Method to Label Proteins Inside the Living Cell for Super-Resolution Fluorescence Microscopy


Super-resolution microscopy has gained increasing interest due to its ability to visualize single protein molecules and sub-diffraction limited cellular structure. However, studying the dynamics of intracellular proteins in living cells using super-resolution microscopy has remained difficult due to the poor permeability of fluorescent probes across the cell membrane. Thus, imaging of intracellular protein in a living cell is often accomplished by using fluorescent proteins, which comes with caveats such as over-expression of target protein and poor photostability. CPLC graduate student Kai Wen Teng and the Selvin group developed a method to deliver fluorescent probes efficiently up to the size of a full antibody for targeting intracellular proteins. The method uses a pore-forming toxin called Streptolysin O, which reversibly creates ~20-30 nm pores on the cell membrane without killing the cells. Using this method, virtually any fluorescent dye can be delivered and any protein targeted using affinity reagents such as fluorescent ligands, nanobodies, or antibodies. In addition, the live-cell friendly oxygen scavenger Oxyrase can be applied to prolong the photostability of fluorescent dyes. By combining the advantages of the delivery method with Oxyrase, the team tracked individual kinesin molecules by single-molecule fluorescence, imaged actin filament dynamics using super-resolution microscopy (dSTORM) over an hour, and measured various steps associated in cancer, all unprecedented in live-cell fluorescence imaging. Refer to: Labeling proteins inside living cells using external fluorophores for microscopy 

1/22/2014 Liz Ahlberg, U of I News Bureau

Living cells are ready for their close-ups, thanks to a new imaging technique that needs no dyes or other chemicals, yet renders high-resolution, three-dimensional, quantitative imagery of cells and their internal structures – all with conventional microscopes and white light.

Called white-light diffraction tomography (WDT), the imaging technique opens a window into the life of a cell without disturbing it and could allow cellular biologists unprecedented insight into cellular processes, drug effects, and stem cell differentiation.

The team of University of Illinois researchers, led by ECE Associate Professor Gabriel Popescu, published their results in the journal Nature Photonics. Popescu is also an assistant professor in the Department of Bioengineering.

“One main focus of imaging cells is trying to understand how they function, or how they respond to treatments, for example, during cancer therapies,” Popescu said. “If you need to add dyes or contrast agents to study them, this preparation affects the cells’ function itself. It interferes with your study. With our technique, we can see processes as they happen and we don’t obstruct their normal behavior.”

A new 3-D imaging technique for live cells uses a conventional microscope to capture image slices throughout the depth of the cell, then computationally renders them into one three-dimensional image. The technique uses no dyes or chemicals, allowing researchers to observe cells in their natural state.

Photo by Gabriel Popescu
A new 3-D imaging technique for live cells uses a conventional microscope to capture image slices throughout the depth of the cell, then computationally renders them into one three-dimensional image. The technique uses no dyes or chemicals, allowing researchers to observe cells in their natural state. Photo by Gabriel Popescu
Because it uses white light, WDT can observe cells in their natural state without exposing them to chemicals, ultraviolet radiation, or mechanical forces – the three main methods used in other microscopy techniques. White light also contains a broad spectrum of wavelengths, thus bypassing the interference issues inherent in laser light – speckles, for example.

The 3-D images are a composite of many cross-sectional images, much like an MRI or CT image. The microscope shifts its focus through the depth of the cell, capturing images of various focus planes. Then, the computer uses the theoretical model and compiles the images into a coherent three-dimensional rendering.

The greatest potential of WDT, according to the researchers, is the ability to study cells in three dimensions over time. Since the cells are not altered, they can be imaged repeatedly, allowing researchers a glimpse into the dynamics of a cell as it goes about its life – or as it is treated with a new drug.

“As a cell grows, we can see the change in all three dimensions,” said Tae Woo Kim, a graduate student and first author of the paper. “We can see the dynamics of the cell in 3-D, which hasn’t been done in a quantitative manner. For example, we could see, in the span of a minute or over a cell’s lifetime, how it grows and how the things in the cell move around.”

To see a video showcasing examples of the 3-D images, please click here.

“With this imaging we can tell at what scale things within the cell are transported randomly and at what scale processes are actually organized and deterministic,” Popescu said. “At first glance, the dynamics looks pretty messy, but then you look at it – we stare at movies for hours and hours – and you realize it all makes sense. Everything is organized perfectly at certain scales. That’s what makes a cell alive. Randomness is just nature’s way to try new things.”

WDT uses a component that adds onto a conventional phase contrast microscope, a common piece of equipment in biology labs, without altering the microscope itself. The researchers used conventional microscopes with the intention of making these new optics principles easily accessible for biologists. The researchers hope that this will allow rapid large-scale adoption of WDT, and Popescu founded a startup company, Phi Optics, to help achieve that goal.

In addition to biological applications, the WDT technique has implications in the broader field of optics as the researchers pushed the boundaries of physics by applying scattering theory to imaging optics.

“The physics behind this technique is another thing we were fascinated about,” Kim said. “Light propagation in general is studied with approximations, but we’re using almost no approximation. In a very condensed form, we can perfectly show how the light changes as it passes through the cell.”

“We started on this problem two years ago, trying to formulate mathematically the sectioning effect observed in spatial light interference light microscopy (SLIM),” said Renjie Zhou, a graduate student and co-first author of the paper. “We came up with equations which eventually described WDT. The final equation is beautiful and the theory opens opportunities for solving other optics problems in a new theoretical language.”

Next, the researchers hope to pursue cross-disciplinary collaborations to explore applications of WDT in biology as well as expansions of the imaging optics demonstrated in WDT. For example, they are using WDT to watch stem cells as they differentiate in hopes of better understanding how they turn into different cell types. Since stem cells are so sensitive, only a chemical-free, non-invasive, white-light technique such as WDT could be used to study them without adverse effects.

The National Science Foundation supported this work. ECE Associate Professors P. Scott Carney and Lynford L Goddard, graduate student Mustafa Mir and former postdoctoral fellow S. Derin Babacan also were co-authors of the paper. All authors are affiliated with the Beckman Institute for Advanced Science and Technology.

Popescu, Zhou and Goddard are also affiliated with the university’s Micro and Nanotechnology Laboratory.