1066 CHINESE OPTICS LETTERS / Vol. 7, No. 12 / December 10, 2009
The multi-functional aspect of scanning holographic
microscopy: a review
Invited Paper
Guy Indebetouw
Physics Department, Virginia Tech, Blacksburg, VA 24061-0435, USA
∗
E-mail: gindebet@vt.edu
Received March 5, 2009
Recent developments in scanning holographic microscopy th at offer the prospects of new quantitative tools
and imaging modalities in bio, micro, and nano sciences are reviewed. The versatility of the method
is emphasized. Scanning holography can operate in an incoherent mode for fluorescence imaging, in a
coherent mode for quantitative phase imaging, or in a tomographic mode for axial sectioning and rejection
of the out-of-focus haze. Possible applications are illustrated with examples, and future prospects are
discussed.
OCIS codes: 090.1995, 110.6880, 180.0180, 180.6900.
doi: 10.3788/COL20090712.1066.
1. Introduction
Digital ho lography is an emerging technique that utilizes
modern technology to implement in convenient and of-
ten useful ways, ideas that had b e en propose d during
the early days of holography
[1,2]
. A successful example
of this development takes advantage of the availability
of high-resolution imag ing detectors (charge-coupled de-
vice (CCD) or complementary metal-oxide se miconduc-
tor (CMOS) chips) to record the hologram, and of deep
digital memory and powerful processing algorithms to
reconstruct and manipulate the holographic data
[3]
. The
application of digital holog raphy to micro scopy has the
distinct advantage that it gives access to a quantita-
tive measure of the phase of the object
[4,5]
. The phase
information is invaluable for characterizing changes of
thickness, density, or refractive indices in unstained liv-
ing cells, for example. However , one limitation of digi-
tal holographic microscopy is that it requires recording
the interference of the light scattered by the specimen
with a mutually coherent reference beam
[6−11]
. This need
for spatial and temporal coherence prohibits the holo-
graphic recording of incoherently scattered fields such
as from fluorescent s pecimens. Yet fluorescence has be-
come an e ssential tool in modern biological research.
It is also an imp ortant diagnostic tool in other fields
of micro and nano sciences. Scanning holographic mi-
croscopy overcomes this limitation by using a two-pupil
interaction method
[12]
that shifts the phas e information
needed to capture a hologram from the spatial domain
to the temporal domain
[13,14]
. The drawback is that a
two-dimensional (2D) scan with a tempor ally modulated
three-dimensional (3D) interference pattern is required to
realize the spatial-to-temporal transformation. The pat-
tern is usually a Fresnel pattern obtained with a point
and a spherical wave as pupils. It should be mentioned
that a scanless incoherent digital holographic method has
recently been developed
[15]
. It has b e e n shown that scan-
ning holographic microscopy offers a number of benefits
in addition to being able to capture the 3D holographic
information of incoherently scattering specimens. Per-
haps the mo st significant benefit is that scanning holog-
raphy can oper ate in an incoherent mode or a coherent
mode simply by varying the detector size
[16]
. The incoher-
ent mode uses a spatially integrating detector and leads
to a hologram o f the 3D incoherently scattered inten-
sity of the specimen (for example the 3D distribution of
fluorophores)
[17,18]
. The cohere nt mode use s a pinhole de-
tector placed on axis in the conjugate imag e plane of the
point pupil, and leads to a hologram of the complex am-
plitude of the specimen from which quantitative phase
information can be measured
[19]
. An equally significant
attribute of scanning holography is that it can operate
either in a holographic mode or in an axially sectioning
tomographic mode by varying the size of the source
[20]
.
With a spatially coherent (point) source, the fringes of
the scanning interference pattern are not spatially lo-
calized, and the entire 3D scattering distribution of the
object is recor ded holographically. With a broad, spa-
tially inco herent source filling the pupil of the objective,
the interference fringes are axially localized in the focal
plane of the objective, and the collected modulated signal
represents the hologram of a single tomo graphic section
through the specimen. Firstly, the method of scanning
holographic microscopy is briefly reviewed. Various ex-
amples illustrating the versatility and possibility of the
method are then discussed. Finally, a brief summary and
mention of future prospects concludes the paper.
2. Holographic modes
Scanning holographic microscopy has been described in
several pap e rs and books
[16,21]
. The main idea is to cre-
ate a s patially structured interference pattern, modulate
it in time, and scan it in a 2D raster over the speci-
men. In its simplest implementation, which leads to the
recording of an in-line single-sideband Gabor hologram,
the interference of a plane wave and a spherical wave
matching the numerical ape rture (NA) of the objective
is used to produce a Fresnel zone interference pattern
(FZP)
[22,23]
. The temporal modulatio n needed to shift
the holographic phase from the spatial to the tempora l
domain is achieved by shifting the frequency of one of the
interfering waves us ing an acousto-optic or an electro-
1671-7694/2009/121066-06
c
2009 Chinese Optics Letters
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