Fig 1.
ATLIS: an affordable system for time-lapse imaging and incubation of cells.
(a) The basic version of ATLIS was assembled by the combination of four modules. The imaging module, composed of a smartphone, a 3D-printed holder, and a motorized shutter, was designed to capture images of cells at a fixed interval minimizing their exposure to light. The heating unit was created to warm up the air introduced into a 3D-printed onstage incubator where cells are kept. Finally, the control unit was programmed to maintain a constant temperature inside the incubator by adjusting the power supplied to the heating unit. (b) A photograph of the ATLIS mounted on a Nikon TMS microscope fitted with a Samsung Galaxy S I9000 smartphone. (1) Nikon TMS microscope; (2) Samsung Galaxy S I9000 smartphone; (3) smartphone holder; (4) onstage incubator; (5) heating unit; (6) power supply; (7) control unit; (8) shutter; (9) temperature sensor; (10) light source.
Fig 2.
(a) The smartphone holder was designed to allow for stable fixation of most currently existing smartphones to microscopes having oculars of up to 42 mm in diameter. Sliding frames (that could be fixed by fastening the nut in each frame) in the holder facilitated the alignment of the smartphone’s camera to the ocular. (b) The shutter was built from a servomotor, a plastic shutter disc, and a 3D-printed connector. The servomotor was used to open or close the shutter in response to commands from the control unit. (c) Examples of two different microscopes fitted with the smartphone holder and an iPhone 5. (d) Images of MDA-MB-231 breast cancer cells captured with an iPhone 5 and the smartphone holder using the 10X- and 20X-objectives of each indicated microscope.
Fig 3.
Heating unit and incubation module.
(a) The heating unit was constructed using a fan, a custom-built heating core, and a 3D-printed case designed to connect to the incubator using a heat-resistant hose. As a safety measure, a temperature sensor was placed close to the heating core to monitor its temperature so that the heating could be turned off when the temperature exceeded a preset safety limit. (b) Photograph of the heating unit where the fan has been removed to show the position of the heating element (1), aluminum plates (2), the anchoring wires (3), and the limiting temperature sensor (4). (c) Exploded view of the onstage incubator. The 3D-printed incubator was built from three parts: a lid (yellow), a main body (gray), and a bottom plate (blue). Four different bottom plates were designed to fit the most common types of cell culture ware. (d) Main dimensions (in mm) of the incubator lid and main body as well as for the different bottom plates designed to fit a multi-well plate, a 60 mm Petri dish, a 35 mm Petri dish or a microscope slide, respectively.
Fig 4.
The control unit and the ATLIS Control app.
(a) Photograph of the ATLIS control unit. (1) The power outlets for the shutter’s servomotor (A), the temperature sensors (B), the fan (C), and the heating element (D) are indicated. (2) Power supply to the control unit. (3) Arduino Nano 3.0 ATmega328 microcontroller. (4) Bluetooth communication module. (5) Power transistor for heating element control. (6) Voltage regulator. (b) Overview of the temperature control loop. The temperature inside the incubator was continuously monitored by a sensor and compared to the target temperature in order to adjust the power supplied to the heating element. (c) Temperature curves for three independent experiments showing the temperature inside the incubator during a 16 h period. Inset (gray box): magnification of the temperature behavior during the first hour of the three experiments. The time to reach 37.0°C, the time to temperature stability, as well as the maximum air temperature reached have been indicated for each experiment. (d) Screenshot of the ATLIS Control app designed to allow the user to set the main parameters for each experiment (e.g. target temperature and time interval between images) and to display temperature reads and the last picture taken.
Fig 5.
Time-lapse imaging using the ATLIS.
(a) HEK cells grown in a 6-well plate were monitored using time-lapse imaging for 12 h. Scale bars: white 100 μm; black 50 μm. (b) Scratch-wound assay performed with HEK cells grown in a 6-well plate. Yellow lines delimit the original wound area while white lines show the collective cell migration front at each time point. Scale bars represent 100 μm. For both experiments the ATLIS was mounted on a Nikon TMS microscope using the 20X objective together with 10X magnification in the eyepiece, and images were captured using a Samsung Galaxy S I9000.
Fig 6.
ATLIS setup with air humidification.
(a) Without humidification the ATLIS reached and maintained the target temperature (37.0°C) in a one-pass (blue line) or a recirculating-loop configuration (yellow line). When humidification was added, the system was incapable of reaching the desired temperature in the one-pass configuration (red line). However, in a recirculating-loop configuration with humidification (green line) the system reached and maintained the target temperature. (b) Without humidification, the relative humidity inside the incubator stabilized around 10% regardless of system configuration. The humidifying module increased humidity up to 35% in the one-pass configuration and up to 70% when the ATLIS was operated in a recirculating-loop configuration. (c) Photograph of the ATLIS mounted on a Nikon TMS microscope in the recirculating-loop configuration with the humidifying module. (d) Photograph showing all the components of the humidifying module. (e) The temperature in the liquid inside a vessel placed in the incubator over time in three independent experiments using a recirculating loop together with the humidifying module. (f) Analysis of the temperature at different positions in the incubator in a recirculating-loop configuration with additional humidity. (g) Simulation of the airflow inside the incubator unit. Black dashed lines mark the edges of the multi-well plate; dashed circles indicate the positions of the temperature sensor.
Fig 7.
Imaging of HEK cells grown in microfluidic channels.
HEK cells were imaged for 4 h with a Samsung Galaxy S I9000 using the ATLIS with the humidifying module (recirculating-loop configuration) mounted on a Nikon TMS microscope with 10X magnification in the eyepiece and using a 20X objective. No signs of evaporation were detected in the microfluidic system and the cells moved and proliferated normally. White boxes delineate magnified areas shown in the bottom row. A proliferating cell can bee seen in the area marked by a yellow box. The lower channel was 250 μm across, while the narrower channels at the top were 125 μm wide. All channels were 100 μm in height. Scale bars: white 100 μm; black 20 μm.