Deconvolution and Denoising for Confocal Microscopy - Modeling in Computational Biology and Biomedicine - page 123

online interactive and elegant virtual tutorials, from several microscope vendors,
that compares WFM and CLSM imaging. These are available in the resources given
in Sect. 4.4 .
The development of the confocal microscope allowed the efficient removal of
blur by physical means by filtering out the emission light that does not contribute to a
well focused and blur-free image. The PMT can only detect light that passes through
the pinhole. Since the diameter of the pinhole aperture can be adjusted, more or
less out-of-focus light can be eliminated from the detected light. The physical
elimination of the out of-focus light by the aperture placed in front of the detector
is explained in Fig. 4.2 b (a schema illustrating the confocal ray path). It is basically
this configuration that gives a CLSM the possibility to create an image representing
the emission fluorescence intensities corresponding to a thin optical slice or a single
plane out of a thick fluorescent specimen. Depending on the objective lens used, this
so called “optical sectioning property” can generate slices as thin as 500 nm.
Unlike in Fig. 4.3 a, in the confocal image in Fig. 4.3 b, a large fraction of the blur
is eliminated and image details inside the sample become visible. This can be further
illustrated by looking at the 2-D Fourier transform [ 32 ] of a single section. We notice
that, for the WFM in Fig. 4.3 c, the high frequency information is unavailable. It is
also impossible to image a single focal plane using a WFM, because in the 3-D
Optical transfer function (OTF), there is a cone of frequencies that are missing (the
missing cone problem [ 14 ]). By comparing Fig. 4.3 c with Fig. 4.3 d, it might seem
like there is more information in the low frequencies in Fig. 4.3 cthaninFig. 4.3 d.
However, the CLSM image in Fig. 4.3 d retains much of the higher frequencies,
shown by the rays along the horizontal and vertical axes, providing sharp details.
The resolut io n of the CLSM in terms of cut-off frequency can be improved by a

factor of √ 2 [ 7 0 ], and the Full-width at half maximum (FWHM) is improved by a

factor of 1 / √ 2

0 . 707 for a very small diameter. In practice, as the signal level is
very low, the level decreases with the square of the diameter.
Although the original design of Minsky [ 49 ] scanned the object by moving the
specimen stage, current commercial adaptation of a CLSM scans the specimen by
using galvanometric mirrors to tilt the laser beam as it passes through the back focal
plane of the objective.

≈

4.2.1.2 Fundamental Imaging Challenges

When using very thin samples obtained after chemical fixation and histological
processing, sharp in-focus images can be obtained with high NA objective lenses.
Nevertheless, imaging intact living cells or tissues that largely surpass the thickness
of the imaging plane (DOF) suffers from a greatly reduced contrast since most
fluorescence observed is out-of-focus light blurring in-focus details and hence
reducing image contrast [ 56 , 74 ].

Diffraction Barrier. Apart from fluorescence from nearby planes, the most impor-
tant source of blur is diffraction . When light from a point source passes through a