Theo Lasser

Confining the sampling volume for Fluorescence Correlation Spectroscopy using a sub-wavelength sized aperture

For the observation of single molecule dynamics with fluorescence fluctuation spectroscopy (FFS) very low fluorophore concentrations are necessary. For in vitro measurements, this requirement is easy to fulfill. In biology however, micromolar concentrations are often encountered and may pose a real challenge to conventional FFS methods based on confocal instrumentation. We show a higher confinement of the sampling volume in the near-field of sub-wavelength sized apertures in a thin gold film. The gold apertures have been measured and characterized with fluorescence correlation spectroscopy (FCS), indicating light confinement beyond the far-field diffraction limit. We measured a reduction of the effective sampling volume by an order of magnitude compared to confocal instrumentation.

Image formation in fluorescence coherence-gated imaging through scattering media.

Recently, we have experimentally demonstrated a new form of cross-sectional, coherence-gated fluorescence imaging referred to as SD-FCT (spectral-domain fluorescence coherence tomography). Imaging in SD-FCT is accomplished by spectrally detecting self-interference of the spontaneous emission of fluorophores, thereby providing depth-resolved information on the axial positions of fluorescent probes. Here, we present a theoretical investigation of the factors affecting the detected SD-FCT signal through scattering media. An imaging equation for SD-FCT is derived that includes the effects of defocusing, numerical-aperture, and the optical properties of the medium. A comparison between the optical sectioning capabilities of SD-FCT and confocal microscopy is also presented. Our results suggest that coherence gating in fluorescence imaging may provide an improved approach for depth-resolved imaging of fluorescently labeled samples; high axial resolution (a few microns) can be achieved with low numerical apertures (NA<0.09) while maintaining a large depth of field (a few hundreds of microns) in a relatively low scattering medium (6 mean free paths), whereas moderate NAs can be used to enhance depth selectivity in more highly scattering biological samples.

Chapter 4 Cross Talk in Full-Field Optical Coherence Tomography

The formation of cross-sectional images of biological tissues requires the discrimination between light conveying useful information—that is, propagating directly from object to image— from the abundant parasitic light caused by multiple scattering inherent to turbid media [1, 2]. In optical coherence tomography (OCT) [3], selective detection of light undergoing a single backscatter event (reflection imaging) and rejection of multiply scattered light (MSL) has been successfully achieved by combining temporal coherence gating and confocal spatial filtering [4, 5].