Correlation of histology and linear and nonlinear microscopy of the ...

Journal of
BIOPHOTONICS
128
1. Introduction
B. R. Masters: Correlation of histology and linear and nonlinear microscopy of the living human cornea
The history of the linear and nonlinear optical microscope
is inextricably linked to the development of
new clinical instruments that play important roles in
our understanding of cells and tissues and in the
early diagnosis and the prevention of disease. In the
domain of linear optics the response of the induced
polarization to the incident electromagnetic field is
linear with the amplitude of the field. Linear optical
effects include one-photon absorption, scattering,
and fluorescence. Exchange of energy and momentum
can occur in linear interactions, e.g. absorption
and scattering. Linear optical phenomena are characterized
by a induced polarization PðtÞ in the material
that depends in a linear manner on the electric
field strength EðtÞ:
PðtÞ ¼"0 c ð1Þ EðtÞ ; ð1Þ
the constant of proportionality c ð1Þ is known as the
linear susceptibility and "0 is the permittivity of free
space.
Nonlinear optical effects are characterized by the
nonlinear response of the induced polarization to
the incident electromagnetic field; the response is
nonlinear in the amplitude of the field [1, 2]. For the
case of nonlinear optics, the optical response is described
by expressing the induced polarization PðtÞ
as a power series of the electric field strength:
PðtÞ ¼"0½c ð1Þ EðtÞþc ð2Þ E 2 ðtÞþc ð3Þ E 3 ðtÞþ...Š : ð2Þ
The quantities c ð2Þ and c ð3Þ are known as the secondand
the third-order nonlinear optical susceptibilities.
In writing these two equations an important assumption
is made: the polarization of the material at
time t, only depends on the instantaneous value of
the electric field strength. It follows from this assumption,
through the Kramers-Kronig relations,
that the medium must be lossless and dispersion less.
Typically nonlinear optical phenomena occur in the
presence of laser-matter interactions in the presence
of very high electric field strengths. At high light
intensities, the incident light modifies the induced
polarization such that light waves can interact and
exchange energy and momentum. Examples of
nonlinear optical effects include the Pockels and
Kerr electro-optic effects, multiphoton excitation
(MPE) [3], second-harmonic generation (SHG),
third-harmonic generation (THG) [4], and coherent
anti-Stokes Raman scattering (CARS) [5].
Correlative microscopy is based on the use of
different optical techniques to study the same specimen,
ideally at the same location within the specimen,
in order to increase the functional and/or morphological
understanding of the specimen. Typically,
correlative microscopy could include linear (confocal
microscopy, optical low-coherence reflectometry)
together with nonlinear microscopy (MPE, SHG,
THG, and CARS).
The purpose and the emphasis of this paper is to
point out the limitations, and the multiple confounding
factors that make the interpretation of corneal
functional and morphological images difficult.
1.1 Confocal microscopy
In the last decades there have been improvements in
both the resolution and the contrast of optical microscopes.
The invention of various types of confocal
microscopes, that generate contrast from the processes
of linear absorption, scattering, and fluorescence,
provides researchers in cell biology with
extremely useful tools to investigate the biology of
cells in thick, highly scattering tissues. Confocal microscopes
are based on the principle of spatial filtering
and permit live cell imaging of cells and thick,
highly scattering tissues with improved “optical sectioning”
and improved contrast as compared to traditional
epi-fluorescence microscopes [6].
The first applications of confocal microscopy
were applied to cells and tissues that were fixed, and
stained with immunochemical stains of high specificity
to delineate the fine structures in cells such as
the cytoskeleton. Only later did researchers apply
confocal microscopy to live cells and tissues both ex
vivo and in vivo, i.e. three-dimensional imaging the
in vivo human cornea [7] and in vivo human skin
[8].
Two modes of contrast are implemented in confocal
microscopy. The first mode generates contrast
in the images with reflected and scattered light
that occurs at the interfaces with different refractive
indexes [6]. A registered stack of optical
sections could then be transformed in a digital
computer into a three-dimensional volume that visualized
the structure of cells and tissues, and these
computer generated structures could be visualized
from any arbitrary orientation. The second mode
generates the image contrast by exciting the fluorescence
from either intrinsic naturally occurring
fluorescent molecules, for example, reduced pyridine
nucleotides, NAD(P)H, and oxidized flavoproteins
[6]. Both the reduced nicotinamide adenine
dinucleotide, NADH, and the reduced nicotinamide
adenine dinucleotide phosphate, NADPH, are denoted
as NAD(P)H. The fluorescence of NAD(P)H
is in the range of 400–500 nm. Alternatively, contrast
in confocal microscopy could be based on
fluorescent probes that are genetically over-expressed
such as green fluorescent proteins (GFP),
or molecular probes that were externally applies to
the cells and tissues [6].
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