Focal molography ("molography" in short) is a sensitive implementation of a diffractometric biosensor and has emerged as a new platform technology to study biomolecular interactions label-free in complex fluids and living cells. In contrast to established refractometric biosensors, in particular surface plasmon resonance, molography is almost insensitive to environmental noise, i.e. temperature gradients and nonspecific binding. Molography achieves this by modulating the analyte binding at a high spatial frequency and reads it out in Fourier space via diffraction of light at the bound molecules, i.e. molography applies the spatial lock-in principle for discrimination of the binding signal from disturbing effects on the sensor surface. In previous implementations of focal molography, the sensor was illuminated by a waveguide mode. While this arrangements has an outstanding resolution, it suffers from several drawbacks such as wavefront instabilities of the guided mode, the relatively high refractive index contrast at the waveguide interfaces and the manufacturing cost of waveguide and grating couplers. In this paper, we propose a simpler and more robust configuration for focal molography. Instead of a waveguide mode, it is based on darkfield illumination by total internal reflection (TIR) of a free space mode. We derive the coherent binding pattern, describe the fabrication process, show that its intensity distribution is as expected, derive the quantitative readout formula and perform a background and noise analysis. Real-time binding curves of streptavidin in buffer and concentrated bovine serum albumin solution show that TIR molography exhibits excellent resolution and robustness.

Focal molography（简称“molography”）是衍射生物传感器的一种灵敏实现，并已成为研究复杂流体和活细胞中无标记生物分子相互作用的新平台技术。与已建立的折光生物传感器（特别是表面等离子体共振）相比，分子学几乎对环境噪声（即温度梯度和非特异性结合）不敏感。Molography 通过以高空间频率调制分析物结合并通过结合分子处的光衍射在傅立叶空间中读出它来实现这一点，即molography 应用空间锁定原理来区分结合信号与传感器表面上的干扰效应。在之前的焦摩尔成像实现中，传感器由波导模式照明。虽然这种布置具有出色的分辨率，但它存在一些缺点，例如导模的波前不稳定性、波导界面处相对较高的折射率对比度以及波导和光栅耦合器的制造成本。在本文中，我们提出了一种更简单、更健壮的焦摩尔成像配置。它不是波导模式，而是基于通过自由空间模式的全内反射 (TIR) 进行的暗场照明。我们推导出相干结合模式，描述制造过程，表明其强度分布符合预期，导出定量读数公式并进行背景和噪声分析。链霉亲和素在缓冲液和浓缩牛血清白蛋白溶液中的实时结合曲线表明 TIR 分子成像具有出色的分辨率和稳定性。