Time-stretched multi-hit 3D velocity map imaging of photoelectrons.

The 2D photoelectron velocity map imaging (VMI) technique is commonly employed in gas-phase molecular spectroscopy and dynamics investigations due to its ability to efficiently extract photoelectron spectra and angular distributions in a single experiment. However, the standard technique is limited to specific light-source polarization geometries. This has led to significant interest in the development of 3D VMI techniques, which are capable of measuring individual electron positions and arrival times, obtaining the full 3D distribution without the need for inversion, forward-convolution, or tomographic reconstruction approaches. Here, we present and demonstrate a novel time-stretched, 13-lens 3D VMI photoelectron spectrometer, which has sub-camera-pixel spatial resolution and 210 ps (σ) time-of-flight (TOF) resolution (currently limited by trigger jitter). We employ a kHz CMOS camera to image a standard 40 mm diameter microchannel plate (MCP)/phosphor anode detector (providing x and y positions), combined with a digitizer pick-off from the MCP anode to obtain the electron TOF. We present a detailed analysis of time-space correlation under data acquisition conditions which generate multiple electrons per laser shot, and demonstrate a major advantage of this time-stretched 3D VMI approach: that the greater spread in electron TOFs permits for an accurate time- and position-stamping of up to six electrons per laser shot at a 1 kHz repetition rate.

Oblique angle transient-reflectivity laser-scanning microscopy for mineral imaging in natural ores.

The microscopic arrangement of different minerals in ores is of high interest for mine planning, mineral processing and extractive metallurgy. Many economically important, naturally occurring minerals are highly absorbing semiconductors. To characterize these materials, we have implemented pump-probe laser scanning microscopy (LSM) in a two-lens reflective configuration that offers efficient collection of signal light by using a combination of galvanometer and sample stage scanning. We show that the short-time (∼10 ps) pump-probe response of a material allows us to distinguish economically important sulfide minerals.

Amplitude and polarization modulated hyperspectral Stimulated Raman Scattering Microscopy.

We present a simple hyperspectral Stimulated Raman Scattering (SRS) microscopy method based on spectral focusing of chirped femtosecond pulses, combined with amplitude (AM) and polarization (PM) modulation. This approach permits the imaging of low concentration components with reduced background signals, combined with good hyperspectral resolution and rapid spectral scanning. We demonstrate, using PM-SRS in a Raman loss configuration, the spectrally resolved detection of deuterated dimethyl sulfoxide (DMSO-d6) at concentrations as low as 0.039 % (5.5 mM). In general, background signals due to cross-phase modulation (XPM), two-photon absorption (TPA) and thermal lensing (TL) can reduce the contrast in SRS microscopy. We show that the nonresonant background signal contributing to the SRS signal is, in our case, largely due to XPM. Polarization modulation of the Stokes beam eliminates the nonresonant XPM background, yielding high quality hyperspectral scans at low analyte concentration. The flexibility of our combined AM-PM approach, together with the use of variable modulation frequency and lock-in phase, should allow for optimization of SRS imaging in more complex samples.

Hyperspectral multimodal CARS microscopy in the fingerprint region.

A simple scheme for multimodal coherent anti-Stokes Raman scattering (CARS) microscopy is based on the spectral focusing of ultrafast-oscillator-derived pump/probe light and synchronous photonic crystal fiber (PCF) fiber-generated broadband Stokes light. To date, such schemes allowed rapid hyperspectral imaging throughout the CH/OH high frequency region (2700-4000 cm(-1) ). Here we extend this approach to the middle (1640-3300 cm(-1) ) and fingerprint regions (850-1800 cm(-1) ) of the Raman spectrum. Our simple integrated approach to rapid hyperspectral CARS microscopy in the fingerprint region is demonstrated by applications to label-free multimodal imaging of cellulose and bulk bone, including use of the phosphate resonance at 960 cm(-1) .

Fluorescence lifetime imaging of alterations to cellular metabolism by domain 2 of the hepatitis C virus core protein.

Hepatitis C virus (HCV) co-opts hepatic lipid pathways to facilitate its pathogenesis. The virus alters cellular lipid biosynthesis and trafficking, and causes an accumulation of lipid droplets (LDs) that gives rise to hepatic steatosis. Little is known about how these changes are controlled at the molecular level, and how they are related to the underlying metabolic states of the infected cell. The HCV core protein has previously been shown to independently induce alterations in hepatic lipid homeostasis. Herein, we demonstrate, using coherent anti-Stokes Raman scattering (CARS) microscopy, that expression of domain 2 of the HCV core protein (D2) fused to GFP is sufficient to induce an accumulation of larger lipid droplets (LDs) in the perinuclear region. Additionally, we performed fluorescence lifetime imaging of endogenous reduced nicotinamide adenine dinucleotides [NAD(P)H], a key coenzyme in cellular metabolic processes, to monitor changes in the cofactor's abundance and conformational state in D2-GFP transfected cells. When expressed in Huh-7 human hepatoma cells, we observed that the D2-GFP induced accumulation of LDs correlated with an increase in total NAD(P)H fluorescence and an increase in the ratio of free to bound NAD(P)H. This is consistent with an approximate 10 fold increase in cellular NAD(P)H levels. Furthermore, the lifetimes of bound and free NAD(P)H were both significantly reduced--indicating viral protein-induced alterations in the cofactors' binding and microenvironment. Interestingly, the D2-expressing cells showed a more diffuse localization of NAD(P)H fluorescence signal, consistent with an accumulation of the co-factor outside the mitochondria. These observations suggest that HCV causes a shift of metabolic control away from the use of the coenzyme in mitochondrial electron transport and towards glycolysis, lipid biosynthesis, and building of new biomass. Overall, our findings demonstrate that HCV induced alterations in hepatic metabolism is tightly linked to alterations in NAD(P)H functional states.

Spatial-spectral coupling in coherent anti-Stokes Raman scattering microscopy.

Coherent anti-Stokes Raman scattering (CARS) microscopy is a third-order nonlinear optical technique which permits label-free, molecule-specific hyperspectral imaging. The interference between coherent resonant and non-resonant terms leads to well known distortions in the vibrational spectrum, requiring the use of retrieval algorithms. It also leads to spatial imaging distortions, largely due to the Gouy phase, when objects are smaller than the Rayleigh range. Here we consider that the focal position and spectral contributions to the nonlinear image formation are intrinsically coupled and cannot be corrected by conventional retrieval methods.

A combined vibrational sum frequency generation spectroscopy and atomic force microscopy study of sphingomyelin-cholesterol monolayers.

A combination of vibrational sum frequency generation spectroscopy and atomic force microscopy is used to study the changes in morphology and conformational order in monolayers prepared from three natural sphingomyelin (SM) mixtures as a function of surface pressure and cholesterol concentration. The most homogeneous SM gave monolayers with well-ordered acyl chains and few gauche defects with relatively small effects of either increasing surface pressure or cholesterol addition. Heterogeneous SM mixtures with a mixture of acyl chain lengths or with significant fractions of unsaturated acyl chains had much larger contributions from gauche defects at low surface pressure and gave increasingly well-ordered monolayers as the surface pressure increased. They also showed substantial increases in lipid chain order after cholesterol addition. Overall, these results are consistent with the strong hydrogen bonding capacity of SM leading to well-ordered monolayers over a range of surface pressures. The changes in acyl chain order for natural SMs as a function of cholesterol are relevant to formation of sphingolipid-cholesterol enriched domains in cell membranes.

Forward-collected simultaneous fluorescence lifetime imaging and coherent anti-Stokes Raman scattering microscopy.

We demonstrate the simultaneous collection and separation of femtosecond-laser-based forward-collected coherent anti-Stokes Raman scattering (F-CARS) and two-photon-excitation-induced fluorescence lifetime images (FLIM) using time-correlated single photon counting (TCSPC). We achieve this in a nondescanned geometry using a single multimode fiber without significant loss of light, field of view, and most importantly, TCSPC timing fidelity. In addition to showing the ability to separate CARS images from FLIM images using time gating, we also demonstrate composite multimodal epicollected FLIM imaging with fiber-collected F-CARS imaging in live cells.

Multimodal CARS microscopy of structured carbohydrate biopolymers.

We demonstrate the utility of multimodal coherent anti-Stokes Raman scattering (CARS) microscopy for the study of structured condensed carbohydrate systems. Simultaneous second-harmonic generation (SHG) and spectrally-scanned CARS microscopy was used to elucidate structure, alignment, and density in cellulose cotton fibers and in starch grains undergoing rapid heat-moisture swelling. Our results suggest that CARS response of the O-H stretch region (3000 cm(-1)-3400 cm(-1)), together with the commonly-measured C-H stretch (2750 cm(-1)-2970 cm(-1)) and SHG provide potentially important structural information and contrast in these materials.

All-fiber CARS microscopy of live cells.

Using an all-fiber laser system consisting of a femtosecond Er/Yb fiber oscillator as the pump and an ultra-highly nonlinear fiber for Stokes generation, we demonstrate multimodal (TPF+SHG+CARS) non-linear optical microscopy of both tissue samples and live cells. Multimodal imaging was successfully performed with pixel dwell times as short as 4 micros at low laser powers (< 40 mW total).

Optimally chirped multimodal CARS microscopy based on a single Ti:sapphire oscillator.

We demonstrate high performance coherent anti-Stokes Raman scattering (CARS) microscopy of live cells and tissues with user-variable spectral resolution and broad Raman tunability (2500 - 4100 cm(-1)), using a femtosecond Ti:Sapphire pump and photonic crystal fiber output for the broadband synchronized Stokes pulse. Spectral chirp of the fs laser pulses was a user-variable parameter for optimization in a spectral focusing implementation of multimodal CARS microscopy. High signal-to-noise, high contrast multimodal imaging of live cells and tissues was achieved with pixel dwell times of 2-8 micros and low laser powers (< 30 mW total).