title: Pulse-shaped multiphoton excitation: a new approach to the single-molecule detection of DNA
Creators: name: Nobis, David
Publisher: name: University of Glasgow
Year: 2020

description: Single-molecule fluorescence microscopy is a method that allows the fluorescence signal from individual molecules to be detected. This can reveal information that is normally hidden in the average signal, produced by conventional ensemble methods. This work seeks to advance the field of single-molecule methods, by combining single molecule fluorescence microscopy with multiphoton excitation. Due to their central role in biology, the direct investigation of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) with fluorescence microscopy would be very attractive but has not been possible due to the negligible fluorescence quantum yield of the bases. Many fluorescent nucleobase analogues have been developed to overcome this problem. However, due to their low brightness a routine detection of these molecules at the single-molecule level is not yet possible. Another issue is that most of the developed analogues are excited in the UV region of the light spectrum, which can inflict photodamage in biological tissue. In this work new nucleobase analogues are examined with multiphoton microscopy, in order to assess their potential to advance the field of single-molecule microscopy with DNA and RNA. Multiphoton excitation offers many advantages over traditional resonant excitation, such as three-dimensional restriction of the excitation volume, less out of focus photo-bleaching or reduced background (He et al. 2008). For some instances, also reduced photobleaching in general was reported (Brand et al. 1997b; Eggeling et al. 1998; Lane and Magennis 2012). However, the signal to background ratio (SBR) is often not good enough for single-molecule experiments. Here a setup is presented that uses pulse-shaper-assisted pulse compression to overcome these issues. Furthermore, the options of phase and amplitude shaping are explored as a way to increase the SBR, in order to realise the full potential of multiphoton excitation combined with single-molecule microscopy. The newly built setup comprises an ultrabroadband, pulsed laser source, a pulse shaper, a microscope and a home-built detection unit including two avalanche photodiodes. The shaper-assisted compression uses Multiphoton Intrapulse Interference Phase Scan (MIIPS) (Coello et al. 2008) and Chirp Reversal Technique (CRT) (Loriot et al. 2013) to measure the accumulated phase which is then compensated for by the pulse shaper. This leads to pulse lengths of around 8 fs at the focal plane of the objective and to a more than 80 times increase in peak excitation power compared to the uncompressed pulse. It is demonstrated with a model system (the fluorescent dye rhodamine 110) that the compression of the pulses leads to a three-fold increase in detected single-molecule events at only 30% of the average excitation power. It is also shown that with additional phase shaping a pulse can be found with only 40% of the peak intensity of the compressed pulse, but that leads to the same SBR as the compressed pulse. The investigated nucleobase analogues are pA (a naphthalene scaffold attached to an adenine molecule) and 1f and 1d (both members of a family of molecules called extended aza-uridines). With the new multiphoton setup and fluorescence correlation spectroscopy (FCS), it is possible to detect pA integrated into a single-stranded DNA at an average of 5 molecules in the laser focus. This makes pA the first nucleobase analogue that can be recorded with multiphoton FCS while incorporated internally into an oligonucleotide. For 1f, which was measured as a ribonucleoside, only a small fraction (1%) of the molecules are in a bright state at any one time. This bright state, however, is bright enough to make it possible to detect single molecules as they diffuse through the laser focus. This makes it the first nucleobase analogue to be detected at the single-molecule level upon multiphoton excitation. Measurements with 1d, integrated into an oligonucleotide, suggest that the dark state might be quenched upon integration into an oligonucleotide. If this behaviour is true for 1f as well it would be a very interesting candidate for single-molecule experiments of a nucleobase in oligonucleotides. With the measurement of these three nucleobase analogues close to and at the single molecule level, it is demonstrated that multiphoton excitation offers a very interesting alternative to traditional one-photon (1P) excitation. Furthermore, the presented data show the successful application of the newly built setup and demonstrate that the pulse-shaper-assisted approach broadens the realm of applications for multiphoton excitation in single-molecule microscopy.