High-Throughput, Time-Resolved Mechanical Phenotyping of Prostate Cancer Cells.

Worldwide, prostate cancer sits only behind lung cancer as the most commonly diagnosed form of the disease in men. Even the best diagnostic standards lack precision, presenting issues with false positives and unneeded surgical intervention for patients. This lack of clear cut early diagnostic tools is a significant problem. We present a microfluidic platform, the Time-Resolved Hydrodynamic Stretcher (TR-HS), which allows the investigation of the dynamic mechanical response of thousands of cells per second to a non-destructive stress. The TR-HS integrates high-speed imaging and computer vision to automatically detect and track single cells suspended in a fluid and enables cell classification based on their mechanical properties. We demonstrate the discrimination of healthy and cancerous prostate cell lines based on the whole-cell, time-resolved mechanical response to a hydrodynamic load. Additionally, we implement a finite element method (FEM) model to characterise the forces responsible for the cell deformation in our device. Finally, we report the classification of the two different cell groups based on their time-resolved roundness using a decision tree classifier. This approach introduces a modality for high-throughput assessments of cellular suspensions and may represent a viable application for the development of innovative diagnostic devices.

What lies beneath? Scanning probe tomography may have the answer.

Scanning probe microscopy facilitates high-resolution noninvasive imaging of surface topography on even the most delicate of biological structures. Moreover, the local probe nature of the instrument architecture lends itself to the measurement of many important physical properties. To date, biological investigations have largely been constrained to imaging surface (membrane)-borne phenomena; however, the advent of extremely high aspect-ratio 'needle' probe tips, as reported by Beard et al. (2013), suggests that the approach can now be extended to address the particular challenges associated with measuring subsurface microscopic targets, including the intracellular components of the stratum corneum.

The spontaneously adhesive leukocyte function-associated antigen-1 (LFA-1) integrin in effector T cells mediates rapid actin- and calmodulin-dependent adhesion strengthening to ligand under shear flow.

Integrins in effector T cells are highly expressed and important for trafficking of these cells and for their effector functions. However, how integrins are regulated in effector T cells remains poorly characterized. Here, we have investigated effector T cell leukocyte function-associated antigen-1 (LFA-1) regulation in primary murine effector T cells. These cells have high LFA-1 integrin expression and display high spontaneous binding to intercellular adhesion molecule-1 (ICAM-1) ligand under static conditions. In addition, these cells are able to migrate spontaneously on ICAM-1. Atomic force microscopy measurements showed that the force required for unbinding of integrin-ligand interactions increases over time (0.5-20-s contact time). The maximum unbinding force for this interaction was ∼140 piconewtons at 0.5-s contact time, increasing to 580 piconewtons at 20-s contact time. Also, the total work required to disrupt the interaction increased over the 20-s contact time, indicating LFA-1-mediated adhesion strengthening in primary effector T cells over a very quick time frame. Effector T cells adhered spontaneously to ICAM-1 under conditions of shear flow, in the absence of chemokine stimulation, and this binding was independent of protein kinase B/Akt and protein kinase C kinase activity, but dependent on calcium/calmodulin signaling and an intact actin cytoskeleton. These results indicate that effector T cell integrins are highly expressed and spontaneously adhesive in the absence of inside-out integrin signaling but that LFA-1-mediated firm adhesion under conditions of shear flow requires downstream integrin signaling, which is dependent on calcium/calmodulin and the actin cytoskeleton.

On the accuracy of framing-rate measurements in ultra-high speed rotating mirror cameras.

Rotating mirror systems based on the Miller Principle are a mainstay modality for ultra-high speed imaging within the range 1-25 million frames per second. Importantly, the true temporal accuracy of observations recorded in such cameras is sensitive to the framing rate that the system directly associates with each individual data acquisition. The purpose for the present investigation was to examine the validity of such system-reported frame rates in a widely used commercial system (a Cordin 550-62 model) by independently measuring the framing rate at the instant of triggering. Here, we found a small but significant difference between such measurements: the average discrepancy (over the entire spectrum of frame rates used) was found to be 0.66 ± 0.48%, with a maximum difference of 2.33%. The principal reason for this discrepancy was traced to non-optimized sampling of the mirror rotation rate within the system protocol. This paper thus serves three purposes: (i) we highlight a straightforward diagnostic approach to facilitate scrutiny of rotating-mirror system integrity; (ii) we raise awareness of the intrinsic errors associated with data previously acquired with this particular system and model; and (iii), we recommend that future control routines address the sampling issue by implementing real-time measurement at the instant of triggering.