In this work, we present a single-pole magnetic tweezers (MT) device designed for integration with substrate deformation tracking microscopy and/or traction force microscopy experiments intended to explore extracellular matrix rheology and human epidermal keratinocyte mechanobiology. Assembled from commercially available off-the-shelf electronics hardware and software, the MT device is amenable to replication in the basic biology laboratory. In contrast to conventional solenoid current-controlled MT devices, operation of this instrument is based on real-time feedback control of the magnetic flux density emanating from the blunt end of the needle core using a cascade control scheme and a digital proportional–integral–derivative (PID) controller. Algorithms that compensate for a spatially non-uniform remnant magnetization of the needle core that develops during actuation are implemented into the feedback control scheme. Through optimization of PID gain scheduling, the MT device exhibits magnetization and demagnetization response times of less than 100 ms without overshoot over a wide range of magnetic flux density setpoints. Compared to current-based control, magnetic flux density-based control allows for more accurate and precise magnetic actuation forces by compensating for temperature increases within the needle core due to heat generated by the applied solenoid currents. Near field calibrations validate the ability of the MT device to actuate 4.5 μm-diameter superparamagnetic beads with forces up to 25 nN with maximum relative uncertainties of ±30% for beads positioned between 2.5 and 40 µm from the needle tip.

Elevated expression of osteopontin (OPN), a matricellular, adhesive glycoprotein, is associated with numerous cancer types. We have reported that ultraviolet B (UVB)-induced OPN is a critical driver for cutaneous squamous cell carcinoma (cSCC) development in the photocarcinogenesis model. In human skin cancer, UVB exposure is the major risk factor on the development of nonmelanoma skin cancer. Immunohistochemical analyses indicated that OPN protein is markedly increased in actinic keratoses, cSCC, and differentiated basal cell carcinoma (BCC), but not in solid basal cell epitheliomas when compared with no sun-exposed skin. This suggests OPN may play an important role in the maintenance of the pre-malignant and malignant keratinocytes in cSCC and differentiated BCC. Whether OPN splice variants, shown to have distinct functions, are expressed in normal and nonmelanoma skin cancer is not known. This study assessed whether 1) OPN splice variants differ in normal skin, cSCC, and BCC, and 2) their expression is altered in human adult keratinocytes (HaCaT) treated in vitro with UVB or 1, 25-dihydroxyvitamin D 3 (calcitriol), by real-time qPCR and Western blot analyses. Normal skin expressed minimal OPN-a, OPN-b and OPN-c mRNAs. In contrast, the transcripts of OPN variants were significantly (p<0.001) elevated in cSCC and BCC. In cSCC, each OPN isoform expression was at similar levels. In BCC, OPN-a was significantly (p<0.05) higher than OPN-c. Consistent with normal skin, HaCaT keratinocytes also expressed low levels of OPN-a and OPNb, however, OPN-c was not detected. UVB did not induce OPN expression, whereas calcitriol stimulated OPN-a and OPN-b only. Collectively, these results indicate elevated expression of the three OPN isoforms in nonmelanoma skin cancer compared to normal skin. Additionally, adult keratinocytes expressed only basal and induced level of OPN-a and OPN-b, suggesting that other cell types may be responsible for OPN-c expression.

In this work, we present a new experimental methodology that integrates magnetic tweezers (MT) with substrate deformation tracking microscopy (DTM) and traction force microscopy (TFM). Two types of MT-DTM/TFM experiments are described: force-control mode and displacement-control mode experiments. In model bead-on-gel experiments for each mode, an MT device is used to apply a controlled force or displacement waveform to a fibronectin-coated superparamagnetic bead attached to a fibrillar type I collagen gel containing a layer of covalently attached red-fluorescent microspheres. Serial fast time-lapse differential interference contrast and epifluorescence image acquisition steps are used to capture displacements of the bead and microspheres, respectively, in response to the applied force or displacement. Due to the large number of acquired images and the dynamic nature of the experiment, new quantitative approaches are implemented to adapt TFM for the analysis of the data, including (i) a temporospatial correction algorithm for improved tracking of microsphere displacements, (ii) a method for the objective determination of L2 regularization parameters for computing incremental traction stress solutions, and (iii) an empirical means for identifying time intervals within the data that can be approximated by elastostatic conditions. We also illustrate how force and energy balances in a force-control mode bead-on-gel experiment can be used to estimate the elastic modulus of a collagen substrate. Finally, in a proof-of-concept, bead-on-cell demonstration, measurements of incremental cell–matrix traction stresses are used to observe how a force applied to a focal contact on the apical surface of a keratinocyte is transmitted to the collagen substrate below the cell.

In this work, we present a single-pole magnetic tweezers (MT) device designed for integration with substrate deformation tracking microscopy (DTM) and/or traction force microscopy (TFM) experiments intended to explore extracellular matrix rheology and human epidermal keratinocyte mechanobiology. Assembled from commercially available off-the-shelf electronics hardware and software, the MT device is amenable to replication in the basic biology laboratory. In contrast to conventional solenoid current-controlled MT devices, operation of this instrument is based on real-time feedback control of the magnetic flux density emanating from the blunt end of the needle core using a cascade control scheme and a digital proportional-integral-derivative (PID) controller. Algorithms that compensate for an apparent spatially non-uniform remnant magnetization of the needle core that develops during actuation are implemented into the feedback control scheme. Through optimization of PID gain scheduling, the MT device exhibits magnetization and demagnetization response times of less than 100 ms without overshoot over a wide range of magnetic flux density setpoints. Compared to current-based control, magnetic flux density-based control allows for more accurate and precise magnetic actuation forces by compensating for temperature increases within the needle core due to heat generated by the applied solenoid currents. Near field calibrations validate the ability of the MT device to actuate 4.5 μm-diameter superparamagnetic beads with forces up to 25 nN with maximum relative uncertainties of ±30% for beads positioned between 2.5 and 40 μm from the needle tip.

In this work we demonstrate the integration of magnetic tweezers (MT) with substrate deformation tracking microscopy (DTM) and traction force microscopy (TFM) for the investigation of extracellular matrix rheology and human epidermal keratinocyte mechanobiology in the context of human blistering skin diseases. Two model bead-on-gel experiments are described in which an MT device is used to apply a prescribed force or displacement waveform to a fibronectin-coated superparamagnetic bead attached to a type I collagen gel containing a layer of covalently attached red-fluorescent microspheres. Serial fast time-lapse DIC and epifluorescence image acquisitions are used to capture displacements of the bead and microspheres, respectively, in response to the applied force or displacement. Due to the large number of acquired images and the dynamic behavior of substrate microspheres observed during the experiment, new quantitative methods are developed for the tracking and filtering of microsphere displacement data, the selection of L2 regularization parameters used for TFM analysis, and the identification of time intervals within the overall image set that can be approximated as being subject to elastostatic conditions. Two major proof-of-concept applications are described in which integrated MT-DTM/TFM experiments are used to (i) estimate the elastic properties of a fibrillar type I collagen gel substrate and (ii) demonstrate how a force applied to a focal adhesion contact on the apical surface of a living keratinocyte is directly transmitted to basal cell-matrix anchoring junctions as observed by substrate deformations and incremental traction stresses that develop within the collagen subjacent to the cell.