Jeff Squier

Picosecond-milliangstrom lattice dynamics measured by ultrafast X-ray diffraction

Fundamental processes on the molecular level, such as vibrations and rotations in single molecules, liquids or crystal lattices and the breaking and formation of chemical bonds, occur on timescales of femtoseconds to picoseconds. The electronic changes associated with such processes can be monitored in a time-resolved manner by ultrafast optical spectroscopic techniques, but the accompanying structural rearrangements have proved more difficult to observe. Time-resolved X-ray diffraction has the potential to probe fast, atomic-scale motions. This is made possible by the generation of ultrashort X-ray pulses, and several X-ray studies of fast dynamics have been reported,. Here we report the direct observation of coherent acoustic phonon propagation in crystalline gallium arsenide using a non-thermal, ultrafast-laser-driven plasma — a high-brightness, laboratory-scale source of subpicosecond X-ray pulses. We are able to follow a 100-ps coherent acoustic pulse, generated through optical excitation of the crystal surface, as it propagates through the X-ray penetration depth. The time-resolved diffraction data are in excellent agreement with theoretical predictions for coherent phonon excitation in solids, demonstrating that it is possible to obtain quantitative information on atomic motions in bulk media during picosecond-scale lattice dynamics.

Spatial mapping of integrin interactions and dynamics during cell migration by Image Correlation Microscopy

Image correlation microscopy methodology was extended and used to determine retrospectively the density, dynamics and interactions of α5-integrin in migrating cells. α5-integrin is present in submicroscopic clusters containing 3-4 integrins before it is discernibly organized. The integrin in nascent adhesions, as identified by the presence of paxillin, is ∼1.4 times more concentrated, ∼4.5 times more clustered and much less mobile than in surrounding regions. Thus, while integrins are clustered throughout the cell, they differ in nascent adhesions and appear to initiate adhesion formation, despite their lack of visible organization. In more mature adhesions where the integrin is visibly organized there are ∼900 integrins μm–2 (about fivefold higher than surrounding regions). Interestingly, α5-integrin and α-actinin, but not paxillin, reside in a complex throughout the cell, where they diffuse and flow together, even in regions where they are not organized. During adhesion disassembly some integrins diffuse away slowly, α-actinin undergoes a directed movement at speeds similar to actin retrograde flow (0.29 μm min–1), while all of the paxillin diffuses away rapidly.

All-Optical Histology Using Ultrashort Laser Pulses

As a means to automate the three-dimensional histological analysis of brain tissue, we demonstrate the use of femtosecond laser pulses to iteratively cut and image fixed as well as fresh tissue. Cuts are accomplished with 1 to 10 microJ pulses to ablate tissue with micron precision. We show that the permeability, immunoreactivity, and optical clarity of the tissue is retained after pulsed laser cutting. Further, samples from transgenic mice that express fluorescent proteins retained their fluorescence to within microns of the cut surface. Imaging of exogenous or endogenous fluorescent labels down to 100 microm or more below the cut surface is accomplished with 0.1 to 1 nJ pulses and conventional two-photon laser scanning microscopy. In one example, labeled projection neurons within the full extent of a neocortical column were visualized with micron resolution. In a second example, the microvasculature within a block of neocortex was measured and reconstructed with micron resolution.

High resolution nonlinear microscopy: A review of sources and methods for achieving optimal imaging

This article reviews the latest instrumentation used in high resolution nonlinear microscopy and techniques for the temporal and spatial calibration of this instrumentation. This includes an overview of currently available ultrashort laser sources, the dispersion characteristics of microscopes, methods for pulse measurement at high numerical aperture, dispersion compensation techniques, and finally a brief overview of a number of nonlinear imaging methods presently used in these systems.

Microfluidic sorting system based on optical waveguide integration and diode laser bar trapping

Effective methods for manipulating, isolating and sorting cells and particles are essential for the development of microfluidic-based life science research and diagnostic platforms. We demonstrate an integrated optical platform for cell and particle sorting in microfluidic structures. Fluorescent-dyed particles are excited using an integrated optical waveguide network within micro-channels. A diode-bar optical trapping scheme guides the particles across the waveguide/micro-channel structures and selectively sorts particles based upon their fluorescent signature. This integrated detection and separation approach streamlines microfluidic cell sorting and minimizes the optical and feedback complexity commonly associated with extant platforms.

Optical trapping, manipulation, and sorting of cells and colloids in microfluidic systems with diode laser bars.

We demonstrate a new technique for trapping, sorting, and manipulating cells and micrometer-sized particles within microfluidic systems, using a diode laser bar.

Invited Review Article: Imaging techniques for harmonic and multiphoton absorption fluorescence microscopy

We review the current state of multiphoton microscopy. In particular, the requirements and limitations associated with high-speed multiphoton imaging are considered. A description of the different scanning technologies such as line scan, multifoci approaches, multidepth microscopy, and novel detection techniques is given. The main nonlinear optical contrast mechanisms employed in microscopy are reviewed, namely, multiphoton excitation fluorescence, second harmonic generation, and third harmonic generation. Techniques for optimizing these nonlinear mechanisms through a careful measurement of the spatial and temporal characteristics of the focal volume are discussed, and a brief summary of photobleaching effects is provided. Finally, we consider three new applications of multiphoton microscopy: nonlinear imaging in microfluidics as applied to chemical analysis and the use of two-photon absorption and self-phase modulation as contrast mechanisms applied to imaging problems in the medical sciences.

Temporally focused femtosecond laser pulses for low numerical aperture micromachining through optically transparent materials

Temporal focusing of spatially chirped femtosecond laser pulses overcomes previous limitations for ablating high aspect ratio features with low numerical aperture (NA) beams. Simultaneous spatial and temporal focusing reduces nonlinear interactions, such as self-focusing, prior to the focal plane so that deep (~1 mm) features with parallel sidewalls are ablated at high material removal rates (25 µm3 per 80 µJ pulse) at 0.04-0.05 NA. This technique is applied to the fabrication of microfluidic devices by ablation through the back surface of thick (6 mm) fused silica substrates. It is also used to ablate bone under aqueous immersion to produce craniotomies.

Visualization of mitochondria in cardiomyocytes

The simultaneous detection of third harmonic (THG), and multiphoton excitation fluorescence (MPF) or second harmonic (SHG) from the same focal volume has led us to the development of a nonlinear multimodal microscopic biological imaging tool. The multimodal microscope has been applied for imaging of isolated live cardiomyocytes, and investigation of structural origin of the THG and SHG signals has been performed. By employing the different image contrast mechanisms, differentiation of structures inside a single live adult rat cardiomyocyte has been achieved. Based on structural crosscorrelation image analysis between NAD(P)H fluorescence and THG, and morphology of cardiomyocytes we were able to assign large part of the structure revealed by THG to the mitochondria. The crosscorrelation of THG with fluorescence of tetramethylrhodamine methyl ester (TMRM) labeled cardiomyocytes confirmed the mitochondrial origin of THG. The SHG generated structures were anticorrelated with THG and possessed the characteristic pattern of the myofibrils in the myocyte in accordance with the literature. Possible visualization of mitochondria with THG microscopy appeared due to enhancement of the third harmonic by multilayer arrangement of cristae.

Simultaneous imaging of multiple focal planes using a two-photon scanning microscope

Despite all the advances in nonlinear microscopy, all existing instruments are constrained to obtain images of one focal plane at a time. In this Letter we demonstrate a two-photon absorption fluorescence scanning microscope capable of imaging two focal planes simultaneously. This is accomplished by temporally demultiplexing the signal coming from two focal volumes at different sample depths. The scheme can be extended to three or more focal planes.