If the mention of holograms makes you think only of Princess Leia’s pleading image projected by R2D2 in Star Wars, you are not alone. Most biologists are not familiar with holographic technology, even though it’s been around for decades.
For years, biologists have dreamed of a microscope that allows them to study living, intact cells with the high resolution of an electron microscope. Electron microscopy’s sample conditions are toxic to live cells, and light microscopy has limited resolution because of the diffraction limit of light. While super-resolution microscopy methods work around this diffraction limit, those methods are not always cell-friendly either.
But now researchers such as Depeursinge’s group are developing imaging methods based on holography that allow the high-resolution study of living cells—in real time and without fluorescent markers.
How Holograms Work
For Depeursinge and his colleagues at the EPFL, the road to digital holographic microscopy (DHM) began when they encountered a challenge: They needed to collect 3-D images of small organs or cells by endoscopy and microscopy without using a complex confocal scanning instrument. The team simply didn’t have the space for a confocal microscope in their lab. The researchers also needed to use short illumination times, in order to minimize blurring of the image taken in vivo. At the time, the idea of digital holography was just emerging, and they saw the possibilities.
“Holography—the invention of which is credited to Dennis Gabor—is based on the idea of reconstructing the full wavefront of the optical beam diffracted by the illuminated object,” explains Depeursinge.
So Depeursinge’s group began developing DHM methods, which combine holographic phase microscopy with advanced computational processing. To start with, the team positions a cell sample between two oil-immersion objectives and then shines a diode laser down through the top objective at a steep angle. A digital camera records the hologram that results from combining the light that passes through the sample and bottom objective with the light from a reference beam. Then the researchers rotate the diode laser beam slightly and repeat the process to collect a series of holograms from different angles. Lastly, the group uses advanced computer processing to turn these sets of holograms into high-resolution images of planes through the sample.
Depeursinge sees the innovative aspects of DHM applied to imaging, where it can offer nanometer accuracy and sub-wavelength resolution. “These features are allied to rapidity, real-time imaging, and robustness,” he says. “Thanks to the capability of reconstructing the 3-D image from a single hologram taken in a very short interval of time, immunity to movement blurs and vibrations can be achieved. Reconstructing the wave fields at different distances provides images focused at various depths, a property sometimes called 'extended depth of focus,' or 'electronic focusing.'”
One of the possibilities that has been realized is DHM’s ability to image cells without fluorescent markers. “Normally, researchers have to label the cell with fluorescent dyes,” says Yann Cotte, a post-doc in Depeursinge’s group and first author of the study published in Nature Photonics (1). “[DHM] allows you to look directly at living cells, giving contrast without the need for invasive means to the cells.”
Not only does this obviate the need for an invasive filling process, it also eliminates the caveats that accompany the introduction of dyes or markers—the mere presence of those exogenous molecules may have unintended effects. Illumination of some fluorescent compounds makes them phototoxic to cells as exposure times increase. The absence of dyes and the extremely short exposure times of DHM are much more cell-friendly. This means that longer-term studies of living cells at high-resolution are also feasible.
Another important feature of DHM, notes Cotte, is its ability to make a tomographic reconstruction of a cell. He likens the importance of studying 3-D images to the Greek philosopher Plato’s Allegory of the Cave from the Republic, where people tried to identify objects according to their 2-D shadows on a cave wall, never able to view their true 3-D forms.
“That’s pretty much the situation still with phase imaging,” says Cotte. “If you look at the 2-D objects of cells, you’re basically missing the point because you see only the shadows, and not the object. Here we have a high resolution that gets us closer to the reality.”
And a little reality could be useful for a variety of examples of complex cellular specialization and differentiation, such as neurons, which are a challenge for any type of microscopy or imaging. The mechanisms of activity-dependent synaptic plasticity that occurs in neurons during important processes like learning and memory are all but inaccessible in vivo, but technologies like DHM can bring us closer.
In their Nature Photonics paper, Cotte and colleagues demonstrated that DHM can enable researchers to study dynamic processes occurring at dendritic spines and interactions between neurons in localized neuronal networks. With a lateral resolution of 70 nm, DHM can resolve the morphologies of dendritic spines, as well as intracellular membrane-bound organelles such as vesicles, mitochondria, the nucleus, and the Golgi apparatus.
Now, Depeursinge’s group is developing methods to apply DHM to thin brain slices and have obtained images of intricate networks of neurons. Slices pose a greater imaging challenge due to the presence of extra tissue and cell layers, which scatter light. Cotte predicts that even with moderate light scattering DHM could potentially give better resolution than normal illumination.
Improvements and Sacrifices
Despite its strengths, DHM still has some weaknesses, one of which is time; in the Nature Photonics paper, one acquisition took 18 seconds. But Cotte thinks that might be one of the easiest issues to address. “Now we are targeting sub-second time resolution,” he says. “But I think for research tools, there’s no real reason why it shouldn’t be real time, so that means something like 25 to 50 frames per second.” All that is required is a high-speed camera and scanning mechanism, both of which are commercially available but come at an increased cost.
Another point is the wavelength of light. In their paper, Cotte’s group used 405 nm blue light, but the method would be less phototoxic for live cells if they used a longer wavelength. The conundrum is that, due to the laws of optics, resolution is directly proportional to the wavelength of light used. So using longer, more cell-friendly wavelengths of light would sacrifice some resolution. The balance of these factors varies for different researchers and different experiments.
And finally, the mechanics of setting up DHM aren’t exactly easy: it’s difficult to position a sample between two oil-immersion objectives. The complexity of the DHM system makes it challenging to use, so the team is working to simplify it so that other researchers can apply it in various fields. “For now, I think we proved the technique’s feasibility,” Cotte says. “So now the question is: What are the applications that can actually benefit from it?”
1. Cotte, Y, Toy F, Jourdain P, Pavillon N, Boss D, Magistretti P, Marquet P, Depeursinge C. (2013) Marker-free phase nanoscopy. Nature Photonics 7: 113–117.