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Interferometric Technique Adds Third Dimension to PALM Microscopy

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Scientists at the National Institutes of Health and the Janelia Farm Research Center campus of the Howard Hughes Medical Institute have developed a new imaging technology that they claim produces the best three-dimensional resolution of cells with an optical microscope.

The technique, called interferometric photoactivated localization microscopy, or iPALM, can locate fluorescent labels in images within 10 nm to 20 nm, a Janelia Farm researcher told CBA News this week.

The iPALM technique, described online last week in the Proceedings of the National Academy of Sciences, also opens a third dimension to standard photoactivated localization microscopy, said study co-author Harald Hess, a research fellow and director of the applied physics and instrumentation group at Janelia Farm.

According to study co-author Jennifer Lippincott-Schwartz, chief of the section of organelle biology at the cell biology and metabolism branch of the NIH's National Institute of Child Health and Human Development, iPALM "allows us to be able to see how molecules comprising intracellular structures — which are normally only seen at an ultrastructural level using electron microscopy — are distributed within these structures.

"We can see it in three dimensions," she said. Previously, researchers have been "limited" in their ability to see in the z dimension, Lippincott-Schwartz said.

According to the PNAS paper, iPALM gives investigators an opportunity to observe how molecular components are distributed in a cell, such as the radial arrays of specific proteins that surround nuclear pores.

With iPALM, which enables researchers to observe molecules at the same resolution as electron microscopy, "you should easily be able to see these individual molecular components of that pore in a three-dimensional context, which is important for trying to understand how different molecules are actually functioning within these structures," Lippincott-Schwartz told CBA News this week.

She added that the technology can enable researchers to "easily investigate important questions related to structures such as the mitochondria, how the inner and outer membranes are localized and related to each other, or how complex organelles such as Golgi bodies might be organized in terms of their protein arrangement."

Before iPALM, electron microscopy was the only tool that could enable scientists to observe these subcellular components, but a weakness of the technology is its inability to show a cell's structural components.

"You have to use very laborious antibody labeling if you want to identify where a particular molecule is distributed in a structure that you are looking at," said Lippincott-Schwartz.

She said one of the current goals of her research with Hess is to perform correlative electron microscopy with iPALM in order "to try to see how images that are obtained with iPALM appear in the electron microscope." The result would be "an electron readout" of structures within the cell, without any type of molecular description, she said.

iPALM Pilot

The investigators used the principle of PALM to switch on photoactivatable fluorescent molecules one at a time, which they observed in 3D.

"Whenever a molecule gives off a photon, the photon can radiate as a wave in all directions, and the iPALM microscope collects the wavefront into two different objectives that are on the top or the bottom of the sample," said Lippincott-Schwartz. "And that wavefront goes through the objectives and is directed back down into a crystal, a three-way beam-splitter."

She said that if the molecule that gives off that photon is perfectly positioned between the two objectives at the time the wavefront goes through the two objectives and finds itself "reunited" in the beam splitter, there would be no interference in the wavefront.

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"But if the fluorophore that you are imaging is shifted either up or down relative to being perfectly centered between the two objectives, when the wavefront is reunited in this beam-splitter, it either constructively or destructively interferes with itself," Lippincott-Schwartz said. "That interference changes the intensity of the light seen by cameras that are positioned in three different places to detect the interfering light paths."

She said that depending on the location of a molecule that is emitting photons in the z plane, the imaging plane, it will constructively or destructively interfere with itself. From that, scientists can deduce where the photon came from in that z space.

iPALM gives "phenomenal resolution" in the z plane, she said. In traditional live-cell imaging, a single molecule would appear as a blurry spot that is 500 nm in size. With iPALM, that spot is reduced to 10 nm.

"Molecules are 2 nm to 4 nm in size, so you are really close to the actual size of the molecule," said Lippincott-Schwartz.

She added that "because you can build up an image one molecule at a time, [iPALM allows you to] define where all of the molecules are distributed within a particular sample."

And if one is expressing a photoactivatable fluorescent protein that is localized to a particular structure, such as a microtubule or a plasma membrane component, "you can now have amazing visualization of these structures within cells."

In the PNAS paper, "we show the distribution of tubulin and microtubules, and come very close to the actual size of the microtubules based on what we are seeing using the photoactivatable fluorescent proteins," Lippincott-Schwartz said.

In the paper, the investigators looked at a plasma membrane protein and observed "for the first time, at the cell periphery, the bottom and the top of the plasma membrane, which come very close to each other," Lippincott-Schwartz said.

As cells flatten out, their top and bottom plasma membranes were observed "within 50 nm" of each other. "That [proximity] prevents any type of normal imaging using widefield microscopy from telling you what you are looking at, in terms of the top and the bottom of the cell," said Lippincott-Schwartz. "With this approach, you can easily see how molecules are distributed in those different parts of the cells."

Asked about licensing or commercializing iPALM, Hess said, "For the moment, it is still a pretty complex instrument, and we are trying to get … the rough edges off of it. The PALM instrument is going through commercialization right now. If all goes well, we might go there as well in the future with iPALM."

Commercialization of the iPALM technique depends on simplifying and refining the instrument, and the potential scope of applications, he said.

Hess said that he has applied for a patent on the iPALM technique. The application was submitted in July 2008, and it is now in a quiet phase, but "I am sure it will be granted," Hess said.

Live iPALM Reading

In 2006, Hess and his colleague Eric Betzig, a group leader at the Janelia Farm campus, published a paper in Science describing PALM.

That technique allows localization in one image plane, but it gets "about one order of magnitude better resolution than you would get with an optical microscope in that direction," Hess said.

After that study was published, Hess said that he and his colleagues wanted to see, "'What is the ultimate that one can do to pull out the third dimension?' If we can localize where certain proteins are in three dimensions to a precision of 20 nm, that would be very significant."

Now, after the iPALM paper, Hess said the next step would be to use iPALM with a two-color technique. Gleb Shtengel, a senior scientist at Janelia Farm and a co-author on the PNAS paper, told CBA News that the original work was done with a single color dye because "we were targeting a single fluorescent protein. Next we would like co-localize different proteins."

This would involve a two-color technique that would allow the researchers to measure the relative location of two different proteins.

"I think we need to make the system compatible for doing two colors," said Lippincott-Schwartz. "I think it would be great if the system could go live so that we could do live-cell imaging and look at the movement of molecules in three dimensions. It is capable of this; we just have not done it yet."

Added Shtengel, "We have not done it yet, but we are certainly working towards it." He did state that the iPALM technique is "fundamentally" compatible with live-cell imaging.

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