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Received 17 October 2016; revised 17 November 2016; accepted 20 November 2016; posted 21 November 2016 (Doc. ID 278850);
published 15 December 2016
An automatic method that fully compensates the quantitative phase measurements in off-axis digital holographic
microscopy (DHM) is presented. The two main perturbations of the quantitative phase measurements in off-axis
DHM are automatically removed. While the curvature phase flaw introduced by the microscope objective is
avoided by the use of an optimized telecentric imaging system for the recording of the holograms, the remaining
phase perturbation due to the tilt of the reference wave is removed by the automatic computation of a digital
compensating reference wave. The method has been tested on both nonbiological and biological samples with and
improving on the quality of the recovered phase maps. 2016 Optical Society of America
OCIS codes: (090.1995) Digital holography; (090.1000) Aberration compensation; (100.5070) Phase retrieval; (110.0180)
Microscopy; (070.4790) Spectrum analysis; (100.3010) Image reconstruction techniques.
https://doi.org/10.1364/AO.55.010299
1. INTRODUCTION was shown that even though the good quantitative quality of the
Why do we do digital holographic microscopy (DHM)? Maybe recovered phase images with the numerical methods, they can-
the most likely answer is because the possibility it provides of not recover the information lost in the recording stage leaving
making label-free quantitative phase images of nonbiological the imaging system spatially variant [15]. This finding points out
and biological samples [16]. This very attractive feature that that the most effective method to avoid phase perturbations due
constitutes the hallmark of DHM, seems to be concealed by the to the imaging system of the microscope is to use an infinity-
artifacts that are added to the phase images recovered from corrected microscope objective in telecentric configuration with
DHM; this fact has been recognized since the very onset of a tube lens to record the digital holograms [1517]. The phase
this microscopy technique [1]. Two are the main deleterious due to the tilt of the reference wave also can be removed by
sources that ruin the phase images: (1) the use of microscope numerical compensation of the unwanted phase factors [5] or
objectives to enlarge the diffracted wave field by the object, in- by using an in-line geometry for the recording of the holograms
troduces a curvature phase that is superimposed with the proper and their processing through a phase shifting approach [18,19].
phase of the object, and (2) as DHM is performed in an off-axis Despite that this latter method effectively removes the tilt phase,
architecture, a tilt phase due to the angle between the reference it needs at least three holograms to be processed, which limits its
and object waves of the microscope is merged with the actual application in, for instance, the imaging of the dynamic process.
phase of the sample and the phase curvature cited in (1). The Dynamic processes like cell motility, in which it has been re-
result is a nonsense phase map that has to be corrected to get ported that some cells can reach up to 1 mm/s speeds [20], ac-
the authentic phase of the object. Therefore, multiple methods, quisition rates on the order of video rates are demanded for the
both numerical and physical, have been proposed in the imaging systems [20].
literature to remove those nuisances on the phase maps that The above paragraphs indicate that if one desires to have a
ruin the quantitative phase images [714]. The phase curvature single-shot quantitative phase image with DHM with the less
introduced by the use of microscope objectives, was initially possible perturbations, the microscope must operate in an op-
removed by means of different types of numerical fitting of timized telecentric architecture [17], and some correction of the
the unwanted phase [79,13] or physically by the use of a twin tilt phase should be implemented. In this paper, we present an
imaging system in both arms of the DHM [12]. Thereafter it off-axis DHM that operates in the optimized telecentric regime
to avoid any curvature phase perturbation introduced by an selection are: (1) In the transmission setup, the Mach
imaging system, and that also includes an automatic method Zehnder configuration requires only one pass of the object
to compensate for the tilt phase introduced by the angle be- wave through the sample and the imaging system, thus dimin-
tween the object and reference waves of the microscope. ishing the losses in the intensity of the object wave, reducing
The fully automatically compensated off-axis DHM is tested the added perturbation to the same and therefore improving the
on imaging transparent nonbiological and biological specimens recovery of the phase information; and (2) The MachZehnder
and fully reflective nonbiological samples. The paper is organ- operating in transmission setup can be easily transformed into
ized as follows: in Section 2 the basics of the off-axis DHM are the reflection setup of Fig. 2 by only two flip mount adapters
revised. In Section 3 the automatic method for tilt compensa- placed at the position of M1 in Fig. 1 and at the position of M3
tion is presented. The experimental validation of the proposed in Fig. 2. In the reflection setup, the object wave Ox; y passes
method is illustrated in Section 4. The text finishes with the twice the microscope system composed of the MO and the TL.
conclusion in Section 5. The reference wave Rx; y is directed by the set of mirrors M4
and M5 that are mounted on a translation stage to match the
optical path length of both Rx; y and Ox; y waves. These
2. OFF-AXIS DIGITAL HOLOGRAPHIC waves are recombined in BS2 to interfere over the surface of
MICROSCOPY the digital CMOS (complementary metaloxidesemiconduc-
The off-axis DHM can be utilized to image both transmission tor) camera. An important matter in the acquisition process of
and reflecting samples. For transmission samples, a Mach this interference pattern is the visibility of its interference
Zehnder based interferometer is usually utilized, see Fig. 1. In fringes. When the proper visibility of the interfering fringes
this configuration, the light source, usually a laser beam, is ini- is reached, the phase information of the sample is recovered
tially collimated by BE and then divided into two coherent waves with the highest quality. Experimentally we have found that
by a beam splitter (BS1). One of these coherent waves impinges to achieve such visibility, the ratio of the intensity of the refer-
the sample originating at the object wave Ox; y. The scattered ence wave to the intensity of the object wave must be 21. This
wave by the object is then magnified by a microscope system, ratio is obtained from a calibration process prior to the acquis-
which is composed of the microscope objective (MO) and a tube ition of the DHM holograms. In the first step of the calibration
lens (TL). The other coherent wave, called the reference wave process, over an image of the reference wave, the mean intensity
Rx; y, travels along the other arm of the interferometer to reach of this image is calibrated to reach 60% of the dynamic range of
the second beam splitter (BS2). After BS2, both waves recom- the CMOS camera; in an eight-bit image, a gray value of 153 is
bine and interfere in a plane where a digital camera is located. adjusted. This adjustment is executed by changing the acquis-
The digitally recorded intensity corresponding to the amplitude ition parameters of the camera and by using variable neutral
superposition of Ox; y and Rx; y is the DHM hologram. In density filters (VNDF) located before the BS1. In a second step,
the off-axis configuration, a controlled angle between both the the mean intensity of an image of the object wave, without a
reference wave and the object wave is produced by tilting BS2 or sample in place, is adjusted through a similar process in which
one of the mirrors M1 or M2. This tilting angle leads to a DHM the adjustment is carried out only with a VNDF located along
hologram composed of straight fringes with distortions that the object arm of the setup. The result of this second step is a
account for the information of the sample. mean intensity of the image adjusted at 30% of the dynamic
For reflecting objects, the usual DHM configuration is a range of the sensor. The latter process is executed for both the
modified Michelson-based interferometer [5]. However, in this reflection and the transmission setups to optimize the phase
work, the DHM holograms of reflective samples are recorded in retrieval process.
the setup illustrated in Fig. 2, which is a modification of a For both setups, transmission and reflection, the use of
MachZehnder interferometer. The reasons underlying this an infinity-corrected microscope objective in telecentric
Fig. 1. Typical setup for transmission off-axis DHM. BS1 and BS2 Fig. 2. Reflection DHM setup utilized in this work. BS1 and BS2
are the beam splitters and M1 and M2 are the mirrors. The microscope are the beam splitters, and M1M6 are the mirrors. The microscope
objective (MO) and the tube lens (TL) compose the microscope sys- objective MO and the tube lens TL compose the microscope system
tem working in telecentric configuration. The existence of an angle working in telecentric configuration. The existence of an angle be-
between the reference wave Rx; y and the object wave Ox; y leads tween the reference wave Rx; y and the object wave Ox; y leads
to an off-axis transmission setup. to an off-axis reflection setup.
Engineering and Laboratory Note Vol. 55, No. 36 / December 20 2016 / Applied Optics 10301
configuration with a tube lens leads to the recording of the holo- with * denoting the complex conjugate. The corresponding
grams with no phase perturbation due to the imaging system; Fourier transform (F ) of Eq. (2) in the space of spatial frequen-
the quadratic phase factor introduced in the recording of the cies f x ; f y is therefore given by
hologram is eliminated, and the imaging system is spatially
F fH x; yg F fjRj2 jOx; yj2 g Of x ; f y R f x ; f y
and invariantly producing isoplanatic phase maps [15]. To avoid
the noise perturbations due to the multiple reflection surfaces in O f x ; f y Rf x ; f y
the imaging system, the infinity-corrected microscope objectives
utilized for the acquisition of both transmission and reflection DC Of x ; f y R f x ; f y
samples are those manufactured with antireflection coatings. O f x ; f y Rf x ; f y ; (3)
Once the DHM hologram has been recorded in the transmis-
sion or reflection setup operating in telecentric configuration, a with the convolution operation, DC F fjRj2
numerical processing of the hologram to recover the information jOx; yj2 g, and denoting the Fourier transform of . The
of the sample is required. If the digital camera is located at the DC term corresponds to the zero diffraction order and is placed
focal distance of the tube lens, the recorded hologram exhibits an at the very center of the Fourier spectrum [24]. Because usually
in-focus amplitude image of the sample, hence the retrieved the reference wave is chosen as a plane wave impinging upon
complex amplitude requires no propagation. This type of the digital camera with given angles x with respect to the x
DHM, known as image-plane DHM, reduces the numerical coordinate and y with respect to the y coordinate, its
reconstruction process to a spatial filtering of the hologram Fourier transform or the Fourier transform of its complex con-
[2123]. The latter can be done by a digital realization of a spa- jugated, are Diracs deltas whose locations in the Fourier space
tial filtering typically done in a 4f architecture [24] as it was first are determined by the cited angles. Those Fourier transforms of
proposed by Takeda et al. [23] or by using the added versatility the reference wave lead, for instance in the case of the second
of the numerical processing of the hologram. term of Eq. (3), to:
Before illustrating the spatial filtering process, a brief sin x sin y
description of the proper experimental adjustment of the im- F fOR g O f x ;f y : (4)
aging system to work in tight telecentric configuration in the
image plane is presented. This description is useful for both the The size of the second (F fOR g, 1 diffraction order) and
reflection and transmission setups. Initially, the TL is located third (F fO Rg, 1 diffraction order) terms of Eq. (3) is there-
somewhere in the object arm, just before the BS2. With no fore the size of Of x ; f y or O f x ; f y . If the object wave is
sample in the object arm, the wavefront impinging the TL considered with support a, the size of the DC is 2a because the
is a plane wave which is focused into the focal plane of this zero diffraction order can be read as the autocorrelation of the
lens; in this location the camera sensor must be placed. This object wave [25]. According with the formerly mentioned sizes,
location can be determined by translating the camera in the the optimum configuration of off-axis DHM sets the angles of
axial direction until the tiniest spot is imaged. Once the the reference wave such that the location of the 1 and 1
image-plane configuration is achieved, the telecentric configu- diffraction orders is such that the complete available space
ration of the MO with respect to the TL is adjusted. This is bandwidth product is utilized [17,26] with no overlapping
achieved by placing the MO just before the TL in the object of the three terms; furthermore, this condition automatically
arm. By moving the MO in the axial direction, a location in leads to the fulfilling of the sampling requirements [27].
which the resulting wavefront after the TL is a plane wave, can Another condition that has to be taken into consideration in
be determined. The latter can be done by using a shear plate the DHM acquisitions is the proper sampling of the interfer-
collimation tester just after the TL and before the BS2 and ence fringes; following the NyquistShannon theorem, at least
observing the resulting interference pattern. When a plane two pixels per the smallest interference fringe in the DHM
wavefront is obtained after the TL and before the camera hologram are mandatory. In our acquisitions, we guarantee this
sensor, the tight telecentric configuration is correctly adjusted. proper sampling by assuring that the angle between the refer-
To illustrate the spatial filtering process, let us consider the ence and object wave follows the well-known condition
DHM recorded hologram in either transmission or reflection MAX 2 with the pixel size of the recording camera
setup. This hologram is the result of the superposition in am- [28]. As the just-mentioned conditions are accomplished,
plitude of the reference Rx; y and the object Ox; y waves, the spectrum of the Fourier transform of the off-axis DHM
which leads to an intensity distribution H x; y hologram recorded in tight telecentric configuration can be
schematically represented as in Fig. 3.
H x; y jRx; y Ox; yj2 : (1) Because the DC term and one of the 1 or 1 diffraction
Rx; y R e ir x;y
is the complex amplitude of the refer- orders represent noise that perturbs the optimum recovery of
ence wave with R a homogenous amplitude level and Ox; y information from the DHM hologram, the spatial filtering
Oe io x;y the corresponding amplitude for the object wave. process consists of a proper selection, in the spatial frequency
Expanding Eq. (1), we have domain, of the frequencies associated with only one of the 1
2 2 or 1 diffraction orders [23]. Once the proper frequencies of
the chosen diffraction order are determined, for instance those
H x; y R Ox; y Ox; yR x; y for the 1 diffraction order, all the others are removed from the
Fourier spectrum. The 1 diffraction order is centered and
Rx; yO x; y; (2) then inverse Fourier transformed. The result of this process
10302 Vol. 55, No. 36 / December 20 2016 / Applied Optics Engineering and Laboratory Note
Fig. 7. Borojoa Patinoi cell imaged through off-axis DHM: (a) the
wrapped nonwell compensated phase image of the sample, (b) the
wrapped best compensated phase image of the automatic method,
(c) the unwrapped phase image of panel (b), and (d) a topographic
view of the Borojoa Patinoi cell imaged in panel (c).
5. CONCLUSION
In summary, in this paper we have presented an automatic full
phase compensation method for image-plane off-axis DHM
operating in tight telecentric configurations. The main advan-
tages of our proposal are: (1) the compensation through a
reference wave is performed with continuous values leading
to a more accurate compensation process than the traditional
centering method, and (2) the proposal is free from phase aber-
ration present in a shift variant system because it is imple-
mented with a microscope system working in telecentric
configuration. Our proposal has been tested and validated with
experimental DHM holograms of a biological sample in a trans-
mission setup and with nonbiological samples in reflection and
transmission setups. The robustness of the method and its low
computational complexity lead to trustable quantitative phase
imaging in off-axis image-plane DHM.
Fig. 8. Reflecting USAF 1951 test target imaged through off-axis Acknowledgment. C. Trujillo acknowledges the support
DHM: (a) a nonwell compensated phase image of the sample, by the National Ph.D. Studies Fellowship from Colciencias-
(b) the best compensated phase imaged delivered by the automatic Universidad Nacional de Colombia.
method, and (c) the topographic view of panel (b).
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