Optical Microscope With Atomic Accuracy

This illustration shows an array of apertures with a spacing of 5000 nanometers ± 1 nm. The apertures pass light through a metal film on a glass slide. Imaging the aperture array with an optical microscope results in apparent errors in the spacing between apertures. Knowledge of the true spacing allows correction of these imaging errors. (Source: NIST)

Over the last two decades, scientists have dis­covered that the optical micro­scope can be used to detect, track and image objects much smaller than their traditional limit about half the wave­length of visible light. That pio­neering research, which won the 2014 Nobel Prize in Chemistry, has enabled researchers to track proteins in ferti­lized eggs, visualize how molecules form electrical connec­tions between nerve cells in the brain, and study the nanoscale motion of miniature motors. Now, research developments at the National Institute of Standards and Tech­nology NIST enable the micro­scopes to measure these nano­meter-scale details with a new level of accuracy.

“We put the optical micro­scope under a micro­scope to achieve accuracy near the atomic scale,” said Samuel Stavis, who served as the project leader for these efforts. Because optical micro­scopes have not tradi­tionally been used to study the nanometer scale, they typically lack the cali­bration necessary to obtain infor­mation that is accurate at that scale. A micro­scope may be precise, consis­tently indi­cating the same position for a single molecule or nano­particle. Yet, at the same time, it can be highly inaccurate – the location of the object identi­fied by the micro­scope to within a billionth of a meter may be millionths of a meter off due to unac­counted-for errors. “Precision without accuracy can be very mis­leading,” said Jon Geist, a co-author of the study.

To address the problem, NIST has developed a new cali­bration process that closely examines and corrects these imaging errors. The process uses reference materials – objects with charac­teristics that are well-known and stable – that have the potential for mass production and widespread distri­bution to indi­vidual labora­tories. This is important because optical micro­scopes are common labora­tory instruments that can easily magnify different samples, ranging from delicate biological specimens to electrical and mechanical devices. As well, optical micro­scopes are becoming increa­singly capable and economical as they incor­porate scientific versions of the lights and cameras in smart­phones.

The team relied on nano­meter-scale fabri­cation processes to develop the reference material. The researchers used electron beams and ion milling to form an array of pinhole apertures through a thin film of platinum on a glass slide. The process enabled the team to space the apertures 5,000 nano­meters apart, to within an accuracy of about 1 nanometer. In this way, the researchers built a measure of accuracy into the aperture posi­tions. Shining light through the array of apertures creates an array of points for imaging. But because all micro­scope lenses have imper­fections, errors inevitably occur during imaging that change the apparent positions of the points, making the spacing between the apertures appear to be larger or smaller than the actual spacing engineered by the team. Knowledge of the true spacing allows correction of the imaging errors and cali­bration of the microscope for measure­ments of position with high accuracy across a wide field of view.

Even a small error can lead to a large problem. Consider, for example, a micro­scope having an actual magni­fication of 103 times when the expected magni­fication, as specified by the manu­facturer, is 100 times. The resulting error of 3 percent adds up over large distances across a micro­scope image. Because of lens imper­fections, a subtler problem also occurs – the micro­scope magni­fication changes across the image, causing image distortion. To solve this problem, the team designed aperture arrays and cali­bration processes that worked across large fields of view.

The aperture arrays, which would enable individual researchers to perform cali­brations in their own labora­tories, could improve by a factor of 10,000 the ability of optical micro­scopes to accurately locate the position of single molecules and nano­particles. “We have identi­fied and solved an under­appreciated problem,” said Craig Copeland. Having cali­brated their optical micro­scope using the arrays, the team reversed the process, using their micro­scope to identify imper­fections in the proto­type arrays from the nanofabri­cation process. “We tested the limits of nanofabri­cation to control the aperture spacing,” noted Rob Ilic, manager of NIST’s NanoFab. The ease and speed of optical micro­scopy could faci­litate quality control of aperture arrays in a production process.

Finally, the team exploited the inherent stabi­lity of the aperture arrays to evaluate whether fluores­cent nano­particles, often used as fixed points of reference in optical micro­scopy, actually remained fixed to a parti­cular point or if they moved around. The researchers found that while uninten­tional motions of their optical micro­scope made views of the nano­particles blurry, using the aperture array showed that the nano­particles were not actually moving at atomic scales. (Source: NIST)

Reference: C. R. Copeland et al.: Subnanometer localization accuracy in widefield optical microscopy, Light: Sci. & App., online 16 May 2018; DOI: 10.1038/s41377-018-0031-z

Link: Center for Nanoscale Science and Technology, National Institute of Standards and Technology NIST, Gaithersburg, USA

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