Quantitative high dynamic range beam profiling for fluorescence microscopy.

Modern developmental biology relies on optically sectioning fluorescence microscope techniques to produce non-destructive in vivo images of developing specimens at high resolution in three dimensions. As optimal performance of these techniques is reliant on the three-dimensional (3D) intensity profile of the illumination employed, the ability to directly record and analyze these profiles is of great use to the fluorescence microscopist or instrument builder. Though excitation beam profiles can be measured indirectly using a sample of fluorescent beads and recording the emission along the microscope detection path, we demonstrate an alternative approach where a miniature camera sensor is used directly within the illumination beam. Measurements taken using our approach are solely concerned with the illumination optics as the detection optics are not involved. We present a miniature beam profiling device and high dynamic range flux reconstruction algorithm that together are capable of accurately reproducing quantitative 3D flux maps over a large focal volume. Performance of this beam profiling system is verified within an optical test bench and demonstrated for fluorescence microscopy by profiling the low NA illumination beam of a single plane illumination microscope. The generality and success of this approach showcases a widely flexible beam amplitude diagnostic tool for use within the life sciences.

Micro-endoscope for in vivo widefield high spatial resolution fluorescent imaging.

In this paper we report the design, testing and use of a scannerless probe specifically for minimally invasive imaging of deep tissue in vivo with an epi-fluorescence modality. The probe images a 500 µm diameter field of view through a 710 µm outer diameter probe with a maximum tissue penetration depth of 15 mm specifically configured for eGFP imaging. Example results are given from imaging the pituitary gland of rats and zebrafish hearts with lateral resolution of 2.5 µm.

Eigenmodes of a hydrodynamically coupled micron-size multiple-particle ring.

We use a continuous acquisition, high-speed camera with integrated centroid tracking to simultaneously measure the positions of a ring of micron-sized particles held in holographic optical tweezers. Hydrodynamic coupling between the particles gives a set of eigenmodes, each one independently relaxing with a characteristic decay rate (eigenvalue) that can be measured using our positional data. Despite the finite particle size, we find an excellent agreement between the measured eigenvalues and those numerically predicted by Oseen theory applied to the two-dimensional (2D) ring geometry. Particle motions are also analyzed in terms of the alternative eigenmode set obtained by wrapping onto the ring the eigenmodes of a 1D periodic chain. We identify the modes for which the periodic chain is a good approximation to the ring and those for which it is not.