WO2009148407A1

WO2009148407A1 - A digital holographic microscopy system and a method of digital holographic microscopy

Info

Publication number: WO2009148407A1
Application number: PCT/SG2008/000206
Authority: WO
Inventors: Vijay Raj Singh; Oi Choo Chee; Yong Chin Eddy Sim; Anand Krishna Asundi
Original assignee: Aem Singapore Pte Ltd
Priority date: 2008-06-06
Filing date: 2008-06-06
Publication date: 2009-12-10

Abstract

A digital holographic microscopy system and a method of digital holographic microscopy. The system comprises a radiation source to provide a radiation beam; a splitter to split the radiation beam into an illumination beam and a reference beam; a first optical device to modulate the illumination beam; a second optical device to modulate the reference beam; a microscopic objective system to magnify the illumination beam in transmission through or reflection from a sample; a combiner to interfere the modulated and magnified illumination beam with the modulated reference beam to form a hologram; an optical signal detection system to record the hologram formed; and a processing unit to reconstruct at least one object wavefront from the recorded hologram.

Description

A DIGITAL HOLOGRAPHIC MICROSCOPY SYSTEM AND A METHOD OF DIGITAL HOLOGRAPHIC MICROSCOPY

FIELD OF INVENTION

The present invention relates broadly to a digital holographic microscopy system and a method of digital holographic microscopy.

BACKGROUND

Holography is an important tool for microscopy and is a two step imaging process for wavefront reconstruction. With the growing development of digital computers and Charged Coupled Devices (CCDs), digital holography has been proposed to overcome the problems of classical holography [U. Schnars and W. Jϋptner, " Direct recording of holograms by a CCD target and numerical Reconstruction", Appl. Opt, Vol. 33, No. 2, pp. 179, 1994.].

Digital recording devices (CCD sensors) provide flexibility to record holograms directly in the digital form. The reconstruction process can then be performed numerically giving quantitative access to the amplitude and the phase of the wavefront. This offers new possibilities for a variety of applications, which in classical holography were done only qualitatively. The advancement of digital holography methods can be useful in developing microscopy methods particularly in the area of digital holographic microscopy [E. Cuche, P. Marquet, and C. Depeursinge, "Simultaneous amplitude- contrast and quantitative phase-contrast microscopy by numerical reconstruction of Fresnel off-axis holograms", Appl. Opt, Vol. 38, No. 34, pp. 6994, 1999.; D. Carl, B. Kemper, G. Wernicke, and G. Bally, "Parameter-optimized digital holographic microscope for high-resolution living-cell analysis", Appl. Opt, Vol. 43, No. 36, pp. 6536, 2004.; P. Ferraro, G. Coppola, D. Alfieri, S. De Nicola, A. Finizio, G. Pierattini, "Recent advancements in digital holographic microscopy and its applications", Proc. SPIE, Vol. 5457, pp. 481-491 , 2004.]. For the numerical reconstruction of wavefronts in digital holography, there is a need to understand the physical behaviour of each component placed in the optical system. The algorithms have to be developed such that they exactly represent the behaviour of each component in numerical terms. For example, the phase information is very sensitive to the reconstruction parameters and, hence, requires an accurate numerical representation of each component. In digital holographic microscopy, a microscopic objective system is usually used to get the magnification of the object wavefront. The use of the microscopic objective system introduces a spherical phase factor in the wavefront and this phase factor in turn needs to be digitally compensated during the digital reconstruction process for phase reconstruction. Hence, for current digital holographic microscopy systems, when there is a change in the microscopic objective system, it is also necessary to change the reconstruction algorithm accordingly.

On the other hand, typically prior art systems employ discrete components to propagate and control the light in a free space optical manner from a light source for digital holography. Examples of such systems include United States Patent 20050036181 : Apparatus and method for digital holographic imaging, 2005.; United States Patent 20060132799: Digital Holographic Microscope, 2006.; United States Patent 6262818: Method for simultaneous amplitude and quantitative phase contrast imaging by numerical reconstruction of digital holograms, 2001.; United States Patent 20080018966: Digital Holographic Microscope for 3D imaging and process using it, 2008.; Hologram recording method and hologram recording apparatus, 2007.; United States Patent 7221490: Holographic method with numerical reconstruction for obtaining an image of a three-dimensional object in which even points out of the depth of field are in focus, and holographic apparatus using such a method, 2006.; United States Patent 20070216906: Method and apparatus for recognition of microorganism using holographic microscopy, 2007.

These prior art systems typically require a numerical compensating phase mask in the reconstruction algorithm taking intα account the various discrete optical components used. Hence, in view of the above, there exists a need for a digital holographic microscopy system and a method of digital holographic microscopy which seek to address at least one of the above problems.

SUMMARY

In accordance with a first aspect of the present invention there is provided a digital holographic microscopy system comprising a radiation source to provide a radiation beam; a splitter to split the radiation beam into an illumination beam and a reference beam; a first optical device to modulate the illumination beam; a second optical device to modulate the reference beam; a microscopic objective system to magnify the illumination beam in transmission through or reflection from a sample; a combiner to interfere the modulated and magnified illumination beam with the modulated reference beam to form a hologram.; an optical signal detection system to record the hologram formed; and a processing unit to reconstruct at least one object wavefront from the recorded hologram.

The radiation source may comprise a laser beam.

The optical device may comprise one or more of a group of a spatial light modulator, electro-optic modulator, acousto-optic modulator and hardware optics.

The microscopic objective system may comprise a lens system.

The optical signal detection system may comprise a charge coupled device or a CMOS sensor.

The system may further comprise a fiber coupler to couple at least one optical fiber to the radiation source, for directing the illumination beam, the reference beam or both to the first optical device, the second optical device, or both.

In accordance with a second aspect of the present invention there is provided a method for digital holographic microscopy, the method comprising the steps of providing a beam using a radiation source; splitting the radiation beam into an illumination beam and a reference beam using a splitter; modulating the illumination beam using a first optical device; modulating the reference beam using a second optical device; magnifying the illumination beam in transmission through or reflection from a sample using a microscopic objective system; interfering the modulated and magnified illumination beam with the modulated reference beam to form a hologram using a combiner; recording the formed hologram using an optical signal detection system; and reconstructing at least one object wavefront from the recorded hologram using a processing unit.

The method may further comprise the step of coupling at least one optical fiber to the radiation source using a fiber coupler, for directing the illumination beam, the reference beam or both to the first optical device, the second optical device, or both.

The step of reconstructing the at least one object wavefront from the recorded hologram using the processing unit may further comprise the steps of converting the hologram into a two-dimensional array of discrete signals using the sampling theorem; multiplying the hologram, a reconstruction wave and an impulse response function; and performing a Fourier Transform onto the product of the hologram, the reconstruction wave and the impulse response function to obtain the at least one reconstructed object wavefront.

The step of reconstructing the at least one object wavefront from the recorded hologram using the processing unit may further comprise the steps of converting the hologram into a two-dimensional array of discrete signals using the sampling theorem; multiplying the hologram and a reconstruction wave; and convoluting the product of the hologram and the reconstruction wave with an impulse response function to obtain the at least one reconstructed object wavefront.

Modulating the illumination beam and the reference beam using the first and second optical devices may further comprise the step of creating diffractive optical elements such as computer generated holograms. Modulating the illumination beam and the reference beam using the first and second optical devices may further comprise the step of creating refractive optical elements such as Fresnel's lenses.

Modulating the illumination beam and the reference beam using the first and second optical devices may further comprise the steps of performing Fourier spectrum analysis of the recorded hologram in the processing unit; and sending signals from the processing unit to at least one optical device control unit to modulate the illumination and reference beams using the first and second optical devices such that the higher frequency spectrum in the Fourier spectrum is minimized.

The step of modulating the illumination and reference beams using the first and second optical devices such that the higher frequency spectrum in the Fourier spectrum is minimized may further comprise the step of modulating the illumination and reference beams such that the wavefronts of the illumination and reference beams are the same.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention will be better understood and readily apparent to one of ordinary skill in the art from the following written description, by way of example only, and in conjunction with the drawings, in which:

Figure 1 shows a schematic block diagram illustrating a transmission digital holographic microscopy system according to an embodiment of the present invention.

Figure 2 shows a schematic block diagram illustrating a reflection digital holographic microscopy system according to an embodiment of the present invention.

Figure 3 shows a set of planes for a digital hologram recording and reconstruction process according to an embodiment of the present invention. Figure 4 shows a flowchart illustrating a method for digital holographic microscopy according to an embodiment of the present invention.

Figures 5(a)-(d) show images illustrating experimental results that present the limitations using the existing method for digital holographic microscopy system.

Figures 6(a)-(d) show images illustrating experimental results using the method for digital holographic microscopy and the digital holographic microscopy system according to an embodiment for the present invention.

Figure 7 shows a flowchart illustrating a method of digital holographic microscopy according to an embodiment of the present invention.

DETAILED DESCRIPTION

Embodiments of the present invention can be used for the study of both reflection and transmission samples based on digital holographic microscopy methods. In addition, the example embodiments can be used to explore methods for digital holographic microscopy systems based on optical devices such as spatial light modulators (SLMs), optical fibers or fiber splitters.

The example embodiments of the present invention include a radiation source to provide a radiation beam, for example a laser beam, coupled to an optical fiber or a fiber splitter. The optical fiber can be used to control the physical behaviour of the radiation beam for example, the direction of propagation, polarization, division into different ratios etc. The example embodiments also include optical devices such as SLMs (or other optical devices for example electro-optic modulator, acousto-optic modulator, hardware optics etc.) to modulate the laser light beams to any desired fashion. The example embodiments also include a microscopic objective system such as a lens system for the magnification of the optical wavefront. The example embodiments also include an optical signal detection system such as a charge coupled device (CCD), or any other kind of optical signal detection systems for example, a CMOS sensor to record the optical signals incident on it. In one example, most of the optical components of conventional digital holographic microscopy are replaced by optical fibers and SLMs. The light coming from the source for example, lasers, can be coupled to an optical fiber which can be directly incident to a SLM.

Optical fiber technology is based on total internal reflection and is basically designed to guide light along its length [J. Hecht, "City of Light, The Story of Fiber Optics", New York: Oxford University Press, p114, 1999.]. Optical fibers can be used in applications such as in telecommunications, medicine, military, automotive, and industrial applications. Optical fibers can also be used for illumination applications by controlling the light path in a non-linear fashion. Interferometer methods have also been developed based on optical fibers. In order to couple the laser light to an optical fiber, the light beam is typically focussed to the centre part of the cross-section of the fiber (also known as the core). There is, thus, flexibility in the set-up and the propagation of the light beam can be controlled.

A SLM is a device that creates spatially-varying modulations to a light beam [U. Efron, "Spatial light modulator technology", Marcel Dekker Inc, USA, 1995.]. Generally, a SLM modulates the intensity of the light beam. However, it is also possible to modulate the phase of the light beam or both the intensity and the phase simultaneously. SLMs are available for the modulation of a light beam for both transmission (transmission SLM) and reflection (reflection SLM) applications. The performance of a SLM can be controlled by a control unit and/or a computer.

In the example embodiments, the illumination beam is magnified after transmission through, or after reflection from a sample and interference of the illumination beam with the reference beam is then implemented. The interference of the illumination and reference beams, also known as a hologram, is digitally sampled, and information of these optically interfering beams is stored in the form of matrices. Numerical processing of the hologram is then performed to simulate the object wavefront. Furthermore, in the example embodiments, a methodology is developed for an automatic compensation in the reconstruction parameters. Thus, the numerical reconstruction parameters can preferably be adjusted automatically for different levels of magnification without prior knowledge of the components in the system.

Figure 1 shows a schematic block diagram illustrating a transmission digital holographic microscopy system 100 according to an embodiment of the present invention. In Figure 1, the laser beam from the laser source 102 is transferred to optical fibers, 106 and 108, using a fiber coupler/splitter 104. In the transmission system 100, the laser beam is split into two parts using the fiber coupler/splitter. One end of the fibers, 106 and 108, is connected to the fiber coupler/splitter while the other end of the fibers, 106 and 108, is connected to transmission SLMs 110 and 112 respectively. The SLMs 110 and 112 are controlled using a processing unit such as the computer 122 such that the laser beams emerging from the SLMs 110 and 112 are modulated accordingly. The modulated light beam emerging from the SLM 112, referred to herein as the modulated illumination beam, illuminates the sample 114 and is then magnified by the microscopic objective system 116. The other modulated beam emerges from the SLM 110 and is also known as the modulated reference beam. The modulated light beams from the SLM 110 and SLM 112, after magnification of the object beam coming from SLM 112 by microscopic objective 116, are combined using a beam combiner 118 and these two beams interfere at, and are recorded by, the CCD 120.

Figure 2 shows a schematic block diagram illustrating a reflection digital holographic microscopy system 200 according to an embodiment of the present invention. In Figure 2, the laser beam from the source 202 is transferred to the optical fiber 206 using a fiber coupler 204. One end of the fiber 206 is connected to the fiber coupler 204 while the other end of the fiber 206 is connected to the transmission SLM 208. The modulated beam emerging from the SLM 208 is divided into two parts (an illumination beam and a reference beam) using a beam splitter/combiner 212. The illumination beam illuminates the sample 216 after passing through the microscopic objective system 214 whereas the reference beam is incident onto the reflective SLM 218. The SLMs 208 and 218 are controlled using a processing unit such as the computer 220 such that the laser beams emerging from the SLMs 208 and 218 are modulated accordingly. The illumination beam reflected from the sample 216 is magnified by the microscopic objective system 214. It then interferes with the reference beam reflected from the SLM 218 at, and is recorded using, a CCD sensor 210. The use of fiber in the example embodiments is to control the propagation of the light beam. In different embodiments not using optical fiber, optical components such as mirrors and lenses can be used to control the path of light in such a way that the light is incident on the SLM(s).

Figure 3 shows a set of planes 300 for digital hologram recording and reconstruction process according to an embodiment of the present invention. In

Figure 3, (*^',/) is the object plane and {ξ, ή) is the hologram plane. The hologram, which is the interference of the illumination beam O(ξ, ή) ar\ά reference beam R(ξ,η) , can be written according to Equation (1). In Equation (1), O^* and R^* are the complex conjugate of O and R respectively.

H(ξ,η)

+ O(ξ,η)R'(ξ,η) (1)

A CCD placed at the hologram plane records this interference pattern H(ξ,η) . The recorded pattern is then converted into a two-dimensional array of discrete signals by using the sampling theorem. The digitally sampled holograms H(m,n) , can be written according to Equation (2). In Equation (2), Mx N is the total number of pixels of the CCD with corresponding size Δ^ and Aη and ® represents the two-dimensional convolution and (or, β) e [0,1] are the fill factors of the CCD pixels.

In the example embodiments, the reconstruction of the hologram is a diffraction process and numerical reconstruction is performed by simulating the diffraction process with a software. In one example, two approaches are used for the numerical reconstruction process. These approaches called Fresnel and Convolution approaches are defined as follows. In the Fresnel approach in the example embodiments, the hologram H(ξ, ή) is illuminated by the reconstruction wave R(ξ, ή) with wavelength λ . The reconstructed wavefield U{x, y) at the image plane (x,y) at distance d (see Figure 3) is given by the Fresnel's approximation according to Equation (3).

U(x, y) = ξ)² + (y - η)² }]dξdη (3)

The reconstructed field is simply the Fourier transform of the product of the hologram, the reconstruction wave and the impulse response function g(ξ, η) as shown in Equation (4). The impulse response giξ,ή) is defined according to Equation (5).

U(x,y) = 3{H(ξ,η)R(ξ,η)g(ξ,η)} (4)

The image intensity I(x,y) can be calculated by squaring the wavefield according to Equation (6) whereas the phase φ(x,y) is calculated according to Equation (7).

I{x,y) =\ U{x,y) γ (6)

The pixel size (Δx , Ay) of the numerically reconstructed image using the Fresnel approach varies with the reconstruction distance d and is given by Equation (8).

Ax = -^- , and Ay = ^- (8)

MAξ NAη The convolution approach in the example embodiments is useful if the pitch of the reconstructed image is independent of the reconstruction distance. It can be shown that the diffraction integral in Equation (3) becomes a convolution for linear space invariant system. The numerically reconstructed wavefield can then be written according to Equation (9). The pixel size of the reconstructed image by the convolution method is the same as the pixel size of the CCD and does not vary with reconstruction distance, i.e. Δx = Δ£ , and Ay = Aη .

U{x ,y^') = [H(<f, η)R{ξ, η)] ® [g(ξ, η)] (9)

Figure 4 shows a flowchart illustrating a method 400 for digital holographic microscopy according to an embodiment of the present invention. In step 402, the fiber is coupled with the laser source and the laser beam is split into two parts, an illumination beam and a reference beam, using a fiber coupler/splitter. In step 404 the illumination beam is modulated by a SLM whereas in step 406, the reference beam is modulated by a SLM. Steps 404 and 406 are implemented for both the transmission and reflection systems. In step 408, the illumination beam is magnified and in step 410, the magnified illumination beam is interfered with the reference beam to form a hologram that is then digitally sampled. The digitally sampled hologram is then sent to the computational process executed in step 412.

In one example, numerical reconstruction of the hologram is performed according to the approaches described with Equations (3) - (9) to obtain a reconstructed object wavefront. In addition, a Fourier spectrum analysis of the hologram is performed. The Fourier transform of the hologram shows the frequency spectrum of the interference pattern of the illumination and reference beams. Since the interference pattern is a cosine pattern in the example embodiments, the frequency spectrum shows mainly two non-zero frequency bands. By using the computational process in this example, the modulation of light through the SLM is controlled in such a way that the higher frequency spectrum is minimized. This can be achieved when the wavefronts of the illumination beam and the reference beam are the same. In the example embodiments, the illumination and reference wavefronts can be controlled by the SLM using different ways, such as by creating computer generated holograms or by creating Fresnel's lenses. Shrinking the higher frequencies of the Fourier spectrum into a smaller area improves the system capabilities and performance by achieving for example, automated phase compensation or a higher imaging resolution. Also, the convolution approach can be effectively utilized for the reconstruction of the object wavefront.

The advantages of the embodiments in the present invention include the following.

In contrast to the existing digital holographic microscopes employing discrete components to propagate and control the light, the example embodiments use optical fibers and SLMs to handle light for digital holographic microscopy applications and can be applied for both transmission and reflection digital holographic microscopic geometries.

The method for a digital holographic microscopy system based on optical fibers and SLMs in the example embodiments and the corresponding proposed system can improve the capabilities and flexibility of the digital holographic microscopy system. The example embodiments can provide a better resolution, an automatic compensation of phase mask corresponding to different magnifications and an optical geometry with less optical components.

Furthermore, the SLM in some example embodiments can control the modulation of the illumination and reference beams such that it advantageously removes the need to use a numerical compensating phase mask in the reconstruction algorithm. In addition, the wavefront aberrations of the magnifying optics, for example the microscopic objective system, can be compensated for using the SLM in such example embodiments. This can be achieved by using the computer generated hologram (CGH) method to modulate the wavefront of the light. Also, the use of optical fibers in the example embodiments can control the propagation of the light without complex alignment problems.

In addition, in the example embodiments, the algorithm is developed such that it can control the SLM as well as the reconstruction of the recorded digital holograms. The SLM control can also be used in some example embodiments to automatically compensate for the phase aberration incorporated with different magnifications of the microscopic objective system.

Embodiments of the present invention can be used for the commercial development of digital holographic microscopy systems (both transmission and reflection) with more flexibility for different magnifications and multi-focus phase imaging applications.

Hence, embodiments of the present invention can provide an optical system geometry of a digital holographic microscopy system based on optical fibers and SLMs.

In addition, the embodiments of the present invention can also provide a software development for analyzing the Fourier spectrum of holograms. The method in some example embodiments is developed for studying the spatial frequencies and for minimizing the higher frequencies by controlling the SLM. Such an analysis can result in substantially the same illumination and reference wavefronts so that the phase aberrations can compensate automatically. Furthermore, the example embodiments can provide a method for the SLM to control the modulation of light. This method basically controls the wavefront of the light coming from the SLM by using a computer generated holography (CGH) method.

Advantages of example embodiments can be further illustrated by experimental results as shown in Figures 5 and 6.

Figures 5(a)-(d) show images illustrating experimental results that present the limitations using the existing method for digital holographic microscopy system. In Figure 5(a), the Fourier transform for a digital hologram recorded using a digital holographic microscopy system according to an embodiment of the present invention for a collimated reference beam is shown. In Figure 5(b), a reconstructed amplitude image is shown. In Figure 5(c), a reconstructed phase contrast image is shown and in Figure 5(d), a phase contrast image at a different focus plane is shown.

The experiments have been performed with collimating and diverging reference beams. In these experiments, the Fourier spectrum of the digital hologram for an object (i.e. 1951 US Air Force (USAF) Glass Slide Resolution Target) was recorded using magnified illumination beams (by using a 6OX microscopic objective system) and with collimated reference beams and is shown in Figure 5(a).

In order to perform the reconstruction process, one frequency order was selected and its inverse Fourier transformation was then calculated. In the example embodiments, the reconstruction of such a filtered hologram can provide an image that is free from the zero-order term and twin image waves. The reconstructed amplitude image of the hologram is shown in Figure 5(b).

For phase reconstruction in the embodiment, to compensate the phase aberration created by the microscopic objective system at the image plane a digital phase mask is created along with the reconstruction algorithm. The reconstructed phase contrast image using such a digital phase mask is shown in Figure 5(c).

If the phase is compensated only at the image plane, the phase mask is typically not able to compensate for the phase aberration of the microscopic objective system when there are changes in the reconstruction distance. In Figure 5(d), the reconstructed phase contrast image using the digital phase mask at a non-image plane is shown, illustrating this problem. A similar problem arises if there is a change in the microscopic objective system in the example embodiments. Thus for a system using different microscopic objective systems according to this embodiment, different phase masks are created with each phase mask corresponding to a microscopic objective system. The Fresnel approach can be used for such a reconstruction process in this embodiment.

Figures 6(a) - (d) show images illustrating further experimental results using the method for digital holographic microscopy and the digital holographic microscopy system according to an embodiment of the present invention. Figure 6(a) shows the Fourier Transform for a digital hologram recorded using a digital holographic microscopy system according to an embodiment of the present invention for a modulated diverging reference beam. Figure 6(b) shows a reconstructed amplitude image. Figure 6(c) shows a reconstructed phase contrast image and Figure 6(d) shows a phase contrast image at a different focus plane. In this example embodiment, the wavefronts of the illumination and reference beams are controlled using the SLMs in such a way that the frequency spectrum is minimized. The Fourier spectrum of the hologram of the object (1951 USAF Glass Slide Resolution Target) after controlling the wavefronts of the illumination and reference beams is shown in Figure 6(a). It can be seen from Figure 6(a) that the range of frequencies has been shrunk to a smaller region. Minimizing the frequency range to a smaller region is controlled by the software in the example embodiment, by controlling the modulation of the wavefronts in such a way that the first order diffraction, as seen from the Fourier spectrum of Figure 6(a), shrinks to as tight a spot as possible. In one example, this can be achieved when both the illumination and reference wavefronts are the same.

In the computational process in this example embodiment, the algorithm is built such that the frequency spectrum is controlled by the computer through the SLM so that the frequency spectrum is minimized. For the reconstruction of the holograms with this approach in the example embodiments, both the Fresnal approach and the convolution approach can be used. The reconstructed amplitude image is shown in Figure 6(b).

One advantage of controlling the wavefronts of the illumination beam and the reference beam is that the phase aberration of the microscopic objective system can be automatically compensated. Thus, there is no need to create a phase mask. The resulting reconstructed phase contrast image is shown in Figure 6(c).

Since there is no phase aberration in the hologram, the phase remains constant regardless of the change in the reconstruction distance in this example embodiment.

The phase contrast image for a different focus plane is shown in Figure 6(d). It can be seen that the method in this example embodiments can provide more flexibility for the digital holographic microscopy system for different magnifications and multiple focal planes via the control of the frequency spectrum using the SLM. When different magnifications are effected for example, by changing the microscopic objective system, the algorithm in the example embodiments can automatically compensate for the phase aberrations and can reconstruct the phase contrast image without using any phase mask. Figure 7 shows a method 700 for digital holographic microscopy according to an embodiment of the present invention. In step 702, a radiation beam is provided using a radiation source. In step 704, the radiation beam is split into an illumination beam and a reference beam using a splitter. In step 706, the illumination beam is modulated using a first optical device and in step 708, the reference beam is modulated using a second optical device. In step 710, the illumination beam is magnified in transmission through or reflection from a sample using a microscopic objective system. In step 712, the modulated and magnified illumination beam is interfered with the modulated reference beam to form a hologram using a combiner. In step 714, the formed hologram is recorded using an optical signal detection system and in step 716, at least one object wavefront is reconstructed from the recorded hologram using a processing unit.

It will be appreciated by a person skilled in the art that numerous variations and/or modifications may be made to the present invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects to be illustrative and not restrictive.

Claims

1. A digital holographic microscopy system comprising: a radiation source to provide a radiation beam; a splitter to split the radiation beam into an illumination beam and a reference beam; a first optical device to modulate the illumination beam; a second optical device to modulate the reference beam; a microscopic objective system to magnify the illumination beam in transmission through or reflection from a sample; a combiner to interfere the modulated and magnified illumination beam with the modulated reference beam to form a hologram; an optical signal detection system to record the hologram formed; and a processing unit to reconstruct at least one object wavefront from the recorded hologram.

2. The system as claimed in claim 1 , wherein the radiation source comprises a laser beam.

3. The system as claimed in any of the preceding claims, wherein the optical device comprises one or more of a group of a spatial light modulator, electro-optic modulator, acousto-optic modulator and hardware optics.

4. The system as claimed in any of the preceding claims, wherein the microscopic objective system comprises a lens system.

5. The system as claimed in any of the preceding claims, wherein the optical signal detection system comprises a charge coupled device or a CMOS sensor.

6. The system as claimed in any of the preceding claims further comprising a fiber coupler to couple at least one optical fiber to the radiation source, for directing the illumination beam, the reference beam or both to the first optical device, the second optical device, or both.

7. A method for digital holographic microscopy, the method comprising the steps of: providing a beam using a radiation source; splitting the radiation beam into an illumination beam and a reference beam using a splitter; modulating the illumination beam using a first optical device; modulating the reference beam using a second optical device; magnifying the illumination beam in transmission through or reflection from a sample using a microscopic objective system; interfering the modulated and magnified illumination beam with the modulated reference beam to form a hologram using a combiner; recording the formed hologram using an optical signal detection system; and reconstructing at least one object wavefront from the recorded hologram using a processing unit.

8. The method as claimed in claim 7, further comprising the step of coupling at least one optical fiber to the radiation source using a fiber coupler, for directing the illumination beam, the reference beam or both to the first optical device, the second optical device, or both.

9. The method as claimed in any one of claims 7 or 8 wherein the step of reconstructing the at least one object wavefront from the recorded hologram using the processing unit further comprises the steps of: converting the hologram into a two-dimensional array of discrete signals using the sampling theorem; multiplying the hologram, a reconstruction wave and an impulse response function; and performing a Fourier Transform onto the product of the hologram, the reconstruction wave and the impulse response function to obtain the at least one reconstructed object wavefront.

10. The method as claimed in any one of claims 7 - 9 wherein the step of reconstructing the at least one object wavefront from the recorded hologram using the processing unit further comprises the steps of: converting the hologram into a two-dimensional array of discrete signals using the sampling theorem; multiplying the hologram and a reconstruction wave; and convoluting the product of the hologram and the reconstruction wave with an impulse response function to obtain the at least one reconstructed object wavefront.

11. The method as claimed in any one of claims 7 — 10, wherein modulating the illumination beam and the reference beam using the first and second optical devices further comprises the step of creating diffractive optical elements such as computer generated holograms.

12. The method as claimed in any one of claims 7 - 11 , wherein modulating the illumination beam and the reference beam using the first and second optical devices further comprises the step of creating refractive optical elements such as Fresnel's lenses.

13. The method as claimed in any one of claims 7 - 12, wherein modulating the illumination beam and the reference beam using the first and second optical devices further comprises the steps of: performing Fourier spectrum analysis of the recorded hologram in the processing unit; and sending signals from the processing unit to at least one optical device control unit to modulate the illumination and reference beams using the first and second optical devices such that the higher frequency spectrum in the Fourier spectrum is minimized.

14. The method as claimed in claim 13, wherein the step of modulating the illumination and reference beams using the first and second optical devices such that the higher frequency spectrum in the Fourier spectrum is minimized further comprises the step of modulating the illumination and reference beams such that the wavefronts of the illumination and reference beams are the same.