Two-photon microscopy of acoustofluidic trapping for highly sensitive cell analysis.

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.

Comprehensive Investigation of Parameters Influencing Fluorescence Lifetime Imaging Microscopy in Frequency- and Time-Domain Illustrated by Phasor Plot Analysis.

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.

Correlative two-color two-photon (2C2P) excitation STED microscopy.

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 (SNR) greater than 10 was achieved in 2C2P excitation mode and approximately 70 nm details were resolved in 2P-STED.

Resolution of spectral focusing in coherent Raman imaging.

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.

Electronically controlled coherent linear optical sampling for optical coherence tomography.

Electronically controlled coherent linear optical sampling for low coherence interferometry (LCI) and optical coherence tomography (OCT) is demonstrated, using two turn-key commercial mode-locked fiber lasers with synchronized repetition rates. This novel technique prevents repetition rate limitations present in previous implementations based on asynchronous optical sampling. Adjustable scanning ranges and scanning rates are realized within an interferometric setup by full electronic control of the mutual time delay of the two laser pulse trains. We implement this novel linear optical sampling scheme with broad spectral bandwidths for LCI, optical filter characterization and OCT imaging in two and three dimensions.

Nonlinear microscopy with fiber laser continuum excitation.

A compact high-power fiber-based femtosecond laser system is presented for coherent anti-Stokes Raman scattering/second-harmonic generation (CARS/SHG) microscopy, and quantitatively compared with a conventional picosecond optical parametric oscillator (OPO)-based system. While the broad spectral width of the femtosecond pulses results in 2.5 times lower image contrast and limited spectral selectivity, lipid stores, myosin, and collagen filaments in living cells can clearly be identified at 60 times lower excitation powers compared to the picosecond system. Visually the images contain the same information. Together with simple operation, small footprint, and low cost, the capabilities of this high-power all-fiber-based laser system promise a more general use of nonlinear microscopy within the biosciences.

Monitoring of lipid storage in Caenorhabditis elegans using coherent anti-Stokes Raman scattering (CARS) microscopy.

Better understanding of the fundamental mechanisms behind metabolic diseases requires methods to monitor lipid stores on single-cell level in vivo. We have used Caenorhabditis elegans as a model organism to demonstrate the limitations of fluorescence microscopy for imaging of lipids compared with coherent anti-Stokes Raman scattering (CARS) microscopy, the latter allowing chemically specific and label-free imaging in living organisms. CARS microscopy was used to quantitatively monitor the impact of genetic variations in metabolic pathways on lipid storage in 60 specimens of C. elegans. We found that the feeding-defective mutant pha-3 contained a lipid volume fraction one-third of that found in control worms. In contrast, mutants (daf-2, daf-4 dauer) with deficiencies in the insulin and transforming growth factors (IGF and TGF-beta) signaling pathways had lipid volume fractions that were 1.4 and 2 times larger than controls, respectively. This was observed as an accumulation of small-sized lipid droplets in the hypodermal cells, hosting as much as 40% of the total lipid volume in contrast to the 9% for the wild-type larvae. Spectral CARS microscopy measurements indicated that this is accompanied by a shift in the ordering of the lipids from gel to liquid phase. We conclude that the degree of hypodermal lipid storage and the lipid phase can be used as a marker of lipid metabolism shift. This study shows that CARS microscopy has the potential to become a sensitive and important tool for studies of lipid storage mechanisms, improving our understanding of phenomena underlying metabolic disorders.