![\"two](\"https:\/\/www.newsbeep.com\/us-ca\/wp-content\/uploads\/2026\/06\/hemoglobin.jpg\")
Cryo-EM images of the blood protein hemoglobin, showing the much-improved contrast achieved with a laser phase plate. The images, which show many hemoglobin molecules, are analyzed to assemble a detailed 3D structure of the protein.<\/p>\nNearly 100 years ago, a seemingly simple discovery revolutionized the microscope. The introduction of phase-contrast, which garnered a Nobel Prize in 1953, brought into clear view structures inside cells that had previously been too faint or washed out for biologists to study.<\/p>\n
UC Berkeley physicists have now adapted the phase contrast technique to the electron microscope, which has about 10,000 times the magnification of microscopes using optical light.<\/p>\n
The addition of a so-called laser phase plate has the potential to greatly improve cryoelectron microscopy (cryo-EM), a technique for determining the structure of molecules that itself revolutionized the understanding of proteins and accelerated new drug discovery starting a decade ago. Despite its impact, however, cryo-EM still struggles to produce clear images of small molecules \u2014 including most human proteins. A laser phase plate promises clear images of most proteins in the cell down to one-third the size of those that are a challenge for today\u2019s machines.<\/p>\n
The addition of a laser phase plate seems certain to revolutionize a newer technique referred to as cryoelectron tomography (cryo-ET), which assembles a number of different angular views of a molecule or protein into a 3D image. This makes it possible to analyze proteins in their natural environment \u2014 inside cells \u2014 instead of in isolation in a solution.<\/p>\n
Cryo-EM images of the blood protein hemoglobin, showing the much-improved contrast achieved with a laser phase plate. The images, which show many hemoglobin molecules, are analyzed to assemble a detailed 3D structure of the protein.<\/p>\n
Holger M\u00fcller, Jessie Zhang\/UC Berkeley<\/p>\n
\u201cCryo-EM has become the new, fastest-growing method for resolving the structure of biological macromolecules, and cryo-ET is expected to show how these molecules work together in their natural, cellular context,\u201d said Holger M\u00fcller<\/a>, a UC Berkeley professor of physics and faculty scientist at Lawrence Berkeley National Laboratory who led the development effort. \u201cBut because of signal-to-noise limitations, the majority of human and animal proteins are too small to be analyzed by these methods. The increase in signal-to-noise ratio provided by this laser phase plate is expected to overcome these important limitations.\u201d<\/p>\n Crucial to the development is the world\u2019s most intense, focused continuous-wave laser, which interacts with the electron beam to change its phase. This phase change boosts contrast for small molecules, such as hemoglobin, and for molecules and structures inside cells, such as the nucleus and mitochondria.<\/p>\n \u201cWith cryo-ET, we\u2019re looking at small, very complicated cellular material that\u2019s incredibly crowded inside the cell,\u201d said Bridget Carragher, founding technical director of imaging at Biohub<\/a> in Redwood City, California \u201cIt\u2019s like a forest of trees, and you\u2019re trying to find one leaf on one tree in there. Cryo-ET needs a dramatic step forward in contrast, so we can start to see what\u2019s going on inside the cell. That\u2019s what the laser phase plate promises to give us.\u201d<\/p>\n Biohub provided funding to M\u00fcller to purchase a state-of-the-art cryo-EM machine that he then outfitted with a laser phase plate, creating a microscope he calls Theia, named after the ancient Greek Titaness of light and radiance. Carragher is overseeing the development of a similar instrument at Biohub\u2019s imaging lab in Redwood City \u2014 this one featuring a dual-laser system, based on theoretical work<\/a> by M\u00fcller and his colleagues. In this system, the two perpendicular laser beams operate at about half power, making the components less likely to burn out and reducing aberrations.<\/p>\n Both groups are collaborating with the firm Thermo Fisher Scientific<\/a>, the primary manufacturer of cryo-EM machines.<\/p>\n Holger M\u00fcller explains how a laser phase plate works and how it improves the contrast of cryoelectron microscope (cryo-EM) images. (Video credit: Biohub)<\/p>\n \u201cTheia is the Formula 1 microscope,\u201d M\u00fcller said. \u201cIt has extra electron optics that give it better resolution than the standard cryo-EM, even without the laser. With the addition of the laser phase plate, we hope that it really becomes the world\u2019s best instrument overall.\u201d<\/p>\n M\u00fcller and his Berkeley team published their newest images and details of the cryo-EM\u2019s laser phase plate<\/a> today (June 11) in the journal Science. Biohub\u2019s two-laser system is described in an online preprint<\/a>.<\/p>\n Biological imaging<\/p>\n Animal and plant cells are mostly water and thus transparent in a light microscope, which should make it easy to see structures such as the nucleus and mitochondria inside. But these structures are small and scatter only a small amount of light, which makes them only slightly darker than the rest of the cell\u2019s insides. This low contrast has typically been improved by staining the cell, though staining also kills the cell.<\/p>\n Robert Sanders\/UC Berkeley<\/p>\n In 1930, Dutch scientist Frits Zernike realized that the brightness or amplitude of the light was not the only feature affected when passing through a cell. The scattered light is also slowed down in a biological sample, which shifts its phase \u2014 the timing of the peak of the waveform \u2014 by a small amount. While this phase shift is invisible to the human eye, it can be turned into visible contrast by also phase shifting the non-scattered light by 90 degrees. When the scattered and non-scattered light are ultimately focused on the retina, features in the sample are enhanced relative to the background, boosting the contrast. Zernike received the 1953 Nobel Prize in Physics<\/a> for this discovery.<\/p>\n By the early 1940s, the phase-contrast microscope had proved its value and scientists speculated about adapting this technique to increase contrast in the electron microscope, which uses a beam of electrons to image much smaller structures, such as proteins. But attempts to make a phase plate that shifts the phase of an electron beam reduced the beam intensity too much, made the images unstable, or resulted in lower resolution.<\/p>\n In 2010, M\u00fcller and Robert Glaeser<\/a>, now a Berkeley professor emeritus of molecular and cell biology, wrote a paper <\/a>proposing a way to create the phase shift by using an intense laser<\/a>, which would not dim the electron beam.<\/p>\n Glaeser is a pioneer of cryo-EM, a major improvement in electron microscopy and theoretically a simpler method for determining molecular structures than X-ray crystallography, which requires that a molecule actually forms a crystal and that the researcher has access to a bright source of X-rays. But a major problem with electron microscopy is that the electron beam heats up and eventually damages its target, limiting image detail. Coating the specimen with metal to prevent this and enhance the contrast only makes fuzzier images.<\/p>\n In the 1960s, scientists proposed freezing samples to slow down sample destruction. Glaeser demonstrated that freezing samples reduced radiation damage and proposed reducing damage even further by lowering the power of the electron beam while irradiating thousands of frozen molecules simultaneously. Though each molecule in the sample would be in a random orientation, computers could combine all the images to create a highly detailed structure.<\/p>\n The originators of cryo-EM were awarded a Nobel Prize in Chemistry in 2017, and in their acceptance remarks credited Glaeser\u2019s work. According to the Nobel Committee, cryo-EM \u201cboth simplifies and improves the imaging of biomolecules. This method has moved biochemistry into a new era.\u201d<\/p>\n After the publication of the 2010 paper, M\u00fcller spent 15 years realizing the goal of a laser phase plate for cryo-EM, funded in part by a grant from the National Institutes of Health. First, he and his team had to develop a way to focus a continuous laser onto a small spot to create light intense enough to shift the phase of an electron beam by 90 degrees. After 10 years, they achieved this by trapping the laser beam in a spherical, mirrored cavity that both focuses the beam and intensifies it as the light bounces back and forth more than 10,000 times. The entire optical cavity housing the laser phase plate is less than four inches wide, tucked inside a microscope that stand 14 feet tall.<\/p>\n \u201cIt\u2019s 75 kilowatts focused to a few microns,\u201d M\u00fcller said. \u201cThat\u2019s more powerful than what you use for welding. It\u2019s more power than a military laser. It builds up the brightest continuous laser focus ever.\u201d<\/p>\n Holger M\u00fcller, Jessie Zhang\/UC Berkeley<\/p>\n![\"a](\"https:\/\/www.newsbeep.com\/us-ca\/wp-content\/uploads\/2026\/06\/MullercryoEM-crop-714x1024.jpg\")
Holger M\u00fcller standing in front of the Thermo Scientific Krios cryo-EM outfitted with a novel laser phase plate to dramatically improve the microscope\u2019s contrast and ability to image smaller molecules and smaller structures inside cells.<\/p>\n![\"two](\"https:\/\/www.newsbeep.com\/us-ca\/wp-content\/uploads\/2026\/06\/apoferritin.jpg\")
Cryo-EM images of the iron storage protein apoferritin, without and with a laser phase plate. The images, which are of many apoferritin molecules, are analyzed to produce a detailed 3D structure of the protein.<\/p>\n
![\"a](\"https:\/\/www.newsbeep.com\/us-ca\/wp-content\/uploads\/2026\/06\/Imaging-Krios-Lab-5-sm.jpg\")
A Thermo Scientific Krios cryoelectron microscope that Biohub has outfitted with two powerful lasers to improve image contrast.<\/p>\n