Deconvolution and Denoising for Confocal Microscopy - Modeling in Computational Biology and Biomedicine - page 115

chemically-fixed biological samples follows a straightforward goal to preserve
structure while maximizing contrast and resolution. Due to the action of chemicals
like acetone, alcohols or cross-linking aldehydes, cell components are immobilized
and the biological sample is killed. Such a fixation step is optimized to preserve the
fine-morphology of the cells and tissues of the biological samples. Chemical fixation
makes biological samples and its constituents immobile, and therefore removes
any time constraint during image recording. By clearing biological samples before
imaging, most bulk components like water and various soluble components are
extracted from the tissues. These components are then replaced by a homogeneous
medium of refraction index close to 1 . 515, a process that will greately improve
image quality and allow one to obtain near ideal optical imaging conditions.
Interestingly, live cell imaging must seek a continuous balance between image
quality and information content (which requires more signal), and the need to
preserve on cell viability and unaltered biological processes. Simply maximizing
contrast and resolution by extending data collection mostly leads to cell damage and
permanent loss of signal intensities (bleaching). As such, preserving the viability
often implies producing noisy data.
In addition, most botanical samples contain highly refractile cell walls that
surround an aqueous cell content often filled with highly auto-fluorescent and light-
scattering components. This can lead to extreme refraction index heterogeneities
within the sample and seriously compromise deep imaging for 3-D microscopy.

4.1.2.2 Fluorescence

Fluorescence is the phenomenon whereby light is first absorbed by a crystal or
molecule and then rapidly (of the order of nanoseconds) re-emitted at a slightly
longer wavelength (Fig. 4.1 a). The Jablonski fluorescence diagram in Fig. 4.1 a,
was named after the Polish physicist Aleksander Jablonski , and it illustrates the
energy of the electronic states of a molecule and the transitions between them. The
states are arranged vertically by energy arrows and the transitions between them
are given by straight arrows. The event time progresses from left to right. This
process can be explained as follows. The fluorescent molecule creates an excited
electronic singlet state S 2 by absorbing some energy E . When it finally relaxes
back to its native ground state, S 0 it emits a photon having a wavelength longer
than that of the excitation beam. This shift in wavelength towards the red spectrum
occurs because the energy of the emitted beam is on an average lower than that of
the illumination. We recall that the energy and the wavelength are related by the
expression Energy

1 / Wavelength. This relative shift is known as the Stokes
shift and the emitted beam is said to be red-shifted (Fig. 4.1 b).

∝

4.1.2.3 Fluorescence Microscopes

Fluorescence microscopes are optical instruments capable of imaging a specimen
in 3-D. Under ideal conditions, the number of photons emitted are proportional