Simple New System Keeps optical Microscopes in Focus Automatically and robustly

Good news!

"Now a team of scientists ... has developed an inexpensive robust fix for this problem that involves little more than a couple of LED lights and some physics-based processing. They describe the new autofocus technique, which they call Digital Defocus Aberration Interference (DAbI) ...

The underlying concept is fairly simple. When two LEDs illuminate a sample from slightly different angles, the combined signal derived from two photographs (one taken at each source of illumination) reveals a hidden fringe, or pattern of stripes. Those stripes change in a predictable way depending on how far the sample is from the focal point, the sweet spot where an image comes into focus. Therefore, a computer reading the stripes can tell the microscope how to correct for any blurriness in the image. ...

The scientists have tested DAbI on six different types of microscopes—from basic compound light microscopes to more complex systems used for imaging living cells and tissues, or even thick 3D specimens—all with excellent results. When dealing with thin flat samples, DAbI kept images in focus across a range more than 400 times larger than the natural depth of focus of a basic microscope lens. ... "This makes it useful and powerful for automated, high-throughput microscopy."

the DAbI technique is unique in that it can be used to locate the plane in which the focal point exists, even in thick 3D samples. Indeed, for thicker 3D samples up to 150 micrometers deep ..."

From the abstract:

"Automation in optical microscopy is critical for enabling high-throughput imaging across a wide range of biomedical applications. Among the essential components of automated systems, robust autofocusing plays a pivotal role in maintaining image quality for both single-plane and volumetric imaging.

However, conventional autofocusing methods often struggle with implementation complexity, limited generalizability across sample types, incompatibility with thick specimens, and slow feedback.

We observed that the digitally summed Fourier spectrum of two images acquired from two-angle illumination exhibits interference-like fringe modulation when the sample is defocused.

These digital fringes correlate directly with defocus through a physics-based relation.

Based on this principle, we developed an automatic, efficient, and generalizable defocus detection method termed digital defocus aberration interference (DAbI).

Implemented with a simple two-LED setup, DAbI can quantify the defocus distance over a range of 443 times the depth-of-field for thin samples and 296 times for thick specimens.

It can additionally extend the natural depth-of-field of the imaging system by 20-fold when integrated with complex-field imaging.

We demonstrated the versatile applications of DAbI on brightfield, complex-field, refractive index, confocal, and widefield fluorescence imaging, establishing it as a promising solution for automated, high-throughput optical microscopy."

Simple New System Keeps Microscopes in Focus Automatically - www.caltech.edu "Anyone who has ever used a microscope knows that it takes time to bring a sample into sharp focus. Each time you move the slide, the image blurs, and you have to stop and carefully turn a knob to bring everything back into clear view. For scientists and clinicians, even if the motion is semi-automated, that time quickly adds up as they work with dozens or hundreds of samples."

Digital defocus aberration interference for automated optical microscopy (open access of early version)

A schematic image of DAbI integration with an optical microscope setup.