This PDF file contains the front matter associated with SPIE Proceedings Volume 12333, including the Title Page, Copyright information, Table of Contents, and Conference Committee information.

This conference presentation was prepared for the Frontiers in Biophotonics and Imaging II conference at SPIE Photonex, 2022.

This conference presentation was prepared for the Frontiers in Biophotonics and Imaging II conference at SPIE Photonex, 2022.

This conference presentation was prepared for the Frontiers in Biophotonics and Imaging II conference at SPIE Photonex, 2022.

Monitoring the dynamic behaviour of individual molecular motors in solution reveals insights into their catalytic activities. Single-molecule Förster resonance energy transfer (smFRET) allows us to study molecular motors in real time at high spatial and temporal resolution. Combining smFRET with molecular confinement enables us to increase the observation time up to several seconds without tethered surface attachment of the observed molecules. Here, we employed an Anti-Brownian ELectrokinetic trap (ABEL trap) that used variable homogeneous electric fields to actively confine single molecules to a femtolitre sized observation volume by acting on their surface charge. We present a non-invasive confinement method that allows trapping of molecular motors like the Rep helicase and the F_OF₁-ATP synthase. In Escherichia coli the ATP-dependent Rep DNA helicase facilitates the replisome progression and is necessary for the restart of the DNA replication machinery. We selectively trapped active Rep molecules based on their smFRET signal with sub-millisecond temporal resolution recording conformational switching events during observation times of up to several seconds. In addition, we observed the ATP-hydrolysis driven rotation of individual F_OF₁-ATP synthase molecules over numerous consecutive reaction cycles and extracted their kinetic rates.

This conference presentation was prepared for the Frontiers in Biophotonics and Imaging II conference at SPIE Photonex, 2022.

This conference presentation was prepared for the Frontiers in Biophotonics and Imaging II conference at SPIE Photonex, 2022.

This conference presentation was prepared for the Frontiers in Biophotonics and Imaging II conference at SPIE Photonex, 2022.

This conference presentation was prepared for the Frontiers in Biophotonics and Imaging II conference at SPIE Photonex, 2022.

This conference presentation was prepared for the Frontiers in Biophotonics and Imaging II conference at SPIE Photonex, 2022.

Multiphoton microscopies are an invaluable tool in biomedical imaging given their inherent capabilities for label free imaging, optical sectioning, chemical and structural specificity. They comprise various types of Coherent Raman microscopies (CR), such as Coherent Anti-Stokes Raman Scattering (CARS), Stimulated Raman Loss (SRL) or Stimulated Raman Gain, different kinds of Harmonic Generation imaging (HG) such as Second and Third Harmonic Generation (SHG and THG respectively), and Multiphoton Autofluorescence imaging (MA) such as Two and Three Photon Excited Autofluorescence (TPEAF and ThPEAF respectively). Despite their significant advantages, multiphoton microscopies, comparably to all other types of optical microscopies, exhibit limited penetration depth in tissue due to absorption and scattering. In this work we explore the advantages of multiphoton microscopies in hard and soft deep tissue imaging when using excitation wavelengths in the range of Short-Wavelength Infrared (SWIR) windows which occur between 1000 nm and 2500 nm. These spectral windows have notable merits including longer attenuation lengths and none or very low signal absorption observed for almost all kinds of multiphoton microscopy. We show results of using excitations in the SWIR windows, generated by standard as well as novel sources, such as a thulium fibre laser, in different types of multiphoton microscopy on a variety of hard and soft tissue samples (bone, cartilage and other tissue types) and demonstrate the advantages of using excitations in this wavelength range, including longer penetration depth and high resolution for deep tissue imaging.

Raman spectroscopy is an analytical technique that non-invasively provides “chemical fingerprinting” information with high degree of specificity and sensitivity for medical/biological diagnostics. However, to avoid autofluorescence background signals, the measurements need to be carried out in the Near Infrared (NIR), where Raman scattering efficiency is low (proportional to λ_laser^-4). This provides challenges in terms of both detection sensitivity and minimisation of diagnostics time. Traditional high NIR sensitivity Si-based detectors suffer from elevated dark current, which can be minimised through cooling, but at the expense of a competing blue-shifting of sensor Quantum Efficiency (QE). Low Dark Current Deep-Depletion (LDC-DD) CCD technology first introduced by Andor minimises the need for deep-cooling, preserving QE for maximum detection capability and achieving higher signal-to-noise more quickly. The origins of this limitation of Si-based sensors and Andor’s technical solution will be explored. Additionally, the configurability of Andor’s spectrographs for researchers designing custom-built Raman and/or multi-diagnostics spectroscopy systems will be discussed. This instrumentation can facilitate new experiments which were not previously possible to enable new scientific breakthroughs. Higher selectivity, sensitivity, and rapidness of data acquisition offers faster sample screening, better clinical diagnostic measurements and minimisation of patient discomfort.