Sometimes It Is Good to Interfere

As any first-year physics student can remind you, the resolution of photon microscopy is limited by the diffraction of light. Because light travels as waves, an infinitely small point of light cannot be focused to a similar small point; rather, it can only be focused to a small spherical spot, the Airy disk. The size of the Airy disk varies depending on the wavelength of the light, the refractive index of the imaging media, and the angular aperture of image collection. This limits the standard resolution of a microscope using visible light to about 200nm. Below that limit, the information is blurred. However, over the past two decades, biological microscopists have come up with very creative ways to tease out additional information about cell structure and use this information to predict what is happening within the blur. In this way they can partially correct the blurring. These methods, providing corrected resolutions below the 200nm limit, fall under the heading of nanoscopy techniques, with some “super resolution” techniques achieving resolutions in the 20nm range [1]. Although these techniques provide unprecedented information about cell architecture, and with some techniques also about cell function, they are very specialized and do have their drawbacks. Almost all techniques require fluorescence. Thus, you need a fluorescence probe that targets the structures of interest and one that is also amenable to the requirements of your specific technique. Most of the nanoscopy techniques also require long image collection times with strong excitation light, so photobleaching and cell damage becomes an issue. These are only a few of the issues limiting these techniques from becoming universally applicable to a wide array of cell biology questions. However, in a 2026 paper in Light: Science & Applications, Michelle Küppers and W. E. Moerner describe a new approach to live cell imaging that can achieve 120nm lateral resolution without the need for fluorescent probes [2]. They call their technique “interferometric image scanning microscopy” (iISM). It is based on two proven photon imaging techniques: interferometric scattering microscopy, and laser scanning confocal microscopy (LSCM). iISM is a label-free technique that can detect nanoscale structures based on their light scattering [3]. LSCM provides superior axial resolution by blocking out light originating above or below the focal point, but also can increase the lateral resolution by up to a factor of the square root of 2 if the detection pinhole is closed down below one Airy unit [4]. It is not often used that way, however, because the restrictive pinhole severely limits the number of photons reaching the detector, degrading the signal-to-noise ratio (SNR). By replacing the normal single detector with an array detector that can capture the off-axis signals, much of the SNR can be regained by reassigning the off-axis scatter back to the central pixel [5]. Since interferometric scattering does not require specific labels, it can identify multiple structures in a cell by their scattering density without intrusive and restrictive fluorescent labeling. The laser illumination power required for iISM is about 10× less than that used in traditional fluorescence LSCM. This lessens the potential for beam damage to the specimen and facilitates live cell imaging over extended periods to collect dynamic information regarding cell function. Using their technique, including new algorithms for image reconstruction from interferometric data, the authors could identify nuclei, mitochondria, endoplasmic reticulum, cytoskeletal elements, and endocytic vesicles within cells and tracked their movement and structural changes over time. The work of Küppers and Moerner expands the arsenal of nanoscopy techniques and provides exciting opportunities for live cell imaging of cells to capture dynamic processes over a very long-time scale. Moreover, the current generation of laser scanning confocal microscopes, particularly those already having array detectors, can be readily adapted to the iLSM technique.