Researchers at the National Institute of Standards and Technology (NIST) have converted the problem of lens aberrations, which cause imperfect focusing of light by optical microscopes, into an ability to accurately measure the positions of points of light in three dimensions instead of the two typical with conventional microscopes.

This opens up the possibility of gaining considerably more information about 3Dl structures such as biological samples including DNA, tissue, an organ or a microscopic organism.

Other methods that have enabled microscopes to provide detailed information about 3D structures have been expensive or required specialized knowledge. One such example is altering the microscope’s optics with extra astigmatism, which requires reengineering and recalibration of the microscope.

With the new measurement method, the positions of objects can be located with greater accuracy and precision, pinpointing the positions of light-emitting particles within a region one-hundredth the size of what is normally possible with an optical microscope.

The NIST researchers carefully analyzed images of fluorescent particles that they deposited on flat silicon wafers for calibration of the microscope. Lens aberrations as the microscope moved along the third dimension (the vertical axis) made the images appear to change, creating large distortions even with just a few micrometers of movement in the lateral plane or a few tens of nanometers in the vertical dimension. By calibrating the changing appearance and the apparent location of a particle to its vertical position, they could accurately measure positions on three dimensions.

Left: Images of fluorescent particles that are above, at and below (top to bottom) the vertical position of best focus of a microscope. Calibrating the effects of lens aberrations on the apparent shape and position of the particle images enables accurate measurement of the position in all three spatial dimensions using an ordinary optical microscope. Right: Tracking and combining information from many fluorescent particles on a tiny rotating gear tests the results of the new calibration and elucidates the motion of a complex microsystem in all three dimensions. Source: NISTLeft: Images of fluorescent particles that are above, at and below (top to bottom) the vertical position of best focus of a microscope. Calibrating the effects of lens aberrations on the apparent shape and position of the particle images enables accurate measurement of the position in all three spatial dimensions using an ordinary optical microscope. Right: Tracking and combining information from many fluorescent particles on a tiny rotating gear tests the results of the new calibration and elucidates the motion of a complex microsystem in all three dimensions. Source: NIST

"Counterintuitively, lens aberrations limit accuracy in two dimensions and enable accuracy in three dimensions," said Samuel Stavis. "In this way, our study changes the perspective of the dimensionality of optical microscope images, and reveals the potential of ordinary microscopes to make extraordinary measurements."

The calibration method was tested by using the microscope to image a constellation of fluorescent particles randomly deposited on a microscopic silicon gear that rotated in 3D. The model accurately corrected for the lens aberrations and provided full 3D information about the position of the particles. They extended their position measurement to include the full range of motion of the gear: rotating, wobbling and rocking.

Microscopy laboratories could easily implement the new method. "The user just needs a standard sample to measure their effects and a calibration to use the resulting data," added Stavis. Aside from the fluorescent particles or a similar standard, which already exist or are emerging, no extra equipment is needed.

The work described in Nature Communications includes demonstration software that guides researchers in how to apply the calibration.

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