4 Tomographic Diffractive Microscopy …
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tion properties mix to form the final image, and for example, a darker, therefore
apparently more absorbing zone, may in fact simply result from a localized destructive interference phenomenon. Similarly, phase contrast microscopy images [12, 13]
are often affected by halo artifacts surrounding sharp structures of the observed
sample.
Note that computer-based phase reconstruction techniques have been developed,
encountering growing success, thanks to modern computers computational power.
Techniques relying on solving the transport of intensity equation [14–18], DIC image
reconstruction [19–23], and ptychography [24–29], have demonstrated their ability to
reconstruct the phase of recorded field. They do however rely on some assumptions,
which may limit their domain of application.
Use of coherent light illumination, combined with interferometric detection, permits on the contrary to directly record holograms [30], encoding amplitude and phase
of the light interacting with the microscopic specimen [31], therefore delivering complete information about the diffraction phenomenon, but not relying on numerical
reconstruction. Wavefront analyzers also permit to record the amplitude and phase of
the diffracted light, without the need of an interferometer, and with short coherence
illumination [32, 33]. Commercial implementations of this technique are growing
[34]. These approaches, known under the name of Digital Holographic Microscopy
or of Phase Microscopy, have in particular proven to be very sensitive to small sample changes, thanks to unsurpassed precision of interferometric measurements. Phase
imaging is now used for many biological studies [35–67].
Figure 4.1 describes the principle of holographic imaging. Three configurations
are often used in holographic microscopy: Gabor holography, phase-shifting holography, and off-axis holography.
The simplest is Gabor holography [43], or in-line holography, first proposed for
electron microscopy, and adapted to digital optical holography [44]. A coherent wave
illuminates the observed sample, and is partly diffracted by it (Fig. 4.1a). Interference
fringes result from the coherent addition of the diffracted field and the illumination,
modulating phase and amplitude of the diffracted wave. A direct demodulation is
not possible, but numerical reconstruction of the phase and amplitude is feasible
[45]. The main advantage of this technique is its robustness, the system being selfaligned, so very insensitive to perturbation, allowing work in harsh environment
(see for example the system developed by the 4Deep company [34] for submarine
applications).
In order to address the hologram demodulation problem, one can also use phaseshifting holography [46, 52]. In this configuration (Fig. 4.1b), a reference beam is used
to create the holograms, and modulated in phase, usually using a piezoelectric-driven
mirror, or an electro-optic modulator. Recording of series of holograms permits to
solve the holographic equation and extract the amplitude and phase of the diffracted
beam, separated from the reference beam and the non-diffracted component of the
illuminating beam. The holograms are acquired sequentially, which may limit speed,
but parallelization or use of several cameras is possible [49, 50].
Off-axis holography has also been applied to microscopy [31, 51]. In this technique, an inclined reference beam (Fig. 4.1c) directly modulates the interferogram.
87
tion properties mix to form the final image, and for example, a darker, therefore
apparently more absorbing zone, may in fact simply result from a localized destructive interference phenomenon. Similarly, phase contrast microscopy images [12, 13]
are often affected by halo artifacts surrounding sharp structures of the observed
sample.
Note that computer-based phase reconstruction techniques have been developed,
encountering growing success, thanks to modern computers computational power.
Techniques relying on solving the transport of intensity equation [14–18], DIC image
reconstruction [19–23], and ptychography [24–29], have demonstrated their ability to
reconstruct the phase of recorded field. They do however rely on some assumptions,
which may limit their domain of application.
Use of coherent light illumination, combined with interferometric detection, permits on the contrary to directly record holograms [30], encoding amplitude and phase
of the light interacting with the microscopic specimen [31], therefore delivering complete information about the diffraction phenomenon, but not relying on numerical
reconstruction. Wavefront analyzers also permit to record the amplitude and phase of
the diffracted light, without the need of an interferometer, and with short coherence
illumination [32, 33]. Commercial implementations of this technique are growing
[34]. These approaches, known under the name of Digital Holographic Microscopy
or of Phase Microscopy, have in particular proven to be very sensitive to small sample changes, thanks to unsurpassed precision of interferometric measurements. Phase
imaging is now used for many biological studies [35–67].
Figure 4.1 describes the principle of holographic imaging. Three configurations
are often used in holographic microscopy: Gabor holography, phase-shifting holography, and off-axis holography.
The simplest is Gabor holography [43], or in-line holography, first proposed for
electron microscopy, and adapted to digital optical holography [44]. A coherent wave
illuminates the observed sample, and is partly diffracted by it (Fig. 4.1a). Interference
fringes result from the coherent addition of the diffracted field and the illumination,
modulating phase and amplitude of the diffracted wave. A direct demodulation is
not possible, but numerical reconstruction of the phase and amplitude is feasible
[45]. The main advantage of this technique is its robustness, the system being selfaligned, so very insensitive to perturbation, allowing work in harsh environment
(see for example the system developed by the 4Deep company [34] for submarine
applications).
In order to address the hologram demodulation problem, one can also use phaseshifting holography [46, 52]. In this configuration (Fig. 4.1b), a reference beam is used
to create the holograms, and modulated in phase, usually using a piezoelectric-driven
mirror, or an electro-optic modulator. Recording of series of holograms permits to
solve the holographic equation and extract the amplitude and phase of the diffracted
beam, separated from the reference beam and the non-diffracted component of the
illuminating beam. The holograms are acquired sequentially, which may limit speed,
but parallelization or use of several cameras is possible [49, 50].
Off-axis holography has also been applied to microscopy [31, 51]. In this technique, an inclined reference beam (Fig. 4.1c) directly modulates the interferogram.