Traditional far-field optical microscopy is limited by the diffraction limit of light. The spatial resolution that can be achieved is a couple hundred nanometers at a minimum. Improved spatial resolution can be achieved using near-field optics; however, there is always a problem of physical intrusion with near-field probes. Even more problematic is the inefficiency in delivering light to probes of sub-wavelength dimensions. There is a need to develop imaging techniques with sub-diffraction limited spatial resolution in order to study numerous chemical systems with important phenomena that occur in the tens of nanometers regime.
TIR Raman was first described by Ikeshoji in 1973. It can provide spatial resolution on the order of the diffraction limit of light in the focal plane, and spatial resolution below the diffraction limit of light in the direction perpendicular to the focal plane (z plane). As with other TIR techniques, the incident angle of light is varied upon a material with a high index of refraction (n1). At angles beyond the critical angle, the incident light is completely reflected and an evanescent wave is created in the adjacent medium (n2), which must have a lower index of refraction than n1. The penetration depth of the evanescent wave varies with the angle of incidence, the wavelength of light, and the indices of refraction of the two media. The penetration depth for Raman scattering is approximately half the exponential decay length of the electric field.
We are developing a high resolution TIR Raman microspectroscopy and imaging system with angle scanning capabilities. A schematic of this instrument is shown in Figure 1. The instrument will be used to study catalytic reactions that take place within functionalized porous silica materials.
Figure 1. Variable Angle Total Internal Reflection Raman Microscope. DL (785 nm diode laser), M (mirror), BL (beam lifter), L (lens), F (filter), VA (variable angle optics), P (prism), FO (fiber optic), DM (dichroic mirror), MBL (motorized beam lifter), CCD (IR enhanced charge coupled device)
S. Hell and coworkers have described and demonstrated the technique referred to as STED, whereby the diffraction resolution limit in far-field fluorescence microscopy is circumvented. This is achieved by inhibiting the fluorescence at the periphery of a diffraction limited spot by stimulated emission. The net result is a fluorescent spot that is smaller than the dimensions set by diffraction. Resolution in the 10-20 nm range has been reported, with molecular scale resolution fluorescence measurements fundamentally achievable in the far field.3, 4 The basic operating principal of STED microscopy (Figure 2) is a tightly focused scanning excitation laser pulse of tens of picoseconds duration, and a ring shaped STED laser pulse of hundreds of picosecond duration. The STED laser pulse is shaped by a spatial phase modulator, and has zero intensity at the center of the ring. The resolution that can be achieved is governed by the power of the STED laser pulse (up to tens of mWs), which is at much higher power than the excitation pulse. The excitation wavelength corresponds to the fluorophoreâ€™s maximum excitation wavelength, and the STED wavelength is lower in energy in a region where the fluorophore does not have appreciable absorption. The stimulated emission laser pulse depopulates the excited electronic state of the fluorophores within the doughnut beam profile, leaving only a small, lower than diffraction limit, spot of excited fluorophores at the center of the doughnut. Fluorescence from the remaining 10-20 nm spot is recorded with a single channel detector. The sample is mounted on a precision translation stage that is scanned in approximately 10 nm increments at dwell times of a few microseconds per pixel. Scanning the surface is used to create a sub-diffraction limit image, as has been demonstrated for nanoparticles and cellular structures.
An alternative approach to performing STED imaging has recently been demonstrated in which continuous wave (CW) lasers are used. The excitation pulse remains a tightly focused spot and the depletion laser is a doughnut shaped beam. In the case of CW STED, the STED laser is at much higher intensities (0.1 to 1 W) in order to keep stimulated emission rates higher than excitation rates within the doughnut beam profile. Generally lower lateral resolution is achieved. One general limitation of the traditional and CW STED microscopy systems that have been described in the literature is the limited number of fluorophores that can be studied with the fixed wavelength lasersâ€”only a few fluorophores have been studied with CW STED. We are developing a STED microscope system, and investigating classes of fluorophores that can be used in the CW format.
Figure 2. Schematic explanation of STED microscopy. Excitation laser beam excites molecules in a diffraction limited spot. A STED laser pulse depletes the excited species with a ring shaped laser profile. The result is a spot of excited species that is smaller than the diffraction limit light. High spatial resolution imaging is possible with this format. Figure modified from Hell, S. et al.
1. Faculty advisor Iota Sigma Pi, Aurum Chapter
2. Volunteer State Science & Technology Fair of Iowa
3. Volunteer Regional High School Science Bowl
4. Reviewer for journals Analytical Chemistry; Langmuir; Journal of the American Chemical Society; and National Science Foundation, U.S. Department of Energy
5. Co-Inventor on US Patent #7,067,322, â€œFusion Protein Arrays on Metal Substrates for Surface Plasmon Resonance Imagingâ€
Collaborators and Co-editors
Collaborators and co-editors past 4 years. Thomas Bunch, Scott Cowell, Victor Hruby, Henry Yamamura (University of Arizona), Jason Coym (University of South Alabama), Edward Yeung, Jacob Petrich, Victor Lin, Ning Fang, Thomas Lubberstedt (Iowa State University)
Graduate and Postdoctoral Advisors and Advisees
Graduate advisors and postdoctoral sponsors. Michael Natan (Oxonica Inc., Mountain View, California), Robert Corn (University of California, Irvine, Department of Chemistry), Mary Wirth (Purdue University, West Lafayette, Department of Chemistry), Danny Brower (deceased)
Graduate students (8 total): Neha Arora, Deepak Dibya, Michael Murphy, Suzanne Sander. Working on DOE projects: Jason Lupoi, Kristopher McKee, Matthew Meyer, Chien-Ju Shih (Iowa State University).
Postdoctoral scholar (1 total): Nuha Salem (Clarkson University)