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Indebetouw et al.
1. INTRODUCTION
Scanning holography was invented as a clever application
of the two-pupil interaction schemes, which are unique in
extending the incoherent optical processing realm to operations requiring bipolar, or even complex, point-spread
functions.1,2 The principles of the method have been described, and its feasibility has been demonstrated.3
More recently, the application of scanning holography to
three-dimensional microscopy has been contemplated.
The holographic recording of three-dimensional fluorescent specimens was shown possible,4 and the technique
proved promising in locating fluorescent anomalies embedded in turbid media.5
The purpose of this paper is to present an analysis of
the imaging properties of scanning holography, define
theoretically expected performances, and discuss some
practical issues associated with the technique. In Section 2 we briefly review the background of threedimensional microscopy and holographic microscopy.
The primary objective here is to identify the main drawbacks of conventional coherent holographic methods applied to microscopy (i.e., speckle noise and a high-spatialbandwidth requirement) and to show how the scanning
heterodyne method may overcome some of the difficulties.
In Section 3 we analyze the properties of the reconstructed image in detail. For this, the imaging process of
scanning holography is described in terms of its pointspread function as well as its transfer function. Numerical examples are provided as an illustration of the expected performance. Many of the formulas given are
restricted to the paraxial approximation, which often
leads to convenient analytical solutions. It must be
stressed, however, that the analysis presented here is
0740-3232/2000/030380-11$15.00
2. BACKGROUND
A. Three-Dimensional Microscopy
Commonly used methods for three-dimensional imaging
in microscopy make use of optical sectioning, which generally requires a three-dimensional sampling of the specimens volume. The two best-known examples are optical
sectioning microscopy and scanning confocal microscopy.
Optical sectioning uses a conventional microscope to sequentially record a series of images focused at different
depths.6 Suitable algorithms are then used to merge the
images into a three-dimensional representation and re 2000 Optical Society of America
Indebetouw et al.
381
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Indebetouw et al.
object (as shown in Fig. 1, which will be described in Subsection 3.C). The quasi-spherical wave emerges from a
point source created at the focal point of a well-corrected
microscope objective of numerical aperture (NA) uniformly illuminated by a plane wave. Within the domain
of validity of the Debye integral representation of the
field,22,23 the amplitude distribution at a distance z from
the geometrical focus is given by
U r, z, t ikA exp i t
NA
exp ik 1 s 2 1/2z
D. Holographic Microscope
The principles of scanning holography have been experimentally demonstrated for simple, macroscopic objects,3
and the method has been extended to record holograms of
fluorescent specimens,4,5 thus demonstrating the incoherent nature of the process. A true holographic microscope
remains to be constructed, but the purpose of this paper is
to discuss some of its expected properties. Certain limitations and unique properties can already be mentioned.
A holographic microscope, for example, will not perform
the sharp optical sectioning characteristic of a confocal
scanning microscope. This is simply because the holographic information is acquired in a single twodimensional scan, which prevents the possibility of rejecting the out-of-focus information before detection.
However, reconstruction from the holographic data can
benefit from the application of a number of algorithms
that have been developed to process and improve images
in the conventional optical sectioning methods.16,17 Restoration and eventually ultraresolution methods18,19 can
also be used to advantage. A unique property of the
scanning holographic method is that it offers the possibility of correcting, during the reconstruction, the aberrations that may have affected the scanning pattern used in
recording the hologram. This may be of importance at
wavelengths for which well-corrected, high-numericalaperture optics are difficult or expensive to fabricate.
A conventional microscope has the capability of varying
the degree of coherence by changing the size of the source.
A broad source provides incoherent imaging, which minimizes speckle noise and artifacts but is blind to object
phase variations, whereas a point source provides spatially coherent illumination, making it possible to image
phase distributions such as unstained biological specimens. A holographic microscope presents an equivalent
versatility because the imaging property of a scanning optical system can be varied from incoherent to coherent
mode by changing the size of the detector.20,21 A large,
spatially integrating detector leads to incoherent imaging, results in speckle-free images, and is capable of imaging fluorescent samples, whereas a pinhole detector results in coherent imaging capable of rendering phase
objects visible and enabling the implementation of wellknown microscopic techniques such as the Zernike phase
contrast and Nomarski interference contrast methods.
J 0 ksr 1 s 2 1/2s ds
ikAE r, z exp i kz t ,
where
E r, z
NA
(1)
NA
(2)
(3)
Indebetouw et al.
r, z kr 2 /2 z.
(4)
E , ; F 0
F z a 2 z /z NA 2 z/.
(6)
(7)
The calculations in Ref. 23 show that the truncatedspherical-wave approximation is excellent for Fresnel
numbers F 40 and is already quite good for F 10.
Noticeable discrepancies appear only near the boundary
of the pattern, which, in a practical setup such as that
shown in Fig. 1, could be clipped or tapered off by additional apertures. It should be stressed, however, that
this does not mean that a scanning pattern with a smaller
Fresnel number or one that does not satisfy the paraxial
approximation cannot be used. In this case, however, the
amplitude distribution of the scanning field must be calculated exactly to achieve a correct reconstruction. In
the following, but purely for convenience, we assume that
the truncated-spherical-wave approximation is valid.
B. Reduced Coordinates
In the following subsections, we consider a relatively thin,
weakly scattering specimen located at a distance z 0 from
the point source. The depth variable in object space is
measured from that distance, i.e.,
z z z0 .
(8)
r NA/,
(9)
z NA /.
2
(10)
Similarly, we define a dimensionless transverse spatialfrequency coordinate scaled to the expected cutoff frequency max NA/. Thus
/ max s/NA.
J 0 2 1 2 NA 2 1/2 d ,
(12)
and becomes, in the paraxial approximation,
E p , ; F 0
exp i 2 F 0 J 0 2 d .
This field is mixed on the object with a plane wave of amplitude E 0 shifted in frequency by to produce a scanning pattern with amplitude
P r, z, t E r, z E 0 exp it .
exp(i2 1 1 2 NA 2 1/2
F 0 NA 2 )
(5)
383
(11)
With these notations the field amplitude of the quasispherical wave becomes, from Eq. (1),
(13)
When F 0 is large enough and the object depth range is
small compared with the average object distance from the
point source (i.e., F 0 ), the truncated-spherical-wave
approximation leads to a simplified expression for the
field amplitude:
E t , ; F 0 exp i 2 F 0 1 circ /F 0
exp i 2 1 /F 0 /F 0 circ /F 0 .
(14)
C. Holographic Record
We now consider the recording of the hologram of a relatively thin, weakly scattering specimen that can be represented by an amplitude transmittance T( , ). Extension of the following arguments to the case of threedimensional reflecting surfaces is trivial, and their
extension to fluorescent specimens and rough surfaces
will be discussed below.
The scanning pattern with amplitude P( , , t) given
by Eq. (7) is projected through the specimen, which is
scanned in a two-dimensional raster. If rs rs (t) [or
s s (t) in reduced coordinates] represents the instantaneous position of the object, the field amplitude behind
the object is approximately
E obj t
d2 d P , , t T s , .
(15)
As shown in Fig. 1, this amplitude is then Fourier transformed by a lens of focal length f and falls on a spatially
integrating quadratic detector through a mask with intensity transmittance M( ). The resulting detector current, for each instantaneous position s s (t) of the object, is proportional to
i s
d2 F E obj 2 M
d2 d2 d2
d M
exp i2 P ,
P * , T s , T * s , ,
(16)
E obj s exp i2 s d2 s
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Indebetouw et al.
M exp i2 d2
d2 d2 d M P ,
P * , T s , T * s , .
H inc s
(17)
(18)
d2 d E , I s , ,
(21)
where I( , ) T( , ) 2 . The process is linear in intensity and thus, according to conventional wisdom, incoherent. This mode of operation is needed to record holograms of rough objects or rough surfaces without speckle
noise and to record holograms of incoherent objects such
as fluorescent specimens. In both the coherent and the
incoherent mode, a point object is encoded as the same
wave E( , ), which, for relatively large Fresnel numbers
(F 10) and relatively small numerical apertures (NA
0.5), is well approximated by a truncated spherical
wave.
D. Hologram Reconstruction
For the reconstruction of an image focused at a distance
z R from the point source used in the recording, that is, a
distance R into the object, in reduced coordinates, the hologram can be digitally correlated with the pattern
E R ( , R ) matched to the desired depth. Thus the focused reconstruction is, from Eq. (19),
R , R
* s , R d 2 s
H s E R
d2 s d2 d2 d M
* s , R E ,
ER
T s , T * s , .
d2 d2 d M E ,
(22)
T s, T * s, .
(19)
smooth three-dimensional surfaces, as met in the microelectronics industry. If the object is rough, however, we
expect the images to be corrupted by speckle noise. The
second extreme case is that of an incoherent process,
which results from using an open mask and a large spatially integrating detector. Here M( ) 1, and M( )
( ). The holographic record is in this case
d2 d E , T s , ,
(20)
where H 0 d2 T * ( s ) is a constant complex factor. In this case the amplitude T( , ) of each object
point is encoded as a wave E( , ). The process is linear
in field amplitude and is thus coherent according to conventional wisdom. This hologram is sensitive to object
phase variations and thus is capable of recording phase
objects such as thin unstained specimens, as encountered
in biomedical imaging, as well as the topography of
R coh , R
d2 s d2 d E *
R s , R
E , T s , ,
(23)
* s , R
d2 s d2 d E R
E , I s , .
(24)
Indebetouw et al.
d2 d PSF ; , R T ,
(25)
d2 d PSF ; , R I ,
(26)
*
d2 E R
1
2 ,
R E
1
2 ,
.
(27)
385
ning field at with the reconstructing field at R . In particular, the in-focus point-spread function is the autocorrelation of E( , R ).
When the truncated-spherical-wave approximation is
valid, both the scanning wave and the reconstructing
function are given by relation (14), and the point-spread
function can be calculated as
PSFt ,
F 02
F 0 /2
dx
F 0 /2
F 0 2 /2x 2 1/2
F 0 2 /2x 2 1/2
dy
i2
i
exp
x
/2 x 2 y 2 .
F0
F 02
for 2F 0
for 2F 0
(28)
For large enough Fresnel numbers, Eq. (28) is well represented by empirical formulas.24 For the transverse distribution in focus, these empirical formulas give, approximately,
PSFe , 0; F 0
P /2F 0
J 1 2 1 /2F 0
1 /2F 0
for 2F 0
for 2F 0
(29)
where P(x) 1 1.38x 0.031x 0.344x and J 1 is
a Bessel function of the first kind and first order. For the
axial distribution, Eq. (28) gives
2
PSFe 0, ; F 0 sinc /2 ,
(30)
(31)
386
2 or z 2/ NA 2 .
Indebetouw et al.
(32)
Fig. 2. Cross sections of the in-focus point-spread function amplitude. For low numerical apertures, the point-spread function
is nearly independent of the Fresnel number and almost identical with the point-spread function of a clear aperture of equal numerical aperture. For high numerical apertures, the pointspread function has a sharper central lobe than the Airy disk but
higher sidelobes.
Indebetouw et al.
387
* ; 0 E ; ,
TF ; ER
(33)
E R , J 0 2 d .
(34)
(35)
(36)
(37)
(38)
which leads to
TFp ; exp i 2 circ .
(40)
388
Indebetouw et al.
*
ER
1
2 ,
0 E
1
2 ,
0 d2 ,
(41)
* ; 0 circ .
TF ; 0 ER
(42)
Indebetouw et al.
1
2
E t
1
2
1
2 d/d E t ,
B. Practical Considerations
Since we were unable to secure the funds necessary to actually build a microscope, we will share our thoughts in
trying to design one, in the hope that it may trigger someones interest. A possible design for a scanning heterodyne microscope was shown in Fig. 1. The illumination
module produces two quasi point sources of light, P 1 and
P 2 , at the output of two single-mode fibers. An adjustable frequency difference is provided by two acousto-optic
modulators driven in synchronism. The lights from the
two point sources are combined at the beam splitter, and
the beams are shaped to produce, in the approximation
discussed above, a spherical and a plane wave superposed
on the object by means of a microscope objective of numerical aperture NA. An adjustable aperture limiting
the size of the scanning pattern is used to vary its Fresnel
number. The objective images this aperture on the object. Light from P 1 is focused by the objective near its focal point, from which it travels toward the object, where it
forms, in the truncated-spherical-wave approximation, a
spherical wave truncated to a half-cone angle sin1(NA).
Light from P 2 is focused by an intermediate lens at the
center of the objectives entrance pupil. The objective collimates this light to project a plane wave of limited extent
on the object. The Fresnel number of the scanning pattern can thus be changed without affecting the numerical
aperture or the resolving power.
The possibility of using the instrument in transmission
mode, to obtain holograms of unstained specimens or
phase objects, as well as in reflection mode, to form holograms of reflecting objects or of fluorescent specimens, is
illustrated in Fig. 1. A mask in the pupil plane is used to
vary the coherence between an imaging linear in intensity and an imaging linear in amplitude, as discussed
above. For fluorescence imaging the beam splitter is a
dichroic mirror transmitting the laser excitation wavelength and reflecting the fluorescence wavelength. The
reference detector uses a pinhole in a plane conjugate to
the object. The signal from this detector is used as a reference signal for heterodyne detection. In this way,
eventual shifts in signal frequency caused by mechanical
or thermal fluctuations in the illumination stage appear
in both the signal and the reference and can be canceled
out. The sample can be mounted on a computercontrolled, two-dimensional stage, or the scanning can be
accomplished by mirrors for faster scanning rates. The
resulting temporal signal from the detectors is sent to the
data acquisition stage, which can be either digital or analog. For digital acquisition the signal is converted by a
fast analog-to-digital converter (ADC) and filtered digitally to extract the holographic record. In this case we
expect the rate of the ADC to impose a limit on the acceptable scanning rate. For analog acquisition the signal
is mixed with the reference signal and filtered to obtain
two downconverted quadrature signals, which are then
converted from analog to digital and combined digitally to
form the complex holographic record.
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5. SUMMARY
We have analyzed the imaging properties of a scanning
holographic system and compared it with conventional
imaging. The salient points are the following. Varying
the detection mode from pinhole detection to spatially integrating detection allows one to vary the coherence property of the imaging process from linear in amplitude to
linear in intensity. The method is thus suitable for imaging phase objects and relief surfaces, as well as for obtaining incoherent (and thus speckle-free) holograms and
for imaging fluorescent specimens.
The three-dimensional point-spread function of the system was calculated as a function of two parameters,
namely, the numerical aperture and the Fresnel number
of the scanning pattern. The results are valid beyond the
paraxial approximation and are presented in terms of dimensionless coordinates scaled to the theoretical resolution limits of the system. This allows for direct comparisons of systems with different numerical apertures. As
one might expect from theoretical considerations, it is
found that the amplitude distribution of the point-spread
function is nearly independent of the Fresnel number of
the scanning pattern, within the domain of validity of the
field representation by a Debye integral. The Fresnel
number can thus be used as a free design parameter, the
variation of which leaves the resolution unaffected.
For low and modest numerical apertures, the pointspread function is found to be nearly identical with that of
an aberration-free conventional imaging system of equal
numerical aperture. For higher numerical apertures, the
holographic point-spread function, in reduced coordinates, exhibits improved transverse and axial resolution
limits, compared with the Airy pattern.
An attractive feature of scanning holography is that
the aberrations of the scanning pattern can easily be canceled out by reconstructing the hologram digitally with an
appropriate conjugate pattern. This process leads to an
ideal system transfer function equal to unity up to the
cutoff frequency, independent of the aberrations of the
scanning beam. This is valid for the system operating in
either the coherent or the incoherent mode. Scanning
holography lends itself well to postprocessing operations,
since the images are reconstructed digitally. A few such
possibilities were mentioned, and some practical issues
were considered.
Indebetouw et al.
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ACKNOWLEDGMENTS
Ting-Chung Poon and Taegeun Kim acknowledge the
financial support of the National Science Foundation
(grant ECS-9810158) for parts of this work.
Address correspondence to Guy Indebetouw at the location on the title page or by e-mail, gindebet@vt.edu.
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REFERENCES
1.
2.
T.-C. Poon, Scanning holography and two-dimensional image processing by acousto-optic two-pupil synthesis, J.
Opt. Soc. Am. A 2, 521527 (1985).
G. Indebetouw and T.-C. Poon, Parallel synthesis of bipolar point spread functions in a scanning heterodyne optical
system, Opt. Acta 33, 827834 (1986).
25.