Low-cost motility tracking system (LOCOMOTIS) for time-lapse microscopy applications and cell visualisation

Abstract

Direct visualisation of cells for the purpose of studying their motility has typically required expensive microscopy equipment. However, recent advances in digital sensors mean that it is now possible to image cells for a fraction of the price of a standard microscope. Along with low-cost imaging there has also been a large increase in the availability of high quality, open-source analysis programs. In this study we describe the development and performance of an expandable cell motility system employing inexpensive, commercially available digital USB microscopes to image various cell types using time-lapse and perform tracking assays in proof-of-concept experiments. With this system we were able to measure and record three separate assays simultaneously on one personal computer using identical microscopes, and obtained tracking results comparable in quality to those from other studies that used standard, more expensive, equipment. The microscopes used in our system were capable of a maximum magnification of 413.6×. Although resolution was lower than that of a standard inverted microscope we found this difference to be indistinguishable at the magnification chosen for cell tracking experiments (206.8×). In preliminary cell culture experiments using our system, velocities (mean µm/min ± SE) of 0.81 ± 0.01 (Biomphalaria glabrata hemocytes on uncoated plates), 1.17 ± 0.004 (MDA-MB-231 breast cancer cells), 1.24 ± 0.006 (SC5 mouse Sertoli cells) and 2.21 ± 0.01 (B. glabrata hemocytes on Poly-L-Lysine coated plates), were measured and are consistent with previous reports. We believe that this system, coupled with open-source analysis software, demonstrates that higher throughput time-lapse imaging of cells for the purpose of studying motility can be an affordable option for all researchers.

Figure 1. Images of the microscope system.

(a) Schematic diagram of microscope system; (b) CAD model; (c) Photograph of finished system.

Figure 2. Autokams software interface.

Three separate scratch assays of MDA-MB-231 cells displayed simultaneously on Autokams.

Figure 3. Analysis process in ImageJ.

(a) Original image; (b) 8-bit black and white; (c) Background subtraction; (d) Thresholded; (e) MTrack2 cell trajectories; (f) Chemotaxis plot.

Figure 4. Incubator temperature stability over time.

Incubator temperature readings (°C) taken every minute for 20 hours.

Figure 5. Calibration images.

(a) 1 mm graticule with 10 µm subdivisions at 206.8× magnification; (b) 1 µm latex beads at 206.8× magnification; (c) 15 mm ruler at 15.7× magnification with 1 mm subdivisions.

Figure 6. Comparison of MDA-MB-231 cell images between our system and a conventional optical microscope.

(a) 310× image taken at 640×480 with a conventional inverted microscope; (b) 206.8× image taken at 640×480 with our system and enlarged post acquisition by 149% to match the size; (c) 620× image taken at 1280×960 with a conventional inverted microscope, arrows show intra-cellular detail; (d) 1280×960 image taken with our system at full magnification (413.6×) and enlarged post-aquisition by 149% to match the size.

Figure 7. Comparison of digital microscope pixel resolutions between 640×480 and 1280×960.

(a) 640×480 image at full magnification; (b) 640×480 image enlarged post-acquisition to 800%; (c) 1280×960 image at full magnification reduced in size by 50%; (d)1280×960 image enlarged post-acquisition to 400%.

Figure 8. Autokams interface showing the result of illumination from the in-built LEDs alone.

Figure 9. Mean velocity (± SE) in µm per hour of the 4 cell types.

Shared letters indicate no significant difference between cell types according to K-W test pairwise comparisons.

Figure 10. Autokams interface showing individual scratch assays simultaneously recorded on each camera.

(a) cells at 0 hr; (b) cells after 10 hrs; Arrows show the scratch channels.

Figure 11. Ability of the microscope system to image distinct samples in different manners simultaneously.

(a) Cyst, nauplii and adult Artemia at 15.7× magnification; (b) Cyst and nauplii at 206.8×, adult at 15.7×; (c) All three stages at 206.8× magnification.