Coherent Microscopy and Optical Coherence Tomography for Biomedical Applications - Bioanalysis Advanced Materials Methods and Devices - page 105

optical techniques is extended greatly. In these cases light scattered from different
depths can be identified by means of the so-called “coherence gating” or “confocal
gating” effects attributed to the source bandwidth and numerical aperture (NA),
respectively. These are the methods of optical tomography.

8.1 Introduction

Driven mainly by biomedical applications the optical microscope has been in
constant development for over a century. Significant milestones include the imag-
ing theory of Abbe [ 1 ], the phase contrast technique introduced by Zernike [ 2 , 3 ],
and the confocal microscope by Minsky [ 4 ], which for the first time demonstrated
3D sectioned images. More recently we have witnessed the development of digital
holographic microscopy (DHM) and other coherent techniques that have similar 3D
capability. In particular, Optical Coherence Tomography (OCT) has produced truly
remarkable 3D images of the retina and Doppler OCT has been used to estimate
blood flow within tissue [ 5 ].
Coherent methods differ from their incoherent counterparts by their ability to
record and reconstruct both the phase and amplitude of the scattered field, and for
this purpose an interferometer is necessary. Of the 3D imaging methods DHM is the
fastest and most straightforward to implement. The simplest set-up uses a point-
source in an in-line transmission configuration [ 6 ] but many variations are possible
including phase stepping, off-axis and backscatter geometries [ 7 - 9 ]. In all these
cases the definitive characteristic of DHM is that the 3D scattered field can be
estimated from a single measurement of the scattered field . In biomedical
applications DHM can be used to provide bright field images comparable to those
from a conventional microscope even when the image is acquired through a
distorting sample container such as a plastic dish. It is also possible to emulate
other classical microscopy modes such as dark field, differential interference
contrast, and phase and even spiral phase contrast [ 10 , 11 ]. A comprehensive
review of DHM techniques has been published by Ferraro, Wax, and Zelevsky in
2010 [ 12 ].
As we explain later, DHM does not provide a true 3D image. This is apparent
when it is considered that the 3D field is reconstructed from a measurement of a 2D
boundary field and as such the reconstruction must be highly correlated. In the
strictest sense, a 3D image can only be synthesized from a sequence of scattered
field measurements using multiple source wavelengths, illumination directions or a
combination of both.
OCT makes use of a broadband or swept source to measure the response of the
object to light of different wavelengths . Several variants of the technique are
possible but all necessarily collect backscattered light from the object of interest.
Early OCT measurements exploited a broadband source with limited coherence to
record interference as the object was scanned in the object path of an interferometer
[ 13 ]. The limited coherence results in a packet of interference fringes that identifies