by Jake Jones and Britta Frenzel, Associate Product Managers, Evident Scientific
When it comes to upgrading your live-cell microscope, whether you are simply adding on features or replacing it entirely, the options can seem overwhelming. What kind of optics do you need? What kind of incubation system is best for your cells? The beauty of having countless options is that you can build a microscope to fit your specific research needs. Live-cell imaging requires consideration of many important factors for experimental success. This article reviews the most common requirements, including incubation, imaging modalities, sample types, equipment flexibility, and facility readiness.
Experimental success
Live cells require careful maintenance of their environment to grow and thrive, and various microscope-based incubation systems are available to meet evolving research needs. Full enclosure-type incubation systems enable time-lapse observations over a period of several days. These types of incubators enable precise control of CO2 or N2 gas concentrations, temperature, and humidity to maintain constant environmental conditions for cells in dishes or well plates. As materials comprising the microscope frame go through cycles of heat and cooling, the frame can expand or contract, resulting in samples drifting out of focus over time.
Full enclosure incubators can help minimize gradual thermal drift in microscope frame assemblies by heating the whole frame to a constant and steady temperature before experimental imaging begins. Certain full enclosure units also offer sterility controls and air filtration, similar to a cell culture hood, which protects sensitive samples that risk contamination during multi-day time-lapse experiments.
Shorter time-lapse experiments can be completed with stage-top microscope incubation systems that can be fitted to the stage and easily removed when not in use. Stage-top incubators offer similar control of gas composition and temperature to their fully enclosed counterparts but keep humidity control contained inside the stage-top insert. This can help protect the optics and electronics of a microscope by limiting high-humidity exposure to the inside of the stage-top incubator. Many stage-top and enclosure systems can also be used in conjunction with each other to maximize the benefits of each, providing a stable, long-term imaging environment for living cell cultures.
Another important consideration is the type of imaging you need to perform. Are you just looking to see that your cells are thriving? Are you looking for fluorescent markers? Will you need to take and process images? Current solutions vary in complexity. Simple cell culture microscopes give you the ability to observe your cells and capture basic images.
For more advanced experiments, you may want to consider an all-in-one or open-architecture system, both of which provide a wider range of options for your imaging applications. These more complex systems can perform multiple imaging modalities, including brightfield, fluorescence, differential interference contrast (DIC), and phase contrast. They can also be fitted with an incubation chamber for long-term live-cell imaging.
Since most live-cell imaging experiments require long time-lapses to capture dynamic changes in cell cultures, preventing thermal or vibrational drift of the microscope is critical to preserving image quality. Important considerations for counteracting drift include the rigidity of the frame, anti-vibration control, and the addition of a focus drive.
Certain microscope frames offer increased stability due to having a single material between the base and the stage, which minimizes gaps between materials that are more susceptible to thermal expansion. These frames are preferred when multi-day imaging is a priority. Vibration of the microscope can be minimized by placing the imaging system on an air table or anti-vibration plate. These will minimize vibration from the electronics and motors operating in the microscope system, as well as external sources of vibration from the surrounding lab space.
Alternatively, some box-style microscope frames contain built-in anti-vibration elements, an appealing option when laboratory space is limited. While frame stability can keep thermal or vibrational drift minimal over a span of hours, researchers looking to capture multi-day time-lapse images of live cells should consider drift compensation mechanisms.
While the exact method of preventing drift can differ by manufacturer, these devices usually function using an internal near-infrared light source to automatically maintain focus on the coverslips, plates, or culture dishes that contain samples. Drift compensation devices work to counter all kinds of drift during live-cell time-lapse imaging and should be included whenever resources allow them.
Your system’s frame and focus drive should be robust enough to reduce the impact of external factors, such as vibration, on the microscope. This will help maintain the desired focus position on the Z-axis to facilitate reliable time-lapse imaging. Imaging software is now capable of autofocus and object tracking, enabling you to easily keep your cells in focus over longer periods of time and track cell movement. Both features are highly advantageous for research where it is critical to track the survival, migration, and differentiation of the cells under investigation. Some systems go beyond basic cell tracking and incorporate cell segmentation software to classify specific cell types.
Many labs deal with the issue of limited space, so finding a way to make the most of your available space will ensure that the quality of your research goes unaffected. This includes finding an imaging system that is, literally, the right fit for your lab. Benchtop systems, such as an all-in-one microscope, are designed to take up as little space as possible while providing a large array of features. All-in-one systems offer the convenience of multiple imaging modalities with a simple workflow, but are limited as the scopes are self-contained and cannot typically be built on further.
Alternatively, an open-frame architecture microscope can take up as much or as little space as you allow it to. The core microscope frame has a relatively small footprint, but as you add on more features, such as lasers or incubation chambers, that footprint increases. Consider how much lab space you have currently and will need in the future when selecting your imaging system.
There are many factors to consider when preparing for your live-cell experiment. Whether you are upgrading your existing system or starting from scratch, focusing on your research goals will help you build the best system for your lab. With factors ranging from room readiness to environmental conditions and cell tracking, making sure your system can both maintain the life of and image your cells is key to a successful research workflow.
About the authors:
Jake Jones, Ph.D., is an associate product manager for IXplore, TIRF, FRAP, luminescence, and multiphoton microscopy at Evident Scientific. As a member of the product management team, Jake works to identify the needs of scientist and researchers and helps provide imaging solutions that match the growing needs of the imaging community. He holds a doctorate in biomedical engineering from the University of Arkansas where he pursued projects involving biomedical applications of multiphoton microscopy, confocal microscopy, in vivo imaging techniques, and neural network-based image processing. Britta Frenzel is an associate product manager for the APX100, BX51WI, and MVX10 systems at Evident Scientific. As a member of the product management team, Britta works to identify the needs of researchers and helps provide imaging solutions that match the growing needs of the imaging community. She has a strong commercial and technical background with a Master's in biomedical engineering from Clemson University and brings to the table several years of experience in the medical device industry.