Biomedical Engineering Reference
In-Depth Information
4Pi confocal fluorescence microscopy introduced by Stefan Hell [1]. However,
instead of using the 4Pi microscope for 3D image scanning, the emitted signal
is collected by two high NA objectives and coupled to a spectroscopy system
to localize monolayers with nanometer accuracy. Scanning of the tight collec-
tion spot is performed in lateral dimensions and axial profile is extracted form
the spectral fringes similar to SSFM.
In Fig. 5.11b, typical spectral response of a 4Pi SSFM system is shown. The
two distinct spectra are from the same spot of a monolayer that was moved by
5 nm in axial direction between the data acquisition. The fluorescent emitter
is a monolayer of Alexa Fluor 488 dye deposited on a glass cover slip and
placed in the common foci of the two high NA oil immersion objectives of
the 4Pi microscope. The sample was excited by a 488 nm continuous wave
laser using the two objectives. The spectral response clearly shows the shift in
spectral fringes because of the change in the axial position of the monolayer of
fluorophores. A simple fitting algorithm that utilizes a geometrical model and
only includes the position of monolayer with respect to the geometrical focus
and the external path length difference can be used to determine the relative
difference in the axial positions of monolayers. As seen from Fig. 5.11c, the
axial position values that minimize the error in fitting the spectral responses
of two positions of the monolayer were actually 5.1 nm apart, which agrees
well with the 5 nm physical change in the axial position of the layer.
5.7 Conclusions
We have developed a new technique, SSFM, which transforms the variation in
emission intensity for different path lengths used in fluorescence interferome-
try to a variation in the intensity for different wavelengths in emission, encod-
ing the high-resolution information in the emission spectrum. Using SSFM,
we have demonstrated analysis of systems that include monolayers of fluores-
cein, quantum dots, and lipid films. More importantly, we have demonstrated
conformation of surface-immobilized DNA by estimating the shape of coiled
ssDNA, the average tilt of dsDNA of different lengths, and the amount of
hybridization. We have also shown that localization of axial position of fluo-
rescent structures with high precision is possible without sacrificing the lateral
resolution using SSFM in 4Pi configuration.
These data provide important proofs of concept for the capabilities of
novel optical methods for analyzing the molecular disposition of DNA and
protein on surfaces. The determination of DNA conformations on surfaces and
hybridization behavior provide information required to move DNA interfacial
applications forward and thus impact emerging clinical and biotechnological
fields.
