Biomedical Engineering Reference
In-Depth Information
Optical path
488 nm notch filter
Spectrometer
Laser
488 nm
Confocal
aperture
Objective
Fluorophore
monolayer
θ
max
Substrate
Fig. 5.5.
Microscope setup for SSFM.
was imaged onto a thermoelectrically cooled charge-coupled device (CCD).
For WL measurements, normal Koehler illumination with a halogen lamp was
used, whereas for fluorescence measurements, the light source was an argon
ion laser with a 488 nm line. The emission from the fluorophores was collected
with a 5
(
NA
=0.12) objective and transmitted into the spectrometer through
a notch filter, blocking the excitation light (Fig. 5.5).
×
5.4.2 Fitting Algorithm
Self-interference spectra are raw data composed of the envelope function rep-
resented by the emission profile of the free fluorophore, the oscillatory interfer-
ence component, and high-frequency Gaussian noise introduced by the spec-
trophotometer. Both WL reflectivity and fluorescence self-interference spectra
were fitted using a custom-built MATLAB application that separates the os-
cillatory component from the envelope function. This program automatically
calculates the parameters of the system such as the thickness of thin films
or position of the emitters above the mirror. The variation in the index of
refraction of silicon oxide within the used wavelength span was taken into ac-
count in the fitting algorithm. The model also takes into account the complex
reflectivity of the underlying stack of dielectrics and the orientation of the
dipole moments of the emitters. Because the curve-fitting algorithm extracts
the oscillatory term to determine the label height, the measurements are im-
mune not only to potential quenching of the entire spectrum, but also to any
spectral modifications or nonradiative transfer effects.
