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Significance: Recent advances in nonlinear optics in neuroscience have focused on using two ultrafast lasers for activity imaging and optogenetic stimulation. Broadband femtosecond light sources can obviate the need for multiple lasers by spectral separation for chromatically targeted excitation. Aim: We present a photonic crystal fiber (PCF)-based supercontinuum source for spectrally resolved two-photon (2P) imaging and excitation of GCaMP6s and C1V1-mCherry, respectively. Approach: A PCF is pumped using a 20-MHz repetition rate femtosecond laser to generate a supercontinuum of light, which is spectrally separated, compressed, and recombined to image GCaMP6s (930 nm excitation) and stimulate the optogenetic protein, C1V1-mCherry (1060 nm excitation). Galvanometric spiral scanning is employed on a single-cell level for multiphoton excitation and high-speed resonant scanning is employed for imaging of calcium activity. Results: Continuous wave lasers were used to verify functionality of optogenetic activation followed by directed 2P excitation. Results from these experiments demonstrate the utility of a supercontinuum light source for simultaneous, single-cell excitation and calcium imaging. Conclusions: A PCF-based supercontinuum light source was employed for simultaneous imaging and excitation of calcium dynamics in brain tissue. Pumped PCFs can serve as powerful light sources for imaging and activation of neural activity, and overcome the limited spectra and space associated with multilaser approaches.

Significance: 2019 SPIE Photonics West conference hosted over 5000 presentations. Some important presentations in the Industrial Laser, Laser Source and Application (LASE) and Optoelectronics, Photonic Materials and Devices (OPTO) sections of the SPIE Photonics West conference have a risk of being overlooked by the biomedical community despite their implications for the field of biophotonics. We review some recent advances in the area of development coherent radiation sources in the infrared (IR), ultraviolet (UV), and terahertz (THz) regimes. Aim: Recent advances in coherent radiation sources in the IR, deep UV, and THz regimes were outlined, and the importance of each presentation to one or more promising biomedical applications was assessed. Approach: Presentations and proceedings from the LASE and OPTO sections were reviewed for inclusion. Emphasis was placed on talks from the Nonlinear Frequency Generation and Conversion: Materials and Devices XVIII conference, and the Terahertz, RF, Millimeter, and Submillimeter-Wave Technology and Applications XII conference. Conference sections that directly focused on biomedical applications were excluded. Results: Enhanced IR supercontinuum generation with compact supercontinuum sources may allow for real-time biomarker detection and create new opportunities for imaging tissues using the third biological window (1600 to 1850 nm). Efficient methods to generate deep UV (200 to 260 nm) radiation allow for the study of biologically important molecules through techniques such as resonance Raman spectroscopy while avoiding fluorescence overlap. Likewise, novel and improved THz generation methods seek to bridge the “THz gap” that has previously limited biomedical applications. Conclusions: Advances in coherent radiation sources in the IR, UV, and THz regimes have created new opportunities for biomedical optics research.

Significance: The large background, narrow dynamic range, and detector saturation have been the common limiting factors in stimulated emission (SE)-based pump-probe microscopy, attributed to the very small signal overriding the very intense laser probe beam. To better differentiate the signal of interest from the background, lock-in detection is used to measure the fluorescence quenching, which is termed spontaneous loss (SL). The advantages are manifold. The spontaneous fluorescence signal can be well separated from both the pump and the probe beams with filters, thus eliminating the background, enlarging the dynamic range, and avoiding the saturation of the detector. Aim: We propose and demonstrate an integrated pump-probe microscopy technique based on lock-in detection for background removal and dynamic range enhancement through SL detection. Approach: The experimental setup is configured with a pulsed diode laser at a wavelength λpu=635 nm, acting as a pump (excitation) and a mode-locked Ti:sapphire laser at a central wavelength λpr=780 nm, serving as the probe beam (stimulation). Both pulse trains are temporally synchronized through high precision delay control by adjusting the length of the triggering cables. The pump and probe beams are alternatively modulated at different frequencies f1 and f2 to extract the stimulated gain (SG) and SL signal. Results: SG signal shows saturation due to the irradiation of the intense probe beam onto the photodetector. However, the detector saturation does not occur at high probe beam power for SL detection. The fluorescence lifetime images are acquired with reduced background. The theoretical signal-to-noise ratios for SG and SL are also estimated by photon statistics. Conclusion: We have confirmed that the detection of SL allows the elimination of the large background without photodetector saturation, which commonly exists in SG configuration. This modality would allow unprecedented manipulation and investigation of fluorophores in fluorescence imaging.

In this paper we describe the synthesis, the electrochemical behaviour as well as the linear and nonlinear optical (NLO) properties of two push-pull derivatives bearing pyranylidene electron donating fragment, pyrimidine/methyl pyrimidinium electron withdrawing moiety and a ferrocene part in the π-conjugated bridge. The properties of these two compounds were compared to their analogues without ferrocene or pyranylidene fragments. Experimental results were completed with DFT calculations to gain further insight into the intramolecular charge transfer (ICT). All the results indicate a significant charge transfer through the ferrocene unit. The ICT is however more limited than in all organic analogues.

Coherent anti-Stokes Raman scattering (CARS) generates a strong label-free signal in the long wavenumber C─H stretching region. Lipid-rich myelinated tissues, such as brain and spinal cord, would appear to be ideal subjects for imaging with CARS laser-scanning microscopy. However, the highly ordered, biochemically complex, and highly scattering nature of such tissues complicate the use of the technique. A CARS microscopy approach is presented that overcomes the challenges of imaging myelinated tissue to achieve chemically and orientationally sensitive high-resolution images.