Research Article

Vol. 56, No. 13 / May 1 2017 / Applied Optics

F1High brightness, low coherence, digitalholographic microscopy for 3D visualization of
an in-vitro sandwiched biological sample
D. G. ABDELSALAM1,* AND TAKESHI YASUI2,3
1

Engineering and Surface Metrology Lab, National Institute of Standards, Tersa St., El Haram, El Giza, Egypt
Graduate School of Science and Technology, Tokushima University, 2-1, Minami-Josanjima, Tokushima 770-8506, Japan
3
JST, ERATO, MINOSHIMA Intelligent Optical Synthesizer Project, 2-1, Minami-Josanjima, Tokushima 770-8506, Japan*Corresponding author: dahi.abdelsalam@nis.sci.eg
2Received 28 October 2016; revised 20 December 2016; accepted 20 December 2016; posted 21 December 2016 (Doc. ID 279673);
published 26 January 2017

We achieve practically a bright-field digital holographic microscopy (DHM) configuration free from coherent
noise for three-dimensional (3D) visualization of an in-vitro sandwiched sarcomere sample. Visualization of such
sandwiched samples by conventional atomic force microscope (AFM) is impossible, while visualization using
DHM with long coherent lengths is challenging. The proposed configuration is comprised of an ultrashort pulse
laser source and a Mach–Zehnder interferometer in transmission. Periodically poled lithium niobate (PPLN)
crystal was used to convert the fundamental beam by second harmonic generation (SHG) to the generated beam
fit to the CCD camera used. The experimental results show that the contrast of the reconstructed phase image is
improved to a higher degree compared to a He–Ne laser based result. We attribute this improvement to twothings: the feature of the femtosecond pulse light, which acts as a chopper for coherent noise suppression,and the fact that the variance of a coherent mode can be reduced by a factor of 9 due to low loss through anonlinear medium. © 2017 Optical Society of America
OCIS codes: (070.6110) Spatial filtering; (090.1995) Digital holography; (100.5090) Phase-only filters; (170.0180) Microscopy.
https://doi.org/10.1364/AO.56.0000F1

1. INTRODUCTION
The study of coherent noise suppression in digital holographic
microscopy (DHM) is an important matter in the fields of bothengineering and biological science [1]. DHM enables the extraction of both intensity and phase information of a wave diffracted by a specimen from a single hologram with highresolution [2,3]. In usual DHM, optical sources with long coherent lengths such as a He–Ne laser have been widely used tofeature the non-sandwiched biological specimens. Because of
the high degree of coherence of the He–Ne laser light, harmful
coherent noise often appears [4–6]. This noise influences thequality of the holograms and a subsequent error in phase measurement is produced. The bigger the phase noise, the less accurate the measurement will be. The harmful coherent noise ismainly produced from two sources. The first is the random
diffraction patterns (speckle noise), which is attributed tothe scattered light. The second is the formation of undesirable
interference fringes (spurious noise) due to stray light. The
spurious noise is formed when light reflected or scatteredfrom various surfaces in the optical path is coherent withthe original beam. The amplitude of the scattered light as1559-128X/17/1300F1-06 Journal © 2017 Optical Society of America

combines vectorially to the amplitude a of the original beam,
resulting in a phase error Δφ, as illustrated in Fig. 1(a) [7,8].
The phase φ is given by (2π∕X ), X being the extra path traversed by the stray light. In the case of sandwiched samples,
where the sample is inserted in between two thin glass plates
to avoid dehydration, the phase error Δφ is considerable. Some
practical solutions such as introducing a wedged beam splitter, arotating diffuser, and anti-reflection coating to the optical component surfaces in the optical system setup were proposed toreduce this phase error [8]. Although these practical solutions
are effective and may reduce the coherent noise, they have somedemerits in terms of blurring the fringe visibility and hindering
the fringe production in DHM, which requires perfect alignment. Numerical techniques have been proposed to suppress
such coherent noise. Abdelsalam and Kim [9] used a numericalmethod based on flat fielding with apodized apertures to minimize the coherent noise in the phase-contrast image by an order of 65%. Although this numerical technique is effective in
reducing such coherent noise in the reconstructed object-wave(amplitude and phase), it offers tradeoffs between the apodized
aperture size and the quality of the formed noise level.

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Research Article

Fig. 1. (a) Phase error Δφ (background spurious noise) created byscattered long coherent light as , and (b) sliced biological sample
mounted in between two thin glass plates to avoid dehydration.

Furthermore, that technique was applied for non-sandwichedsamples. Optical sources with short coherent lengths such as
LEDs were suggested to avoid the harmful coherent noise[10–12]. However, the limited coherence length of LED
and its insufficient brightness hinders its application in an
off-axis DHM, since just a limited number of interference
fringes with low visibility appear in the field-of-view (FOV).
Zhang et al. [13] used a wide-field microscope based on an
in-line digital holography configuration with LEDs as opticalsources for imaging non-sandwiched biological samples.
Although the in-line configuration of holography can make fulluse of the resolving power of the camera used, it suffers fromthe existence of both zero order and twin image in the reconstructed object-wave. In contrast, using DHM with long coherent laser sources such as He–Ne to visualize the sandwichedbiological samples is challenging because of the existence ofthe harmful coherent noise (speckle and spurious). It is worth
mentioning that visualization of sandwiched biological samples
is considered difficult to achieve by mechanical microscopes
such as AFM. Therefore, in this paper, we present an off-axis
DHM scheme using femtosecond pulse light with ultrashortcoherent length, which makes it possible to visualize sandwiched samples with no spurious or speckle noises in the reconstructed object-wave. PPLN crystal was used to produce
bright light by the SHG conversion process. We used the
PPLN crystal for two reasons: to convert the fundamental beamof wavelength of 1550 nm into the generated beam of wavelength 777.8 nm fit to the camera used, and because the variance of the coherent mode of the fundamental beam can be
reduced by a factor of 9 due to low loss through a nonlinear
medium [14]. This generated bright light with a Mach–
Zehnder interferometer in transmission is used to visualize asandwiched microstructured sarcomere sample. The sarcomeresare isolated fibers dissected from rabbit muscle and mounted in
between two thin glass plates (gap distance t ! 15 μm) to
avoid dehydration. A schematic diagram to place a fresh, sliced
biological specimen in DHM is shown in Fig. 1(b).
2. EXPERIMENTAL SETUP
The experimental setup consisted of two parts: generation ofbright femtosecond pulse light in the near-infrared region anda Mach–Zehnder interferometer in transmission, as demonstrated in Fig. 2. A mode-locked Er-doped fiber laser light (center
wavelength λc ! 1550 nm, spectral bandwidth Δλ ! 73 nm,
pulse duration Δτ ! 100 fs, mean power P mean ! 380 mW,
and repetition frequency f rep ! 250 MHz) was converged by

Fig. 2. Experimental setup. PPLN, periodically poled lithium niobate crystal; F, bandpass filter at 775 nm; M, mirror; NF, neutral density filter; NPBS1 and NPBS2, nonpolarizing beam splitters; MO1,
MO2, and MO3, microscope objectives with (50×, NA ! 0.45),(50×, NA ! 0.45), and (20×, NA ! 0.1), respectively.

a focusing lens on a periodically poled lithium niobate(PPLN) crystal to convert the wavelength by SHG into the operating wavelength region of a charged coupled device (CCD)
camera. To generate the bright light by SHG process, we used
a narrow bandpass optical filter with a specification of F, passband wavelength ! 775 " 5 nm. The generated bright light by
SHG mechanism has λc of 777.8 nm, Δλ of 10 nm, Δτ of
120 fs, and P mean of 14 mW. The output power was sufficient
to illuminate the sample and form off-axis holograms with highcontrast in the entire field of the CCD camera. The coherence
length of the generated bright pulse light by SHG process wasmeasured to be 30 μm. The generated bright pulse light beam
was enlarged to a diameter of 20 mm by a telescope system. This
enlargement of the falling pulse beam is important to overcome
the vignetting of the lenses used in the optical setup. In the
Mach–Zehnder off-axis scheme in transmission, a pair of
non-polarized beam splitters (NPBS1 and NPBS2) was used
to divide the two SHG pulse beams into reference (R) and object(O) beams and combine them again at the plane of NPBS2.
Disparity of optical path length in the two arms of the interferometer was precisely adjusted within the coherence length of
30 μm by a scanning mirror, equipped with a precise mechanicaltranslation stage, in the reference arm of the interferometer. Notethat the object that is a sandwiched microstructured sarcomere
sample is positioned carefully in the object arm to check the region of interest first before capturing the off-axis hologram. The
idea is to visualize the same region or an area very close to theregion that is tested by phase contrast microscope. Photographsof the investigated sample taken by both the phase contrast microscope and the proposed DHM with blocking the reference
arm are shown in Figs. 3(a) and 3(b), respectively. The numberof sarcomeres in Fig. 3(a) is 25, while the number of sarcomeresin Fig. 3(b) is 30. The difference in the number of sarcomeres isdue to the difference in magnification. Testing sarcomere lengthand thickness has an important determinant and indicator of
cardiac mechanical function [15]. To test the thickness of thesarcomeres, off-axis fringes should be formed to cover the region

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Vol. 56, No. 13 / May 1 2017 / Applied Optics

Fig. 3. Photographs of the investigated sarcomere sample (a) taken
by phase contrast microscope, and (b) taken by the proposed opticalsetup with blocking of the reference arm.

F3

in the Fourier domain [17,18], and the last two are the interference terms.
The reconstructed wave front ψ of the recorded hologram in
the observation plane 0ξη has been obtained by computing
the Fresnel integral of the digitized hologram I #k; l $ and isgiven by [16]!
"iπ 2 22
2
ψ#m; n$ ! A exp#m Δξ % n Δη $
λd
#
!
"$
iπ 2 22
2× FFT R D #k; l $I#k; l $ × exp#k Δx % l Δy $
;
λd
m;n

(2)

of interest shown in Fig. 3(b). Off-axis fringes are formed when
the interference of the object and the reference beams are tilted ata small angle with respect to each other. The off-axis hologram isformed at the plane of the beam splitter NPBS2. The off-axis
hologram was recorded by making the reference wave (insteadof the object wave) subtending the off-axis angle with the opticalaxis. Such arrangement is not only easier to align, but also makes
the image plane parallel to the sensor surface. The formed offaxis hologram was transferred to a black-and-white CCD camera(640 pixel by 480 pixel, pixel size ! 4.3 μm) using a microscopelens MO3 (20×, NA ! 0.1).
3. RECONSTRUCTION METHOD
The light coming from the beam expander is assumed to be an
ideal plane wave. The microscopic objective (MO2) should be
identical to the microscopic objective (MO1) to cancel out
the net sphericity and maintain the ideality of the plane wave.
As shown in Fig. 4, the object wave O propoagates along 0z
direction with normal incidence on the CCD plane 0xy.
The reference wave R propagates with an angle respect
to the 0z. The off-axis digital hologram intensity I #k; l $ recorded by the CCD camera can be written by the following
expression [16]:I #k; l $ ! jOj2 % jRj2 % R & O % RO& ;(1)

where k, l are discrete coordinates in the hologram plane and *stands for the complex conjugate and. In Eq. (1), the first two
intensity terms are of zero order, which can be directly filteredFig. 4. Off-axis geometry at the incidence on the CCD camera.
The 0xy plane is parallel to the CCD camera.

where m; n; k, and l are integers and λ is the wavelength of thelaser light. A is a complex constant given as A ! exp#i2π∕λ$∕#iπλ$, and FFT denotes a two-dimensional (2D) fast Fouriertransform. Δx and Δy refer to the sampling intervals in the hologram plane. The digital reference wave R D #m; n$ is defined as
R D #m;n$ ! AR exp'i#2π∕λ$#kx mΔx %ky nΔy$(. The samplingintervals Δξ and Δη in the observation plane are related to sizeof the CCD (L) and to the distance d as Δξ ! Δη ! λd ∕L.
After the FFT of the hologram is computed, three spectra are obtained. One specrum is selected by applying a spatial filtering
technique and the inverse FFT is computed. The amplitudecontrast image can be obtained by using the following intensity'Re#ψ#m; n$$2 % Im#ψ#m; n$$2 ( and the phase-contrast image
can be obtained by the argument arctan'Re#ψ#m; n$$∕Im#ψ#m; n$$(. To reconstruct the object-wave in the observation
plane, the two components of the wave vectors kx and ky are adjusted such that the propagation direction of R D #m; n$ coincides
as closely as possible with that of the experimental reference wave.
4. EXPERIMENTAL RESULTS AND DISCUSSION
Figures 5(a) and 5(b) show the off-axis holograms of the sarcomere sample obtained by a He–Ne laser light with wavelength 632.8 nm and femtosecond pulse light withwavelength 777.8 nm, respectively. Note that the off-axis hologram demonstrated in Fig. 5(a) was produced by the same experimental setup shown in Fig. 2 by replacing the femtosecond
pulse light with a He–Ne laser light. A layered structure ofaround 15 sarcomeres was captured at nearly the same regionof FOV. Comparison of these images indicated that the image
brightness of DHM with the femtosecond pulse light was
higher than that with the He–Ne laser light due to temporally
localized energy of the femtosecond pulse light. Figures 6(a)and 6(b) show one filtered spectrum Ψ ! R & O from thethree spectra in 2D-FFT of Figs. 5(a) and 5(b), respectively.

Fig. 5. Off-axis holograms of layered structure of sarcomeres takenby (a) He–Ne laser light, and (b) femtosecond pulse light.

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Fig. 6. Filtered 2D-FFT spectrum of off-axis holograms of the sarcomere sample with (a) He–Ne laser light, and (b) femtosecond pulse light.

Note that the three spectra appear in the filtered one spectrum
are attributed to the off-axis fringes of the sarcomere sample
itself. The spurious and speckle noises are clearly observedin the frequency domain of Fig. 6(a), while such noise completely disappeared when we used our setup, which is shownin Fig. 6(b).
Figure 7 shows the sequential reconstruction steps of the offaxis hologram with femtosecond pulse light. The reconstructed
amplitude and phase was recorded by selecting the appropriate
values of the two components of the wave vector k x !
0.00999 mm−1 and k y ! −0.5239 mm−1 for the off-axis hologram with He–Ne laser and k x ! 0.02145 mm−1 and k y !
−0.51570 mm−1 for that with the femtosecond pulse light.
To see the effectiveness of our setup compared to the conventional DHM with He–Ne laser light, we have not applied
any of the numerous image enhancement techniques [19,20] tosignificantly improve the perceived image quality for biologicalapplications. Figures 8(a) and 8(b) show the reconstructed 3D
pseudocolor amplitude map of Figs. 5(a) and 5(b), respectively.
It is noted that these 3D reconstructed amplitude maps were
flipped upside down to see the sarcomeres from a different side.
In Fig. 8(a), the existence of spurious noise in the backgroundimage [see Fig. 5(a)] makes it difficult to visualize the structure

Fig. 7. Steps of reconstruction: (a) off-axis hologram obtained byfemtosecond pulse light, (b) Fourier transformed spatial frequency domain spectra, (c) filtered one spectrum, (d, e) reconstructed amplitudeand phase-contrast images after computing the inverse FFT of the filtered spectrum, (f ) simulated digital reference wave, and (g, h) final
amplitude- and phase-contrast images.

Research Article

Fig. 8. 3D pseudocolor reconstructed amplitude-contrast image of(a) He–Ne laser light, and (b) femtosecond pulse light.

of the sarcomeres clearly. On the other hand, Fig. 8(b) shows
the high quality and contrast of the structure detail on the sarcomeres, and provides accurate profile edges of the hexagonal
shape of the sarcomeres. We believe these results show that our
proposed technique is effective and preferable in featuring the
sandwiched biological samples.
Our deductions were verified by applications of the apodization technique [21] to evaluate the coherent noise level inthe off-axis holograms of Figs. 5(a) and 5(b). Apodization is
the same topic as windowing in signal processing. The transmission of the apodized aperture function is perfectly transparent in the large central part of the profile. At the edges, thetransmission varies from zero to unity following a curve definedby a cubic spline interpolation. A 2D transmission window thatis 480 × 480 pixels and shown in Fig. 9(a) is multiplied with theoff-axis holograms in Figs. 5(a) and 5(b), respectively. Note thatthe strong profile shape of the apodized function used approaches the triangle shape to clearly discriminate the intensity
level at the middle of the image (small area occupies aroundfour sarcomeres). Normalization of intensities distribution offour sarcomeres at the middle of the off-axis holograms in
Figs. 5(a) and 5(b) before and after application of apodization
are shown in Figs. 9(b) and 9(c), respectively.
As seen from Fig. 9(b), the variation in intensities before andafter the application of apodization is considerable. This indicates that there is coherent noise in the reconstructed object
wave (amplitude and phase). Such coherent noise totallydisappears in Fig. 9(c), whereas there is no variation in theintensities distribution before and after the application of
apodization. This validates our deductions that the reconstructed object wave created by the proposed technique isfree from coherent noise and this technique is preferable to investigate sandwiched biological samples. Because of the low
coherence bright pulse light used, the proposed techniqueFig. 9. Coherent noise estimation by apodization: (a) 2D transmission function, (b) 2D intensity distribution with and without apodization at the middle of Fig. 5(a), and (c) 2D intensity distribution
with and without apodization at the middle of Fig. 5(b).

Research Article

Fig. 10. Synthesized image (a) generated four off-axis holograms,and (b) synthesized reconstructed amplitude-contrast image.

may suffer from a limited field of view. In this paper, we obtained full-field image by applying the synthesis image technique, which is used to superimpose many reconstructions
from many off-axis holograms by a direct linear addition.
Figure 10(a) shows four off-axis holograms generated by varying the optical path length of the interferometer. The gray regions demonstrate high-contrast fringes, while the non-grayregions demonstrate low-contrast fringes. The full field synthesized amplitude contrast-image shown in Fig. 10(b) was obtained by adding the reconstruction of the amplitude-contrast
image of each off-axis hologram of Fig. 10(a).
Because phase images tend to suffer from coherent noise to asignificantly less degree compared to the amplitude images, theresults of phase distributions reconstructed from Figs. 5(a) and5(b) were converted to height distributions [22] and compared.
Figures 11(a) and 11(b) show 3D pseudocolor height distribution images of the stripe structures of 10 sarcomeres ofFigs. 5(a) and 5(b), respectively. As seen in Fig. 11(a), the sarcomeres are hardly viewed in Fig. 11(a) because of the existenceof coherent noise in the background. In contrast, Fig. 11(b)shows that the stripe structures of the sarcomeres are clearlyviewed because there is no coherent noise in the backgroundimage. The 2D height profile extracted along the red and bluelines in Figs. 11(a) and 11(b) are shown in Fig. 11(c), indicating the cross-sectional profile of one sarcomere.
The height profile extracted by the proposed technique wasestimated to be 0.72 μm along one sarcomere [blue line in
Fig. 11(c)]. On the other hand, the height profile extracted
along one sarcomere [red line in Fig. 11(c)] was estimatedto be 0.53 μm. We believe that the estimated height may
be varied from one sarcomere to the other, so many sarcomeresFig. 11. 3D pseudocolor height map converted from the reconstructed phase-contrast image of stripy sarcomere with (a) He–Ne laser
light, (b) femtosecond pulse light, and (c) 2D height profiles along the
red and blue lines in (a) and (b).

Vol. 56, No. 13 / May 1 2017 / Applied Optics

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Fig. 12. (a) 3D height map converted from the reconstructed phaseof the white rectangular area of Fig. 5(b) after removing the zero orderby subtractions among the successive four holograms in Fig. 10(a), and(b) 2D phase profile along the white line of (a).

were measured at different positions and the ratio is still considerable. We attribute this phase difference to the coherent
noise (speckle and spurious) and the instability of He–Ne laser
light. It is due to the fact that stray light or reflected ambient
light (spurious noise from He–Ne) generally causes a reductionof contrast [23], as shown in Fig. 11(a). From these results, it
could be said that the proposed technique is free from noise andthe sarcomere cross-section can be calculated precisely with noneed for time-consuming image enhancement techniques. A
further improvement in contrast was applied to the proposed
technique by removing the zero order of the synthesized
hologram [24] produced from the four holograms shown in
Fig. 10(a). Because of the holograms’ shifting process, the reference and object beam may have some phase shifts. When thesecond hologram in Fig. 10 is subtracted from the first hologram, the zero order is removed. The subtraction process wasdone for the third to the second and the fourth to the third.
Subtractions among the successive four holograms of Fig. 10(a)were superimposed using a direct linear addition method toform a high-quality synthesized hologram. The synthesized
hologram was reconstructed by the same procedures demonstrated in Fig. 7 with no zero order in the frequency domain.
Figure 12 shows the 3D pseudocolor height map converted
from the reconstructed phase of the white rectangular of
Fig. 5(b) after removing the zero order by subtractions among
the successive four holograms in Fig. 10(a). The height profile
extracted along the white line in Fig. 12(a) is shown in Fig. 12(b),indicating the cross-sectional height profile of one sarcomere andestimated to be 0.74 μm. As seen from Fig. 12, the contrast is
improved and we attribute this improvement to the zero orderelimination in addition to any random noise during the scanning
process. The average thickness measurement of the sarcomere isapproximately consistent with the results published in [25].
The repeatability of the optical setup, which is defined as theroot mean square (RMS) of the height difference for two measurements on the sample-free setup, is around 10 nm. We believe that the repeatability of the setup can be further improvedby lessening the exposure time of the CCD camera used, in
addition to reducing the environmental disturbance.
5. CONCLUSION
In this paper, we presented the use of the femtosecond laser
bright light in a Mach–Zehnder interferometer to enhance
the image contrast while suppressing the coherent noise inF6Vol. 56, No. 13 / May 1 2017 / Applied Optics

DHM. The presented method was effectively applied to investigate the structure of a sarcomere sample mounted between
two thin glass plates. Compared to the DHM with long coherent He–Ne laser light, the proposed setup is insensitive to coherent noise. This insensitivity to coherent noise can be
attributed to the features of the femtosecond pulse light with
an ultrashort coherent length, as well as the high brightness
light generated by the nonlinear system, which reduces the coherent mode of the fundamental light by a factor of 9. Further
improvement in contrast was achieved by applying the synthesis image technique. The proposed technique can provide a fast
inspection of the uniformity of 3D nanopatterns as well as enhanced image contrast with no need for image enhancement
techniques for coherent noise suppression.
Funding. Japan Science and Technology Agency (JST),
Exploratory
Researchfor
Advanced
Technology,
MINOSHIMA Intelligent Optical Synthesizer Project;
Ministry of Education, Culture, Sports, Science, andTechnology of Japan (Grants-in-Aid for Scientific Research
No. 26246031).
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