarity measures. Overall, tracking was successful and the Rayleigh technique performed better than the Laplacian or Gaussian technique. One goal of this research was to non-invasively monitor FDS tendon displacement in the wrist for the purposes of controlling a prosthetic device. An additional goal was to obtain pre- and post-operative clinical information.

Localized, macromolecular transport for thin, adherent, single cells via an automated, single cell electroporation biomanipulator.

Single cell electroporation (SCE), via microcapillary, is an effective method for molecular, transmembrane transport used to gain insight on cell processes with minimal preparation. Although possessing great potential, SCE is difficult to execute and the technology spans broad fields within cell biology and engineering. The technical complexities, the focus and expertise demanded during manual operation, and the lack of an automated SCE platform limit the widespread use of this technique, thus the potential of SCE has not been realized. In this study, an automated biomanipulator for SCE is presented. Our system is capable of delivering molecules into the cytoplasm of extremely thin cellular features of adherent cells. The intent of the system is to abstract the technical challenges and exploit the accuracy and repeatability of automated instrumentation, leaving only the focus of the experimental design to the operator. Each sequence of SCE including cell and SCE site localization, tip-membrane contact detection, and SCE has been automated. Positions of low-contrast cells are localized and "SCE sites" for microcapillary tip placement are determined using machine vision. In addition, new milestones within automated cell manipulation have been achieved. The system described herein has the capability of automated SCE of "thin" cell features less than 10 µm in thickness. Finally, SCE events are anticipated using visual feedback, while monitoring fluorescing dye entering the cytoplasm of a cell. The execution is demonstrated by inserting a combination of a fluorescing dye and a reporter gene into NIH/3T3 fibroblast cells.

Machine vision-based localization of nucleic and cytoplasmic injection sites on low-contrast adherent cells.

Automated robotic bio-micromanipulation can improve the throughput and efficiency of single-cell experiments. Adherent cells, such as fibroblasts, include a wide range of mammalian cells and are usually very thin with highly irregular morphologies. Automated micromanipulation of these cells is a beneficial yet challenging task, where the machine vision sub-task is addressed in this article. The necessary but neglected problem of localizing injection sites on the nucleus and the cytoplasm is defined and a novel two-stage model-based algorithm is proposed. In Stage I, the gradient information associated with the nucleic regions is extracted and used in a mathematical morphology clustering framework to roughly localize the nucleus. Next, this preliminary segmentation information is used to estimate an ellipsoidal model for the nucleic region, which is then used as an attention window in a k-means clustering-based iterative search algorithm for fine localization of the nucleus and nucleic injection site (NIS). In Stage II, a geometrical model is built on each localized nucleus and employed in a new texture-based region-growing technique called Growing Circles Algorithm to localize the cytoplasmic injection site (CIS). The proposed algorithm has been tested on 405 images containing more than 1,000 NIH/3T3 fibroblast cells, and yielded the precision rates of 0.918, 0.943, and 0.866 for the NIS, CIS, and combined NIS-CIS localizations, respectively.

Development of an autonomous biological cell manipulator with single-cell electroporation and visual servoing capabilities.

Studies of single cells via microscopy and microinjection are a key component in research on gene functions, cancer, stem cells, and reproductive technology. As biomedical experiments become more complex, there is an urgent need to use robotic systems to improve cell manipulation and microinjection processes. Automation of these tasks using machine vision and visual servoing creates significant benefits for biomedical laboratories, including repeatability of experiments, higher throughput, and improved cell viability. This paper presents the development of a new 5-DOF robotic manipulator, designed for manipulating and microinjecting single cells. This biological cell manipulator (BCM) is capable of autonomous scanning of a cell culture followed by autonomous injection of cells using single-cell electroporation (SCE). SCE does not require piercing the cell membrane, thereby keeping the cell membrane fully intact. The BCM features high-precision 3-DOF translational and 2-DOF rotational motion, and a second z-axis allowing top-down placement of a micropipette tip onto the cell membrane for SCE. As a technical demonstration, the autonomous visual servoing and microinjection capabilities of the single-cell manipulator are experimentally shown using sea urchin eggs.