Microscopes have driven scientific progress since the 16th century, but major improvements have often required increasingly specialized tools. As techniques became more advanced, they also faced tradeoffs in what they could measure. Quantitative phase microscopy (QPM) uses forward-scattered light to visualize structures at the microscale (in this study, over 100 nanometers), which makes it useful for capturing still images of complex cell features. However, QPM cannot detect very small particles. Interferometric scattering (iSCAT) microscopy works differently by capturing back-scattered light and can detect structures as tiny as single proteins. While iSCAT enables researchers to "track" individual particles and observe rapid changes inside cells, it lacks the wider view offered by QPM.

Capturing Two Directions of Light at Once

"I would like to understand dynamic processes inside living cells using non-invasive methods," says Horie, one of the first authors.

Motivated by this goal, the team examined whether collecting light from both directions at the same time could bridge the gap and reveal activity across a broad range of sizes and motions in a single image. To explore the idea and confirm that their microscope performed as expected, they observed how cells behaved during cell death. In one experiment, they captured an image that contained information from both forward- and backward-traveling light.

Separating Overlapping Signals

"Our biggest challenge," Toda, another first author, explains, "was cleanly separating two kinds of signals from a single image while keeping noise low and avoiding mixing between them."

The researchers succeeded in identifying the movement of larger cell structures (micro) as well as much smaller particles (nano). By comparing the patterns in forward- and back-scattered light, they could estimate each particle's size and its refractive index, which describes how strongly light bends or scatters when it passes through a material.

Future Applications for Smaller Particles

"We plan to study even smaller particles," Toda says, already thinking about future research, "such as exosomes and viruses, and to estimate their size and refractive index in different samples. We also want to reveal how living cells move toward death by controlling their state and double-checking our results with other techniques."