Lights! Camera! Action!

Thu, 02/07/2008 - 8:53am
James Netterwald, PhD, MT (ASCP), Senior Editor

Live-cell imaging offers the power of capturing the dynamics of biological action in live cells and in real-time, something not previously available with biochemical approaches. But is this tool perfect yet?

Live-Cell Image of a Mouse Embryo 
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A live-cell image of a mouse embryo. (Source: Gloria Kwon, Weill Graduate School of Medical Sciences-Cornell University and the Memorial Sloan-Kettering Institute)

Capturing a biological event is a lot like taking a photograph. When one wants to capture a live event, they typically use a camera to record it on film or save it to digital media. Such a snapshot might suffice if one just simply needed to capture the event in a rough image, but did not care much about all of the detail in that image. To capture the level of detail to create a great image, one needs to adjust things like lighting to reach a desired final look.

However, even with great lighting, it is still difficult to capture what is truly happening in a given event. This is an inevitable result of trying to capture motion or time-based events by taking a static snapshot with a still camera. The photographer misses the point of the event: that it is constantly changing over time. To fully understand what has occurred in an event, the photographer should record a video. And there in lies the essence of live-cell imaging: the act of capturing all of the dynamics of a biological event in a live cell, in real-time, with a microscope, a camera, and video-recording software.

Experts cite that the main advantage of live-cell imaging is the fact that it can yield information about the dynamics of cellular processes. And although the idea of imaging a cell in action is not a new idea, there were issues that restricted an understanding of cellular dynamics before live-cell imaging came to fore. “Previously, a lot of work had been done in cells that were fixed in, let’s say, formaldehyde. That leads to many artifacts and it gets rid of any dynamic info,” says Christine K. Payne, PhD, assistant professor, School of Chemistry and Biochemistry—Georgia Institute of Technology, Atlanta, Ga.

By taking cues from the biochemist that isolates a protein and its substrate, mixes them, and calculates their reaction rate based on the reaction he/she observes, Payne tries to measure reaction rates. The only difference is she does all of the measurements in live cells, where all of the factors necessary for the process to occur are in their natural environment.

Specifically, Payne is studying the interaction between two types of vesicles in cells. The first type, the endosome, carries the substrate for the chemical reaction under study; the second, the lysosome, carries the enzymes. Payne is interested in how these two populations of vesicles locate each other and fuse, and how this fusion results in a chemical reaction between their respective cargoes.

To do the kind of time-dependent tracking necessary to study the dynamics of vesicle movement in cells, Payne uses a fluorescent protein to label one population of endosomes and a small fluorescent molecule that localizes inside the other population of vesicles. After labeling the cells and setting up the experimental conditions, Payne records all of the live-cell action using a home-built microscope based on an Olympus microscope and digital camera set-up.

To take all of these great live-cell images, one must start with good lighting in the form of a good reporter molecule. But selecting an appropriate reporter system for an experiment has its own challenges.

The good reporter
There is a big push in modern biology to understand the processes occurring  inside the organism without perturbing the system’s delicate, physiological balance. “I think it is very difficult to definitely say that you are watching what the cell is doing without perturbing what the cell is doing,” says Peter Lassota, PhD, divisional vice president of imaging biology and oncology, Caliper Life Sciences, Hopkinton, Mass. A well-designed reporter molecule can give one the detection method one needs to capture cellular events, while not perturbing the biological system in the process.

click to enlarge 
Spread of an infection is shown as light emission coming from lux-operon-labeled bacteria on the top panels. Also, shown here is the resulting intracellular process triggered by infection, measured by induction of GFAP protein detected as expression of luciferse (resulting in light emission) from the reporter construct where luciferse is controlled by the GFAP promoter. (Source: Caliper Life Science)

According to Lassota, there are two basic types of reporter used in optical imaging–fluorescent reporters and bioluminescent reporters. The fluorescent reporter is a molecule that, upon being hit with excitation light of one specific wavelength from a fluorescent microscope, will emit light of a different wavelength. The light emitted can be is then recorded by a camera system attached to the microscope.

The other type of reporter system used for optical imaging is based on bioluminescence. Similar to firefly luciferase protein, bioluminescent reporters emit light only in the presence of their substrate; the emitted light can also be detected by a microscope and camera system. The same principles apply to whole-animal imaging.

Both reporters systems have the requirement of being introduced into the cell in order to utilize them to study cellular processes. The main difference between them is that, in most cases, the gene for the bioluminescent reporter needs to be introduced into the genome of cell in order to detect reporter protein activity. In contrast, fluorescent reporters do not have to be inserted into the genome; they can be brought into the cell by injection or passive diffusion, or attached to an antibody to target a process that occurs on the outside of the cell.

An obstacle inherent in the development of reporter systems is that cells usually produce some level of background fluorescence. Because these naturally-occurring auto-fluorescent proteins are omnipresent, one needs to design fluorescent reporters that are brighter than the background auto-fluorescence. “In some ways, it’s lucky that those [auto-fluorescent] proteins mainly fluoresce in the blue and green region of the spectrum. So if you work with red-emitting probes, you are able to discriminate against the auto-fluorescence of the cell,” says Payne. Of course, background fluorescence is not a problem when using reporters that are bioluminescent.

According to Lassota, there is a lot of information to obtain by looking at single cells, but by taking the cell out of the organism, one might miss some of the vital information from cellular processes that don’t behave the same way in vitro as they do in vivo. So, to study the cell in its natural state, Caliper has moved toward performing live, whole-animal, optical imaging, not just live-cell imaging.

Imaging, HTS-style 
Live-cell imaging is not only used to solve problems in basic biology, there are now companies trying to scale it up to allow the pharmaceutical industry to perform high-throughput screening with it. Fluxion Biosciences, Inc., San Francisco, Calif., is one of those companies. Fluxion is focused on performing cell-based assays in a standard well-plate format. “The reason we do cell-based assays is that we noticed that the pharmaceutical industry is moving away from doing biochemical screening and moving toward doing functional screening. And the only way to do functional screening on a large-scale is to use a small number of cells,” says Cristian Ionescu-Zanetti, PhD, chief technology officer at Fluxion. The readouts for most of the cell-based assays they perform are image-based.
Many of these cell-based assays look at cell adhesion. Integration of microfluidics into the assay design allows for accurate control of flow, which is necessary for the cell adhesion assays they perform. “The reason that it is important to have fluidics is that you need to apply it to shear in these situations. So the idea here is to look at cells that adhere to other substrates or to a layer of other cells,” says Zanetti. This assay models the adherence of inflammatory cells to human endothelium. Such a model is useful when trying to develop ways to modulate the immune response (e.g., in autoimmune disease).

Cells used in this assay are labeled with a fluorescent molecule and either imaged using an inverted, confocal microscope or placed on a plate-reader that reads fluorescence intensity. Images are taken as the cells attach to the substrate and spread out. A shearing force is then applied to the cells to determine the number of truly adherent cells.

But how are the images recorded in a high-throughput fashion? “We’re definitely doing real-time recording for a lot of the things we do. What you typically have is some kind of a trade-off between recording and throughput,” says Zanetti. “If you’re looking at 48 wells, it is hard to look at them all at once. So, people end up doing real-time imaging on eight wells, and recording an endpoint assay for the remaining wells.” A variation on the assay is being used to look at antibiotic-resistance in bacterial biofilms. In all cases, the assays can be used to perform high-throughput screening to find new drugs.

Along the same lines as Caliper’s whole-animal imaging is the work of Gloria Kwon, who performs a variation on live-cell imaging in transgenic mouse embryos. “My primary research project involves studying the morphogenesis of the early mammalian embryo with respect to the cellular and molecular mechanisms underlying embryo growth, differentiation and patterning,” says Kwon, a PhD student in neuroscience at Weill Graduate School of Medical Sciences-Cornell University, who is working in the laboratory of Anna-Katerina Hadjantonakis at the Memorial Sloan-Kettering Institute, New York. “In order to better understand these processes as they take place in vivo, we use live-cell imaging of fluorescent protein reporters to observe the specific events that occur during development, in real-time.” Kwon was the winner of Nikon’s 2007 Small World competition. 

Sweet, sweet imaging
Getting back to imaging in single cells. Mark A. Rizzo, PhD, assistant professor of physiology, University of Maryland, Baltimore, Md., uses live-cell imaging to study the activation of glucokinase—the glucose-sensing protein that sets the rate for insulin secretion in the pancreas. “Defects in glucokinase regulation or signaling is associated with type 2 diabetes and so our goal is to try to find out how to manipulate activation of this protein and try to use glucokinase as a new drug target for diabetes.”

Rizzo has developed fluorescent biosensors to track the real-time activation of glucokinase and to test how this activation sensitizes a culture of pancreatic beta cells to glucose. The biosensor in this case comes in the form of a fluorescent protein. And what Rizzo typically does is create a recombinant glucokinase protein with two different fluorescent protein tags on it. In beta cell culture, stimulation of the cells with glucose causes the recombinant proteins to interact and produce an energy transfer reaction called FRET. The energy transfer is then measured from images captured using a standard, laser-scanning, fluorescence, confocal microscope.

Future improvements
As outlined in the previous sections, live-cell imaging is a powerful tool for understanding the dynamics of cellular processes, but it can always use some improvement, especially in the area of reporter molecules. “It would be possible to label a protein with a single fluorescent probe, but under typical conditions you would not be able to image a single probe,” says Payne. The exception to this rule occurs with use of quantum dots. Where extremely bright fluorescence is needed, Payne turns to quantum dots for labeling because they are much brighter than a single organic dye or a single fluorescent protein. One drawback of using quantum dots, she says, is that they are “very large and are difficult to get into the cell.” The purpose of one of her research projects is to try to find ways to get quantum dots into cells. 

Another researcher who uses live-cell imaging for cell biology research is Gaudenz Danuser, PhD, associate professor, Laboratory for Computational Cell Biology, Department of Cell Biology, The Scripps Research Institute, La Jolla, Calif. Danuser, who uses live-cell imaging to study the dynamics of actin function during cell migration, sees that there are two major improvements needed in live-cell imaging. The first is resolution. Danuser would like to see the resolution of live-cell imaging lowered to the point where it can be bridged to electron microscopy (EM). And he says: “There are a number of groups and a number of techniques out there that are very promising, and that researchers will crack this current resolution gap between traditional live-cell microscopy and EM within the next 10 years.” The second, he feels, is that live-cell imaging, in particular, and biology in general, are becoming essentially problems of information technology. While the hardware to acquire raw data is rapidly advancing on all fronts, including imaging, the development of methods to extract valuable and robust information on biological mechanisms from these data is lagging behind by at least a decade. The result is an over-accumulation of unanalyzed data.

It is obvious that live-cell imaging is the future of cell biology, but there are still some kinks to be worked out. However, with brighter reporters illuminating cellular structures and single molecules and more EM-compatible resolution, live-cell imaging will likely be used to capture cellular events for years to come.

This article was published in Drug Discovery & Development magazine: Vol. 11, No. 2, February, 2008, pp. 18-22.


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