A recent study has examined a label-free microscopy technique, interferometric image scanning microscopy (iISM), which is designed to deliver high resolution and contrast in live cells with substantially reduced light dose.
The research was conducted by Dr Michelle Kueppers, lead author, and Dr W. E. Moerner, professor of chemistry and by courtesy of applied physics at Stanford University.
“By parallelising 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,” said Kueppers.
Fluorescent labels can photobleach, cause phototoxicity and sometimes alter the processes researchers aim to observe. Many label-free methods avoid these issues by imaging native optical signals from cells. However, they typically struggle with sensitivity, contrast and resolution in the crowded, highly scattering environment inside living specimens.
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.
Although iSCAT can detect very small scatterers, applying it inside cells is challenging because background scattering from many intracellular components can overwhelm the signal of interest. While confocal implementations suppress out-of-focus background, they use a small pinhole that discards many photons, forcing either higher illumination power or slower aging.
“Confocal iSCAT already gives excellent optical sectioning, but a single pinhole detector forces a tough tradeoff between background rejection, resolution and photon efficiency,” said Kueppers
iISM replaces the single confocal pinhole detector with an array detector, such as a camera. This enables the microscope to record the full interferometric point-spread function (iPSF) at each scan position and capture many “off-axis pinholes” in parallel.
The researchers then combined these parallel measurements into a reconstructed image with improved resolution and contrast-to-noise ratio through a modified pixel reassignment algorithm.
“Our next goal is to push iISM toward faster dynamics and enable widespread adoption, by improving acquisition speed and making the technique widely accessible,” said 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.”
