Total internal reflection focal molography (TIR-M)

Blickenstorfer et al. propose a simpler configuration of focal molography that uses darkfield illumination by total internal reflection (TIR) instead of a waveguide mode. Their real-time streptavidin experiments show high resolution and robustness.

The Challenge: Limits of Waveguide Illumination

Earlier focal molography illuminated the sensor with a guided mode. This gives very good resolution, but it brings wavefront instabilities and a high refractive index contrast at the waveguide interfaces. Waveguides and grating couplers also add manufacturing cost.

The Approach: Total Internal Reflection Focal Molography (TIR-M)

The authors derive the coherent binding pattern, describe the fabrication process and derive the quantitative readout formula. They then perform a background and noise analysis. Real-time binding of 500 pM streptavidin was measured in buffer and in 100 µM bovine serum albumin (BSA), in triplicates.

Key Results for TIR-M Noise and Robustness

• Baseline noise: root mean square values of 0.051 to 0.070 pg/mm² over 100 seconds, without drift correction. Guided-wave molography (GW-M) reported about 0.08 pg/mm².
• Long experiments: noise as low as 4 fg/mm² over almost 20 minutes, which corresponds to about 6500 streptavidin molecules on the mologram.
• Compared with GW-M: GW-M noise was up to a factor of 3 higher for experiment durations of 100 to 1000 seconds.
• Non-specific binding: a control with 100 µM BSA and no analyte showed only a slight drift upon injection.

Why It Matters for Diffractometric Sensors

TIR-M has advantages in measurement stability, quantification accuracy, substrate complexity and multichannel fluorescence microscopy. GW-M keeps advantages in shot noise, measurements faster than 1 second and contact-free chips. The authors conclude that the better choice depends on the application.