Deciphering single- and multi-particle trapping dynamics under femtosecond pulsed excitation with simultaneous spatial and temporal resolution

Recent theoretical and experimental studies have shed light on how optical trapping dynamics under femtosecond pulsed excitation are fine-tuned by optical and thermal nonlinearities. Here, we present experimental results of nonlinear optical trapping of single and multiple polystyrene beads (of 1 μm diameter). We show how integration and synchronization of bright-filed video microscopy with confocal detection of backscatter provide both spatial and temporal resolution required to capture intricate details of trapping dynamics. Such spatiotemporal detection is promising to have far-reaching applications in exploring controlled optical trapping and manipulations harnessed by optical and thermal nonlinearities.

Wide-field fluorescent nanodiamond spin measurements toward real-time large-area intracellular thermometry

Measuring optically detected magnetic resonance (ODMR) of diamond nitrogen vacancy centers significantly depends on the photon detectors used. We study camera-based wide-field ODMR measurements to examine the performance in thermometry by comparing the results to those of the confocal-based ODMR detection. We show that the temperature sensitivity of the camera-based measurements can be as high as that of the confocal detection and that possible artifacts of the ODMR shift are produced owing to the complexity of the camera-based measurements. Although measurements from wide-field ODMR of nanodiamonds in living cells can provide temperature precisions consistent with those of confocal detection, the technique requires the integration of rapid ODMR measurement protocols for better precisions. Our results can aid the development of camera-based real-time large-area spin-based thermometry of living cells.

Metabolic Imaging by Simultaneous FLIM of NAD(P)H and FAD

We describe a metabolic imaging system based on simultaneous recording of lifetime images of NAD(P)H and FAD. The system uses one-photon excitation by ps diode lasers, scanning by galvanometer mirrors, confocal detection, and two parallel TCSPC FLIM recording channels. Two lasers, with wavelengths of 375nm and 410 nm, are multiplexed to alternatingly excite NAD(P)H and FAD. One FLIM channel detects in the emission band of NAD(P)H, the other in the emission band of FAD. For both channels, the data analysis delivers images of the amplitudes of the decay components, a1 and a2. We show that these are robust parameters to characterize the metabolic state of cells. FLIM results obtained from excised human-bladder tissue were in perfect agreement with histology.

Fast multi-directional DSLM for confocal detection without striping artifacts

In recent years light-sheet fluorescence microscopy (LSFM) has become a cornerstone technology for neuroscience, improving quality and capabilities of 3D imaging. By selectively illuminating a single plane, it provides intrinsic optical sectioning and fast image recording, while minimizing out of focus fluorescence background, sample photo-damage and photo-bleaching. However, images acquired with LSFM are often affected by light absorption or scattering effects, leading to un-even illumination and striping artifacts. In this work we present an optical solution to this problem, via fast multi-directional illumination of the sample, based on an acousto-optical deflector (AOD). We demonstrate that this pivoting system is compatible with confocal detection in digital scanned laser light-sheet fluorescence microscopy (DSLM) by using a pivoted elliptical-Gaussian beam. We tested its performance by acquiring signals emitted by specific fluorophores in several mouse brain areas, comparing the pivoting beam illumination and a traditional static one, measuring the point spread function response and quantifying the striping reduction. We observed real-time shadow suppression, while preserving the advantages of confocal detection for image contrast.