Gary R. Holtom

Coherent anti‐stokes Raman scattering microscopy: A biological review

Microscopic imaging of cells and tissues are generated by the interaction of light with either the sample itself or contrast agents that label the sample. Most contrast agents, however, alter the cell in order to introduce molecular labels, complicating live cell imaging. The interaction of light from multiple laser sources has given rise to microscopy, based on Raman scattering or vibrational resonance, which demonstrates selectivity to specific chemical bonds while imaging unmodified live cells. Here, we discuss the nonlinear optical technique of coherent anti‐Stokes Raman scattering (CARS) microscopy, its instrumentation, and its status in live cell imaging.

Mode-locked Yb:KGW laser longitudinally pumped by polarization-coupled diode bars

Mode-locked operation of a simple Yb:KGW (potassium gadolinium tungstate) oscillator is described, providing 10 W at 1039 nm with a 290 fs pulse width. A polarization-coupled scheme is used for efficient longitudinal pumping by a pair of reshaped laser diode bars. With changes in cavity dispersion, the pulse width is adjustable from 134 to 433 fs, in a high-quality circular mode. A saturable absorber mirror provides self-starting operation, and the cavity is stabilized by the Kerr-lens effect.

Proton transfer reaction rates as a probe of size-dependent properties of large water clusters

Abstract Solvent-controlled excited state intermolecular proton transfer kinetics in 1-naphthol·(H2O)n (n⩽800), clusters were observed by picosecond time-resolved fluorescence in a molecular beam. Fast (

Simultaneous 1H PFG-NMR and confocal microscopy of monolayer cell cultures: Effects of apoptosis and necrosis on water diffusion and compartmentalization

We induced apoptosis and necrosis in monolayer cultures of Chinese hamster ovary cells using okadaic acid and hydrogen peroxide (H2O2), respectively, and examined the effect on water diffusion and compartmentalization using pulsed‐field‐gradient (PFG) 1H‐NMR and simultaneous confocal microscopy. In PFG experiments characterized by a fixed diffusion time (<4.7 ms) and variable b‐values (0–27000 s/mm2), 1H‐NMR data collected with untreated cells exhibited multiexponential behavior. Analysis with a slow‐exchange model revealed two distinct cellular water compartments with different apparent diffusion coefficients (ADCs; 0.56, 0.06 × 10−3 mm2/s) and volume fractions (0.96 and 0.04). During the first 12 hr of necrosis or apoptosis, the amount of water in the smallest compartment increased twofold before significant changes in cell density or plasma membrane integrity occurred. Over the same period, water content in the largest compartment decreased by a factor of >2 in apoptotic cells, in accordance with observed cell shrinkage, and changed little in necrotic counterparts, where only slight swelling was evident. These results indicate that PFG 1H‐NMR serves as a sensitive indicator of early cell death in monolayer cultures, and can be used to distinguish apoptosis from necrosis. Measurements of restricted diffusion and water exchange are presented to elucidate the compartment origins and justify the model assumptions. Magn Reson Med 52:495–505, 2004.

Combined confocal and magnetic resonance microscopy

Confocal fluorescence optical microscopy and magnetic resonance microscopy are each used to study live cells in a minimally invasive way. Both techniques provide complementary information. Therefore, by examining cells simultaneously with both methodologies, more detailed information is obtained than is possible with each microscope individually. In this paper two configurations of a combined confocal and magnetic resonance microscope are described. The first configuration is capable of studying large single cells or three-dimensional cell agglomerates, whereas the second configuration is designed for the investigation of monolayers of mammalian cells. In both cases the sample compartment is part of a temperature regulated perfusion system. Images obtained with the combined system are shown forXenopus laevis oocytes, model JB6 tumor spheroids, and a single layer of Chinese hamster ovary cells. Finally, potential applications of the combined microscope are discussed.

Multichannel multiphoton imaging of metal oxides nanoparticles in biological system

Near-IR ultrafast pulse laser and confocal microscope are combined to create a multiphoton multichannel non-linear imaging technique, which allows in situ 3-D characterization of nonfluorescent nanoparticles in biological systems. We observed intense CARS signals generated from various metal oxides due to their high third-order nonlinear susceptibilities (Chi(3)), which do not depend on the vibrational resonance but on the electronic resonance. We show that fine and ultrafine particles of metal oxides in alveolar macrophage cells may be imaged in vitro using CARS and multiphoton fluorescence microscopy with highest optical resolution for extended periods without photobleaching effects. The advantage of the epi-detection over the forward detection for imaging sub-micron particles has been investigated.

Application of coherent antistokes Raman scattering (CARS) to imaging mammalian cells: a means for gaining molecular selectivity in multiphoton imaging

Virtually all laser based microscopy imaging methods involve a single laser, with ultrafast lasers emerging as the enabling tool for a variety of methods. Two-photon fluorescence is a high sensitivity method with selectivity depending on a chromophore that is either added or produced by genetic engineering. While there are fundamental advantages over white light or other fluorescence microscopies, there are unavoidable limitations such as bleaching, photoinduced damage to the cell, and the inability to label some major constituents of the cell, particularly the abundant species. Raman imaging affords chemical selectivity but application is limited due particularly to its low sensitivity and unavoidable fluorescence background. Adding a second laser beam, shifted from the first laser by a molecular vibrational frequency, increases the detected Raman signal by many orders of magnitude and in addition shifts the detected signal to the high energy (blue) side of both lasers, removing fluorescence artifacts. Signal levels sufficient to acquire high signal-to-noise ratio images of 200 by 200 pixels in one minute requires sub-nanojoule pulse energy. A convenient, tunable source of the Stokes-shifted beam is provided by an Optical Parametric Amplifier (OPA), which requires an amplified laser. 250-kHz sources have ample energy and in addition keep the average sample power on the order of 0.1 mW, a level that even sensitive biological systems tolerate at the focal spot diameter of 0.3 micrometers . Long-term viability of mammalian cells has been demonstrated during dozens of scans in a single plane. Two-photon fluorescence provides a useful complimentary data channel that is acquired simultaneously with the Raman image. Several dyes and green fluorescence protein have been used for this purpose. Interpretation of images, acquiring three dimensional images, and identification of cellular features are ongoing activities.

A simple mode-locked Yb:KGW laser pumped by polarization-coupled diode bars

Direct-diode-pumped crystalline Yb lasers are simple and efficient but low powered. We use reshaped diode bars and a polarization-coupled scheme to produce as much as 6.3 W in 100 to 225 fs pulses.

An integrated confocal and magnetic resonance microscope for observing living cells

We have combined a confocal fluorescence optical microscope and a magnetic resonance microscope into one instrument to image simultaneously a living cell system. The high spatial resolution confocal and high spectral resolution magnetic resonance microscope produces a set of closely linked, complementary images. To enhance the value of this image set, we have also developed a statistical method, local linear modeling, to predict a high spatial resolution magnetic resonance image from the confocal optical and magnetic resonance image pair. This high spatial, high spectral resolution image offers insights not available in images obtained from separate studies, nor available in the viewing the image pair as is. This paper presents first results from application of this instrument and local linear modeling, a statistical prediction technique, to a calibration phantom and a Xenopus laevis oocyte cluster.