Stanford built a microscope that watches inside living cells at about 120 nanometers without dyes

Stanford chemists have built a microscope that images the inside of living cells at about 120 nanometers without any fluorescent dye or stain. The instrument, called interferometric image scanning microscopy, or iISM, comes from the lab of W.E. Moerner, who shared the 2014 Nobel Prize in Chemistry for super-resolution fluorescence microscopy. The new tool dispenses with fluorescence altogether.

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Three collaborations are lined up to use it, from cancer drug uptake to malaria, and the researchers want it alongside fluorescence rather than in place of it.

Two imaging methods fused into iISM

The design combines interferometric scattering microscopy, which detects light scattered by cellular structures, with the array-detector scanning approach used in advanced confocal microscopes. The pairing is described in the Stanford news story on the instrument and in the paper by first author Michelle Küppers, a postdoctoral scholar in Moerner’s lab whose doctoral work focused on interferometric scattering microscopy, and Moerner, published in the Nature-portfolio journal Light: Science and Applications.

Conventional scattering-based imaging can yield high-contrast pictures of unlabeled cells, but it tends to demand more light than a living cell tolerates. The iISM design aims for a better trade by recording many detector pixels at every scan position and then realigning them in software. The preprint describes an adaptive pixel-reassignment algorithm adapted to coherent detection, which the authors use to recover the sharper image from the array detector without adding illumination.

A bioRxiv preprint of the work supplies the technical detail. Lateral resolution measured 120 nanometers, with a spread of 4 nanometers, similar to the roughly 122 nanometers of a closed-pinhole confocal version of the technique, while contrast roughly doubled. The authors report a contrast-to-noise ratio of about 38, compared with about 10 for closed-pinhole and about 14 for open-pinhole confocal interferometric scattering. Live-cell imaging used about 0.5 microwatts of incident power, which the authors report as about ten times less than conventional confocal microscopy per diffraction-limited spot.

Why skipping the stain matters

Fluorescent labels have two known drawbacks. They can bleach under illumination, which limits how long a cell can be watched, and they may alter the behavior of the structures they tag. A label-free method sidesteps both problems and can image many structures at once over long periods. The School of Humanities and Sciences summary says the lower illumination power also reduces the risk of photodamage to living cells.

The preprint tested the approach on COS-7 cells, a standard cell line, imaging the endoplasmic reticulum, mitochondria, vesicles and actin in living cells. Moerner, the Harry S. Mosher Professor of Chemistry, said the instrument gives a fantastic new view into the cell. In another remark in the Stanford coverage, he called it a wonderful look into the complex little cellular boxes that drive life.

The practical payoff is time. A cell watched with fluorescent labels can only be observed for as long as the dye survives and the light does not harm it, whereas a method that works at roughly half a microwatt, as the preprint reports, can in principle keep looking. Researchers studying slow events, such as a drug working its way into a cell or an organelle changing position, benefit most from that patience.

Complement to fluorescence, with limits

Küppers is explicit that iISM is no replacement for fluorescence. She said every method has advantages and disadvantages and that the team envisions a complementary use of the two, which can be combined on the same sample. In the preprint, fixed cells labeled with a fluorescent dye were imaged with both methods for exactly that kind of comparison.

The limitations are spelled out. The resolution of about 120 nanometers is not as fine as that of some specialized microscopes, and scattering from surrounding structures can obscure fine features such as the actin mesh that fluorescence imaging shows more clearly. The preprint is also frank about its sample: calibration relied on a single set of 60-nanometer polystyrene nanoparticles. It also notes that circular polarization was required, that temporal resolution is limited by the camera frame rate, and that results rest on a limited number of imaged cells, so independent groups will need to test how well the approach carries over to other cell types.

Cancer drugs, malaria and plant microbes

Three collaborations are described in the Stanford School of Humanities and Sciences announcement. One watches plant cells, fungi and bacteria interact in real time. A second follows how a cancer drug enters a cell. A third, at the planning stage per the ScienceDaily version, will examine how red blood cells change shape during malaria infection. Küppers said the technique is not niche and has broad applications.

The work was funded by the U.S. National Institute of General Medical Sciences. Moerner’s own prize, listed by the Nobel Prize organization as awarded in 2014 for the development of super-resolved fluorescence microscopy, was earned with the very labeling approach that this instrument now complements. The ScienceDaily report carried the story to a wider audience on October 16.

This article was produced with the assistance of AI and reviewed by Morning Overview editors prior to publication.

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