A Digital in-line Holographic Microscope using Fresnel Zone Plate

Yonghao Liang, Yilei Hua

and Changqing Xie

Laboratory of Microelectronics Devices and Integrated Technology, Institute of Microelectronics,

Chinese Academy of Sciences, Beijing 100029, China

Keywords: Holography, Fresnel Zone Plate, Microscopy.

Abstract: A digital in-line holographic microscope is presented using Fresnel zone plate. The light impinging on the

Fresnel zone plate is divided into a number of diffraction orders. We use the 0rd light, which propagate

along the original direction, as the reference beam. And the first order focus is used as a virtual point source

after which the sample is placed. The light transmitted through the sample is scattered by the object and the

structure information is carried by the light. The interference fringes created by the first and zero order

diffraction are recorded by a digital camera. Afterwards, the object information is retrieved using

reconstruction algorithm. With the aid of the Fresnel zone plate, an image with higher lateral resolution and

lower noise could be obtained. This holographic microscope is tested with several samples and the results

show that the lateral resolution is good, and for the phase object, the measured phased difference is accurate

compared with the AFM test result.

1 INTRODUCTION

Digital in-line holographic microscopy (DIHM) is a

type of lensless Fourier transform digital

holographic, which the object light and reference

light are coaxial, interference pattern are recorded

digitally by CCD camera. (Depeursinge, 2011;

Kreuzer, 2007; Kreuzer, 2010) The amplitude and

phase distributions of the object are obtained by

simulating the reconstruction process numerically.

Compared with traditional microscopy, DIHM is

excellent in simple optical path, speed, real-time,

wide field, non-contact, and differential-

interference-contrast imaging etc. It is widely

applied to measure the three-dimensional shape of

the diffraction optical components, interference,

deformation, vibration, flow field and particle

tracking ,etc.

The most widely used the pinhole DIHM is

among the numerous methods. (Garcia-Sucerquia,

2006; Xu. 2002; Granero, 2011; Kuznetsova, 2007)

Its main factors affecting the imaging have the

following several aspects: the structure size and

shape of pinhole; area and pixel pitch of image

sensor; zero - order image and twin image. In

addition to select a more suitable sensor, composite

image is an effective way to expand the area through

nine images collected by moving CCD.

Twin image is another factor which affects the

performace of the DIHM. Many ways have been

proposed to reduce its effect on reconstructed image,

for instance, off-axis digital holographic, phase shift,

twice positions measurement etc. However, these

optical system or process of measurement is

complicated. So, we proposed a simple way, using

conventional diffraction optical element of Fresnel

zone plate (FZP) to solve this problem.

The FZP is laid behind the pinhole to separate

the point source into the 0th beam and the 1st beam.

The sample is put between the focus of the 1st beam

and the CCD along the optical axis. The interference

fringe pattern, formed by the two beams, is recorded

digitally by CCD. In a different way from point

diffraction interferometers, the interference fringe

pattern is a Fresnel hologram. A novel

reconstruction algorithm is proposed to present the

object image. Besides, it also has the following

advantages: the third problem proposed in previous

paragraph will be solved just through single

inversion; expands field of view, improves lateral

resolution, increases the space around sample plate

to be easy integrated.

Liang, Y., Hua, Y. and Xie, C.

A Digital in-line Holographic Microscope using Fresnel Zone Plate.

DOI: 10.5220/0007388802090212

In Proceedings of the 7th International Conference on Photonics, Optics and Laser Technology (PHOTOPTICS 2019), pages 209-212

ISBN: 978-989-758-364-3

209

2 FZP DIHM CONFIGURATION

AND RECONSTRUCTION

ALGORITHM

2.1 DIHM Configuration

The configuration of the FZP DIHM is shown in

fig.1. The laser reaches pinhole through attenuation

sheet, reflection in turn. A spherical wave of

wavelength λ, emanating from the pinhole which is

regarded as the point source in pinhole DIHM,

illuminates an object. Without FZP, sample is

typically a distance of a few thousand wavelengths

from the source, and forms a highly magnified

diffraction pattern on a CCD much farther away.

The point source is major divided into 0rd scattered

spherical wave along the original direction and 1rd

gathered spherical wave by FZP. Sample is laid

behind the 1rd focus so that object information can

be scattered and form interference fringes with 0rd

reference beam. In order to satisfy the Nyquist

sampling criteria, CCD must be far away from

sample to ensure the finest interference fringe can be

digitally recorded by CCD.

Figure 1: The configuration of the FZP DIHM: laser is

focused onto pinhole after attenuation sheet and reflection

mirror. The emerging spherical wave is separated major

0rd scattering beam along the original direction as

reference light and the scattering object beam after 1rd

gathering. The interference pattern or hologram is

recorded digitally by CCD.

2.2 Interference Pattern

Ideally the reference wave emanating from the

pinhole is a spherical wave, irrelevant with FZP,











  



, where    is the

wavenumber. 



 denotes the object beam, the

interference pattern as follows :

ref scat

ref scat ref scat ref scat

I A A

A A A A A A

( )=| ( )+ ( )|

=| ( )| +| ( )| + ( ) ( )+ ( ) ( )

r r r

r r r r r r

(1)

As Eq.1, the third term in the second line is the

holographic diffraction pattern in pinhole DIHM,

because it arises from the superposition of the

interference terms between the along the original

direction reference wave from point source and the

scattered wave from the object. The fourth term is

the conjugate function of previous term and also

contains the interference between the scattered

waves.

The structure of FZP can be expressed as a

coordinate transformation structure as follows

( , ) exp( )

FZP n

t r c jnar











(2)

0 1 2 3

1/ 2 1/ 0 1/ 3 ...c c c c



  

    ，，，

Where,

The intensity of higher order diffraction is far

below 0rd and 1rd, moreover, 0rd is the reference

beam and 1rd is the object beam. So Eq.2 can be

simplified only including C

and C

. Then Eq.1 can

be rewritten as follows:

( ) exp( )/2 ( )/

scat

I j A



rr

(3)

It is obvious that first term in the second line is

constant, does not affect the results of the restoration

object. Second term is self-modulation image nearly

constant. According to third term, object information

can be acquired through reverse diffraction

transform. In regard to fourth term, the conjugate

image will appear if we do the forward diffraction

transform.

2.3 Reconstruction Algorithm

We use the angular spectrum method to reconstruct

the complex amplitude of the object. The O(x, y, 0)

stands for the optical field on the object plane and

O(x, y, z) stands for the optical field on the CCD

plane. The standard angular spectrum theory give us

   

 

( , ,0) ( , , ) exp 1

O x y O x y z jkz f f









  









(4)

Where f

and f

are spatial frequencies.

For amplitude object, the structure can be

recovered by reckoning the amplitude. Phase object

must remove the phase envelope, because the

reference light is spherical wave, that the object

phase contains a phase envelope of a spherical wave

PHOTOPTICS 2019 - 7th International Conference on Photonics, Optics and Laser Technology

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in general. Moreover, maybe there are some dirty

points on the CCD. Therefore, we use a symmetrical

hologram to restore the object. The equation is as

follows:

( , ) ( , ) ( , )I x y I x y I x y

(5)

The former is the hologram with object, the

latter not. Regardless of the type of object, light

changes only in the object exist. It is efficient to

recover the amplitude or phase object.。

3 EXPERIMENT RESULTS AND

DISCUSSION

In order to ensure good coherence on the surface of

CCD, 532nm laser is selected for illumination. After

all, green light is sensitive for our eyes and CCD.

CCD camera comes from Lumenera Company (pixel

pitch of 3.5um, 3000×2208 pixels, size of

10.5mm×7.7mm). The distance between CCD and

pinhole is 130±10mm (10mm is used to adjust Z

axis of CCD). The purpose of such a design is to

manufacture prototype consistent with the ordinary

microscope in size. The experimental setup is shown

in fig.2.

Figure 2: The experimental setup.

One key feature of the DIHM is that it can detect the

transparent phase object. Phase object, which is

quite common in biological microscopy, does not

change the amplitude of the light, but change the

phase of the light. Many method, such as Zernike

phase contrast or differential interference contrast,

has been proposed. However, accurately measure the

phase change is still difficult.

To test the capability of our DIHM for phase

object detection, we fabricated a silica sample,

which is transparent, and with only steps on it, it is a

pure phase object. The steps are firstly measure by a

atomic force microscope (AFM), the test result is

shown in fig.3. According to the test result, the step

height is 111nm.

Figure 3: The profile of the silica sample with etched bars.

Then we use the DIHM to measure the height

of the bars. The hologram captured by CCD camera

is shown in fig.4. Auxiliary spokes is shown in the

hologram, for the bars in the center are relatively

small and difficult to find .

Figure 4: The hologram of the silica phase object.

After the hologram is recorded, we use the

numerical method discuss in section II to reconstruct

the profile of the bars. The reconstructed image is

shown in fig.5 , and the height of the step is also

shown, the result is 100nm, which is very close to

the result measured by AFM.

To verify the capability of our DIHM, many other

samples were observed using our experimental setup.

In fig.6, the wings of a fly and a bee is observed in

detail, the reconstructed image of the wings shows

that our DIHM can be applied to observe varies type

of samples.

A Digital in-line Holographic Microscope using Fresnel Zone Plate

211

Figure 5: The reconstructed image of the bars and the

measured height.

Figure 6: The hologram and the reconstructed image of a

fly wing (a) and bee wing (b).

4 CONCLUSIONS

Digital in-line holographic microscopy using Fresnel

zone plate is a quick, large visual field and no

damage measurement method based on pinhole

DIHM. 0rd scattered spherical wave and 1rd object

spherical wave, emerging from FZP, form the

interference fringes on the surface of CCD. 1rd

spherical wave is gathered to a virtual point source,

and sample is putted behind the focus. So the

measurement object is easy to replace, because the

distance between FZP and sample is farther than

pinhole to sample in pinhole DIHM. Furthermore,

the field of view is expanded and the resolution is

improved.

Continuing to improve resolution of the

optical system, identification of interference fringes

must be promoted. Using a smaller pixel pitch CCD,

a shorter focal length FZP and composite picture

through moving the CCD to collect nine images are

efficient ways to make the resolution into Nano-

scale at present. Moreover, structured light may be a

way to increase contrast ratio between interfere

pattern and background and narrowband filter is

possible to remove unwanted lights.

The microscopy measurement system can be

used in detection of Micro / Nano element,

measurement and reconstruction of three-

dimensional shape of optical element, biological

recognition, path tracking of plankton etc.

ACKNOWLEDGEMENTS

This work was funded by National Key Research

and Development Program of China (Grant

No.2017YFA0206002)

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