WHITE PAPER

Expert Q&A: Live-Cell Imaging in Drug Discovery

Introduction

The future of drug discovery requires automated tools that can enhance the understanding of cells at a dynamic, microscopic level. Live-cell imaging provides these advantages, applicable to small- and large-molecule research facilities, academic institutions, CROs, and more.

In this interview, Dr. Jonas Schwirz, a PerkinElmer Field Application Scientist specializing in Cellular Imaging, describes the benefits of live-cell imaging tools in drug discovery. Dr. Schwirz also discusses the benefits of automated tools, tips during use, and features to consider when purchasing.

PerkinElmer: Dr. Schwirz, can you explain some of the advantages and disadvantages of live-cell imaging for drug discovery over traditional types of imaging? What types of imaging are now possible with live-cell systems?

Dr. Schwirz: Live-cell assays provide greater physiological relevance than traditional fixed end-point assays, so they support strategies to reduce costly late-stage attrition rates in drug discovery. Understanding dynamic cellular events, such as proliferation, migration, signaling, apoptosis, cytotoxicity, and morphology, is key for gaining a deeper understanding of disease mechanisms and responses to treatments.

Unlike traditional fixed-endpoint cell assays that give a point-in-time snapshot of cellular responses, live-cell imaging allows scientists to determine when and how fast something happened. These insights help scientists better understand cellular responses.

A major challenge of long-term live-cell imaging is keeping cells alive and functional as normally as possible for the duration of the experiment. Cells need to be incubated under defined environmental conditions of temperature, CO2 concentration, and humidity with minimal disruption. Typically, scientists can overcome these challenges by careful selection of live-cell imaging systems.

Another particular challenge in drug discovery is that scientists are usually not looking at a single treatment, but at multiple treatments at a time. Imaging several hundred samples at once in certain time intervals requires a fast and precise imaging system. In drug discovery, and in particular during time course experiments, scientists create large data sets, often consisting of hundreds of thousands of individual images. Live-cell imaging systems need powerful, flexible and easy-to-use software solutions to help scientists draw a conclusion from these large amounts of data.

Techniques such as brightfield, phase contrast, digital phase contrast, differential interference contrast (DIC), or holotomographic imaging help minimize light exposure to reduce phototoxicity. A key feature of these techniques is that they work without additional labeling. This means the use of potentially toxic stains and the exposure to high doses of excitation light are both avoided, as is usually used during fluorescent microscopy.

However, for many applications fluorescent microscopy is still the method of choice due to the high contrast and unrivaled multiplexing options. Technologies like spinning disc confocal microscopy reduce phototoxic stress on the cells, provide excellent image quality, and remove background.

PerkinElmer: Can you give some examples of which drug discovery applications benefit most from live-cell imaging? How has automation impacted these live-cell imaging systems and applications?

Dr. Schwirz: There are many applications for live-cell imaging. During the assay development stage, live-cell imaging is often used to optimize the timing of an endpoint assay, which is then run in a cost-effective, high-throughput screen. However, there are alternative applications where the user needs to look at living cells. Cardiotoxicity and mitochondrial assays are just two prominent examples of studies that are often performed using living cells. Other applications are patient-derived primary cells such as neurons or cancer cells. These studies aim to understand neurodegenerative diseases, find new cancer treatments, or optimize current cancer treatments.

Integrating screening microscopes into fully automated environments that include robotic arms, incubators, and pipettors has opened the field to a much higher throughput and complexity of live-cell imaging experiments. For example, for 3D applications, you can now automate the whole workflow from spheroid growth, drug treatment, and labeling up to image acquisition and analysis. This provides the efficiency and productivity many drug discovery labs need.

PerkinElmer: What are some of the most important or recommended features that users should look for when investing in a live-cell imaging system?

Dr. Schwirz: When selecting a live-cell imaging system, it is important to consider how you will keep the cells healthy during your experiments. Look for systems that minimize photobleaching and phototoxicity. Choose a system that keeps unnecessary excitation to a minimum, while capturing the maximum of the emitted photons. From a technical perspective, this means you need lenses that capture a maximum of light emanating from the sample.

Water immersion lenses are usually optimal for this purpose, since they allow a high numerical and match the refractive index of the lens immersion medium to the aqueous environment of the cells. This also minimizes image deteriorations, which increase with deeper focus onto the sample and therefore are of particular importance when imaging thick samples like organoids.

Compared to endpoint assays, the number of cells and hence data points that can be imaged in a given time is limited during live-cell experiments. If you are imaging several different samples at a time, then you need a fast and accurate stage to always return to your exact cells of interest. The imaging system should also contain a fast and precise autofocus.

The specific requirements for the system can be quite different, depending on the study. For fast kinetic studies like calcium imaging or cardiomyocyte beating, a fast and sensitive camera is necessary to capture enough photons per timepoint at high speeds.

An automated pipettor also may be required to dispense reagents so that the full cell response can be captured. In contrast, when imaging for several hours or days, the environmental control needs to be very accurate and the imaging needs to be gentle to minimize photobleaching and phototoxicity caused by illumination of the cells.

Finally, ease-of-use of the instrument and software is paramount, yet often overlooked. The instrument should be trouble-free and simple to use without the potential of damaging parts or selecting the wrong setup. Ensuring ease-of-use of the hardware through decent software development enables scientists to focus on getting high quality images and data rather than troubleshooting the system.

PerkinElmer: What types of live-cell imaging are best for 3D cultures?

Dr. Schwirz: 3D cell models pose special requirements to the imaging system. You usually need a confocal or light-sheet microscope to image these samples. Light-sheet microscopy is hard to do in a higher throughput such as a high-content screen. Due to the superior speed, spinning disc confocal systems are the method of choice for screening campaigns of 3D cultures. They are also gentler on the cells.

The best confocal systems, especially for 3D cultures, utilize microlens enhanced spinning discs. These systems use the excitations light more efficiently and are hence much faster. They also provide much better confocal performance, especially for thick samples typical of 3D cultures, compared to traditional spinning disc design.

PerkinElmer: Can you share any practical tips for live-cell imaging and troubleshooting that might be useful for discovery scientists?

Dr. Schwirz: Time dedicated to assay development is usually time well spent. Different cell lines can react to the process of imaging in different ways, so it is important to spend ample time on assay development to learn about the cell lines of interest: how they react, what is the optimum time interval to answer the scientific question, and so on. Start with small setups to optimize the experimental conditions before starting the “real” treatments. It is important to make sure that perturbations seen during the experiment are caused by the treatment and not the imaging.

Scientists need to include all the necessary controls, including a sample of cells that are not imaged. After conclusion of the experiment, it is good practice to keep the imaged cells in the incubator for another couple of days to check if they develop differently from the cells that have not been imaged.

A common pitfall is to start with non-optimal cells. Live-cell imaging usually involves quite a lot of cell treatment: harvesting, seeding, and often staining. Sometimes, the cells react badly. Therefore, always monitor the cells before harvesting and directly before imaging to ensure they are still healthy. If the cells don’t look suitable at any point, then it is usually a good idea not to proceed with the experiment.

PerkinElmer: Finally, what live-cell imaging technology would you recommend for new users in drug discovery and for those who want to do more high-throughput imaging?

Dr. Schwirz: We have three in our portfolio.

Opera Phenix® Plus High Content Screening System is the premier confocal solution for the most demanding high-content applications. The system is designed for high-throughput high-content assays, phenotypic screening, assays using complex disease models such as live cells, primary cells, and microtissues, and fast-response assays such as Ca2+ flux.

Operetta CLS™ High-Content Analysis System combines speed and sensitivity with the powerful and intuitive Harmony® high-content analysis software that empowers biologists to run everyday (and complex) analyses right away, from fixed-cell to live-cell and 3D assays.

MuviCyte™ Live-Cell Imaging System is designed to operate inside a cell-culture incubator, enabling cells to be maintained under optimal conditions for long-term live-cell imaging. The system enables labs to study cellular behaviors and pathways to gain a deeper understanding of functions, disease mechanisms, and responses to treatments.

PerkinElmer: Thank you for taking the time to discuss the benefits of live-cell imaging tools in drug discovery, Dr. Schwirz.