In this manuscript, we present a detailed investigation of the impact of dispersion on the spectral resolution achievable by the application of spectral focusing in coherent Raman imaging. Our results reveal the detrimental effect of third order dispersion that limits the resolution for group delay dispersion of 100 000 fs² and more. Experimental examples for the exact determination of the described effects are given as well as a condensed presentation of the known equations. We introduce useful approximations to the latter, which serve to facilitate the straightforward integration of spectral focusing into any multimodal microscope.

In this Letter, we present an all-fiber, easy-to-use, wavelength-tunable, ultrafast laser, based on soliton self-frequency shifting in an Er-doped polarization-maintaining very large mode area fiber. The system is tunable over 370 nm, starting at 1620 nm, with an average power of up to 1.5 W that emits 120 fs short laser pulses directly out of the fusion-spliced fiber without using bulky pulse compression optics. The output is subsequently frequency doubled to a wavelength range covering 810 nm up to almost 1000 nm with more than 500 mW average power and 120 fs pulse width. Having a polarization extinction ratio better than 40 dB and a $M^2$ of 1.1, the fully computer-controlled laser system presents a robust and compact laser source. These parameters make the laser perfectly suited for multimodal imaging in nonlinear microscopy.

Deep tissue imaging with high contrast close to or even below the optical resolution limit is still challenging due to optical aberrations and scattering introduced by dense biological samples. This results in high complexity and cost of microscopes that can facilitate such challenges. Here, we demonstrate a cost-effective and simple to implement method to turn most two-photon laser-scanning microscopes into a super-resolution microscope for deep tissue imaging. We realize this by adding inexpensive optical devices, namely a cylindrical lens, a field rotator, and a sCMOS camera to these systems. By combining two-photon excitation with patterned line-scanning and subsequent image reconstruction, we achieve imaging of sub-cellular structures in Pinus radiata, mouse heart muscle and zebrafish. In addition, the penetration depth of super-resolved imaging in highly scattering tissue is considerably extended by using the camera’s lightsheet shutter mode. The flexibility of our method allows the examination of a variety of thick samples with a variety of fluorescent markers and microscope objective lenses. Thus, with a cost-efficient modification of a multi-photon microscope, an up to twofold resolution enhancement is demonstrated down to at least 70μm deep in tissue.

Having access to fluorescence lifetime, researchers can reveal in-depth details about the microenvironment as well as the physico-chemical state of the molecule under investigation. However, the high number of influencing factors might be an explanation for the strongly deviating values of fluorescent lifetimes for the same fluorophore reported in the literature. This could be the reason for the impression that inconsistent results are obtained depending on which detection and excitation scheme is used. To clarify this controversy, the two most common techniques for measuring fluorescence lifetimes in the time-domain and in the frequency-domain were implemented in one single microscopy setup and applied to a variety of fluorophores under different environmental conditions such as pH-value, temperature, solvent polarity, etc., along with distinct state forms that depend, for example, on the concentration. From a vast amount of measurement results, both setup- and sample-dependent parameters were extracted and represented using a single display form, the phasor-plot. The measurements showed consistent results between the two techniques and revealed which of the tested parameters has the strongest influence on the fluorescence lifetime. In addition, quantitative guidance as to which technique is most suitable for which research task and how to perform the experiment properly to obtain consistent fluorescence lifetimes is discussed.

We present a two-color two-photon stimulated emission depletion microscopy technique (2C2P-STED) that correlates a confocal image with a super-resolved image employing the inherent self-referencing mechanism of nonlinear excitation. The novel approach overcomes the substantial challenge posed by two different imaging modalities in laser-scanning fluorescence microscopy for colocalization on the nanometer scale. Demonstrating the principle of 2C2P-STED, we show for the first time super-resolved images of the gram-positive bacteria Streptococcus pneumoniae TIGR4 pilus type- 1 . A signal-to-noise ratio (SINIR) greater than 10 was achieved in 2C2P excitation mode and approximately 70 nm details were resolved in 2P-STED. (C) 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

We combine two-photon-excited fluorescence microscopy and acoustofluidic trapping in a spherical microchamber to in vitro study cells and cell clusters three-dimensionally close to in vivo conditions. The two-photon microscopy provides the in-depth 3D analysis of the spherical microchamber dimensions as well as the positions of trapped samples therein with high spatial precision and high temporal resolution enabling even tracking of the fast moving particles. Furthermore, optical sectioning allows to gather information of individual cells in trapped cell clusters inside the chamber. We demonstrate real-time monitoring of osmosis in A549 lung cells and red blood cells as one possible biomedical application. The observed osmosis reduced the cell membrane diameter by approximately 4 μm in the A549 cells and by approximately 2 μm in the red blood cells. Our approach provides an important optical tool for future investigations of cell functions and cell–cell interactions avoiding wall-contact inside an acoustofluidic device.