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Type of the Paper (Review)
Resolution Enhancement in Phase Microscopy: a

Review
Juanjuan Zheng 1, Vicente Micó 2,*, Peng Gao 1, 3, 4,*
1 School of Physics and Optoelectronic Engineering, Xidian University, Xi’an, 710071, China
2 Departamento de Óptica y de Optometría y Ciencias de la Visión, Universitat de Valencia, C/Doctor
Moliner 50, Burjassot 46100, Spain
3 Institute of Applied Physics, Karlsruhe Institute of Technology, 76131 Karlsruhe, Germany
4 Institute of Nanotechnology, Karlsruhe Institute of Technology, 76344 Eggenstein-Leopoldshafen,
Germany
* Correspondence: vicente.mico@uv.es (VM) and peng.gao@kit.edu (PG);
Abstract: Quantitative phase microscopy (QPM), a technique combining phase imaging and
microscopy, enables visualization of the 3-D topography in reflective samples as well as the inner
structure or refractive index distribution of transparent and translucent samples. However, as in
conventional optical microscopy, QPM provides either a large field of view (FOV) or a high resolution
but not both. Many approaches such as oblique illumination, structured illumination and speckle
illumination have been proposed to improve the spatial resolution of phase microscopy by restricting
other degrees of freedom (mostly time). Therefore, the space bandwidth product (SBP) of QPM
becomes enlarged. This paper aims to provide an up-to-date review on the resolution enhancement
approaches of QPM, discussing the pros and cons of each technique as well as the confusion on
resolution definition claim on QPM and other coherent microscopy.
Keywords: Quantitative phase microscopy; Digital holographic microscopy; lensless microscopy;

resolution enhancement; space bandwidth product
1. Introduction
Despite other high-resolution microscopy technologies, e.g., scanning electron microscopy
(SEM), scanning tunnel microscopy (STM) and atomic force microscopy (AFM), optical microscopy
acts as a mainstream inspection tool in the imaging community, especially, in biological studies. The
main reason is because optical microscopy is a minimally invasive technique and therefore
compatible to live cell or tissue. When being used with fluorescence specific labeling strategy, a
certain kind of biomolecule on targeted structures can be observed. However, due to the diffraction-
limit imposed by the wave nature of light, conventional optical microscopy has a spatial resolution
around half of the used wavelength (typically ~200 nm) and thus, cannot resolve the subcellular
structures finer than this limit. Since the 90s of the 20th Century, this limitation has been broken by
the development of super-resolution optical microscopy [1-3]. The key of super-resolution
microscopy is to switch single molecules “on” and “off” by light and therefore, a high-resolution
image can be described by these single molecules.
There is no doubt that the usage of fluorescence markers has revolutionary advantages since it
enables observation of specific targeted molecules. Nevertheless, it is also desirable to directly
observe cells or tissues for their morphological structures in their natural condition, since
fluorescence probes are harmful, or at least of disturbance, to biological systems. Of note, the
determination of the 3-D shape and refractive index of biological samples has recently experienced a
© 2018 by the author(s). Distributed under a Creative Commons CC BY license.

Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 30 March 2018 doi:10.20944/preprints201803.0273.v1
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growing interest in the biomedical community [4-6]. However, looking directly at biological samples
is tremendously challenging since they are transparent or translucent under the visible light. Luckily,
when a light propagates through a cell, its phase is distorted as consequence of the thickness profile
and the refractive index distribution of the cell. And this information can be exploited to visualize
(qualitative imaging) and characterize (quantitative imaging) those transparent samples.
There are many approaches to retrieve the phase information of a sample. Wavefront sensing,
employing a Shack-Hartmann sensor [7] or a pyramid sensor [8], is a simple and useful approach to
investigate slightly distorted wavefronts. The basic idea is to spatially divide the tested wavefront
into a series of sub-regions and focus them separately onto a CCD camera typically with a micro-lens
array. Then, by analyzing the location of the spots on the CCD, the local slope of the wavefront over
each region is computed. Besides wavefront sensors, interferometric approaches are the most
commonly used techniques to retrieve phase information. Here, a reference wave is superimposed to
the object wave and the phase is reconstructed from the generated interference pattern [9-27].
Alternatively, beam-propagation-based methods can also retrieve the phase distribution by
recording a series of diffraction patterns under different conditions or applying different
perturbations [28-36]. For example, a diffracted beam by a phase sample influences the propagation
of the beam and results in different intensity distributions at different planes. Vice versa, the phase
can be recovered by analyzing the intensity distributions of a diffracted beam recorded at different
distances [28] or by using different illumination wavelengths [33-36].
Quantitative phase microscopy (QPM) emerges as a combination between phase imaging and
microscopy. Thus, QPM has similar constraints than conventional optical microscopy regarding basic
properties such as resolving power, available field of view (FOV), working distance, depth of focus
and global costs. Of special interest are the spatial resolution limit and the FOV provided by QPM
which directly come from the used microscope lens and that are closely related one to each other
since the higher the former, the lower the latter (and vice versa). The ratio between these two
parameters defines the space-bandwidth product (SBP) degree of freedom of the QPM imaging
system [37]. The SBP concept generalizes the theorem of invariance of information capacity [38]
because not only degrees of freedom are considered but also the shape of the SPB of the imaging
system. Just as an example, a standard 20X microscope lens with a resolution limit of 0.8 m and a
circular FOV of 1.1 mm in diameter, provides a SBP of 7 megapixels [39]. However, microscopists
always expect to have higher SBP in the sense of increasing resolution while maintaining FOV or, in
other words, to provide enhanced resolution under the same FOV. This can be accomplished from a
theoretical point of view by matching the SBP of the sample with the SBP of the imaging system in
order to allow effective transmission range of the sample’s image through the microscope system
[40,41].
In the past decades, there were many efforts to improve spatial resolution without scarifying the
FOV size. This paper aims to complete and update previous manuscripts dealing with overviews
about superresolution [42,43] by providing an up-to-date review on the approaches to enhance the
resolution in QPM. Moreover, it includes an overview about enhanced resolution in lensless QPM.
This paper will also cover the pros and cons of each technique, as well as the confusion on resolution
definition claim on QPM and other coherent microscopy techniques. This article is organized as
follows: Section 2 summarizes the applications of QPM; Section 3 reviews resolution enhancement
approaches in lens-based interferometric QPM; Section 4 overviews resolution enhancement
approaches in lensless interferometric QPM; Section 5 presents resolution enhancement approaches
of reference-less QPM. Section 6 describes the criterion for evaluating the resolution of QPM; Finally,
Section 7 summarizes this review.
2. Quantitative Phase Microscopy (QPM) and its Applications
When a beam is reflected by the sample’s surface or passes through a transparent sample, the phase
of the beam contains information about, respectively, the 3-D sample’s profile or the refractive index
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distribution of the sample. This information can be exploited thus providing not only high-contrast
images of transparent samples (qualitative improvement), but also quantitative evaluation on their
3-D profiles or refractive index distributions [44]. In that sense, QPM provides a versatile tool to
observe or investigate the microscopic world quantitatively. Hence, QPM has been widely used in
industrial inspection [11,45], biomedical observation [46-50], special beam generation [51-54], air or
liquid flow monitoring [55,56], and adaptive imaging [57]. Figure 1 summarizes some of these
applications.
Figure 1. Applications of QPM. The three images on the top are adapted from Ref. [11]
In conventional bright field microscopy, illumination is provided by an incoherent source (such

as a thermal light) and the diffraction-limited resolution is defined as min=/NA where and NA
indicate the illumination wavelength and the numerical aperture of the objective lens. However, in
QPM, a coherent or partially-coherent light is used for illumination and the resolution limit becomes
in min=/NA [58,59] (vide infra, section 5). This limit can be improved by using shorter
illumination wavelengths [60,61], or by synthesizing a larger numerical aperture [62-65] using
different illumination strategies such as oblique illumination (see Ref. [42] for an extensive reference
list on synthetic aperture generation by tilted beam illumination), structured illumination [32,66-71],
grating projection [72-75], and illuminating with random patterns [76-81]. Depending on whether a
reference wave is needed or not in QPM, these resolution enhancement approaches can be
categorized into two groups: Resolution enhanced QPM via interferometric approaches and
Resolution enhanced by single-beam QPM.
3. Resolution enhancement approaches in lens-based DHM
The background layout for interferometric QPM is defined by digital holographic microscopy (DHM).
Figure 2 presents a typical DHM setup in transmission mode where the architecture is based on a
standard Mach-Zehnder interferometer. The laser output is split into the object and reference waves
by the beam splitter BS1. In the object wave path, the beam is expanded by the beam expander BE1
and illuminates a sample located in the front focal plane of the objective MO. After passing through
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the specimen, the object wave curvature is magnified by the MO and collimated by the lens L before
reaching the digital sensor. This object wave interferes with the reference wave which is also
expanded by a second beam expander BE2 in order to equalize beam curvature. Both beams generate
hologram (Figure. 2(b)) which is recorded by a charge-coupled device (CCD) or complementary
metal–oxide–semiconductor (CMOS). After digitally reconstruction from the recorded hologram
[9,11,12,56], the complex amplitude distribution (intensity and phase) of the sample is recovered
(Figures 2(c, d)). Unlike conventional microscope which only provides in-focus sample images, DHM
does not need an image plane hologram to achieve imaging since it is capable of digitally refocus the
sample afterwards [13,21,82-84] by using numerical propagation tools.
Figure 2. Basic configuration of DHM used with transparent or translucent specimens. (a) Schematic optical
setup; (b) Recorded hologram example; (c) and (d) the reconstructed amplitude and phase images of the imaged
sample. BE1 and BE2, beam expanders; BS1 and BS2, beam splitters; L, lens; MO, microscopic objective; NF,
neutral variable attenuator. Scale bar in (c), 45 m. Images taken from Ref. [21].
3.1 DHM with oblique illumination
In a regular DHM layout (Figure 2) where a microscope lens is used to image a specimen which is
illuminated by a non-tilted plane wave, only the spatial-frequencies diffracted by the sample and up
to a maximum value derived from the NA (NA/) of the objective will be transmitted through the
limited aperture of the microscope lens (Figure 3, upper part). The high spatial frequency content is
diffracted at higher angles falling outside the limited lens aperture. However, when a plane but tilted
wave is used as illumination (Figure 3, lower part), part of this higher spatial-frequency range is
diffracted on-axis and, thus, will pass through the limited lens aperture. This downshift of the high
spatial frequency range is recovered in a similar way than the centered spectrum aperture by DHM
does. After multiplexing different incidence directions, a complementary set of apertures containing
different spatial-frequency content is available and a synthetic aperture is generated as the coherent
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addition of such aperture set. Figure 3 depicts the addition of 4 external apertures for synthetic
aperture generation.
The reconstruction process is done digitally and the downshifted frequencies are back assembled
to their original positions in the object’s spectrum, thus synthesizing a wider spectrum than the one
defined by the regular limited aperture. Final enhanced resolution image is achieved by inverse
Fourier transform of the generated synthetic aperture. It is worthy to mention that the synthetic
aperture synthesis is a coherent process where different phases need to be equalized and properly
adjusted. These phase-mismatches come from different recording conditions of the different
holograms, different aberrations terms per each tilted beam, and slightly mismatches in the
experimental layout.
Using this strategy and despite that the spatial resolution of the regular imaging lens is modest,
synthesized image can contain sub-resolution information. Here sub-resolution means that it
contains details incoming from the spatial frequencies that are outside the conventional aperture
under plane wave illumination. This is the reason why this type of techniques for resolution
enhancenment are also named superresolution methods because it is related with the capability to
overcome the resolution limit imposed by diffraction without changing the NA value of the lenses.
So, given that the resolution limit in DHM under plane wave illumination is 0.82/NA, it is improved
to 0.82𝜆/[𝑁𝐴 + 𝑠𝑖𝑛(𝛼𝑖𝑙𝑙𝑢𝑚 )] by using tilted beam illumination where 𝛼𝑖𝑙𝑙𝑢𝑚 is the tilted beam
illumination angle.
Figure 3. Schematics of oblique illumination and synthetic aperture generation.
Since the first implementations of superresolution by tilted beam illumination [85-88], many
ways to synthesize a larger aperture via oblique illumination have been extensively reported in the
literature [49,89-114]. Schwarz et al. used off-axis illumination to downshift the high-frequency
components of the object’s spectrum, and a reference beam is reinserted just at the proper angle to
upshift the image content back to its proper spatial frequency region [65]. This process is performed
twice for orthogonal illumination directions and sequentially in time, thus allowing the transmission
of different frequency bands of the 2-D object’s spectrum. The authors obtained a resolution gain
factor of 3 for both orthogonal directions. Subsequent works demonstrate resolution improvement
until the maximum limit for air-immersed optical imaging systems [90] as well as considering
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evanescent waves [102]. Also with optical rearrangement of the additionally transmitted spectral
ranges, Mico et al. reported on the use of a vertical-cavity surface-emitting laser (VCSEL) array for
resolution improvement [113]. In this case, the resolution improvement was 1-D but it was obtained
with a single camera snap-shot with a resolution gain factor of 5. Extension to 2-D imaging was
straightforward using different interferometric configurations [105,108-110] and synthetic numerical
apertures above the maximum limit for air immersed imaging systems (NA = 1) was also reported
[101]. Additional implementations considered wavelength multiplexing [95-97], and adaptation of
tilted beam illumination techniques has been recently implemented into a regular upright Olympus
microscope [92]. The use of scanning elements has also been reported as a technological improvement
for providing tilted beam illumination [90,98,115,116], and sample rotation instead of tilt the
illumination direction was also validated [74,100,102,107]. Tilted beam illumination for resolution
enhancement has also been applied to differential interference contrast (DIC) microscopy [117] and
Zernike phase contrast microscopy [118]. Moreover, axial rather than transversal resolution
improvement has also been validated from the synthetic aperture generation [119] and an application
of the technique to edge processing was also reported [120].
Just as an example of the results provided by the tilted beam illumination technique, Figure 4
shows the experimental results provided by the methods reported in Refs. [105,121] for different type
of samples. Images in Figure 4(a,c) correspond to the central part of a high-resolution negative USAF
test target, and (d-h) images are human red blood cells (RBCs). In the USAF case [105], a 0.14-NA,
long working distance, infinity corrected microscope lens is synthetically expanded till
approximately 0.45-NA. The generated synthetic aperture [Figure 4(b)] expands up by a factor of 3
the cut-off frequency of the regular lens (dashed white circle in Figure 4(b)) for every direction in the
Fourier domain, thus allowing full 2-D resolution improvement. For the bio-sample, 2-D full space
coverage of the sample’s spectrum is again achieved (Figure 4(e)). A microscope objective with 0.1-
NA is used to generate a synthetic aperture with an approximated value of 0.27-NA which enables
RBCs visualization (Figure 4(f)) in comparison with the low-resolution images provided by the
objective under conventional on-axis illumination (Figure 4(d)). Moreover, superresolved QPI is
available since complex amplitude distribution is retrieved. This fact is demonstrated through
Figures 4(g)-(f) where a 3-D view of the optical phase is represented.
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Figure 4. Demonstration of resolution-enhanced imaging using tilted beam illumination: (a-c) coming from Ref.
[105] and (d-f) derived from Ref. [121].
3.3 DHM with structured illumination
Although structured illumination is typically used to obtain resolution improvement of intensity

images [122,123], it has also been applied to resolution enhancement in DHM [66-71,124]. Figure 5(a)
shows a setup used for the DHM with structured illumination [124], where the phase-shifting of the
structured illumination was firstly implemented. In the implementation, four binary phase gratings
rotated by m×45° (Figure 5(b)) and generated by a spatial light modulator (SLM) are projected onto
the sample, so the sample is illuminated sequentially by 4 sinusoidal fringe patterns. After passing a
telescope system comprised by the microscope objective MO and the lens L, the object wave interferes
with a tilted reference wave R and the generated holograms are recorded by a CCD camera. We
denote with  mn the structured illumination wave generated by the grating having mth orientation
and nth phase shifting, and with mn the resultant wave transmitted through the object when
becomes illuminated by  mn . Thus, mn interferes with the tilted reference wave R on the detector
plane generating an intensity hologram in the form of I mn  R  mn . From this intensity, the
2
wave mn can be reconstructed by using standard methods as in off-axis DHM. Generally, mn can
be decomposed into three waves Am,-1, Am,0 and Am,1 along the -1st, 0th and +1st diffraction orders of
the illumination wave. When we assume that the phase increment for each shifting of the grating is
, then mn can be written as:
mn   1 exp( in ) Am, 1   0 Am,0   1 exp(in ) Am,1 , (1)

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where -1, 0 and 1 denote the magnitudes of the diffraction orders. From Equation (1) we can calculate the
reconstructed object waves Am,-1, Am,0 and Am,1 coming from the different diffraction orders and
combined in the Fourier plane to yield the synthetic spectrum as:
1
 Am, 1    1 exp( i )  0  1 exp( i )   m1 
      (2)
 Am,0    1 exp( i 2 )  0  1 exp( i 2 )  m 2 
 Am,1   1 exp( i 3 )  0  1 exp( i 3 )   m 3 
Finally, a focused image with enhanced resolution is retrieved by inverse Fourier transform of the
synthetic spectrum. As in previous tilted beam illumination approaches, the enhanced resolution
limit in structured illumination DHM is determined by the illumination angle illum of the +1 and -1
diffraction orders. Assuming the angular aperture of the imaging system (limited by the objective
MO) is NAMO, the synthetic NA of the structured illumination DHM is: NA=NAMO+sinillum (Figure 5(c)).
And this synthetic NA means an enhancement in spatial resolution, compared with the on-axis plane wave
illumination whereillum=0. Specifically, in the experiment [124], the NAMO =0.25 of the objective lens
limits the resolution of the set-up to plan=1.55m for the on-axis plane wave illumination. Using the structured
illumination (with illum=10 degrees), the resolution was improved to str=0.9 m. Particle-cluster on a glass
plate (amplitude and phase object) have been used to validate the resolution enhancement of the described
method (Figure 6). The phase images obtained by on-axis plane wave and structured illumination are
shown in Figure 6(a,b), respectively. The phase image in Figure 6(b) has a better resolution compared
with that in Figure 6(a) and two particles separated by 1.4 m become distinguishable as shown in
Fig. 6(c). The full width at half maximum (FWHM) of a single particle is 1 m, which is slightly larger
than the theoretical resolution value.
Figure 5. DHM with structured illumination. (a) Schematic setup; (b) four groups of structured illuminations
with different directions; (c) the synthesized spectrum. Images taken from Ref. [124].
Structured illumination in DHM is completely analogous to the case of simultaneous

illumination with two oblique plane waves at two different angles and provides the same effect
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achieved by illuminating first at one angle and then at the other one. In fact, there is a complete
parallelism between structured illumination and tilted beam illumination [85-121]. Although
structured illumination leads to a larger amount of detectable sample frequencies than any single-
beam illumination scheme, it does not contain more information beyond the diffraction limit [125].
Recently, structured illumination with unknown sinusoidal structured illumination has been exploited
for DHM, for which an iterative reconstruction is needed to obtain resolution-enhanced images [69,126].
Structured illumination has also been applied in differential interference phase contrast (DIC) microscopy [127].
Figure 6. Experimental results for resolution enhancement in DHM with structured illumination. (a) and
(b) reconstructed phase images by using plane wave illumination and structured illumination; (d) the phase
distributions along the two lines drawn in (a) and (b). Images taken from Ref. [124].
3.4 DHM with speckle illumination
Speckle illumination can also be used to improve the spatial resolution of DHM. A speckle beam,
which is generated by illuminating temporally coherent source through a diffuser such as a ground
glass or a holographic diffuser, can be seen as a dozen of oblique illuminations with different angles.
In incoherent microscopy, a dynamic diffuser changes the speckle patterns in time and, by utilizing
full condenser aperture to improve spatial resolution and to reduce the effect of unwanted
diffractions, it provides confocal-equivalent sectioning [128]. However, the phase information is lost
during the time-averaging of the speckles. In order to retrieve phase information, each speckle field
(including the information of both the targeted sample and the illumination pattern) should be
recorded by interfering with a reference wave [128-130].
A typical setup of DHM with sequential speckle illumination [75] is shown in Figure 7. In the
object wave path, a holographic diffuser (HD) is used to generate speckle field illumination. A
galvanometric mirror (RM) before the HD controls the incident angle of the laser beam onto the
diffuser and generates angle-dependent speckle fields. Two sets of angle-dependent speckle images
were recorded, one with the sample and other without it. And two sets of object waves with and
without the sample are reconstructed from the phase-shifted DHM holograms, which are obtained
by utilizing the acoustic optic modulators AOM1 and AOM2. Then, a set of net angle-dependent
electric-field images were obtained after dividing one set with the sample by the other without it and
the sample-induced complex field image is retrieved. However, along with each speckle field, there
are singular points (vortex) in the resulting amplitude and phase maps. To solve this problem,
hundreds of speckle fields were recorded with and without the sample, and the reconstructed image
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from these speckle illuminations were averaged to remove the speckle in reconstructed images.
Compared to the phase images obtained with plane wave illumination (Figure 7(b,c)), the
reconstructed images by using speckle illumination (Figure 7(d,e)) verify image resolution
enhancement. Quantitatively, the full width half maximum (FWHM) of the point spread function
was 516 nm for plane illumination and 305 nm for the speckle illumination, which are very close to
the theoretical expectations.
Figure 7. Schematics of DHM with sequential speckle illumination. (a) Experimental setup; (b)-
(d) Phase maps of polystyrene beads (200nm diameter) under plane wave illumination (b-
c) and speckle illumination(d-e). Scale bar in (d,e), 500 nm. Images taken from Ref. [76].
By using the aforementioned method, 3-D images of a sample with high spatial resolution, free of
diffraction noise and with improved depth sectioning can be obtained. This method combines the
advantages of incoherent imaging in resolution and image cleanness, with the merit of coherent
imaging in complex electric-field recording and 3-D imaging. For the averaging operation, it is
preferable to use a speckle field having the same weight or power along the whole frequency
spectrum. Therefore, a highly-scattering device such as ground glasses or holographic diffusers are
often used for speckle pattern generation.
Instead of this, an iterative reconstruction [80] was proposed. Figure 8 shows the optical setup
used for that study and containing a spatial light modulator (SLM) for generating the speckle fields.
The speckle fields illuminate the object after passing through the lens L1 and the microscope objective
MO1. We denote with 𝐴𝑖𝑜 the complex amplitudes of the fields diffracted by the object illuminated
with the ith pattern. When no object is inserted in the setup the fields corresponding to the ith pattern
𝑖
are denoted with 𝐴𝑆𝑝𝑒𝑐𝑘𝑙𝑒 . Holograms are recorded by superimposing a reference beam R to the
𝑖
object 𝐼𝑜𝑖 = |𝐴𝑖𝑜 + 𝑅|2 and speckle fields 𝐼𝑆𝑝𝑒𝑐𝑘𝑙𝑒 = |𝐴𝑖𝑆𝑝𝑒𝑐𝑘𝑙𝑒 + 𝑅|2 from which 𝐴𝑖𝑜 and 𝐴𝑆𝑝𝑒𝑐𝑘𝑙𝑒
𝑖
can
be reconstructed.
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Figure 8. DHM with sequential speckle illumination. Images taken from Ref. [80].
As previously stated, speckle fields can be understood as a combination of plane waves with
various illumination directions. Each one of those tilted illumination beams will shift the spectrum
of the object in the reciprocal plane (see Figure 9(a)) allowing access to extra spatial frequencies. This
extra information is integrated by an iterative method in order to synthesize the NA. The flowchart
is shown in Figure 9(b) and essentially involves the following steps: 1) initialize the object wave O(x,y)
by averaging the complex amplitude of the reconstructed object wave Oi =1/N ∙ ∑𝑁 𝑖 𝑖
𝑖=1 𝐴𝑜 /𝐴𝑆𝑝𝑒𝑐𝑘𝑙𝑒 for
𝑖
i =1 to N; 2) multiply O(x,y) with the speckle illumination complex amplitude 𝐴𝑆𝑝𝑒𝑐𝑘𝑙𝑒 to get the
complex amplitude of the speckle-based object wave 𝐴𝑆𝑖 ; 3) perform the Fourier transform of 𝐴𝑆𝑖 , and
replace its central part (circle confined to a radius equal to NA/), with the corresponding part of the
spectrum of 𝐴𝑖𝑜 . The replacement operation bring new high frequencies which are downshifted by
𝑖
the speckle illumination 𝐴𝑆𝑝𝑒𝑐𝑘𝑙𝑒 ; 4) determine the resolution-enhanced object wave O(x,y) by taking
𝑖 𝑖
the inverse Fourier transform, and dividing the retrieved complex wave 𝐴𝑆𝑦𝑛 by 𝐴𝑆𝑝𝑒𝑐𝑘𝑙𝑒 ; 5)
continue the iteration by replacing the object wave O(x,y) with the newly-reconstructed one. The
iterative method enhances not only the resolution but also the signal to noise ratio (SNR) when the
reconstructed object wavefronts from different speckle-based holograms are averaged. The use of an
SLM for speckle field illumination avoids mechanical movements and allows high repeatability in
comparison with dynamic devices.
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Figure 9. Iterative reconstruction of DHM with speckle illumination. (a) Frequency spectrum evolution in the
imaging process; (b) iterative reconstruction with resolution enhancement. The red-dashed circles in (a) and the
black-dashed circles in (b) represent the NA of the optical system. Images taken from Ref. [80].
3.5 DHM with randomly-moving particles
Another possibility for achieving resolution enhancement in DHM is by using particles in the close
proximity of the specimen. This strategy can be subdivided into two main categories. In the first
category, random movement of sparse particles is used for localizing with sub-resolution accuracy
the transmittance change introduced by the sample’s structure [131-134]. The superresolved image is
retrieved after scanning the whole sample’s area with the randomly-moving particles), and localizing
their positions with a sub-resolution accuracy. In a few words, the random high-resolution pattern
provided by the sub-resolution particles attached to the sample allows to shift the sub-resolution
information in a similar way than tilted beam illumination produces. The constantly changing of the
random distribution allows separation between the sub-resolution information and the original
diffraction limited content. Thus, a proper resolving of the folded sub-resolution features is
accomplished by multiplying each image in the stored sequence by the decoding pattern that is
similar to the encoding one. The decoding pattern can be digitally extracted frame by frame from the
set of low resolution recorded images since the particles are sparse. In any case, the particles should
have a size below the resolution limit imposed by the imaging system. This type of methods can be
seen as an evolution of the original idea reported by Françon in the middle of the past century [135]
and based on synchronized moving pinholes, one over the object and another one at the image plane
through an optical imaging system.
Figure 10 includes the experimental results reported using gold nano-particles (200 nm diameter)
in Brownian motion for enhanced resolution imaging [133]. Figure 10(a) presents a SEM image of the
resolution test target used in the experiment while Figure 10(b) shows the diffraction-limited image
of the resolution-test target and corresponding with the conventional resolution image provided by
the microscope lens (60X, 1.0-NA, water immersion, , Center Valley, PA, USA). The target is
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illuminated with the white light source of a standard Olympus BX51 upright microscope, but a broad
band interference filter (950 nm CWL, 50 nm FWHM, 25 mm Mounted Diameter, Edmund Optics,
Tucson, AZ, USA) was previously inserted for experimental layout matching. According to the
theoretical prediction, the resolution limit provided by the system is 475 nm, so the last resolved
element is marked with a white rectangle at Figure 10(b) and corresponds to the bars having a pitch
of 600 nm coming from a bar width of 350 nm and separated by a gap of 250 nm. Element S6 (with
pitch equal to 471.3 nm) which is just in the limit of resolution but not resolved. Finally, Figure 10(c)
depicts the superresolved image obtained by using the proposed approach. One may see that sub-
resolution features corresponding with the elements labeled as S6-S5-S4, which cannot be seen in
Figure 10(a), become now distinguishable in Figure 10(b). In order to further demonstrate the
resolution improvement in the lower part of the Figure 10(a) as well as 10(b) we zoom in several
demonstrative features of the obtained images. It is found that some bars of the superresolved image
are resolved while the others are not (see for instance the vertical bars on Element S4). The reason is
due to the random Brownian motion of the nanoparticles does not sweep enough such area.
Figure 10. Resolution enhanced DHM with randomly-moving particles. (a) SEM image of the fabricated
resolution-test target (left) and magnification of the elements S4-S5-S6 marked with a white square in the left
image (right); (b) diffraction-limited image of the resolution-test target obtained when averaging the entire set
of captured images; and (b) superresolved image containing sub-wavelength details. Images taken from Ref.
[133].
As the second category, microsphere-based microscopy systems have garnered lots of recent
interest mainly due to their capacity in focusing light and imaging beyond the diffraction limit
[136,137]. The microspheres are placed on top of the sample and provide resolution enhancement by
transforming the object wave from the higher frequencies to the lower ones. This new type of
microscopy has been successfully applied to intensity-based microscopy [138,139] as well as to DHM
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[140,141]. However, one of its main drawbacks is the very restricted FOV provided by the method
since, essentially, the FOV is limited by the size of microsphere which is in the sub-wavelength range
for having superresolution effect.
4. Resolution enhancement approaches in lensless DHM
Lensless holographic microscopy (LHM) is a relatively modern development raising from the same
holographic principles as classical holography but using a solid-state image recording device
(typically a CCD or CMOS camera) instead of the holographic plate recording media by. Nowadays,
LHM is an emerging imaging modality with widespread interest where imaging is performed using
a lensfree configuration. LHM derives from a digital implementation of the Gabor’s invention [142]
where a point source of coherent light illuminates the sample and the diffracted wavefront is
recorded by a digital sensor [143].
LHM is typically implemented using two opposite layouts [144]. In the first layout, the sample
is closely placed to the illumination point source and farther in comparison with the digital sensor
[143,145-147]. And in the second layout, the illumination source is faraway while the sample is on
top of the digital sensor [148-151]. The former configuration introduces a magnification factor
(typically ranging from 5X to 20X) by geometrical projection of the sample’s diffraction pattern at the
digital sensor plane, and provides similar field of view (FOV) and resolution limit as reported in lens-
based DHM with a medium NA (e.g., in the range of 0.4-0.5). The latter layout provides no geometric
magnification (approximately 1X range) but an extremely improved FOV since the whole sensitive
area of the detector is available. This second configuration provides a modest resolution limit
incoming from lower NA values (0.2-0.3 NA range), mainly because of the geometrical constraints
imposed by the detector. For the sake of simplicity, let us call the first implementation as digital in-
line holographic microscopy (DIHM) while the second one will be referred as on-chip microscopy.
4.1 DIHM with synthetic aperture
In order to improve general capabilities (such as resolution in particular) of both previously

commented configurations, many approaches have been reported mainly in the last decade. In the
first case (sample magnified in the 5X-20X range), the resolution limit is ruled by diffraction in a
similar way to that in DHM: there is an aperture in the system that limits the maximum range of
spatial frequencies passing through the system. In LHM, the NA of the imaging system is defined by
the ratio L/D with L and D being the half size of the digital sensor and the distance between the
sample and the sensor. Thus, the resolution limit in DIHM can be improved scanning a larger
hologram by applying different approaches. By scanning we mean a relative motion between the
object and the digital camera and it implies the generation of an expanded hologram containing a
bigger portion of the diffracted object wavefront compared to the non-scanning case.
The generation of the expanded hologram can be implemented using, in essence, 4 different
strategies. The first strategy (and maybe the most appealing one) deals with the displacement of the
camera at the recording plane [152-164] thus synthesizing a larger hologram in comparison with that
one provided by only one camera position. The second strategy performs synthetic aperture
generation by angular multiplexing the spectral object information [97,165-172]. Angular
multiplexing is implemented by tilted beam illumination over the object in a similar way as in DHM
and allows the recovery of additional spatial frequencies falling outside the digital camera sensible
15 of 39
area when on-axis illumination is used. The third strategy is related with the use of additional optical
elements (typically diffraction gratings) that are placed between the object and the digital camera
[76,173-179]. The diffraction grating directs towards the camera an additional portion of the diffracted
object wavefront allowing the generation of the synthetic aperture hologram. And finally, the fourth
strategy is based on object movement instead of shifting the digital camera for aperture synthesis
[180-183].
As one can notice, the four strategies are equivalent and the effective outcome of each one of the
four encoding strategies is the same one: to downshift extra content of the object spectrum that
usually falls outside the limited system aperture with conventional illumination. This downshift
allows the recovery of such extra apertures using different decoding methods and depending on the
used encoding one. Figure 11 includes 4 cases of experimental results regarding synthetic synthesis
in DIHM when considering the 4 previously reported strategies. By rows and from up to down,
Figure 11 includes (a) camera shifting, (b) tilted beam illumination, (c) diffraction grating insertion
and (d) object scanning for superresolution in DIHM. And by columns and from left to right, Figure
11 presents the conventional low-resolution image (left), the generated synthetic aperture (center)
coming from the addition of elementary apertures (white dashed rectangles) in comparison with the
conventional aperture (central blue solid line rectangle), and the superresolved image as inverse
Fourier transform of the synthetic aperture (right). Notice that in DIHM the conventional aperture
shape is rectangular rather than circular as in DHM since it is the CCD the element defining the
system’s aperture.
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Figure 11. Experimental results for resolution enhancement of DIHM via (a) shifting the camera at the recording
plane (first row – images taken from Ref. [159]), (b) using tilted beam illumination (second row – images taken
from Ref. [169]), (c) inserting a grating in the experimental layout (third row – images taken from Ref. [176]),
and (d) considering object movement at the input plane (fourth row – images taken from Ref. [181]). Inside
each column, the conventional image without aperture synthesis (left images), the generated synthetic aperture
(central images), and the finally retrieved superresolved image (right images). In each row, the central parts was
magnified to allow easy identification of the superresolution effect.
The first row of Figure 11 shows the experimental results obtained by shifting the camera [159]
doubling the resolution of the conventional imaging system. The camera was shifted at the image
plane in the horizontal and vertical directions since most relevant information of the object is in those
directions. But the synthetic aperture shape can be customized according to such a priori object
information. As an example, a cross-shape synthetic aperture (central image) was generated with
expanded frequency coverage that finally yields in an isotropic resolution-enhanced image (right
image) in comparison with the conventional one (left image). The resolution limit is expanded from
12.4 m (Group 6-Element 2 of the USAF test) until 6.9 m (Group 7-Element 2) for the horizontal
CCD direction, revealing a resolution gain factor close to 2.
Tilted beam illumination downshifts the spatial frequency information of the object so the
synthetic enlargement of the system’s aperture can be achieved without camera displacements. Here,
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an illumination pinhole is shifted to different off-axis positions in order to provide tilted beam
illumination. To allow this, the sample should be static between the different recordings, so the
technique is using time multiplexing for aperture synthesis. Figure 11 (second row) includes the
experimental results reported in Ref. [169] and concerning a fixed swine sperm bio-sample. In this
case, the 4 apertures in the vertical and horizontal directions and the 4 apertures in the oblique
directions were considered. As a consequence, full 2-D coverage at the Fourier domain is achieved
when generating the synthetic aperture. Using this strategy, a synthetic numerical aperture close to
0.7 was experimentally validated for the largest CCD direction.
Figure 11 (the third row) depicts the experimental results concerning enhanced resolution
imaging by properly inserting a diffraction grating in the lensless Fourier holographic experimental
layout [176]. Depending on the Fourier spectrum of the diffraction grating, the superresolution effect
can be 1-D or 2-D. Two possibilities can also be considered for retrieving the complex amplitude
distribution of each band-pass image depending on the mode that the reference beam is introduced
in the recording plane: on-axis recording with phase shifting algorithm and off-axis recording with
Fourier domain filtering. Thus, it is possible to choose between a superresolved image over a large
object FOV or a higher number of band-pass by limiting/masking the object´s FOV. Here, a cross-
shape synthetic aperture is generated yielding in a resolution gain factor of 2 in both directions.
According to the images included at Figure 11, the resolution limit is improved from 31.25 m (Group
5-Element 1 of the USAF test) until 15.6 m (Group 6-Element 1) for the horizontal CCD direction.
Last but not the least, aperture synthesis has also been reported by using the object translation
as key tool for image quality improvement in LHM [181,182]. Lensless Object Scanning Holography
(LOSH), profits of the linear movement of the object to record a set of reflective digital lensless Fourier
holograms. Because of the linear object displacement, different spatial information coming from
different object’s regions of interest (ROIs) is recorded in each hologram and retrieved later on.
Meanwhile, each single ROI passes along the whole FOV in front of the CCD and becomes
illuminated with a different tilted beam for every position, thus allowing for retrieve different spatial
frequency content of every ROI. Those two characteristics produce object FOV enlargement and
resolution improvement, respectively, when considering the whole set of recorded holograms.
Moreover, the SNR of the final image is also enhanced due to the averaging of the coherent noise
when adding the whole set of recorded holograms. As a result, the final image provided by LOSH
resembles an image obtained under white light illumination but considering that not only intensity
but also phase information is accessible. Thus, an extended DOF image can be also synthesized owing
to the coherent nature of LOSH by numerical propagation to different axial distances. Figure 11 (last
row) shows the experimental results provided by LOSH where an impressive image quality
improvement concerning FOV, resolution and SNR is achieved for a 2-D resolution test. The scanning
was performed using a regular raster composed by 20 rows and 20 holograms per each row. As a
consequence, the conventional FOV (1.5x1.5 mm, approx.) is expanded up to a synthetic FOV 3.7 time
higher (5.5x5.5 mm, approx.), the resolution limit is enhanced from 44 m to 14 m meaning a
resolution gain factor equal to 3.2, approx., and the SNR value is improved from 2.34 (single hologram)
to 10.16 (superresolved image) after applying LOSH yielding in a SNR gain factor of 4.34.
4.2 On-chip microscopy with geometrical superresolution

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Coming back to our two opposed layouts in LHM, resolution enhancement methods have also been
reported using on-chip microscopy (1X sample’s magnification). In the on-chip microscopy, sample
are placed really close to, or, directly contact with the imaging sensor, so the limiting factor in
resolution is essentially the pixel size of the digital sensor that defines the sampling requirements and
the coherence properties of the illumination source which defines the maximum angle that a
diffracted beam will interfere with the non-diffracted reference beam. In this configuration, an
immersion medium with the refractive index higher than 1 fills the gap between the sample and the
sensor since there is glass coming from the coverslips containing the sample. Such immersion scheme
enhances the resolution in comparison with the previous DIHM layout where air is filling the gap.
Nowadays, there are available sensors having a pixel size at the micron range yielding resolution
limits that are similar to that of the DHM systems having microscope lenses with 0.25-NA for a visible
illumination. Despite the spatial resolution can be improved by reducing the pixel dimensions, doing
this the signal-to-noise ratio (SNR) will be reduced. To circumvent this conflict between the resolution
and the SNR, pixel superresolution (or geometrical superresolution) was proposed and successfully
applied to on-chip microscopy [144,184-197]. This strategy recods sequentially multiple images of the
same sample when the sample is shifted along the horizontal and vertical directions for a sub-pixel
distance each time. These sub-pixel shifts synthetically reduce the effective pixel size of the camera,
so the final resulting image exhibits a higher resolution limit from a sampling point of view.
Similar to the previous DIHM configuration, the shift is relative between the sample and the
digital recording device. So, it is possible to shift the digital sensor [196,197], the sample [194,195], or
the illumination [190-193] but keeping static the rest of the setup. However, the most practical
technique is to shift the illumination source because precise subpixel shifts can be generated from
less-precise illumination shifts due to the geometrical characteristics of the layout (distance’s ratio
between the illumination-to-sample and the sample-to-digital sensor). Nevertheless, other
approaches have been reported using wavelength scanning [188,189] or multiple intensity images at
different sample’s distances [186,187]. Using these types of approaches, resolution limit in LHM has
approached diffraction limited resolution limit with half-pitch resolutions equivalent to those from
0.8-0.9 NA microscope lenses [144, 190].
Finally, similar to the previous DIHM configuration, synthetic aperture generation has also been
proposed as a way to increase the resolution limit in on-chip microscopy [184]. Here, the illumination
source is not only shifted to achieve geometrical superresolution by subpixel shifts but tilted to allow
oblique illumination and aperture synthesis. Thus, a set of multiple low-resolution holograms
(typically between 16 and 64) for each tilted beam illumination position (11 oblique illuminations for
each orthogonal direction) are recorded and then used to achieve geometrical as well as diffraction
limited superresolution imaging. By digitally combining all those lensfree holographic
measurements, the authors clearly resolved 250-nm grating lines under 700-nm illumination
wavelength which effectively corresponds to a 1.4-NA.
In addition to those holographic approaches, on-chip microscopy with enhanced resolution has
also been applied to other imaging modalities such as fluorescent imaging [198-201], structured
illumination [198], holographic color imaging [188,191,202] and as specific arrangement for cell
culture [203,204] and waterbone parasites analysis [150] in combination with deep learning
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microscopy [205,206] as well as 3-D tracking of sperm cells trajectories [207-209] based on improved
axial resolution [210].
5. Resolution Enhancement of Reference-less QPM
Phase imaging based on interferometric methods is the most commonly used approach to retrieve
phase information. It presents high accuracy, but sometimes the need to introduce an additional
reference wave improves its complexity. Moreover, the measurements are sensitive to environmental
disturbances coming from thermal and/or vibration conditions that are different on both
interferometric beams. To circumvente this difficulty, reference-less phase imaging approaches have
been exploited in both DHM and LHM.
Regarding DHM, a wide range of techniques have been developed in the last years for achieving
QPM without interferometric tools. This category includes, just to cite a few, wavefront sensing based
on Shack–Hartmann sensors [7,211,212], pyramid sensors [8,213], or shearing systems [214,215].
Other approaches are based on common-path interferometric configurations and are capable of
reference beam synthesis from the object beam in order to avoid the external reinsertion of a reference
beam [216-218]. Others utilize a TIE algorithm with slightly defocused images for QPI [219-223] while
others are based on the recording of different defocused images of the sample to iteratively retrieve
phase information [224,225]. Alternatively and in LHM, beam-propagation-based methods can also
retrieve quantitative phase information from the recording of a series of diffraction patterns, which
are usually recorded at different planes [28,29], with different wavelengths [33-36,226], by moving
sub-apertures over the sample plane [30], by modulating the object wave with different phase
patterns [31], or by using modulated illuminations [32].
5.1 Reference-less phase retrieval with modulated illumination
Here we depict the first type of single-beam QPM with resolution enhancement by using modulated
illumination [32,227]. Figure 12(a) shows the setup used for sQPM which is similar to the one used in
Section 3.4 but no reference wave is needed. The random patterns on SLM (insets in Figure 12(a)) are
projected to the sample plane by the relaying system (L1-MO1). After passing through the sample
placed in the focal plane of MO2, the object wave is imaged by the system MO2-L2 and the diffraction
pattern (Figure 12(a), inset) are recorded by a CCD camera located at a distance z from the image
plane IP of the specimen.
Because these random phase patterns are generated using an SLM, they are highly repeatable
and their complex amplitudes can be measured in advance. We denoted them with Aillum
k
, and
k=1,2,…M while Ik denotes the diffraction pattern of the object wave under Aillum
k
. The phase retrieval
is performed using the following steps: (1) multiply the amplitude of the kth diffraction pattern with
a random initial phase factor exp (ik); (2) propagate I k exp(ik) to the object plane; (3) the calculated
wave is divided by the kth illumination amplitude Aillum
k
, and multiplied by the (k+1)th illumination
k 1
amplitude Aillum ; (4) propagate the newly-obtained object wave to the CCD plane; (5) replace the
amplitude of the obtained object wave with I k 1 ; (6) repeat the iteration loop (2)–(5) by using k+1
instead of k, until the difference between two neighboring reconstructions will be smaller than a
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threshold value. Furthermore, the object waves reconstructed from different groups of those random-
phase illuminations are averaged in order to reduce the noise. Figure 12(b) shows a representative
phase image obtained by using sQPI. Notably, the spatially modulated illumination also improves
the spatial resolution of the phase imaging since it scrambles the specimen spectrum and, thus, some
high-frequency components of the object wave beyond the NA of the system are downshifted and
can pass through the limited system aperture. These high-frequency components are shifted back to
their original positions during the iterative process and the resolution of the imaging becomes
improved. Figure 12(c) shows the resolution enhancement of sQPM, compared with the DHM with
plane wave illumination.
Figure 12. Single-beam QPM using modulated illumination. (a) schematic setup; insets in (a) show
the phase patterns displayed on SLM (left) and the diffraction patterns recorded by the CCD camera
(right); (b) Representative phase image obtained by using sQPM; (c) resolution comparison of DHM
with plane wave illumination (I) and sQPI with modulated illumination (II). Images taken from Refs.
[32,227].
5.2 Reference-less QPI with aperture synthesis
Another interesting approach for superresolution in reference-less QPI comes from the combination
of tilted beam illumination and phase retieval using an iterative algorithm [115,228]. The basic idea
of the iterative algorithm is to record a sequence of defocused intensity images at N planes and then
process these images to extract the phase encoded in the defocus. The different intensity planes are
generated by linear translation of the camera [229] or without moving parts using a tunable lens [228].
The procedure is quite similar to the previous references [224,229] but now tilted beam illumination
is considered aside of the on-axis conventional illumination. Thus, the phases at multiple illumination
angles are retrieved and a synthetic aperture can be generated as the coherent addition of the
retrieved information coming from different shifted pupils. The main drawbacks of the method are:
i) the sequential recording of the different N intensity images that prevents the use of the technique
for fast dynamic samples; ii) the need for mechanical scanning of both the tilted beam illuminations
(using galvo mirrors) and the camera shift (linear motorized translation stage); and iii) the inherent
21 of 39
limitation of the method to transmit the background beam at oblique illuminations for phase retrieval
meaing that the maximum resolution gain factor is intrinsically limitted to a factor of 2 (authors
demonstrated a resolution gain factor equal to 1.3). We note that the resolution enhancement
approaches of the on-chip microscopy also belongs to this category.
As we have previously stated, another completely different possibility for QPI in reference-less
approaches is to perform reference beam synthesis from the diffracted object wavefront [219-225,230].
Alternatively, the point-diffraction phase microscopy [23,26] generates a reference beam by pinhole-
filtering on one copy of the object wave. The setup has the advantages of being less sensitive to
environmen-tal vibration and high lateral and phase resolution. In that sense, SMIM (initials
incoming from Spatially-Multiplexed Interferometric Microscopy) proposes a simple and low-cost
way to convert a commercially available regular microscope into a holographic one with only
minimal modifications [231,232]. SMIM implements a common-path interferometric setup into a real
microscope embodiment where: i) the broadband light source is replaced by a coherent one; ii) a 1-D
diffraction grating is properly inserted at the microscope embodiment; and iii) a clear region at the
input plane is saved for reference beam transmission. Using SMIM, a regular microscope is converted
into a holographic one working under off-axis holographic recording with Fourier filtering [231] or
slightly off-axis recording with phase-shifting algorithm [232]. The apealling advantage above the
convetional DHM is the stability (less sensitive to environmental disturbance) due to the common-
path configuration.
Maybe the main drawback of SMIM is the FOV restriction imposed by the need to transmit a
clear transparent reference beam for the holographic recording. Despite the FOV is penalized,the
resolution will be improved in SMIMwithout modifying a given optical imaging configuration. In
order to improve the resolution of SMIM, tilted beam illumination has been recently adapted to the
SMIM architecture [92]. Tilted beam illumination is achieved by lateral displacement of the coherent
source to a set of off-axis positions, thus allowing a complementary spatial-frequency content
diffracted on-axis for each considered tilted beam (a regular procedure in oblique illumination
techniques). And SMIM technique recovers the complementary spatial-frequency content provided
by each tilted illumination. In addition to the on-axis one, the full set of tilted beam illuminations are
used to expand up the system’s aperture by generating a syntehtic aperture having a cutoff frequency
two times higher than that of the conventional one under on-axis illumination. All together,
suprresolved SMIM adapts a commercially available non-holographic microscope into a
superresolved holographic one. When applying SMIM with the 5X/0.15NA lens, the resolution is
doubled and the useful FOV of is reduced to one half [232]. The FOV is still equal to the one provided
by the 10X/0.30NA lens which is often employed to reach the same resolution with suoperresolved
SMIM. Figure 13 depicts the experimental results provided by this method [92] when using a
5X/0.15NA objective lens. The conventional image (left) corresponding with on-axis illumination
mode is restricted in resolution and comes from the inverse Fourier transform of the conventional
pupil (blue circle in the central image). Then, tilted beam illumination procedure is implemented
allowing the recovery of 4 extra off-axis apertures that are used to synthesize the expanded aperture
(center). Finally, the superresolved image (right) showing a resolution enhancement by a factor of 2
is achieved from the syntetic aperture.
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Figure 13. Experimental results for superresolved imaging of SMIM using tilted beam illumination.
The low-resolution image retrieved with on-axis illumination (a), the generated synthetic aperture
(b), and the finally retrieved superresolved image (c). Images taken from Ref. [92].
5.2 Fourier ptychographic microscopy (FPM)
Another reference-lens QPM approach with resolution enhancement capability is termed with
Fourier ptychographic microscopy (FPM) [39,233-237]. This method uses successive illuminations at
different directions to recover a high-resolution and high-SBP output image by iteratively stitching
together a number of low-resolution images in Fourier space.
In FPM, a sample is placed at the focal plane of a microscope objective often with a low-NA
(Figure 14). The light from different LEDs in a source-coded LED array is used to illuminate the
sample at different directions/orientations. A sequence of images is collected with the sample
successively illuminated by plane waves at different propagation angles from different LEDs. Then,
an iterative process is used to reconstruct a high-resolution image from the recorded low-resolution
images. (1), initialize the high-resolution image, √𝐼ℎ 𝑒 𝑖𝜑ℎ with h=0 and Ih as any upsampled low-
resolution image as a starting point. (2) generate a low-resolution image √𝐼1 𝑒 𝑖𝜑1 by selecting a
circular region on the spectrum of √𝐼ℎ 𝑒 𝑖𝜑ℎ and taking inverse Fourier transform. The position of the
circular region corresponds to a particular angle of illumination, and the size of the region given by
the coherent transfer function of the objective lens. (3) replace I1 by the measured intenisty I1m, and
update the corresponding region of √𝐼ℎ 𝑒 𝑖𝜑ℎ in Fourier space. Here the subscript m indicates the mth
measured intensity. (4) repeat steps (2)-(3) for other plane-wave incidences (total of N intensity
images). (5) repeat steps (2)-(4) till a self-consistent solution is achieved. Eventually, the converged
solution in Fourier space is transformed to the spatial domain to recover a high-resolution image of
the targeted sample with a dramatically increased SBP (namely, high-resolution and wide-FOV).
However, the increase of SBP is paid with long acquisition time, typically in the order of minutes.
Faster capture times, once achieved, will improve the imaging speed and consequently, allows
studying live samples, which is continuously evolving at various spatial and temporal scales [237].
And scheems exhibiting the working principle of high-speed aperture synthesis are also recently
reported [238,239]. A smart implementation scheme on source-coded LED array, together with an
improved algorithm, was proposed to increase the speed of FPM with enhanced SBP. In order to
further improve the resolution enhancement range, an oil-immersion condenser was employed to
enlarge the illumination angle [240]. Furthermore, instead of enlarging NAill, FPM is also
implemented by using a high-NA imaging objective, proving a final NAsyn of 1.4536 [235].
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Besides the aforesaid two types of resolution-enhanced single-beam QPM, annular illumination
was used to improve the spatial resolution of on Zernike phase contrast [118] or transport-of-intensity
equation (TIE) [241] based QPM. In addition, LED array was also used in differential phase contrast
microscopy [242].
Figure 14. Schematics of Fourier ptychographic microscopy (FPM, modified from the reference [238]);
(a) experimental setup of FPM with a source-coded LED array; (b) bandwidth extension of FPM.
Images taken from Ref. [238].
6. The criterion for evaluating the resolution of QPM
Figure 15. Comparison of the resolution of incoherent and coherent microscopy. (a) Schematics
illustrating the resolving power of a microscope; (b) intensity distribution of the Airy patterns A’ and
B’ obtained by imaging two infinitely-small point sources A and B through a diffraction-limited
system; superimposed intensity of A’ and B’ in the case of incoherent (c) and coherent microscopy
24 of 39
(d). “In-phase” and “Anti-phase” in (d) indicates the case A and B have the phase difference 0 and p,
respectively.
Here we first examine the resolving power of incoherent microscopy and coherent microscopy with
two neighboring points (with infinitely small diameter), separated by a distance w1 in the object plane
(Figure 15(a)). The imaging system of an incoherent or coherent system produces in the image plane
two Airy patterns A’ and B’, with their complex amplitude:
2 J1 (kaw)
U A ( w)  ,
kaw (3)
2 J (kaw1  kaw)
UB  1 .
k ( w1  w) w
Here kaw indicates a unit-less coordinate, i.e., the spatial dimension normalized by the system
resolution. J1() is the first-order Bessel function. In an incoherent microscope, there is a linear relation
between the optical intensity emitted from the sample and the intensity detected at the image plane.
Therefore, we have the superimposed intensity:
2
 2 J (kaw)   2 J1 (kaw1  kaw) 
2
I in ( w)   1 (4)
   ,
 kaw   k ( w1  w) w 
In a coherent microscope, a linear relation holds between the input complex amplitude and the
output complex amplitude, and the two output Airy patterns A’ and B’ are considered coherent. The
resultant image corresponds to the coherent superposition of the two Airy diffraction patterns. The
intensity at a point situated between A’ and B’ at distance w1 from A’ is given by:
2
 2 J (kaw) 2 J1[ka( w1  w)]  (5)
I co ( w)   1   exp(i diff ) .
 kaw k ( w1  w) w 
Here, diff denotes the phase difference between point A and B. Figure 15(b-d) compares the imaging
process of two points A and B, which are separated by a Rayleigh distance (0.61/NA), for the case
of incoherent and coherent imaging. For this purpose, the intensity distributions were calculated with
Equation (4) (for incoherent microscopy) and Equation (5) (for coherent microscopy), and the results
are shown in Figures (c,d), respectively. In incoherent microscopy (Figure 15(c)), A’ and B’ are
considered to be resolved since the principal intensity maximum of the pattern A’ coincided with the
first minimum of the pattern B’. The intensity at the midpoint between the two maxima is then equal
to 0.735 of the maximum intensity of each pattern, i.e., I(w1)=0.735I(0). In coherent microscopy (Figure
15(d)), the same points, A and B separated with the same Rayleigh distance (0.61/NA), are
considered for the following two cases: A and B having phase difference diff=0 (in-phase) and diff=
(anti-phase) in-between. It is found from Figure 15(d) that, for the case with diff=0 (in-phase), the
Airy patterns of A’ and B’ are considered not being resolved, since a central lobe appears in the
middle of the two points. However, for the case with diff= (anti-phase), the Airy patterns of A’ and
B’ are widely separated, and considered being resolved well. This implies that there is no easy
criterion to evaluate the resolution of coherent microscopy, considering the resolution depends on
both the microscope and sample (phase dependent). Nevertheless, we recommend to use 
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=/NA [58,59] to estimate the resolving power of coherent microscopy, since with this distance
two in-phase points A and B (diff=0) can be resolved, i.e., the intensity maximum of Airy pattern
A’ coincided with the first minimum of Airy pattern B’. To transparently and reliably assess the
merits of coherent microscope techniques, a practical unambiguous resolution standard (Table 1) has
been proposed, considering phase dependent resolution, the presence of computational post-
processing and noise [243].
Table 1 Practice guidelines for resolution determination

1. Report the predicted non-zero area of the coherent transfer function based on a specific
system specifications.
2. Explicitly provide information on any a priori assumptions applied to the sample (for
example, sparsity) and post-processing steps applied to the data (for example,
deconvolution).
3. Image the Siemens-star target and report its amplitude and/or phase.
4. Summarize performance with the minimum periodicity metric, if desired.
5. For wide-field systems, present data for different locations across the sample plane.
7. Discussion and Conclusions
Quantitative phase microscopy (QPM), via interferometric approaches or non-interferometric

approaches, provides not only high-contrast images of transparent samples, but also quantitative
evaluation on their 3-D profiles or refractive index distributions. However, conventional QPM has a
limited space-bandwidth product (SBP) of the optical system and consequently, trade-offs between
the spatial resolution and field of view (FOV) are often needed to be made.
For the purpose of enhancement of SBP, the improvement of the spatial resolution of QPM for a
fixed FOV have been performed by using a short wavelength, or synthesizing a larger hologram (for
lensless QPM) or a larger numerical aperture (for lens-based QPM). Specifically speaking, in lensless
QPM, moving the CCD camera or the sample was used to synthesize a larger hologram. In lens-based
QPM, oblique illumination, structured illumination, and speckle illumination were used to
synthesize a larger numerical aperture to improve the spatial resolution.
It is worthy to note that, the word “super-resolution” was often used for the approaches
reviewed in this paper. Considering plane wave illumination and for air-immersed imaging systems,
the NA is defined by the imaging lens itself and equals to a maximum theoretical value of 1 meaning
that the resolution limit  can go down to a minimum value of  =. However, it is habitual in
microscopy to use lower NA values because these lenses provide attractive additional properties
(longer working distances, larger fields of view, reduced prices…). In this case, optical “super-
resolution” relates with the capability to overcome the resolution limit imposed by diffraction
without changing the NA value of the lenses [244]. That is, to overcome the limitation imposed by 
=/NA. Nevertheless and for clarification, the word “super-resolution” is attributed in other
disciplines in microscopy to surpass the resolution designed by a specific microscope, i.e., 0.61/NA,
rather than the physical limit /2 imposed by the Abbe diffraction limit which was obtained by
integrating in phase all the diffracted waves from a sample with the angle range [0, 180o] in the semi-
plane [245]. Whatever the nomenclature, the resolution-enhancement (or superresolution)
26 of 39
approaches described in this review yield no resolution enhancement over the Abbe diffraction limit
[125].
Acknowledgments: This research was partially supported by National Science Foundation of China
(NSFC) (61475187, 61605150, U1304617); Project funded by China Postdoctoral Science Foundation
(2017M610623), and the Fundamental Research Funds for the Central Universities under Grants No.
JB160511, XJS16005 and JBG160502. Also, part of this work was supported by the Spanish Ministerio
de Economía y Competitividad and Fondo Europeo de Desarrollo Regional under the projects
FIS2007-60626, FIS2010-16646, FIS2013-47548-P and FIS2017-89748-P.
Author Contributions: The authors contributed equally to this work, and all the authors wrote this
paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 30 March 2018 doi:10.20944/preprints201803.0273.

Type of the Paper (Review)

Resolution Enhancement in Phase Microscopy: a

4 Institute of Nanotechnology, Karlsruhe Institute of Technology, 76344 Eggenstein-Leopoldshafen,

Keywords: Quantitative phase microscopy; Digital holographic microscopy; lensless microscopy;

© 2018 by the author(s). Distributed under a Creative Commons CC BY license.

2. Quantitative Phase Microscopy (QPM) and its Applications

In conventional bright field microscopy, illumination is provided by an incoherent source (such

3. Resolution enhancement approaches in lens-based DHM

3.1 DHM with oblique illumination

Figure 3. Schematics of oblique illumination and synthetic aperture generation.

3.3 DHM with structured illumination

Although structured illumination is typically used to obtain resolution improvement of intensity

mn   1 exp( in ) Am, 1   0 Am,0   1 exp(in ) Am,1 , (1)

Structured illumination in DHM is completely analogous to the case of simultaneous

3.4 DHM with speckle illumination

3.5 DHM with randomly-moving particles

4. Resolution enhancement approaches in lensless DHM

4.1 DIHM with synthetic aperture

In order to improve general capabilities (such as resolution in particular) of both previously

4.2 On-chip microscopy with geometrical superresolution

5. Resolution Enhancement of Reference-less QPM

5.1 Reference-less phase retrieval with modulated illumination

5.2 Reference-less QPI with aperture synthesis

5.2 Fourier ptychographic microscopy (FPM)

6. The criterion for evaluating the resolution of QPM

Table 1 Practice guidelines for resolution determination

7. Discussion and Conclusions

Quantitative phase microscopy (QPM), via interferometric approaches or non-interferometric

Conflicts of Interest: The authors declare no conflict of interest.

1. Hell, S.W., Far-field optical nanoscopy. Science 2007, 316, 1153-1158.

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