Resurrecting Microscopes for Live Cell Imaging
by Rodrigo Hernández Vera, Emil Schwan & Johan Kreuger Labtimes 03/2017
The costs of commercial live cell imaging systems easily exceed the budget of ordinarily funded laboratories. However, with a few 3D-printed parts, a smartphone and off-the-shelf electronics, you may reanimate your old microscope for live cell imaging and time-lapse experiments.
Johan Kreuger and his team at Uppsala University constructed a DIY time-lapse imaging and incubation module for inverted light microscopes. Photo: Uppsala University
Are you studying the cell migration and behaviour of unlabelled cells using light microscopy and live imaging, but you are tired of waiting to get access to the often fully booked microscopes in your department? Maybe you dream of your own setup, always available for you and your cells. If this is the case, then we have good news! Chances are that there is an old inverted microscope somewhere in your department. Possibly buried in a storage room, or left to decay in the students’ lab. This old, simple microscope with great objectives, can now be dusted off and given a second chance as part of an affordable system for time-lapse imaging of cells grown in vitro.
Many assays commonly used to study cell migration, such as the in vitro scratch wound assay, do not require fluorescent labelling of cells. It is also sometimes preferred to work with cells that are unlabelled and thus unperturbed. But, in order to study how cells move and behave dynamically over time, you do need to be able to perform time-lapse imaging, often for several hours, or even for days.
In most cases this means that, in addition to a microscope, you must have a cell incubator with temperature and humidity control to keep the cells at 37 °C and to prevent the cultures from drying out. You also need a camera connected to your microscope that is controlled by a computer equipped with software for automatic time-lapse imaging.
Overall, the equipment required for time-lapse imaging can be very expensive. And this is arguably a bottleneck, restricting the use of time-lapse imaging to rather well-funded departments and organizations. We have addressed this problem and developed an Affordable Time-Lapse Imaging and incubation System (ATLIS) based on 3D-printed parts, a smartphone, and off-the-shelf electronics (PLoS ONE 11(12): e0167583). In the article describing ATLIS, we demonstrate how simple, inverted light microscopes can be transformed into high-quality live imaging systems for less than 300 euro. The ATLIS was designed to be modular, and the different modules are briefly described below.
For proof-of-concept studies, a Nikon TMS microscope and a Leitz Laborvert microscope were equipped with the different ATLIS modules. The imaging unit was composed of a 3D-printed smartphone holder, a regular smartphone, and a motorized shutter. The phone holder (based on an original design deposited at Thingiverse, www.thingiverse.com/thing:431168) was compatible with most types of smartphones. It was attached to the eyepiece tube of the microscope where subsequently the phone camera was aligned with the ocular. Pictures of cells were thus taken directly through the microscope ocular.
The motorised shutter was built from a Bluetooth-controlled servomotor equipped with a plastic shutter disc, to protect the cells from harmful light in between exposures. The shutter was attached to the microscope light source using a 3D-printed custom-designed holder.
Next, the microscope was equipped with a 3D-printed onstage incubator, fitted with a transparent plastic cover to allow for the passage of light. The incubator was designed to be compatible with a range of commonly used cell culture dishes and plates, and connected to a heating unit that was built using a 3D-printed case, a standard heating element, and a standard 60 mm fan.
A 3D-printed smartphone holder fixes the smartphone to the eyepiece of the inverted microscope used for live cell imaging. Photo: Uppsala University
The incubator was equipped with a temperature sensor for continuous monitoring and the heating unit with a temperature control failsafe to disable heating if required. The heating as such was controlled using an Arduino Nano-based on the commonly available Atmega328 microcontroller connected to a Bluetooth communication module, a transistor, and a voltage regulator. The microcontroller monitored and regulated the temperature, controlling the power to the heating element.
Of note, the heating unit is essentially a glorified hair dryer, and this turned out to be problematic when analysing cells grown in evaporation-sensitive microfluidic systems, which (surprise!) quickly dried out and died when exposed to hot air. To solve this problem, a simple humidifying module with a water reservoir holding an aquarium air stone was constructed to create a humid atmosphere within the incubator.
The humidifying module was added in between the heating unit and the onstage incubator, and the system additionally operated in a closed-loop configuration to recirculate the warm and humid air: this sufficiently solved the problem with evaporation.
What about the user interface then, and how were time-lapse imaging and other experimental parameters controlled and data collected? To address these issues, we used MIT’s App Inventor to create the ATLIS control app that was installed on the Android smartphone.
The app was used to set the imaging interval and to control both the shutter (via Bluetooth) and the smartphone camera as well as to set and monitor the temperature via the microcontroller (also via Bluetooth). It was constructed to display temperature readings and to show the last picture taken on the smartphone display. Images and temperature logs could also be accessed from an external computer to remotely oversee the experiment’s progression.
Experiments were conducted with both cancer cells and embryonic kidney cells in a variety of assays. The images and time-lapse videos captured were of surprisingly high quality.
So, if you can get hold of an old inverted microscope (from the last decades of the previous century or so) you can, with a little bit of 3D printing, a regular smartphone, and some off-the-shelf electronics (that you can easily order online) put together a simple, but very functional, setup for time-lapse imaging of cells in vitro. Of course, the system described here is far from being as advanced as commercially available systems, but we believe that the ATLIS concept has potential to lower barriers for less well-funded institutions that would like to carry out live imaging of unlabelled cells. Or to enable the inquisitive home brew science geek, making great strides for the bettering of humanity in his/her basement. The simplicity of the system makes it very easy to modify according to the requirements of the user.
Perhaps you may only need some of the ATLIS modules, or you could use the system as inspiration for creating exactly what you need for your imaging experiments. One exciting possibility could also be to combine the ATLIS with emerging DIY solutions for fluorescence imaging, thereby visualising and identifying cells labelled with fluorescent dyes or proteins.
(Johan Kreuger is senior lecturer and group leader at the Department of Medical Cell Biology at Uppsala University)
Last Changed: 26.06.2017