Biomedical Engineering Reference
In-Depth Information
It is interesting to note that for
a
> 1, the sensitivity is increased, as the phase varies more rapidly for
the same Δ
z
displacement. In contrast, the medium for which
a
< 1 reduces the sensitivity. This suggests
a way to tune the sensitivity by appropriate selection of the surrounding medium or laser wavelength.
9.6 Selected Applications
We have, up to now, discussed all the underlying bases of holographic SHG imaging: we have cov-
ered the principles of holography, the implementation of a holographic SHG microscope, the numerical
reconstruction of digital holograms, and the various types of image contrast it yields. In this section,
we describe a few selected applications in which holographic SHG imaging is among those reported in
the actual literature.
9.6.1 imaging of Biological Structures
A good imaging tool should provide contrast specific to the structure of interest with a satisfying signal-
to-noise ratio (SNR), a high enough spatial resolution to resolve it, and a high enough temporal resolu-
tion to observe its dynamics.
In modern microscopy, contrast specificity of biological specimens is very often obtained via fluores-
cence imaging. Sometimes, the fluorescent substance (fluorophore) is the material of interest, but most
often it consists of a marker. Markers may be of exogenous nature, for example, molecules biochemi-
cally functionalized to bind to the material of interest, or of endogenous nature, for example, genetically
encoded to be expressed by the specimen. In any case, there are inconveniences related to the use of
markers. One is that it generally increases the specimen preparation time. Another, more important, is
that markers may alter the normal behavior of the specimen in unexpected ways. For these reasons, it
is favorable, when possible, to exploit a contrast intrinsic to the structure of interest, and thus avoid the
use of markers. One advantage often claimed by SHG imaging, and nonlinear coherent imaging in gen-
eral, is that it does not necessarily require labeling. Indeed, many materials, especially biological struc-
tures like mitotic spindles, actomyosin complexes, microtubules, collagen, and muscles, have intrinsic
nonlinear response to electromagnetic radiation (Campagnola and Loew, 2003; Mertz, 2004), which is
rather fortunate since collagen is the most abundant protein in mammals. SHG microscopy has already
proved especially appropriate for structural investigation of collagen and muscles (Freund et al
.
, 1986;
Kim et al
.
, 1999; Lin et al
.
, 2006; Plotnikov et al
.
, 2006; Teng et al
.
, 2006; LaComb et al
.
, 2008; Matteini
et al
.
, 2009; Nucciotti et al
.
, 2010; Xu et al
.
, 2010a).
Additionally, SHG microscopy is based on an instantaneous nonsaturating interaction with the
specimen, and may thus, in principle, achieve a much higher temporal resolution than its fluorescence
counterpart. Unfortunately, technical limitations have until now prevented from fully exploiting this
advantage. Originally, the limiting technology of light sources and detectors made impossible to record
full-field SHG images with good SNR at high frame rates. For instance, in their first reports of SHG
microscopy (Hellwarth and Christensen, 1974, 1975), Hellwarth and Christensen had to expose ASA
3000 Polaroid films for 20−60 min to produce SHG images. It must be emphasized that, at the time of
SHG microscopy's birth, light sources were either continuous wave lasers or Q-switched lasers deliv-
ering pulses with durations in the hundred of nanosecond range (Hellwarth and Christensen, 1974;
Sheppard et al
.
, 1977). To overcome the insufficient SHG efficiency of such lasers for full-field imaging
systems, scanning SHG microscopes were developed (Sheppard et al
.
, 1977). Yet, because of their nature,
scanning microscopes are intrinsically flawed for high-speed imaging. Recent developments of ultrafast
lasers and digital sensors now make possible nonscanning SHG imaging.
Off-axis holographic SHG imaging is a nonscanning technique and, as such, it is especially suited for
studying dynamics of live tissues or cells. Provided sufficient signal, it may truly exploit the instanta-
neous nature of SHG. Already, label-free holographic SHG imaging of static biological specimens has
