Widely tunable ultrafast lasers have enabled a large number of biological imaging techniques including point scanning multiphoton excited fluorescence (MPEF), SHG/THG and stimulated Raman imaging. Tunable ultrafast lasers offer spectral agility, covering the entire relative transparency window in live tissue (700-1300nnm) and flexibility with multi-color, synchronized outputs to support sophisticated label free techniques (e.g. stimulated Raman modalities). More recently newly available high peak power lasers based on Ytterbium technology drive advances in two-photon light-sheet, 3 photon excited fluorescence and holographic patterning for optogenetics photo-stimulation. These laser platforms offer a unique blend of compactness, ease of use and cost efficiency, and ideally complement tunable platforms typically based on Ti:Sapphire and IR optical parametric oscillators (OPO). We present various types of ultrafast laser architectures, link their optical characteristics to key bio-imaging requirements, and present relevant examples and images illustrating their impact in biological science. In particular we review the use of ultrafast lasers in optogenetics and fast in-vivo Calcium imaging deep in mouse brain.
Neuroscience applications benefit from recent developments in industrial femtosecond laser technology. New laser sources provide several megawatts of peak power at wavelength of 1040 nm, which enables simultaneous optogenetics photoactivation of tens or even hundreds of neurons using red shifted opsins. Another recent imaging trend is to move towards longer wavelengths, which would enable access to deeper layers of tissue due to lower scattering and lower absorption in the tissue. Femtosecond lasers pumping a non-collinear optical parametric amplifier (NOPA) enable the access to longer wavelengths with high peak powers. High peak powers of >10 MW at 1300 nm and 1700 nm allow effective 3-photon excitation of green and red shifted calcium indicators respectively and access to deeper, sub-cortex layers of the brain. Early results include in vivo detection of spontaneous activity in hippocampus within an intact mouse brain, where neurons express GCaMP6 activated in a 3-photon process at 1320 nm.
Two-photon (2P) microscopy based on tunable Ti:sapphire lasers has become a widespread tool for 3D imaging with sub-cellular resolution in living tissues. In recent years multi-photon microscopy with simpler fixed-wavelength femtosecond oscillators using Yb-doped tungstenates as gain material has raised increasing interest in life-sciences, because these lasers offer one order of magnitude more average power than Ti:sapphire lasers in the wavelength range around 1040 nm: Two-photon (2P) excitation of mainly red or yellow fluorescent dyes and proteins (e.g. YFP, mFruit series) simultaneously has been proven with a single IR laser wavelength. A new approach is to extend the usability of existing tunable Titanium sapphire lasers by adding a fixed IR wavelength with an Yb femtosecond oscillator. By that means a multitude of applications for multimodal imaging and optogenetics can be supported. Furthermore fs Yb-lasers are available with a repetition rate of typically 10 MHz and an average power of typically 5 W resulting in pulse energy of typically 500 nJ, which is comparably high for fs-oscillators. This makes them an ideal tool for two-photon spinning disk laser scanning microscopy and holographic patterning for simultaneous photoactivation of large cell populations.
With this work we demonstrate that economical, small-footprint Yb fixed-wavelength lasers can present an interesting add-on to tunable lasers that are commonly used in multiphoton microscopy. The Yb fs-lasers hereby offer higher power for imaging of red fluorescent dyes and proteins, are ideally enhancing existing Ti:sapphire lasers with more power in the IR, and are supporting pulse energy and power hungry applications such as spinning disk microscopy and holographic patterning.
Widely tunable ultrafast lasers have enabled a large number of biological imaging techniques including point scanning multiphoton excited fluorescence (MPEF), SHG/THG and stimulated Raman imaging. Tunable ultrafast lasers offer spectral agility, covering the entire relative transparency window in live tissue (700-1300nnm) and flexibility with multi-color, synchronized outputs to support sophisticated label free techniques (e.g. stimulated Raman modalities). More recently newly available high peak power lasers based on Ytterbium technology drive advances in two-photon light-sheet, 3 photon excited fluorescence and holographic patterning for optogenetics photo-stimulation. These laser platforms offer a unique blend of compactness, ease of use and cost efficiency, and ideally complement tunable platforms typically based on Ti:Sapphire and IR optical parametric oscillators (OPO). We present various types of ultrafast laser architectures, link their optical characteristics to key bio-imaging requirements, and present relevant examples and images illustrating their impact in biological science. In particular we review the use of ultrafast lasers in optogenetics for photo-stimulation of networks of neurons.
In recent years two-photon microscopy with fixed-wavelength has raised increasing interest in life-sciences: Two-photon (2P) absorption spectra of common dyes are broader than single-photon ones. Therefore, excitation of several dyes simultaneously with a single IR laser wavelength is feasible and could be seen as an advantage in 2P microscopy. We used pulsed fixed-wavelength infrared lasers with center wavelength at 1040 nm, for two-photon microscopy in a variety of biologically relevant samples, among these a mouse brain sample, a mouse artery (within the animal, acute preparation), and a preparation of mouse bladder. The 1040 nm laser proved to be efficient not only in exciting fluorescence from yellow fluorescent protein (YFP) and red fluorescent dyes, but also for second harmonic generation (SHG) signals from muscle tissue and collagen. With this work we demonstrate that economical, small-footprint fixedwavelength lasers can present an interesting alternative to tunable lasers that are commonly used in multiphoton microscopy.
An ultrafast optical pump and probe technique known as picosecond ultrasonics is used to generate and detect coherent acoustic phonon pulses in nanostructured films grown on Si wafers. By detecting the phonons after they have diffracted across a millimeter thick wafer, it is possible to measure the scattered phonons in the acoustic far field. Numerical backpropagation algorithms can then be used in order to reconstruct the object which scattered the acoustic phonon pulses. We describe measurements and simulations of experiments performed on surface and sub-surface nanostructures. Results with ~500 nm image resolution are shown, and plans for improving that resolution by an order of magnitude will be described.
A new series of organic light emitting copolymers, poly(dioctyl fluorene - co - diphenyl oxadiazole)s P(DOF-DPO), are reported in this paper. We found organic polymer light emitting devices with comparable device efficiency and more saturated blue color based on this new series of copolymers with less than 10% DPO moiety when compared with poly(dioctyl fluorene) P(DOF) devices. The device structures used in this study were indium tin oxide (ITO) anode/ PEDOT-PSS layer/emissive layer/calcium (Ca)/aluminum (Al) cathode, while the emissive layer was P(DOF) or P(DOF-DPO) copolymer with different DOF and DPO ratio. The AFM data shows that the emissive layer has a smooth surface with RMS roughness about 0.5 ± 0.2 nm over a 2 μm by 2 μm area. The photo-luminescence quantum efficiency of the P(DOF-DPO) copolymer decreases with increasing DPO moiety. Both the photo-luminescence spectra and electro-luminescence spectra are slightly blue shifted when adding less than 10% DPO moiety into P(DOF). Both the emission efficiency (~0.3-0.6 cd/A at 100 cd/m2) and power efficiency (~0.1-0.2 lm/W at 100 cd/m2) of the P(DOF-DPO) based PLEDs with less than 10% DPO moiety are comparable to the P(DOF) based PLEDs.
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