single molecule Experiments

Single molecule spectroscopic methods represent a powerful set of tools that are now providing detailed information on the properties of individual nanoscale environments in a range of thin film materials, including sol-gel-derived silicates. Single molecule spectroscopy experiments are usually performed on a light microscope by first locating the individual molecules in imaging experiments. The molecules are then spectroscopically interrogated by positioning them (one at a time) in the microscope focus.

Single molecule experiments in our labs are performed on a sample scanning confocal microscope. A simplified diagram of the instrument is shown below. The sample sits atop a piezo-electric scanning stage, which in turn is mounted above a high numerical aperture oil immersion objective on an inverted light microscope. Only a few hundred nanowatts are needed to excite the molecules, so we use low-power, air-cooled argon ion lasers, green HeNe lasers and small solid-state laser systems as light sources. Light from the laser source is first directed through appropriate optics to control the power, polarization state and spectrum of the incident light. It is then directed into the epi-illumination port of the microscope, where it is reflected from a dichroic mirror into the back aperture of the microscope objective. A diffraction-limited spot (â 400 nm 1/e² diameter) is produced in the sample.

Light from the sample is collected in reflection and the desired single molecule fluorescence is isolated from residual excitation light by use of appropriate notch and bandpass filters. Fluorescence detection is most often accomplished using a single photon counting avalanche photodiode (APD).

Images are obtained by raster scanning the sample over the incident laser spot, while recording the fluorescence. Time transients depicting the signal fluctuations that often occur in single molecule studies are obtained by positioning the point of interest over the laser focus and recording the spectrally-integrated fluorescence in time. Fluorescence spectra are obtained in an analogous manner, except that the fluorescence is passed through a spectrograph and onto a liquid-nitrogen-cooled charge-coupled device (see above Figure) for detection.

In our single molecule studies, we select specific dyes for use primarily based on their sensitivities to certain matrix properties. The structures of four different dyes that we have employed are shown below. We have made extensive use of the nile red chromophore primarily because of its sensitivity to materials polarity. C.SNARF-1 was chosen because of its sensitivity to the pH of its surrounding environment. These dye molecules are doped into the films to be studied at nanomolar concentrations. They are subsequently individually located and probed in the microscope described above.

SINGLE MOLECULE STUDIES OF Silicate thin films

Recently, we have been collaborating with the group of Prof. Maryanne Collinson to apply single molecule methods to the study of sol-gel-derived silicate thin films. We have employed single molecule spectroscopy to probe (on the nanoscale) silicate film polarity, acidity, and mass transport properties.

Important results from our group include the observation of nonrandom (bimodal or multimodal) distributions in the polarity properties of organically-modified silicate (ORMOSIL) thin films prepared by the cohydrolysis and cocondensation of precursors such as tetraethoxysilane (TEOS) and butyltrimethoxysilane. Nile red was used as the probe dye in these studies. An example single molecule fluorescence spectrum is depicted below.

These spectra are curve fit (as shown) and analyzed using a modified form of Marcus Theory for charge transfer transitions. The results provide information on the static and dynamic polarity properties of the matrix. The environment-dependent shift in the transition energy, designated DDG^o and the local reorganization energy, designated l are obtained. Histograms depicting the values obtained from hundreds of molecules are then prepared for each film. Examples of these data are shown below for ORMOSIL films derived from butyltrimethoxysilane (BTMOS) and cyanopropyltrimethoxysilane (CNS) along with TEOS.

The data depict significant materials heterogeneity and provide strong evidence for the formation of phase separated inorganic and organic-rich domains. The presence of such domains has potentially profound implications on the solubilities and mass transport properties of reagents and analytes encapsulated within ORMOSIL thin films.

Additional investigations we have undertaken on ORMOSIL films include studies of dye molecule diffusion within the matrix. We have observed facile molecular diffusion in certain ORMOSIL films, even for silicate-bound dye molecules, pointing to the presence of liquid-like oligomers within films of high organic content. We obtain such information be recording spectrally integrated time transients from single locations in the films (i.e. in fluorescence correlation spectroscopy experiments). These transients are then mathematically analyzed to obtain diffusion coefficients reflective of the molecular mobility in distinct sample regions. Below is shown an example time transient giving strong evidence for continuous diffusion of molecules into and out of the microscope focus. Also shown is an example ≥autocorrelation≤ of this transient, from which the diffusion coefficient is obtained.

Finally, in studies of nanoscale pH variations in silicate films, we have shown that materials acidity is controlled by variations in the pKas of surface silanols and by residual acid (used to catalyze hydrolysis and condensation) left in the films after gelation. Importantly, while treatment by immersion in external solutions can be used to alter the pH of the local nanoscale environments, some of these environments are inaccessible to external solutions on periods as long as several hours. These results promise to help further our understanding of the functional properties of silicate thin film chemical sensors.

related publications

1. Higgins, D. A.; Collinson, M. M. "Gaining Insight into the Nanoscale Properties of Sol-Gel-Derived Silicate Thin Films by Single Molecule Spectroscopy" Langmuir Feature Article, submitted.

2. Higgins, D. A.; Hou, Y. Single Molecule Spectroscopy Studies to Characterize Nanomaterials. In Encyclopedia of Nanoscience and Nanotechnology; Schwartz, J. A., Contescu, C., Putyera, K., Eds.; Marcel-Dekker: New York, 2004; p. 3575.

3. Martin-Brown, S. A.; Fu, Y.; Saroja, G.; Collinson, M. M.; Higgins, D. A. "Single Molecule Studies of Diffusion by Oligomer-Bound Dyes in Organically-Modified Sol-Gel-Derived Silicate Films" Anal. Chem. 2005, 77, 486.

4. Fu, Y.; Collinson, M. M.; Higgins, D. A. "Single Molecule Spectroscopy Studies of Microenvironmental Acidity in Silicate Thin Films" J. Am. Chem. Soc. 2004, 126, 13838.

5. Higgins, D. A.; Collinson, M. M.; Saroja, G.; Bardo, A. M. "Single Molecule Spectroscopic Studies of Nanoscale Heterogeneity in Organically-Modified Silicate Thin Films" Chem. Mater. 2002, 14, 3734.

6. Hou, Y.; Higgins, D. A. "Single Molecule Studies of Dynamics in Polymer Films and at Surfaces: Effect of Ambient Relative Humidity" J. Phys. Chem. B 2002, 106, 10306.

7. Bardo, A. M.; Collinson, M. M.; Higgins, D. A. "Nanoscale Properties and Matrix-Dopant Interactions in Dye-Doped Organically-Modified Silicate Thin Films" Chem. Mater. 2001, 13, 2713. Erratum Chem. Mater. 2001, 13, 3058.

8. Mei, E.; Bardo, A. M.; Collinson, M. M.; Higgins, D. A. "Single Molecule Studies of Sol-Gel-Derived Silicate Films. Microenvironments and Film Drying Conditions" J. Phys. Chem. B 2000, 104, 9973.

9. Wang, H.; Bardo, A. M.; Collinson, M. M.; Higgins, D. A. "Microheterogeneity in Dye Doped Silicate and Polymer Films" J. Phys. Chem. B 1998, 102, 7231.

10. Hou, Y.; Bardo, A. M.; Martinez, C.; Higgins, D. A. "Characterization of Molecular Scale Environments in Polymer Films by Single Molecule Spectroscopy" J. Phys. Chem. B 2000, 104, 212.

11. Wetzel, D. L.; Striova, J.; Higgins, D. A.; Collinson, M. M. "Synchrotron Infrared Microspectroscopy Reveals Localized Heterogeneities in an Organically Modified Silicate Film" Vibrational Spectroscopy 2004, 35, 153.