The cellular environment is ever dynamic in terms of growth, movement, molecular changes, and response to stimuli. Live-cell imaging has been a boon to scientists looking to capture these cellular nuances in real-time. However, live-cell imaging comes with its own set of nuances, which depend on the biological question to be addressed, the types of samples to be analyzed, and the imaging technique used.
How to pick the right microscopy technique
Different types of microscopy and imaging modalities are available for live-cell imaging. However, there are always tradeoffs in terms of cost, speed, resolution, accuracy, and automation, and the decision is often driven by the sample and the application. For instance, widefield fluorescent microscopy is best for imaging large regions of adherent cells or thin tissue sections, but not for studying spatial and temporal resolution. Confocal laser scanning microscopy offers real-time imaging of dynamic cellular changes but is relatively slow and the lasers used can induce phototoxicity in cells. Multiphoton microscopy uses two longer wavelength photons, which helps with deeper penetration of light into the sample, less scattering and less phototoxicity, but is slow too. For applications that need label-free detection, Raman microscopy is a good choice. Similarly there are other types of fluorescence microscopy that can be used for live-cell imaging such as light sheet fluorescence microscopy, fluorescence resonance energy transfer (FRET), fluorescence recovery after photobleaching (FRAP), and total internal reflection fluorescence (TRIF) microscopy, and they each have their own advantages and limitations.
Meike Tröger, who is a doctoral student in the Institute of Pharmacy at the Free University of Berlin, says that clearly defining the purpose of live-cell imaging is very important. “With the CytoSMART Omni we are monitoring cell growth in our assays, to transfect cells at optimal confluency levels and to evaluate transfection efficiency. We also use the Omni for toxicity studies as it allows the monitoring of up to 96 wells in parallel.” Tröger is also beta-testing the CytoSMART Lux3 FL, which has an integrated fluorescence microscope that enables parallel monitoring in brightfield and in fluorescence mode. “I currently use the Lux3 FL to evaluate transfection efficiency by checking the GFP-related fluorescence in the cells. I am also planning to use it for uptake studies by formulating fluorescence-labelled particles.”
The innovations in live-cell imaging have largely been driven by the need for getting real-time data. Humane Genomics is developing synthetic viruses as personalized cancer therapies and they use the Sartorius’ Incucyte system for live-cell imaging and analysis to inform computer-aided design of viral genomes, confirm assembly and bootup of functional virus particles, and to evaluate the safety and efficacy of therapies. “With the Incucyte system we get immediate, real-time virus quantification and a deeper understanding of viral infection and replication,” explains Chad Moles, CSO and Co-Founder. “We can multiplex measurements and evaluate object counts, area, fluorescence intensity of our viral therapies in real-time.”
Bringing the microscope to the cell
Recent innovations in microscopy are focused on bringing imaging to the incubator by miniaturization of hardware and developing systems that fit comfortably in any standard size incubator. “This has enabled analysis of cellular changes in real-time, reduced data variation, significantly increased throughput and research productivity,” says Moles.
Hsiang-Cheng Chi, Ph.D., a researcher in the Gene Regulation Laboratory at Chang-Gung University, uses Blue-Ray Biotech’s live-cell imaging system to study changes in gastric cancer cells. “We use the live-cell imaging system to explore the effect of DOCK6 expression on cell crawling ability and to record the crawling direction and speed. The system we use is small and easy to install. It can be easily placed in a corner of the CO2 incubator, does not require special materials, and there is no complicated operation interface.”
Image: Sara Imboden, a visiting research graduate works on imaging mesenchymal stromal cell heterogeneities with the Etaluma microscope. Image courtesy of Dr. Neil Lin Lab at UCLA.
Neil Lin, Ph.D., Assistant Professor in the Departments of Mechanical and Aerospace Engineering, and Bioengineering at the University of California, Los Angeles, is developing 3D engineered tissues that recapitulate the functionality of human organs. His lab uses the LS720 automated microscope from Etaluma for examining epithelial cell behavior over time after treatment with various perturbations. With the microscope placed in the incubator, setting up multiplexed time-lapse experiments has become much easier. “The automated imaging system can generate 96 or 384 multi-channel high-quality and high-magnification time-lapse movies. The ability to do many parallel time-lapse experiments in a multi-well plate means one can vary parameters, like seeding density,” says Lin.
Checklist for Live-Cell Imaging
- Did you pick the right microscopy technique to answer the biological question?
- Can the microscopy technique work with the different sample types that you are going to study?
- Do you know what parameters are the most important for the study—speed v/s resolution etc.?
- What enhancements can you make to the instrumentation (microscopy system, camera, filters, illumination devices) to improve sensitivity, speed, accuracy?
- Can you keep the cells alive and healthy during the course of the experiment?
- What types of dyes and detectors do you need to maximize data quality?
- Are the experimental conditions and equipment chosen to provide optimal imaging?
- Do you have the right controls in place to make sure everything is working well during the experiment?
- Have the lab personnel been trained to perform the experiment?
- Do you have the data visualization and analysis tools to get the most from your data?
- Do you have the capabilities to store, secure, access and share the large imaging data files?
“Our lab plates cells at varying densities and then introduces reagents intracellularly through microinjection or via lentiviral vectors to generate stable reporter expressing cell lines,” says Alexander Zambon, Ph.D., Associate Professor of Biopharmaceutical Sciences at the Keck Graduate Institute, who studies cell cycle progression in immortalized human cancer cell lines. “By integrating a microinjection capability onto the Etaluma LS720 microscope, we are able to introduce virtually any agent (e.g. mRNA, CRISPR constructs, DNA, dyes, drugs) while monitoring phenotypic outcomes without moving the field of view, because the cells stay in the incubator during imaging.”
How to keep cells alive and healthy
Cells need the right physiological conditions to grow and function, and it’s critical to maintain the right temperature, oxygen concentration, pH, viscosity, and other parameters throughout the duration of the live-cell imaging experiment. Specially designed culture chambers are available to ensure the correct and consistent levels of carbon dioxide, oxygen, and dissolved gases. Imaging chambers have been designed to allow continuous flow of fluids for experiments that need frequent changes of culture media. There are also custom-designed viewing chambers that provide optimal optics for checking and imaging cells regularly to make sure they don’t show any signs of abnormal morphology such as blebbing, swelling, detachment of substrate, etc. Cells are also prone to photobleaching. Traces of ultraviolet or infrared light, or high-intensity lasers can damage cells. Fluorescent dyes used for imaging can react with oxygen to produce free radicals that can also lead to cellular damage. Shuttering the light, switching off the light when not being used, removing unwanted wavelengths of light, using optimized emission filters, and reducing oxygen levels can reduce photobleaching.
Matteo Santin, Ph.D., Professor of Tissue Regeneration, School of Pharmacy and Biomolecular Sciences, at the University of Brighton, uses live-cell imaging to study the formation of organoids using specific tissue cells to mimic vascularized tissue-like structures. He uses PhenoDrive, a class of cell substrates that mimic the extracellular matrix of tissues in 2D cultures and enables the live imaging of cells driven into the formation of organoids and spheroids. The substrate-driven formation of these structures is monitored using the CytoSMART system. “A desirable feature would be to integrate technologies enabling the real-time monitoring of fluorescent markers,” explains Santin. “Live-cell imaging could also be coupled with equipment generating mechanical stresses in cells.”
How to get the best imaging data
According to Tröger, the biggest advantage of live-cell imaging in comparison to end-point measurements, is the insight in the time-development of the parameter being studied. “The real-time analysis of the data and the ability to check the current status in the cloud (all data is directly uploaded and analyzed) is really useful,” adds Tröger. “I also like to get a notification when confluency reaches a specific value. CytoSMART is currently working on the fluorescence quantification algorithm that will be a nice add-on.”
Image: CytoSMART Multi Lux: Combining four compact Lux2 devices into one system increases throughput and functionality. Image courtesy of CytoSmart.
“With enormous datasets, it’s important to have well-designed data storage and analytical tools and there are plenty of open-source tools such as CellProfiler that allow users to develop custom pipelines to analyze complex datasets,” says Ross Marklein, Ph.D., Assistant Professor in the School of Chemical, Materials, and Biomedical Engineering at the University of Georgia. Marklein’s lab uses live-cell imaging to characterize the heterogeneity of mesenchymal stromal cells (MSCs), which are proving to be a promising cellular therapy. However, there are challenges with manufacturing MSCs with consistent functional properties and the lab is currently developing imaging-based approaches to assess MSC morphology as a predictor of quality. “Machine learning to analyze cell morphology and tracking dynamic cell behavior seems to be exploding and technologies that lower the barrier for entry to non-computational biologists will significantly broaden applications of high content imaging (HCI),” says Marklein.
Advances in Live-Cell Imaging
- Diverse equipment integration
“Some areas of improvement could be additional filters, as well as the ability to image larger vessels (e.g. T175s) or integrate with external bioreactors and microfluidic devices that have integrated flow circuits,” says Marklein. The imaging systems need to be robust and have the ability to incorporate other devices.
- Imaging complex cellular models
“Traditional 2D models and cellular monolayers enable rapid testing and learning, but these are simplistic and lack the physiologic complexity of tumors,” says Moles. “I foresee technological advances in imaging techniques and data analysis for 3D cell culture models to recapture the complex environment and gain deeper insight into how our candidate virus therapies will perform in vivo.”
- Wireless monitoring and real-time video capabilities
“Currently the imaging system needs to be connected and controlled by a cable to the computer outside the CO2 incubator,” says Chi. “If the manufacturer can develop a wireless real-time monitoring model, it will be easier for installation and more flexible for operation.”
“New live-cell probes based on fluorescent protein biosensors are critical,” says Zambon. “Injectable probes, whether they are mRNA for GFP or fluorescence ubiquitination cell cycle indicator (FUCCI) constructs or CRISPR reagents, are very powerful in continuously monitored live-cell imaging experiment.”
- User-friendly data visualization and analysis
“Blue-Ray Biotech’s design allows up to four units of the live-cell imager to be controlled by one computer,” says Chi. “The software provided by the manufacturer can convert the continuously shot images into a video file without any additional image processing software needed. Moreover, all the live-cell views can be displayed on the screen concurrently.”
“Throughput, ease-of-use, and customization are key aspects and we have not felt limited by the technology, only by our own time and personal resources,” says Marklein. “The BioTek Cytation system seems to occupy that space before ‘diminishing returns’ in terms of price and capabilities.”