Fluorescence microscopy is indispensable for biology, but fluorescent labels can photobleach, cause phototoxicity, and sometimes alter the very processes researchers aim to observe. Label-free methods avoid these issues by imaging native optical signals from cells, yet they typically struggle with sensitivity, contrast and resolution in the crowded, highly scattering environment inside living specimens.
In a new study published in Light: Science & Applications (Nature Portfolio), researchers from Stanford University report interferometric Image Scanning Microscopy (iISM) , a label-free microscopy technique designed to deliver high resolution and contrast in live cells with substantially reduced light dose. The research was conducted by Dr. Michelle Kueppers and Dr. W. E. Moerner, Professor of Chemistry and by courtesy of Applied Physics at Stanford University.
iISM builds on interferometric scattering microscopy (iSCAT) , which is exceptionally sensitive because it measures interference between weak light scattered by nanoscale structures and a strong reference reflection. While iSCAT can detect extremely small scatterers, applying it inside cells is challenging because background scattering from many intracellular components can overwhelm the signal of interest. Confocal implementations suppress out-of-focus background, but they typically do so by using a small pinhole that discards many photons, forcing either higher illumination power or slower imaging.
The key advance of iISM is to replace the single confocal pinhole detector with an array detector , such as a camera . This allows the microscope to record the full interferometric point-spread function (iPSF) at each scan position and to capture many “off-axis pinholes” in parallel. The team then developed a modified pixel reassignment algorithm, that accounts for the interferometric phase, in order to combine these parallel measurements into a reconstructed image with improved resolution and contrast-to-noise ratio.
A useful analogy is human vision: with one eye, foreground and background can be harder to disentangle; with two eyes, parallax makes separation easier. In iISM, the camera provides not two but tens to hundreds of “eyes” viewing from different angles, enabling robust estimation and suppression of background contributions that would otherwise dominate interferometric scattering images.
“Confocal iSCAT already gives excellent optical sectioning, but a single pinhole detector forces a tough tradeoff between background rejection, resolution and photon efficiency,” says Michelle Kueppers, Postdoctoral Researcher in the Moerner lab and lead author of this study. “By parallelizing interferometric imaging and adding modified computational reassignment, iISM recovers information that would otherwise be discarded, so we can improve contrast and resolution without increasing the light dose.”
Using iISM, the researchers demonstrate ~120 nm lateral resolution in the label-free mode and report an imaging efficiency improvement that can be traded for either ~10× faster acquisition at the same laser power or ~10-fold lower illumination power at the same imaging speed , an important advantage for live-cell viability and long-term recordings. In demonstrations in live specimens, iISM visualizes intracellular structures and their dynamics including the endoplasmic reticulum, mitochondria, lysosomes, and vesicles without fluorescent labels. Continuous imaging produces a fascinating dance of the motion and rearrangements of many organelles in the cell. Crucially, the approach is also compatible with simultaneous confocal fluorescence microscopy , enabling correlative imaging that combines label-free structural context with molecular specificity when needed.
The authors expect iISM to open new opportunities for observing nanoscale cellular dynamics under near-native conditions, including studies of intracellular trafficking, organelle network remodeling, host–pathogen interactions, and cytoskeletal rearrangements, especially in situations where fluorescence labelling is limited or not readily available.
“Our next goal is to push iISM toward faster dynamics and enable widespread adoption, by improving acquisition speed and making the technique widely accessible,“ says W. E. Moerner. “By combining ultrasensitive scattering detection with the molecular specificity from fluorescence microscopy, we anticipate that these hybrid strategies will deepen our understanding of cellular structure and function and establish iISM as a next-generation label-free microscope method.”
Light Science & Applications
Interferometric Image Scanning Microscopy for label-free imaging at 120 nm lateral resolution inside live cells