What Is Interferometric Image Scanning Microscopy?
A new microscopy technique is changing how scientists view living cells. Researchers at Stanford University have introduced interferometricimage scanning microscopy (iISM) — a next-generation label-free imaging method. It achieves approximately 120 nm lateral resolution inside live cells. Crucially, it does so without fluorescent dyes or genetic tags.
Light microscopy remains essential in life sciences. It helps scientists visualize cellular structures and observe biological dynamics in real time. However, conventional fluorescence imaging carries significant drawbacks. Phototoxicity, limited labeling efficiency, and biological perturbation are persistent challenges. iISM directly addresses these limitations by combining two powerful concepts: interferometric scattering microscopy (iSCAT) and image scanning microscopy (ISM).
Why Label-Free Imaging Matters
The Problem With Fluorescence Microscopy
Fluorescence techniques have dominated cell biology for decades. Super-resolution methods like STORM, PALM, STED, and SIM push spatial resolution below the diffraction limit. Yet each relies on fluorescent labels. These labels introduce several problems.
First, phototoxicity limits how long cells can be imaged. Second, labeling efficiency is never perfect — some structures remain invisible. Third, the labeling process itself can alter cellular behavior. For studies of native biological processes, these are serious constraints.
iSCAT as a Label-Free Alternative
Interferometric scattering microscopy (iSCAT) offers a compelling alternative. It detects nanoscale structures through their light scattering — no dyes needed. iSCAT has detected single proteins and viruses on clean surfaces. More recently, confocal iSCAT has extended this capability into live cells, enabling subcellular imaging with high contrast. iISM builds directly on this foundation, taking label-free resolution to a new level.
How iISM Works
Combining iSCAT With ISM
Image scanning microscopy solves a long-standing trade-off in confocal microscopy. Closing the pinhole improves resolution but discards most detected photons, reducing signal-to-noise ratio (SNR). ISM replaces the single-element detector with an array detector. It then reassigns off-axis signals to their correct positions — a process called pixel reassignment. The result: both high resolution and high SNR at the same time.
iISM applies this concept to interferometric detection. The technique uses a 445 nm diode laser with circularly polarized light. Circular polarization minimizes scattering artifacts and improves the symmetry of the interferometric point-spread function (iPSF). Both reflected light from the coverglass and scattered light from the sample are collected through a high-NA oil immersion objective.
The Adaptive Pixel-Reassignment Algorithm
Standard pixel reassignment works well for incoherent fluorescence signals. Coherent interferometric signals are fundamentally different — they encode both amplitude and phase. Therefore, the team developed a modified adaptive pixel-reassignment (APR) algorithm tailored to coherent detection.
The key innovation is using the radial variance transform (RVT). RVT converts each interferogram into an intensity-only map reflecting local radial symmetry. This effectively removes phase modulations. The resulting RVT maps feed into the APR algorithm, which computes precise shift vectors. These vectors then realign the original interferometric data before summation. The final reconstruction achieves both enhanced resolution and superior contrast.
Key Performance Gains
Resolution at 120 nm
iISM achieves a measured lateral resolution of 120 nm ± 4 nm (FWHM). This matches the theoretical limit of approximately 115 nm for a √2 improvement over the illumination PSF. Furthermore, this resolution is maintained at roughly 10× lower incident illumination power than conventional confocal iSCAT.
Superior Contrast-to-Noise Ratio
The contrast-to-noise ratio (CNR) improvements are striking. Compared to closed-pinhole confocal iSCAT, iISM delivers a CNR approximately four times higher. Compared to open-pinhole confocal, it is roughly three times higher. At the same incident power and photon count, iISM simultaneously achieves both the highest resolution and the lowest noise floor. The team describes this as analogous to the “super-concentration of light” effect reported in fluorescence ISM.
Reduced Photodamage
Operating at 0.5 µW incident illumination power per focused spot, iISM enables essentially unlimited observation times in live cells. No visible photodamage or compromise of cellular integrity is observed. This is particularly valuable for imaging primary cells, where phototoxicity thresholds are typically even lower than in immortalized cell lines.
Live-Cell Imaging Results
Visualizing Intracellular Organelles
The researchers applied iISM to live COS-7 cells. They targeted major intracellular organelles across a single optical section spanning approximately 40 × 80 µm. Without any labels, iISM clearly resolved the following structures:
- Nucleus
- Mitochondria
- Endoplasmic reticulum (ER)
- Vesicles
- Actin cytoskeleton
- Plasma membrane and lamellipodia
Moreover, individual organelles show both positive and negative interference contrast. This reflects small axial displacements relative to the optical section, giving iISM intrinsic sensitivity to three-dimensional nanoscale morphology.
Tracking Organelle Dynamics
After applying a flat-field background correction, the team tracked dynamic events over extended time periods. At 8.2-second frame intervals, they observed vesicle motion and ER tubule remodeling in real time. Unlike fluorescence ISM, iISM has no photophysical upper limit on temporal resolution. Frame rate is limited only by the camera — not by any light-matter interaction. This opens possibilities for faster dynamic imaging with upgraded detectors.
Correlative Imaging Capability
iISM Plus Fluorescence ISM
iISM is compatible with existing confocal fluorescence ISM systems. To demonstrate this, the team performed correlative imaging on fixed COS-7 cells. They labeled actin with Phalloidin-Alexa Fluor 647 and imaged with both 445 nm (iISM) and 628 nm (fluorescence ISM) lasers sequentially.
The results are compelling. iISM reveals extended filamentous actin structures without labeling. Fluorescence ISM provides molecularly specific actin detection. Overlaying both images confirms excellent spatial correspondence between modalities. Furthermore, iISM detects additional unlabeled structures — vesicles and focal adhesions — that remain invisible in fluorescence.
Complementary, Not Competing
The two techniques offer different and complementary information. Fluorescence provides molecular specificity. iISM delivers structural contrast, context, and quantitative scattering information. Together, they present a more complete picture of cellular architecture. This hybrid approach is a key strength of the iISM platform.
Future Applications
Adaptable to Existing Instruments
iISM integrates readily into commercial confocal fluorescence ISM systems. Platforms such as Zeiss Airyscan, Nikon Nsparc, and PicoQuant Luminosa can adopt iISM. The upgrade requires substituting the main dichroic mirror with a polarizing beam splitter and adding a quarter-wave plate. This low barrier to adoption accelerates real-world implementation.
Expanding the Technique
Several exciting extensions are possible. Replacing the sCMOS camera with a SPAD array would improve temporal resolution significantly. Parallelized spinning-disk detection schemes could push imaging toward the millisecond timescale. Additionally, computational staining approaches — using neural networks trained on paired fluorescence and scattering data — could add molecular specificity to purely label-free acquisitions.
Combining iISM with single-molecule fluorescence ISM (SM-ISM) represents perhaps the most ambitious future direction. This hybrid strategy would unite ultrasensitive scattering detection with molecular specificity, potentially transforming how scientists study cell structure and function.
Broader Biological Impact
iISM opens new paths for studying dynamic biological processes. Host-pathogen interactions, intracellular trafficking, and cytoskeletal rearrangements can now be observed under near-native, label-free conditions. As a result, iISM offers a powerful platform for biological discovery with minimal perturbation and maximum resolution.
