Steven D. Colson

Achieving molecular selectivity in imaging using multiphoton Raman spectroscopy techniques

In the case of most optical imaging methods, contrast is generated either by physical properties of the sample (Differential Image Contrast, Phase Contrast), or by fluorescent labels that are localized to a particular protein or organelle. Standard Raman and infrared methods for obtaining images are based upon the intrinsic vibrational properties of molecules, and thus obviate the need for attached fluorophores. Unfortunately, they have significant limitations for live‐cell imaging. However, an active Raman method, called Coherent Anti‐Stokes Raman Scattering (CARS), is well suited for microscopy, and provides a new means for imaging specific molecules. Vibrational imaging techniques, such as CARS, avoid problems associated with photobleaching and photo‐induced toxicity often associated with the use of fluorescent labels with live cells. Because the laser configuration needed to implement CARS technology is similar to that used in other multiphoton microscopy methods, such as two‐photon fluorescence and harmonic generation, it is possible to combine imaging modalities, thus generating simultaneous CARS and fluorescence images. A particularly powerful aspect of CARS microscopy is its ability to selectively image deuterated compounds, thus allowing the visualization of molecules, such as lipids, that are chemically indistinguishable from the native species.

s and d Rydberg complexes of NO probed by double-resonance multiphoton ionisation in the region n* = 5 to n* = 25; multichannel quantum defect analysis. Part II

Optical-optical double-resonance multiphoton ionisation spectroscopy is used to probe the n*s and n*d Rydberg series converging to the first rotational levels of NO+X1Σ+, v = 0. Intermediate values of the principal quantum number probed are 5 ⩽ n* ⩽ 2>5. The initial excitation occurs via one of two rotational-parity levels of the C 2Π state (N′ = 2 and N′ = 6). A description of the rotational-electronic structure of the observed supercomplexes is proposed in terms of multichannel quantum defect theory (MQDT). The observation of ‘forbidden’ n*f levels is tentatively explained both in terms of d-f mixing in the upper levels and of a d-wave contribution to the 3pπ orbital of the C state. Rydberg-valence interactions show up as predissociation, which strongly competes with ionisation in the intermediate n*d supercomplexes with 8 ⩽ n* ⩽ 1>2. This work complements the previous analysis of the s and d Rydberg states of NO corresponding to n* ⩽ 8> and 25 ⩽ n* ⩽ 4>0 [15].

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.

FRET measurements between small numbers of molecules identifies subtle changes in receptor interactions

Overexpression of HER2 alters the cellular behavior of EGF receptor (EGFR) and itself, with great implications on cell fate. To understand the molecular interactions underlying these alterations, we quantified the association between the two receptors by looking at efficiency changes in fluorescence resonance energy transfer (FRET) between a small number of molecules at the membrane of living cells. Human mammary epithelial (HME) cells expressing varying degrees of HER2 were studied, to identify and compare the degree of receptors interactions as a function of HER2 overexpression. A high resolution wide-field laser microscope combined with a high sensitivity cooled CCD camera was used to capture simultaneously donor and acceptor emissions. Alternating between green and red lasers every 80 msec, donor, FRET, and acceptor images were acquired and were used to calculate FRET efficiency. Automated image analysis was developed to create FRET efficiency maps from overlapping donor, acceptor and FRET images, and derive FRET efficiency histograms to quantify receptor-receptor interactions pixel by pixel. This approach enabled us to detect subtle changes in the average distance between EGFR molecules, and between EGFR and HER2. We found pre-existing EGFR homoassociations, and EGFR-HER2 heteroassociations in cells overexpressing HER2, and identified the changes in these interactions with ligand stimulation. These observations demonstrate the power of FRET measurements between small numbers of molecules in identifying subtle changes in molecular interactions in living cell.

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.

Rotationally resolved spectroscopy of the ([Ctilde]-[Xtilde] band of 15NH3 by infrared-ultraviolet double resonance

Double resonance measurements on 15NH3 in a free expansion jet are reported. These were carried out using a continuous wave (CW) CO2 laser and a pulsed ultraviolet (UV) laser system. Infrared radiation was used to prepare molecules in individual rotational levels of the v2=1 vibrational state of the electronic ground state of [15N]ammonia. These molecules were the photoionized via transitions of the [Ctilde]-[Xtilde] band in a (2+1) process using the doubled output of a pulsed dye laser. The ion signal was detected using a time of flight mass spectrometer. Two known coincidences between CO2 laser lines and individual rovibrational transitions of 15NH3 were used to populate the (J, K)=(5, 4) and (3, 0) symmetric levels of v2=1. Double resonance signals corresponding to O, P, Q, R, and S branch two photon transitions in the [Ctilde]-[Xtilde] band were observed and measured accurately. These data were augmented by conventional (2+1) REMPI measurements on the 20/0, 20/1, and 20/2 sub-bands of 15NH3: A complet...

Annual Report 2002. Chemical Structure & Dynamics

This report describes the research and accomplishments of the Chemical Structure and Dynamics (CS&D) Group of the William R. Wiley Environmental Molecular Sciences Laboratory (EMSL) from October 2000 through December 2001. Publications, presentations, and collaborations are listed from October 2000 to September 2002. The EMSL is a national user facility located at the Pacific Northwest National Laboratory, Richland, Washington. The CS&D program supports the Department of Energy?s mission of fostering fundamental research in the natural sciences to provide a basis for new and improved energy technologies and for understanding and mitigating the environmental impacts of energy use and contaminant releases.

A Risk and Outcome Based Strategy for Justifying Characterization to Resolve Tank Waste Safety Issues

This report describes a risk-based decision-making strategy for determining characterization needs and resolving safety issues during the storage and remediation of radioactive waste stored in the 177 underground tanks located on the U. S. Department of Energy’s Hanford Site in Washington State. From 1944 to the late 1980’s, Hanford reprocessed 100,000 metric tons of irradiated nuclear fuel to produce 67 metric tons of plutonium-239 for use in nuclear weapons. The high-level radioactivity and chemical waste generated from this reprocessing is now stored in aging carbon steel tanks. The recommended strategy uses interactive problem evaluation and decision analysis methods commonly used in industry to solve problems under conditions of uncertainty (i. e., lack of perfect knowledge). It acknowledges that problem resolution comes through both the application of high-quality science and human decisions based upon pReferences and sometimes hard-to-compare choices. It recognizes that to resolve a safety problem, the controlling waste characteristics and chemical phenomena must be measurable or estimated to an acceptable level of confidence tailored to the decision being made.