Time-lapse lens-free imaging of cell migration in diverse physical microenvironments

Abstract

Time-lapse imaging of biological samples is important for understanding complex (patho)physiological processes. A growing number of point-of-care biomedical assays rely on real-time imaging of flowing or migrating cells. However, the cost and complexity of integrating experimental models simulating physiologically relevant microenvironments with bulky imaging systems that offer sufficient spatiotemporal resolution limit the use of time-lapse assays in research and clinical settings. This paper introduces a compact and affordable lens-free imaging (LFI) device based on the principle of coherent in-line, digital holography for time-lapse cell migration assays. The LFI device combines single-cell resolution (1.2 μm) with a large field of view (6.4 × 4.6 mm(2)), thus rendering it ideal for high-throughput applications and removing the need for expensive and bulky programmable motorized stages. The set-up is so compact that it can be housed in a standard cell culture incubator, thereby avoiding custom-built stage top incubators. LFI is thoroughly benchmarked against conventional live-cell phase contrast microscopy for random cell motility on two-dimensional (2D) surfaces and confined migration on 1D-microprinted lines and in microchannels using breast adenocarcinoma cells. The quality of the results obtained by the two imaging systems is comparable, and they reveal that cells migrate more efficiently upon increasing confinement. Interestingly, assays of confined migration more readily distinguish the migratory potential of metastatic MDA-MB-231 cells from non-metastatic MCF7 cells relative to traditional 2D migration assays. Altogether, this single-cell migration study establishes LFI as an elegant and useful tool for live-cell imaging.

Fig. 1. Lens-free imaging (LFI) principle of operation and schematic

(A) Principle of operation of holographic in-line LFI. A collimated light source sent through a pinhole encounters a transparent object. The non-interacting reference wavefront and the object wavefront travel to a sensor (in this case, a CMOS chip), creating an interference pattern that is read by the sensor. The interference pattern is then reconstructed to create an image of the object. (B) Schematic of the LFI platform. Laser light travels through a fiber optic cable to illuminate the sample, here represented by a transparent microfluidic chip. The reference wavefront and object wavefront are detected using a CMOS sensor.

Fig. 2. Phase contrast and LFI imaging platforms generate similar results for random 2D motility assays

Time-lapse images of (A) MDA-MB-231 and (B) MCF7 breast adenocarcinoma cells migrating on collagen type I-coated glass slides and imaged using either phase contrast microscopy (10x, 0.45 NA objective) or the LFI platform. Scale bars represent 50 μm. (C) Mean squared displacements observed for the two cell types with each imaging platform. (D) Cell velocity as a function of time lag. Velocities for time lags of (E) 10 min and (F) 120 min are also shown. (G) Persistence ratio as a function of time lag. The persistence ratio at a time lag of (H) 120 min is also shown. (I) Total diffusivity observed for the two cell types with each imaging platform. (J) Primary persistence time observed for the two cell types with each imaging platform. For all metrics, cell trajectories were tracked for the indicated time periods for up to 2 h. N=90 cells/condition, with 30 cells/experiment were analyzed from 3 independent experiments. Statistical significance between phase contrast and LFI imaging results was determined by an unpaired t test if cells passed the D’Agostino and Pearson omnibus normality test, or by Mann-Whitney test if they did not. Differences between MDA-MB-231 and MCF7 cells were assessed by Kruskal-Wallis test with Dunn’s multiple comparisons post-test. n.s., difference not statistically significant; *, p<0.05; ****, p<0.0001.

Fig. 3. Phase contrast and LFI imaging platforms generate similar results for microcontact printing migration assays

Time-lapse images of (A) MDA-MB-231 and (B) MCF7 breast adenocarcinoma cells migrating on 6 μm-wide collagen type I printed lines and imaged using either phase contrast microscopy (10x, 0.45 NA objective) or the LFI platform. Scale bars represent 50 μm. (C) Mean squared displacements observed for the two cell types with each imaging platform. (D) Cell velocity as a function of time lag. Velocities for time lags of (E) 10 min and (F) 120 min are also shown. (G) Persistence ratio as a function of time lag. The persistence ratio at a time lag of (H) 120 min is also shown. (I) Total diffusivity observed for the two cell types with each imaging platform. (J) Primary persistence time observed for the two cell types with each imaging platform. For all metrics, cell trajectories were tracked for up to 2 h. For MDA-MB-231 cells, N=90 cells/condition, with 30 cells/experiment were analyzed from 3 independent experiments. For MCF7 cells, N=60 cells/condition, with 30 cells/experiment analyzed over 2 independent experiments. Statistical significance between phase contrast and LFI imaging results was analyzed by Mann-Whitney test. Differences between MDA-MB-231 and MCF7 cells were assessed by Kruskal-Wallis test with Dunn’s multiple comparisons post-test. n.s., difference not statistically significant; *, p<0.05; ****, p<0.0001.

Fig. 4. Phase contrast and LFI imaging platforms generate similar results for microchannel migration assays

Time-lapse images of (A) MDA-MB-231 and (B) MCF7 breast adenocarcinoma cells migrating through 6 μm-wide, 10 μm-tall collagen type I-coated PDMS microchannels and imaged using either phase contrast microscopy (10x, 0.45 NA objective) or the LFI platform. (C) Mean squared displacements observed for the two cell types with each imaging platform. (D) Cell velocity as a function of time lag. Velocities for time lags of (E) 10 min and (F) 120 min are also shown. (G) Persistence ratio as a function of time lag. The persistence ratio at a time lag of (H) 120 min is also shown. (I) Total diffusivity observed for the two cell types with each imaging platform. (J) Primary persistence time observed for the two cell types with each imaging platform. For all metrics, cell trajectories were tracked for up to 2 h. For MDA-MB-231 cells, N=90 cells/condition, with 30 cells/experiment were analyzed from 3 independent experiments. For MCF7 cells, N=75 cells/condition, with 25 cells/experiment analyzed over 3 independent experiments. Statistical significance between phase contrast and LFI imaging results was analyzed by Mann-Whitney test. Differences between MDA-MB-231 and MCF7 cells were assessed by Kruskal-Wallis test with Dunn’s multiple comparisons post-test. n.s., difference not statistically significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

Fig. 5. Increasing confinement results in more efficient cell migration

Data generated via LFI imaging across differing levels of physical cell confinement were compared. Persistence ratios were calculated (A) as a function of time lag and (B) at 120 min for both MDA-MB-231 and MCF7 cells in different physical microenvironments. Velocity was determined (C) as a function of time lag and at (D) 10 min and (E) 120 min for both MDA-MB-231 and MCF7 cells in each microenvironment. (F) Mean squared displacement, (G) total diffusivity, and (H) primary persistence time were calculated for MDA-MB-231 and MCF7 cells in each microenvironment. Cells were tracked by LFI for up to 120 min. For MDA-MB-231 cells, N=90 cells/condition, with 30 cells/experiment were analyzed from 3 independent experiments. For MCF7 cells, N=90 cells, with 30 cells/experiment were analyzed from 3 independent experiments from 2D assays; N=60 cells, with 30 cells/experiment were analyzed from 2 independent experiments for printed 1D lines; and N=75 cells, with 25 cells/experiment were analyzed from 3 independent experiments for microchannel results. Comparisons between microenvironments for a given cell type were made with Kruskal-Wallis test with Dunn’s multiple comparisons post-test. n.s., difference not statistically significant; **, p<0.01; ***, p<0.001; ****, p<0.0001.