CYTOSource Current Issues for Cytopathology
Sharper Focus Advances in subcellular imaging have researchers envisioning a clearer future for biomedical research or years, optical microscopes were stuck at a resolution limit of approximately 200 nanometers, meaning that anything smaller than a relatively large virus appeared as an indistinct blob. The handicap derived from what was believed to be an insurmountable physical law governing how light waves bend as they encounter a small opening such as a lens. The distortion meant that 2 objects could not be resolved if they were separated by a distance of less than one-half the wavelength of the incoming light. To overcome that diffraction barrier, as it is known, scientists had to kill, modify, or chemically treat cells and use techniques such as scanning electron microscopy or X-ray crystallography to magnify or infer the cellular features. The work-
REPRINTED WITH PERMISSION FROM BIOTECHNOL J. 2009;4:927-938.
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Cancer Cytopathology Cancer Cytopathology
arounds, however, failed to capture cells in their natural, living state. In October 2014, 3 researchers from the United States and Germany shared the Nobel Prize in Chemistry for bypassing these presumed limits and allowing optical microscopy to “peer into the nanoworld,” according to the Royal Swedish Academy of Sciences. The breakthrough relied in large part on using laser light to illuminate fluorescently tagged molecules in the cell, and on using various tricks to turn the fluorescence on and off to focus on specific molecules, a process known as photoswitching. As a result, light microscopes are now getting down to the subcellular level within living cells and could provide an unprecedented view of the mechanisms underlying cancer, the acquired
May May2015 2015
267 2
Nanoscopy’s Sharper Focus
Nanoscopy’s
immunodeficiency syndrome, and other diseases. “Seeing is sometimes believing and enabling,” says Syed A.M. Tofail, PhD, a lecturer in the physics and energy department at the University of Limerick in Ireland and an expert in what is often called nanoscopy. As the science expands, researchers are experimenting with new ways to use fluorescent tags and organic dyes to mark molecules of interest such as beacons, and to zero in on proteins of interest and track their fates across a cell. A new collaboration in the United Kingdom known as Nanoscopy Oxford, or NanO, for example, is aimed at building bespoke super-resolution instruments and optimizing them for biomedical research applications. Christian Eggeling, PhD, a cellular imaging expert, NanO leader, and professor of molecular immunology at Oxford University, is using nanoscopy to visualize previously undetectable molecular interactions such as protein–protein and protein–lipid interactions that lie at the heart of many immune responses. Achieving the best image means minimizing or at least finding a good compromise among 3 potential limitations: spatial resolution, or the imaging clarity at a specific location; temporal resolution, or visual clarity that lasts over time; and light-induced cell death, or phototoxicity. “If your laser light is too high, you destroy the cell and then you cannot observe them for very long,” says Dr. Eggeling, who worked with Nobel Prize co-winner Stefan Hell, PhD, at the Max Planck Institute for Biophysical Chemistry in Gottingen, Germany before joining Oxford. No nanoscopy method has yet solved all 3 issues. With a process called stimulated emission depletion (STED), researchers dampen the fluorescence in the outer part of the laser-illuminated spot, creating a reverse donut that focuses the laser beam on only the central portion of the fluorescence. STED can achieve a spatial resolution of 40 to 50 nanometers but is unable to track a molecule’s movement for very long. Another technique called stochastic optical reconstruction microscopy/ photo-activated localization microscopy (STORM/PALM) instead uses a cameralike method to image the entire cell
CYTOSOURCE
Nucleus of a bone cancer cell using normal high-resolution fluorescence microscopy (left) and 2-color localization microscopy (right).
By Bryn Nelson, PhD Edited By Terence J. Colgan, MD
CYTOSOURCE
Nanoscopy’s Sharper Focus
CONTINUED from previous page
but only turn on a small number of fluorescent spots at any given time. Although blurry, these spots can reveal the position of the fluorescently tagged molecules. By switching on another set of fluorescent labels in a second camera frame and repeating the process thousands of times, a high-resolution picture eventually emerges. Good spatial resolution down to 10 to 20 nanometers, however, comes
laser beam that pulses so that the shorter duration will not overheat the sample. The method also can be tuned to focus on specific vibrational frequencies that act like signatures of the early stages of disease. “Our philosophy is that every pathology is chemical by nature to start with,” Dr. Tofail explains. As a disease process begins to alter more and more molecules, they may vibrate differently
As subcellular microscopy methods become more routine, researchers may be able to expand the technology to enable higher-resolution in situ or in vivo imaging within animal models of disease. With a method known as intravital fluorescence microscopy, for example, researchers are already zooming in on cellular processes deep within the tissues of living mice. at the expense of tracking any molecular movement. Dr. Eggeling’s group favors STED but uses a small window of illumination to avoid killing cells too quickly from the laser light. The super-resolution method, he says, has been particularly useful in investigating the earliest moments of T-cell activation in response to perceived threats.
Good Vibrations? In February, after a session on nanoscopic imaging at the American Association for the Advancement of Science annual conference, Dr. Tofail explained the concept behind another emerging technique called infrared nanoscopy. Within the next 6 months, he says, the prototype tabletop microscopes built by his group could achieve a spatial resolution of up to 70 nanometers, a potential boon for in vivo applications. With infrared nanoscopy, the light alters the frequency of naturally vibrating molecules in a way that creates vibration-based signatures for each of them. “You can see someone else because they have contrast against the background,” he says. In the same way, the infraredcaused vibration of an object is different from its background, allowing that object to be distinguished. However, because high-intensity infrared light can burn the sample, Dr. Tofail’s group has devised a tabletop system that uses a high-intensity
than normal, and sensitive instruments could detect the subtle changes. “If you know the exact location in the tissue where the pathology is at an early stage, you can figure out why it is happening and you can try to stop it from happening,” he says. Research groups are also trying to improve the STED method by scanning cells with up to 1000 laser beams at once. On its own, a single laser can only focus on 1 labeled molecule at a time. A whole army of them, however, could simultane-
The “dream” of cell biologists, he says, is to combine both techniques. Dr. Subramaniam and his colleagues recently used such correlative imaging to describe how the human immunodeficiency virus matures, in particular by imaging the viral core that delivers the viral genome to newly infected cells.1 “That’s very exciting because that lets us essentially get the best of both worlds: the localization and the live cell imaging where you can get the dynamics, and combine that with the ability to image the local, spatial architecture of where these things are in the cell.” As subcellular microscopy methods become more routine, Dr. Subramaniam says, researchers may be able to expand the technology to enable higher-resolution in situ or in vivo imaging within animal models of disease. With a method known as intravital fluorescence microscopy, for example, researchers are already zooming in on cellular processes deep within the tissues of living mice. One key to the future of subcellular microscopy will be its ability to answer biologically relevant questions. “Yes, the technology exists, but what do you use it for? It’s one thing to look at it in tissue culture cells, but it’s a whole different ballgame to look at it in a live animal,” Dr. Subramaniam says. Dr. Eggeling is optimistic about the progress toward useful applications. “This super-resolution microscopy is more and more applicable to living cells,
‘This super-resolution microscopy is more and more applicable to living cells, and for a longer period of time.’ —Christian Eggeling, PhD ously illuminate 1000 spots and greatly enhance the field of view. “I think that’s a very exciting frontier,” says Sriram Subramaniam, PhD, a senior investigator and microscopy expert at the National Cancer Institute in Bethesda, Maryland. Fluorescence-based microscopy methods, he says, have already helped the field move from qualitative descriptions of what is present in a cell to more quantitative descriptions of how many molecules are present and where. Electron microscopy, which Dr. Subramaniam favors, still achieves a higher resolution, although it requires researchers to kill cells in order to observe them.
and for a longer period of time,” he says. He too, however, agrees that the field will need to show off more than just its technological side. “In principle, we have shown that the microscopes work,” he says. The next step is proving that they can live up to their promise by providing new insights in cancer research, immunology, neurobiology, and other biomedical fields. Reference 1. Frank GA, Narayan K, Bess JW Jr, et al. Maturation of the HIV-1 core by a non-diffusional phase transition. Nat Commun. 2015;6:5854. DOI: 10.1002/cncy.21556
Content in this section does not reflect any official policy or medical opinion of the American Cancer Society or of the publisher unless otherwise noted. © American Cancer Society, 2015.
3 268
Cancer Cytopathology Cancer Cytopathology
May 2015 May 2015
Sharper Focus Advances in subcellular imaging have researchers envisioning a clearer future for biomedical research or years, optical microscopes were stuck at a resolution limit of approximately 200 nanometers, meaning that anything smaller than a relatively large virus appeared as an indistinct blob. The handicap derived from what was believed to be an insurmountable physical law governing how light waves bend as they encounter a small opening such as a lens. The distortion meant that 2 objects could not be resolved if they were separated by a distance of less than one-half the wavelength of the incoming light. To overcome that diffraction barrier, as it is known, scientists had to kill, modify, or chemically treat cells and use techniques such as scanning electron microscopy or X-ray crystallography to magnify or infer the cellular features. The work-
REPRINTED WITH PERMISSION FROM BIOTECHNOL J. 2009;4:927-938.
F
Cancer Cytopathology Cancer Cytopathology
arounds, however, failed to capture cells in their natural, living state. In October 2014, 3 researchers from the United States and Germany shared the Nobel Prize in Chemistry for bypassing these presumed limits and allowing optical microscopy to “peer into the nanoworld,” according to the Royal Swedish Academy of Sciences. The breakthrough relied in large part on using laser light to illuminate fluorescently tagged molecules in the cell, and on using various tricks to turn the fluorescence on and off to focus on specific molecules, a process known as photoswitching. As a result, light microscopes are now getting down to the subcellular level within living cells and could provide an unprecedented view of the mechanisms underlying cancer, the acquired
May May2015 2015
267 2
Nanoscopy’s Sharper Focus
Nanoscopy’s
immunodeficiency syndrome, and other diseases. “Seeing is sometimes believing and enabling,” says Syed A.M. Tofail, PhD, a lecturer in the physics and energy department at the University of Limerick in Ireland and an expert in what is often called nanoscopy. As the science expands, researchers are experimenting with new ways to use fluorescent tags and organic dyes to mark molecules of interest such as beacons, and to zero in on proteins of interest and track their fates across a cell. A new collaboration in the United Kingdom known as Nanoscopy Oxford, or NanO, for example, is aimed at building bespoke super-resolution instruments and optimizing them for biomedical research applications. Christian Eggeling, PhD, a cellular imaging expert, NanO leader, and professor of molecular immunology at Oxford University, is using nanoscopy to visualize previously undetectable molecular interactions such as protein–protein and protein–lipid interactions that lie at the heart of many immune responses. Achieving the best image means minimizing or at least finding a good compromise among 3 potential limitations: spatial resolution, or the imaging clarity at a specific location; temporal resolution, or visual clarity that lasts over time; and light-induced cell death, or phototoxicity. “If your laser light is too high, you destroy the cell and then you cannot observe them for very long,” says Dr. Eggeling, who worked with Nobel Prize co-winner Stefan Hell, PhD, at the Max Planck Institute for Biophysical Chemistry in Gottingen, Germany before joining Oxford. No nanoscopy method has yet solved all 3 issues. With a process called stimulated emission depletion (STED), researchers dampen the fluorescence in the outer part of the laser-illuminated spot, creating a reverse donut that focuses the laser beam on only the central portion of the fluorescence. STED can achieve a spatial resolution of 40 to 50 nanometers but is unable to track a molecule’s movement for very long. Another technique called stochastic optical reconstruction microscopy/ photo-activated localization microscopy (STORM/PALM) instead uses a cameralike method to image the entire cell
CYTOSOURCE
Nucleus of a bone cancer cell using normal high-resolution fluorescence microscopy (left) and 2-color localization microscopy (right).
By Bryn Nelson, PhD Edited By Terence J. Colgan, MD
CYTOSOURCE
Nanoscopy’s Sharper Focus
CONTINUED from previous page
but only turn on a small number of fluorescent spots at any given time. Although blurry, these spots can reveal the position of the fluorescently tagged molecules. By switching on another set of fluorescent labels in a second camera frame and repeating the process thousands of times, a high-resolution picture eventually emerges. Good spatial resolution down to 10 to 20 nanometers, however, comes
laser beam that pulses so that the shorter duration will not overheat the sample. The method also can be tuned to focus on specific vibrational frequencies that act like signatures of the early stages of disease. “Our philosophy is that every pathology is chemical by nature to start with,” Dr. Tofail explains. As a disease process begins to alter more and more molecules, they may vibrate differently
As subcellular microscopy methods become more routine, researchers may be able to expand the technology to enable higher-resolution in situ or in vivo imaging within animal models of disease. With a method known as intravital fluorescence microscopy, for example, researchers are already zooming in on cellular processes deep within the tissues of living mice. at the expense of tracking any molecular movement. Dr. Eggeling’s group favors STED but uses a small window of illumination to avoid killing cells too quickly from the laser light. The super-resolution method, he says, has been particularly useful in investigating the earliest moments of T-cell activation in response to perceived threats.
Good Vibrations? In February, after a session on nanoscopic imaging at the American Association for the Advancement of Science annual conference, Dr. Tofail explained the concept behind another emerging technique called infrared nanoscopy. Within the next 6 months, he says, the prototype tabletop microscopes built by his group could achieve a spatial resolution of up to 70 nanometers, a potential boon for in vivo applications. With infrared nanoscopy, the light alters the frequency of naturally vibrating molecules in a way that creates vibration-based signatures for each of them. “You can see someone else because they have contrast against the background,” he says. In the same way, the infraredcaused vibration of an object is different from its background, allowing that object to be distinguished. However, because high-intensity infrared light can burn the sample, Dr. Tofail’s group has devised a tabletop system that uses a high-intensity
than normal, and sensitive instruments could detect the subtle changes. “If you know the exact location in the tissue where the pathology is at an early stage, you can figure out why it is happening and you can try to stop it from happening,” he says. Research groups are also trying to improve the STED method by scanning cells with up to 1000 laser beams at once. On its own, a single laser can only focus on 1 labeled molecule at a time. A whole army of them, however, could simultane-
The “dream” of cell biologists, he says, is to combine both techniques. Dr. Subramaniam and his colleagues recently used such correlative imaging to describe how the human immunodeficiency virus matures, in particular by imaging the viral core that delivers the viral genome to newly infected cells.1 “That’s very exciting because that lets us essentially get the best of both worlds: the localization and the live cell imaging where you can get the dynamics, and combine that with the ability to image the local, spatial architecture of where these things are in the cell.” As subcellular microscopy methods become more routine, Dr. Subramaniam says, researchers may be able to expand the technology to enable higher-resolution in situ or in vivo imaging within animal models of disease. With a method known as intravital fluorescence microscopy, for example, researchers are already zooming in on cellular processes deep within the tissues of living mice. One key to the future of subcellular microscopy will be its ability to answer biologically relevant questions. “Yes, the technology exists, but what do you use it for? It’s one thing to look at it in tissue culture cells, but it’s a whole different ballgame to look at it in a live animal,” Dr. Subramaniam says. Dr. Eggeling is optimistic about the progress toward useful applications. “This super-resolution microscopy is more and more applicable to living cells,
‘This super-resolution microscopy is more and more applicable to living cells, and for a longer period of time.’ —Christian Eggeling, PhD ously illuminate 1000 spots and greatly enhance the field of view. “I think that’s a very exciting frontier,” says Sriram Subramaniam, PhD, a senior investigator and microscopy expert at the National Cancer Institute in Bethesda, Maryland. Fluorescence-based microscopy methods, he says, have already helped the field move from qualitative descriptions of what is present in a cell to more quantitative descriptions of how many molecules are present and where. Electron microscopy, which Dr. Subramaniam favors, still achieves a higher resolution, although it requires researchers to kill cells in order to observe them.
and for a longer period of time,” he says. He too, however, agrees that the field will need to show off more than just its technological side. “In principle, we have shown that the microscopes work,” he says. The next step is proving that they can live up to their promise by providing new insights in cancer research, immunology, neurobiology, and other biomedical fields. Reference 1. Frank GA, Narayan K, Bess JW Jr, et al. Maturation of the HIV-1 core by a non-diffusional phase transition. Nat Commun. 2015;6:5854. DOI: 10.1002/cncy.21556
Content in this section does not reflect any official policy or medical opinion of the American Cancer Society or of the publisher unless otherwise noted. © American Cancer Society, 2015.
3 268
Cancer Cytopathology Cancer Cytopathology
May 2015 May 2015
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