A New Pulse-Shaped Multiphoton Single-Molecule Microscope

Summary form only given. Single-molecule (SM) fluorescence microscopy can reveal information that is otherwise hidden in the average signal produced by conventional ensemble methods. We are interested in studying the structure and dynamics of DNA, which normally involves attaching a bright extrinsic fluorescent probe. Although there would be benefits to using intrinsic probes such as fluorescent nucleobases, photobleaching, UV absorption and background has so far prevented their practical detection at the single-molecule level. Multi -photon excitation could solve many of these problems and also offers deeper penetration of biological tissue due to reduction of out of focus excitation, and large spectral separation of excitation and emission which leads to less scattered background. The reduced cross section for two -photon absorption means that ultra short laser pulses must be used for excitation; but this requires control of the dispersion from optical components. In this work we seek to use the next generation of ultrafast lasers for single -molecule detection. We will describe a new home -built multiphoton fluorescence setup. The setup deploys an ultra -broadband Ti:Sapphire laser (bandwidth 135 nm FWHM) and a pulse shaper for pre -compensation of broadening using the MIIPSmethod. With this method it is possible to greatly enhance the brightness in single -molecule experiments. In our setup we achieve pulses of 8fs length in the focal plane of the microscope. Additionally, chromophorespecific phase and amplitude pulse shaping can be employed to enhance the signal to background ratio. In this work, we will present the characteristics of this setup and how pulse shaping can improve the signal to background ratio. We will further showcase the capabilities of this new setup with recent measurements on new DNA nucleobase analogs.