Environmental Control in Live-Cell Imaging

Imaging cells over time—to create movies as they migrate along a gradient, for example, or document the molecular effects of receptor binding—generally means keeping the cells happy on a microscope stage. Depending on parameters including the nature and duration of the experiment, the type of cell, and whether live cells will be recovered, setups can range from simple open coverslips to atmospherically controlled boxes within microscope-enclosing plexiglass cages, and beyond. This article will examine some stage-top solutions for controlling the environment during live-cell imaging.

Feel the heat

It’s not hard to find elaborate, expensive, dedicated systems that finely control nearly every aspect of the imaging environment. But before taking that leap, Krzysztof (Kris) Hyrc, who manages imaging cores at Washington University in St. Louis, would first ask whether it’s really necessary. A lot of short-term experiments—especially on cells that “are not particularly vulnerable”—can simply be done at room temperature, he points out.

Disposable solutions are available that range from simple open gaskets on optical-quality slides to covered chambers equipped with ports for easy addition and removal of reagents—“ideal for short-term experiments that do not require tightly-controlled environmental conditions,” notes Ilenia Bertipaglia, technical support representative at Grace Bio-Labs.

“Of course, if you want to be more physiological then you need to maintain the culture at the appropriate temperature," Hyrc, who is also an associate professor of neurology, notes.

When he ran experiments on temperature-sensitive mutants, Hyrc would submerge a custom-made heating coil with thermistor-based feedback into a Petri dish. These days he generally puts the dish into a small stage-top chamber that controls the temperature with thermocouple feedback-based control, and free software to record the temperature on his computer.

Some researchers use just a heated stage plate to keep their dishes, plates, or slides warm, and prevent the transfer of heat between the cold stage and the warm sample. “Those tend to be pretty affordable and can be DIY as well,” notes Lauren Alvarenga, product manager for Olympus’ life science microscopy group.

“Once you start to add in gas controls and things like that it becomes a lot more difficult to DIY. But there are tons of different options in terms of commercial manufacturers,” she remarks.

It’s in the atmosphere

An open dish (or plate or slide) will quickly begin to evaporate, altering the osmolarity of the medium, and equilibrate CO₂ with the atmosphere, leading to shifts in pH. As a result, many researchers choose to enclose their experiments in a stage-top microincubation system capable of maintaining a steady humidity and gas composition. These will often look like small, flat discs or boxes with windows and ports and other connections, and house standard formats (such as coverslips or 35 mm dishes), proprietary chambers, or both, in which cells are maintained and imaged.

Traditionally gas will be perfused through water, allowing it to pick up humidity before it enters the chamber. Most simple experiments can use pre-mixed 5% CO₂, although some systems include a gas mixer to control the concentrations of several different gases, for example to simulate an anoxic tissue environment. “Once it finally makes its way over to the sample it has enough moisture in it to actually be able to condense on the top of the lid—so some manufacturers have gotten very creative in terms of what element needs to be at what temperature to prevent things like condensation,” explains Alvarenga.

“If you don’t have gas control, you can buy CO₂-independent media,” explains Erika Wee, manager of McGill University’s advanced bioImaging facility. She emphasizes that this is only good for an hour or two, and even then is not ideal: she only resorts to CO₂-independent media in situations such as “when we run a workshop with many demo systems, and it’s really painful to have CO₂ tanks for each station.”

Many experiments can benefit from perfusion. “At the shortest-term measurement, all you really need is a chamber that you can perfuse solutions through and maintain temperature,” points out Edmond Buck, senior application scientist at Warner Instruments, a part of Harvard Bioscience. The perfusate, warmed via an inline heater, can rapidly carry gas and nutrients, and introduce dyes, agonists, and antagonists through the culture with relatively little mixing. The perfusate can be gravity fed, syringe injected, or mechanically pumped.

Image: Warner Instruments RC-27L Large Bath Chamber with slice supports in a PH-6 platform.

Almost all of our chambers have perfusion and outflow capabilities, says Buck. “As a design principle, you’re better off to have that perfusion capacity there and not use it than to not have it and need it.”

The cage

High-power microscopy generally requires an immersion lens to effectively be in contact with the sample. Regardless of how it is managed, the temperature is usually five to seven degrees lower at the point of contact because “the objective is attached to this massive scope, which is a deep heat sink … and all your heat is going out through the objective,” Buck says. One solution is to use an objective heater. But, he cautions, to minimize focal drift look for a heater that allows the objective to maintain a fairly steady temperature even as the heater cycles.

Another solution is to enclose the microscope in a box, and warm everything including the stage and the objective to the target temperature. These can be as DIY as enclosing the microscope and an egg incubator heater with cardboard and insulation. Sophisticated off-the-shelf and custom enclosure systems (often termed “cages”) are also available.

The McGill facility’s cages provide CO₂ and humidity controls that can be turned on or off as well as temperature stability. “The downside is that the box is really annoying,” notes Wee.

Most full-cage enclosures still make use of a stage-top microenvironment to maintain gas concentrations and humidity, rather than subjecting the entire scope to warm, moist gas, says Alvarenga. She points out another downside: “once your scope is at 37 degrees, you generally want to leave it at 37 degrees … so with a full cage system you’re essentially dedicating an entire scope to live-cell imaging.”

Live-cell imaging at its basis means documenting a culture as it responds to the experimental conditions, while minimizing variables like temperature, humidity, and pH. Things like duration of the experiment, manipulations necessary, optical resolution required, how finicky the culture is, and, of course, budget, will all help to dictate how you’ll set yours up.

Tips to Keep It Simple

Live cell imaging can be complicated, sophisticated, intimidating, and expensive, but it doesn’t always have to be. Here are some tips to ease in while keeping costs down.

• Enclose the scope with insulation and cardboard, and heat it with an egg heater.
• Make a chamber out of a slide, coverslip, and some vacuum grease.
• Use pre-mixed gas instead of mixing your own.
• Bubble the gas through a cup of warm water to pick up humidity.
• Use free software to export data to your computer or smart phone.
• Use a handheld pipette for perfusion.
• Layer mineral oil on the culture to keep down evaporation.

Hero image: A panoramic view of HeLa cells. The cells' nuclei containing the DNA are stained in blue and the cells' cytoskeletons in gray. Image courtesy of NIH Image Gallery/Tom Deerinck, National Center for Microscopy and Imaging Research