Personnel
Michael Mason
University of Maine
Department of Chemical and Biological Engineering
209 Jenness Hall
(207) 581-2344
mmason@umchem.maine.edu
http://www.umche.maine.edu/chb/faculty/mmason.htm
Lab Members:
Venkata Gopal Reddy Chada, Research Associate (IMB)
Thanuprabha Girirajan, Graduate Student (IMB)
Gary Craig, Graduate Student (BLE)
Mathias Uvieghara, Graduate Student (IMB/EE)
Research:
Time-resolved simultaneous PL/Raman Imaging Spectroscopy of single molecules/single nanoparticles in vivo
Single Molecule/Single Nanoparticle Imaging Techniques. Optical imaging, in various forms, has become the standard method for probing the spatial distribution of a wide range of properties within biological systems. In most measurements, in order to obtain sufficient signal-to-noise, a site-specific molecular probe is used in relatively high concentrations to "stain" the sample of interest which is then imaged 1:1 onto a camera (CCD), or imaged pointwise using a confocal geometry onto a photo-multiplier tube (PMT). Generally, this labeling and measurement scheme limits the spatial resolution of the image to the optical diffraction limit (~?/2). A somewhat more recent approach has been to label the system of interest at low (< 1 nM) concentrations where fluors can be observed individually. To achieve sufficient sensitivity to observe individual fluors a scanning confocal system is employed where single photons are recorded using a fast avalanche photodiode (APD). Although the optical diffraction limit is not improved upon, given that the point-spread function of the microscope system is well characterized, the certainty to which the probe position can be determined is proportional to the square root of the number of photons counted (?x,y < 10nm typical). Furthermore, the quantum optical nature of the photon stream from the individual molecular or nanoparticle probe make a detailed statistical analysis possible for the deconvolution of very faint or rapidly fluctuating signals from an otherwise dominant background. These fast sensitive measurements can reveal localized kinetic and thermodynamic information otherwise unattainable using conventional ensemble or time-averaged measurement techniques.
Time-Resolved Photoluminescence/Raman Spectral Imaging. Time-resolved, PhotoLuminescence (PL), and Raman measurements are usually considered to be orthogonal techniques. This is largely due to the 1015 fold difference in the efficiencies of the Raman and PL processes and the long integration times often used to obtain detailed spectra. At the single molecule/probe level, however, a combination of these techniques becomes possible despite dramatic reduction in the photon budget.
This technique makes use of the quantum optical nature of light absorption/emission. Specifically, when a fluorescent molecule absorbs a photon it cannot absorb a second photon until it has returned back to the electronic ground state. This process requires 1-10 ns for typical molecular and quantum dot fluors. Scattering processes such as those measured in Raman spectroscopy, however, are nearly instantaneous (picoseconds). This means that for an intense narrow (< 100ps) excitation pulse, we expect to observe only a single fluorescent photon for each molecule in the focal volume, and a large number of scattered photons proportional to the excitation intensity. Furthermore, the fluorescence photons will be emitted long after the scattered photons. Using a time-gated pump probe technique the "slowly" emitted photons can be electronically separated from the nearly instantaneously scattered photons dramatically improving signal-to-noise in fluorescence measurements. Alternatively, when two-photon excitation is used, the Raman signal and the fluorescence signal can be separated into two detection pathways. One providing < ns timing resolution and limited spectroscopic information (2-8 channels) of the dynamics of the fluor and the other channel yielding detailed vibrational information about the structure and environment of the fluor with limited (100ms) timing resolution.
With this technique the temporal and spectroscopic characteristics of individual probe fluors can be observed, while simultaneously obtaining chemical information about their local environment, with high spatial resolution. This technique promises to be a powerful tool capable of investigating a broad range of properties including: folding kinetics, translational diffusion (transduction), binding and reorganization, chemical and rotational dynamics, charge transfer, small molecule or ion/proton exchange (signal transduction), and potentially excited electronic state dynamics.
PL/Raman Nanoparticle Tags for in vivo Spectroscopic Imaging. Unlike their fluorescent counterparts Raman tags have several inherent advantages. Because photons are being scattered and not absorbed, they do not exhibit power-dependent excitation limitations such as photobleaching or fluorescence intermittency. They can be excited at any wavelength, allowing for the use of red-NIR light sources capable of improved optical penetration into biological samples while simultaneously reducing background fluorescence. Furthermore, they can be designed to have distinct "fingerprint" vibrational spectra with narrow (< 20cm-1) linewidths, compared to typical broad room temperature fluorescence linewidths (~1000cm-1), making it possible to image and distinguish hundreds of tags simultaneously using spectral deconvolution. While scattering cross-sections for individual molecules are extremely small, (10-30 cm2/molecule), intelligent nanoparticle and experimental design can result in signal enhancements of >1015 when using SERS, and as high as 1017 for Resonant SERS (SERRS).
Taking advantage of surface-enhanced Raman effects we use Au and Ag nanoparticles, prepared using redox solution chemical techniques, as a scaffold for a series of molecular Raman tags (nominally 1-2 nm diameter). Raman tags will then be carefully selected, in collaboration with other members of the IMB, where vibrational signatures are optimized to be distinct from those found in the chosen biological system. Using surface chemistry and sol-gel techniques, the Raman tags are then bound to the metallic nanoparticle and imbedded in a thin (1-2 nm) glass shell. A second sol-gel derived glass shell (1-2 nm) is added, passivating the surface of the particle, making the Raman signal invariant to fluctuations in the local environment while reducing nanoparticle toxicity issues. The use of multiple tags per nanoparticle dramatically increases the scattering efficiency such that signal levels can be in excess of 100 times that of comparable fluorescent probes. Unlike their semiconductor quantum dot counterparts, these ~5-10 nm glass capped nanoparticles have been shown to exhibit almost no aggregation in aqueous solutions. The Si-OH terminal surface of the outermost glass shell allows for further chemical modification to include specific active and/or passive biological probes (RNA, DNA, antibody, etc). Using PL/Raman spectral imaging, fluorescence probe (PL, FRET, FLIM, Molecular Beacons) signals can be monitored along with the Raman tag signal, providing time-resolved chemical sensitivity and imaging fidelity of many species simultaneously.
As a newly emerging technology a detailed characterization of nanoparticle preparations must be performed prior to use in complex biological systems. Although the glass-capping layer on the tagged metallic nanoparticle, in principle, renders the Raman signal insensitive to local environment, it is unknown whether well-established sol-gel techniques can produce surface coatings of sufficiently low porosity to completely eliminate diffusion into the nanoparticle over extended periods. This and other issues (size dispersion, absorption/emission, scattering efficiency, polarization effects, nanoparticle dimension optimization, etc) are being investigated as an ensemble (freely diffusing) and as individual species within the ensemble, using single molecule imaging techniques, providing detailed quantitative information about the photophysical processes associated with these nanoparticles, and allowing for the highest level of design control possible.
Recent Publications:
Mason MD, Credo GM, Weston KD, et al. 1998. Luminescence of individual porous Si chromophores. Phys Rev Let 80:5405-5408.
Credo GM, Mason MD, Buratto SK. 1999. “External quantum efficiency of single porous silicon nanoparticles. Appl Phys Let 74:1978-1980.
Michler P, Imamoglu A, Mason MD. 2000. Quantum correlation among photons from a single quantum dot at room temperature. Nature 406:968-970.
Mason MD, Sirbuly DJ, Carson PJ, et al. 2001. Investigating individual chromopores within single porous silicon nanoparticles . J Chem Phys 114:8119-8123.
Schuck PJ, Mason MD, Grober RD, et al. 2001. Spatially resolved photoluminescence of inversion domain boundaries in GaN-based lateral polarity heterostructures. Appl Phys Let 79:952-954.
Michler P, Imamoglu A, Kiraz A, et al. 2002. Nonclassical radiation from a single quantum dot. Physica Stat Solidi B–Basic Res 229:399-405.
Mason MD, Sirbuly DJ, Buratto SK. 2002. Correlation between bulk morphology and luminescence in porous silicon investigated by pore collapse resulting from drying. Thin Solid Films 406:151-158.
Schwarz UT, Schuck PJ, Mason MD, et al. 2003. Microscopic mapping of strain relaxation in uncoalesced pendeoepitaxial GaN on SiC. Phys Rev B 67:045321.
Sirbuly DJ, Schmidt JP, Mason MD, et al. 2003. Variable-ambient scanning stage for a laser scanning confocal microscope Rev Sci Ins 74:4366-4368.
Mason MA, Ray K, Pohlers G, et al. 2003. Probing the local pH of polymer photoresist films using a two-color single molecule nanoprobe. J Chem Phys B 107:14219-14224.
Mason MD, Ray K, et al. 2004. Single molecule acid-base kinetics and thermodynamics. Phys Rev Let 93:(7).
Ray K, Mason MD, Yang C, et al. 2004. Single-molecule signal enhancement using a high-impedance ground plane substrate. Appl Phys Let 85:5520-5522.
Ray K, Mason MD, Grober RD, et al. 2004. Quantum yields of photoacid generation in 193-nm chemically amplified resists by fluorescence imaging spectroscopy. Chem Mat 16:5726-5730.
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