Optical micromirrors reveal the secrets of cell membranes

13 Nov 2018 Sponsored by Mad City Labs

Janice L Robertson with Rahul Chadda (middle), Kacey Mersch (right), and the team's TIRF microscope

Progress in understanding the lipid bilayer – an ingenious two-molecule thick oily barrier that protects all living cells, including our own – has been dramatic over the past 100 years. But there are still puzzles to be solved. In particular, biologists are keen to discover more about the mysterious proteins that exist inside the lipid bilayer. These chains of amino acids assemble into folded structures that change the behaviour of the cell membrane – allowing, for example, the selective passage of salts and sugars in and out of a micro-organism.

In fact, cell membranes are home to a variety of proteins that are essential for life to proceed, including channels, transporters, pumps, and receptors. These forms are relatively well known, but what isn’t understood is how these membrane proteins, which are oily themselves, navigate the oily lipid bilayer to go about their work.

The first step for scientists is to figure out the thermodynamic rules that govern protein assembly in membranes. And research groups such as Janice Robertson and her team, formerly based at the University of Iowa and now at Washington University in St Louis, are using cutting-edge microscopy tools to come up with the answers. “The major goal of my research is to answer the question: why do greasy membrane proteins choose to interact with other greasy proteins, instead of the greasy lipids in the surrounding cell membrane?” she explains.

Optical solutions for biological problems

One of the most useful techniques for looking inside the lipid bilayer is total-internal-reflection fluorescence microscopy (TIRF). The optical arrangement in these systems generates a localized illumination of molecules just above the surface of a glass slide that is situated above an objective lens. Using fluorophores – chemicals that fluoresce when excited by light – as markers, researchers can examine how populations of molecules inside the lipid bilayer change in response to different conditions.

A number of different microscope configurations are available, but what’s special about Robertson’s set-up is the use of micromirrors to direct incoming laser light into the microscope objective. The angle of incidence is adjusted so that the beam internally reflects at the sample interface, sending an evanescent wave into the sample that decays over a distance of a few hundred nanometres. This yields the narrow excitation volume that is ideal for looking at single molecules.

Buying the microscope was a huge time-saver and it meant that we could benefit from all of the engineering and stability that had been designed into the systemJanice Robertson, Washington University in St Louis

The use of micromirrors, compared with the more usual multi-wavelength dichroic filters, improves the signal-to-noise ratio that can be achieved with TIRF microscopy. Two micromirrors are placed underneath the microscope objective, one directing laser light into the lens and the other guiding the radiation out, leave plenty of space around the sample stage – which is useful for placing other equipment such as micropipettes or additional optical detectors. Also, the mirrors are optically compatible with a range of different laser wavelengths, which means that Robertson and her team can use different colours of light to selectively excite a series of fluorophores in a sample without swapping components in the optical path.

“I first learnt about this microscope when I was doing my post-doctoral training and collaborating with researchers at the Gelles laboratory in the Department of Biochemistry at Brandeis University,” says Robertson. “They had built their own microscope – an objective-based TIRF that used micromirrors instead of a dichroic mirror to direct the excitation beam.” This set-up allowed the scientists to collect a strong signal from their sample while reducing background noise.

Flexibility benefits research

When Robertson was in a position to set up her own lab at the University of Iowa, she knew exactly what she wanted from a TIRF microscope and looked for a vendor that could supply a micromirror-based system. “The big advantage of the Mad City Labs MicroMirror TIRF microscope is that it allows you to have the advantages and the flexibility of a home-built system,” she comments.

In principle, her group could have also assembled a microscope from scratch, but the researchers wanted to get up and running as quickly as possible so that they could continue their experiments. “Buying the microscope was a huge time-saver and it meant that we could benefit from all of the engineering and stability that had been designed into the system,” Robertson points out.

After five years at Iowa, Robertson had the opportunity to expand her research again – this time moving to Washington University in St Louis. It meant breaking down the microscope and reassembling it in her group’s new labs, but it also gave the team the chance to further update the apparatus.

The latest set-up features Mad City Labs’ newest MicroMirror TIRF microscope design. The updated design makes the system even more flexible, and allows users to switch easily between different illumination modes (including TIRF) thanks to an automated module. “It’s really useful to have that versatility to flip between studying whole-cell or liposome fluorescence, as well as single-membrane protein molecules, in the same samples,” Robertson comments.

Molecular interactions brought into view

Using the system, the team has been continuing to study the reactions of membrane proteins to shed light on what makes these molecules self-assemble, fold and bind to one another in the lipid bilayer. In water, explains Robertson, protein folding and assembly is driven by strong differences between the protein (oily) and the solvent (water), but these conditions don’t exist within the lipid bilayer and yet protein folding and assembly persists.

To investigate this question, the Robertson laboratory is studying the equilibrium association reactions of proteins inside the membrane. By looking at probability distributions of protein assemblies in data generated by the MicroMirror TIRF microscope, the group has been able to follow populations of transporter proteins as they equilibrate inside membrane layers. “We can do full accounting of the protein into the liposomes, which takes us a step closer to understanding the binding process and the thermodynamic driving force for assembly,” says Robertson.

The data collected can also include movies. The team’s new set-up features two cameras – a charged-coupled device for capturing regular micrographs and a CMOS camera to provide enhanced time resolution – which makes a powerful combination. “It’s possible to look at a lipid bilayer on the glass and see single-molecule diffusion of both the lipids and different membrane protein components, allowing us to potentially visualize those reactions in real time,” Robertson explains.

Robertson also values the support provided by the community. “It’s a very open field and people are willing to help each other, which helps a biologist like me,” she says.

Visit the Mad City Labs website for more information on its MicroMirror TIRF microscope.

13/11/2018 FROM PHYSICSWORLD.COM