title: Single molecule imaging towards 1 nm resolution Raman microscopy
Creators: name: Sanchai, Apitchaya
Publisher: name: The University of Edinburgh
Year: 2025
Subjects: subject: Photo-induced Force Microscopy
subject: PiFM
subject: picosecond pulse lasers

description: Driven by the need for spectroscopic sensitivity at single molecule resolution, various forms of scanning probe microscopy have been combined with light illumination strategies, resulting in a set of powerful tools for generating nano-spectroscopic maps of materials and biological samples. Photo-induced force microscopy (PiFM) combines the advantages of atomic force microscopy (AFM) with vibrational spectroscopy. Instead of light detection, PiFM was developed to detect the mechanical movement of tip–cantilever system due to an attractive force arising from the interaction between a sample dipole and its image dipole in the tip. While recent PiFM studies have utilised continuous wave (CW) lasers. We have designed a new optical path in PiFM system using picosecond lasers which offer a high peak intensity during the pulse. The time-averaged signal level for pulsed lasers is increased by a factor of time between pulses (τrep) to pulse duration (τp) for 2-photon illumination compared to CW illumination by lasers with the same average power. In our case τp = 6.5 ps and τrep = 13 ns, giving a factor of approximately 2,000 times more signals. This enhancement of signal level can offer improvements in spatial resolution and imaging speed. Our PiFM system, with a Stokes beam at 1064 nm and a tuneable Pump beam at 700-1000 nm, was used to excite stimulated Raman scattering (SRS), and the attractive force was measured on an AFM tip sensitive to an individual vibrational mode within sample molecules beneath the tip. With the tuneable laser capability, our system allows imaging across the full biological Raman range (600 – 4,000 cm⁻¹). In order to deepen our understanding of the origins of the photo-induced force in SRS-PiFM (or Raman-PiFM), we introduced a force equation to estimate its magnitude. This equation consists of three main components. The first component is optical enhancement. Finite element models were created with ANSYS Multiphysics software with a high-frequency electromagnetic analysis. These simulations were carried out across a range of tip materials and tip radii in the illumination range of 400 – 1400 nm. The second part is the probability of a molecule gets excited which depends on intrinsic excitation properties of the sample molecules, quantified by the Stimulated Raman cross section (σSRS). The third component is Debye force. We derived the force from Debye interaction (induced dipole and dipole interaction). Subsequently, these three parts were integrated to compute the force value, which was then converted to amplitude change that we expected to observe in the experiments. This equation provides valuable insights into the materials and wavelengths required for force measurement, serving as a fundamental tool for estimating force levels and offering guidance for experimental design and interpretation. Additionally, we examined another contrast mechanism of the photo-induced force: Thermal energy transfer force. A key insight from our study is that while previous Raman-PiFM studies have mainly focused on induced dipole forces, our force modelling results indicate that thermal-related forces are also significant and should be considered. Despite the systematic alignment and experiments conducted for force measurement, our results did not reveal a true force signal from molecules, instead the observed image contrast originated from artefacts. Several factors may contribute to this lack of force measurement. These include low optical enhancement of the tip at the excitation wavelengths, cancellation of the attractive force magnitude of induced dipole force with the repulsive force from thermal energy transfer and radiation pressure forces, high thermal noise in the AFM obscuring the force signal, limitation in the sensitivity of the lock-in amplifier to detect small force signal, and practical issues such as difficulties in laser beams alignment. Additionally, in high humidity environments, water meniscus forms on the sample surface. As the tip approaches, it may easily get damaged due to sudden snap-in to contact with water layer.