Measuring temporal dynamics and advancements in fluorescence microscopy

This event is hosted by Neil Hunt (neil.hunt@york.ac.uk).

A novel approach to measuring temporal dynamics and vibrational spectra with single beam Coherent Anti-Stokes Raman Spectroscopy

By Professor Pieter Neething

Ultrashort laser pulses are temporally controlled, shaped, and arbitrarily manipulated by modulating the spectral phase. The spectral phases can be simply thought of as the relative timing of the various spectral components within the laser pulse. We do this by using a spatial light modulator (SLM) in a setup known as a 4f-pulse shaper. This enables accurate measurement and manipulation of the light’s phase and amplitude. This control over the spectral phase directly translates to control over the temporal shape through its Fourier relationship. We can now compress these laser pulses to their Fourier limit, which is useful for traditional nonlinear spectroscopy and microscopy, or we can tailor the light fields to selectively drive vibrational transitions in molecules.

This leads to a simpler experimental technique to produce high-quality Coherent Anti-Stokes Raman Spectra (CARS), a vibrational spectroscopy technique that is complementary to infrared absorption. We start by dividing compressed broadband ultrashort pulses into two spectral slices. The one slice is used to excite a range of molecular vibrations (pump), while the second narrow slice, acts as a probe. Furthermore, by delaying the probe pulse in time with respect to the pump pulse, we extract temporal dynamics of the excited vibrational states (free induction decay (FID)).

In this talk, I will aim to explain the experimental setup and measurement scheme in an accessible manner while highlighting some of the results obtained. I will end by looking ahead at our efforts to expand the technique to produce 2-dimensional CARS spectra. It is my belief that this will perfectly complement 2D-IR spectroscopy, which is commonly employed to study molecular structure and interaction.

Advancements in Fluorescence Microscopy from Single-Molecule Sensitivity to Imaging of Self-Assembled Cellular structures

By Dr Gurthwin Bosman

Fluorescence microscopy is a powerful optical technique for imaging objects often invisible to the unaided eye, with sensitivities reaching the single-molecule level. This high sensitivity, combined with an inherent high spatial resolution, makes fluorescence microscopy indispensable in many scientific and industrial applications, where sample inhomogeneity is a standard feature rather than an obstacle. I will discuss typical applications of conventional fluorescence microscopy and describe straightforward adjustments that enable high localization imaging with single-molecule resolution and high contrast imaging in cellular and subcellular structures. The adjustments can be divided into two categories.

The first involves spatial shaping of either the excitation or emission light, while the second involves temporal shaping of ultrashort optical pulses. In the first category we include point spread function engineering through spatial tailoring the light field, to achieve high-precision three-dimensional localization of single emitters, which typically surpasses the optical diffraction limit. This allows for real-time measurement and tracking of single molecules in, for instance, thin polymer film samples. By studying an ensemble of these particle tracks, we obtain direct information about the nano-environment.

Metrics such as particle mean square displacement are used to determine diffusion properties and sample viscosity, which, in turn, is used to determine the glass transition temperature of prototypical polymer thin films. Secondly, we incorporate temporal shaping using a new compression algorithm for near-infrared ultrashort pulses for two-photon excitation fluorescence microscopy in a light sheet geometry.

I will demonstrate the notable improvement in optical response and, consequently, image contrast when imaging self-assembled cellular structures. This advanced technique opens new avenues for detailed and dynamic studies of complex biological and material systems.