Optical fluorescence microscopy helps visualize biological cells that are normally invisible under regular light, offering high resolution and precise targeting. In contrast, label-free microscopy doesn’t rely on dyes or markers—it uses natural optical properties like light polarization to study samples.
Mueller Matrix (MM) microscopy is a label-free technique that analyses how polarized light changes after passing through a sample. This change reveals structural details without needing chemical labels. The method uses a setup with a polarization generator and a polarization analyser, often involving rotating polarizers or advanced cameras to capture data faster.
New technologies like metasurfaces and lensless holography are expanding MM microscopy’s capabilities. Some systems use photoelastic modulators or Zeeman lasers to control light polarization quickly and without moving parts.
In this context, researchers at the Italian Institute of Technology propose a new Mueller Matrix microscope scheme. This system uses a scanning setup with a photoelastic modulator to rapidly control light polarization without mechanical movement. A darkfield-style detection method improves image contrast, allowing for high-quality capture of the off-diagonal elements of the Mueller matrix.
Insigths into Muller Matrix microscopy
Label-free microscopy achieves super-resolution by analyzing optical properties—especially light polarization, which is highly sensitive to interactions with matter.
Mueller Matrix (MM) microscopy uses the Mueller-Stokes formalism to detect sample anisotropies by observing changes in the polarization state of light after it interacts with a specimen—no labeling agents required.
A typical MM microscope includes a Polarization State Generator (PSG) and a Polarization State Analyzer (PSA). These can be implemented with rotating polarizers, waveplates, or advanced setups like polarization-sensitive cameras, metasurfaces, and lensless holography.
Although MM microscopy is commonly used for studying tissues, it can detect extremely small structural changes—down to 1/20th of the light’s wavelength. This makes it promising for examining cells and potentially linking MM data with molecular insights from super-resolution fluorescence microscopy.
this scope, researchers Paolo Bianchini, Alberto Diaspro and Nicolò Incardona at the Italian Institute of Technology aimed to link MM microscopy with molecular insights from super-resolved fluorescence microscopy, enhancing biological analysis without the need for fluorescent labels.
Optical setup of the MM microscope proposed by Paolo Bianchini and Alberto Diaspro
The optical setup used is illustrated in Figure 1. It utilizes FYLA´s supercontinuum laser as the light source, with the desired wavelength selected via an acousto-optic tunable filter. A Glan-Thompson prism is employed to isolate vertically polarized light. For scanning, a commercial Nikon C2 unit is used in conjunction with its dedicated control module, allowing image acquisition to be managed through NIS Elements software. Following the scan and tube lenses, half-wave and quarter-wave plates are used to compensate for phase delays caused by mirror reflections. A photoelastic modulator, positioned at a 45° angle, alters the polarization state at approximately 50 kHz, generating a dynamic birefringence.
Figure 1. Optical setup of the MM microscope.
After calibration, tissue samples produced clear Mueller matrix images, but cell samples lacked signal. To improve contrast, a darkfield-like detection scheme, shown in Figure 2 was added using a 3D-printed stop that blocks direct light and captures scattered light.
Figure 2. Darkfield detection scheme.
Testing with octopus sperm heads—helical structures similar to chromatin—confirmed the system’s ability to detect circular intensity differential scattering. The darkfield setup showed strong results, unlike the brightfield system, which failed to capture signal.
FYLA´s supercontinuum laser for non-invasive microscopy of biological samples
The use of FYLA´s supercontinuum laser in Mueller matrix microscopy offers significant benefits due to its broad spectral range and high brightness. This allows for precise wavelength selection, enabling multispectral imaging and enhanced contrast across different sample features
- Broad and flat spectral coverage (450–2300 nm) Enables multispectral and hyperspectral imaging across visible and infrared regions, ideal for analyzing diverse optical properties in complex samples.
- High total output power (>3 W) Provides strong illumination for deep tissue imaging or low-reflectivity materials, improving signal-to-noise ratio and measurement accuracy.
- Strong visible range power (>250 mW) Ensures excellent performance in conventional microscopy wavelengths, supporting high-resolution imaging and polarization analysis.
- Ultrashort pulse duration (<10 ps) Minimizes thermal effects and allows for time-resolved measurements, enhancing contrast and dynamic studies in biological and material samples.
- Exceptional power stability (<0.5% standard deviation) Delivers consistent illumination over time, crucial for reproducible Mueller matrix measurements and minimizing systematic errors.
