## Off-axis setup taking full advantage of incoherent illumination in coherence-controlled holographic microscope |

Optics Express, Vol. 21, Issue 12, pp. 14747-14762 (2013)

http://dx.doi.org/10.1364/OE.21.014747

Acrobat PDF (3954 KB)

### Abstract

Coherence-controlled holographic microscope (CCHM) combines off-axis holography and an achromatic grating interferometer allowing for the use of light sources of arbitrary degree of temporal and spatial coherence. This results in coherence gating and strong suppression of coherent noise and parasitic interferences enabling CCHM to reach high phase measurement accuracy and imaging quality. The achievable lateral resolution reaches performance of conventional widefield microscopes, which allows resolving up to twice smaller details when compared to typical off-axis setups. Imaging characteristics can be controlled arbitrarily by coherence between two extremes: fully coherent holography and confocal-like incoherent holography. The basic setup parameters are derived and described in detail and experimental validations of imaging characteristics are demonstrated.

© 2013 OSA

## 1. Introduction

1. R. Barer, “Interference microscopy and mass determination,” Nature **169**(4296), 366–367 (1952). [CrossRef] [PubMed]

3. H. Janečková, P. Veselý, and R. Chmelík, “Proving tumour cells by acute nutritional/energy deprivation as a survival threat: a task for microscopy,” Anticancer Res. **29**(6), 2339–2345 (2009). [PubMed]

4. F. Dubois, C. Yourassowsky, O. Monnom, J. C. Legros, O. Debeir, P. Van Ham, R. Kiss, and C. Decaestecker, “Digital holographic microscopy for the three-dimensional dynamic analysis of in vitro cancer cell migration,” J. Biomed. Opt. **11**(5), 054032 (2006). [CrossRef] [PubMed]

5. E. Cuche, F. Bevilacqua, and C. Depeursinge, “Digital holography for quantitative phase-contrast imaging,” Opt. Lett. **24**(5), 291–293 (1999). [CrossRef] [PubMed]

8. T. Colomb, N. Pavillon, J. Kühn, E. Cuche, C. Depeursinge, and Y. Emery, “Extended depth-of-focus by digital holographic microscopy,” Opt. Lett. **35**(11), 1840–1842 (2010). [CrossRef] [PubMed]

9. F. Dubois, L. Joannes, and J.-C. Legros, “Improved three-dimensional imaging with a digital holography microscope with a source of partial spatial coherence,” Appl. Opt. **38**(34), 7085–7094 (1999). [CrossRef] [PubMed]

10. P. Klysubun and G. Indebetouw, “A posteriori processing of spatiotemporal digital microholograms,” J. Opt. Soc. Am. A **18**(2), 326–331 (2001). [CrossRef] [PubMed]

11. G. Indebetouw and P. Klysubun, “Optical sectioning with low coherence spatio-temporal holography,” Opt. Commun. **172**(1-6), 25–29 (1999). [CrossRef]

13. E. N. Leith, W.-C. Chien, K. D. Mills, B. D. Athey, and D. S. Dilworth, “Optical sectioning by holographic coherence imaging: a generalized analysis,” J. Opt. Soc. Am. A **20**(2), 380–387 (2003). [CrossRef] [PubMed]

14. M.-K. Kim, “Tomographic three-dimensional imaging of a biological specimen using wavelength-scanning digital interference holography,” Opt. Express **7**(9), 305–310 (2000). [CrossRef] [PubMed]

16. W. Choi, C. Fang-Yen, K. Badizadegan, S. Oh, N. Lue, R. R. Dasari, and M. S. Feld, “Tomographic phase microscopy,” Nat. Methods **4**(9), 717–719 (2007). [CrossRef] [PubMed]

9. F. Dubois, L. Joannes, and J.-C. Legros, “Improved three-dimensional imaging with a digital holography microscope with a source of partial spatial coherence,” Appl. Opt. **38**(34), 7085–7094 (1999). [CrossRef] [PubMed]

17. T. Zhang and I. Yamaguchi, “Three-dimensional microscopy with phase-shifting digital holography,” Opt. Lett. **23**(15), 1221–1223 (1998). [CrossRef] [PubMed]

18. L. Xu, J. M. Miao, and A. Asundi, “Properties of digital holography based on in-line conﬁguration,” Opt. Eng. **39**(12), 3214–3219 (2000). [CrossRef]

5. E. Cuche, F. Bevilacqua, and C. Depeursinge, “Digital holography for quantitative phase-contrast imaging,” Opt. Lett. **24**(5), 291–293 (1999). [CrossRef] [PubMed]

19. D. Carl, B. Kemper, G. Wernicke, and G. von Bally, “Parameter-optimized digital holographic microscope for high-resolution living-cell analysis,” Appl. Opt. **43**(36), 6536–6544 (2004). [CrossRef] [PubMed]

21. P. Girshovitz and N. T. Shaked, “Generalized cell morphological parameters based on interferometric phase microscopy and their application to cell life cycle characterization,” Biomed. Opt. Express **3**(8), 1757–1773 (2012). [CrossRef] [PubMed]

22. B. Bhaduri, H. Pham, M. Mir, and G. Popescu, “Diffraction phase microscopy with white light,” Opt. Lett. **37**(6), 1094–1096 (2012). [CrossRef] [PubMed]

23. F. Dubois and C. Yourassowsky, “Full off-axis red-green-blue digital holographic microscope with LED illumination,” Opt. Lett. **37**(12), 2190–2192 (2012). [CrossRef] [PubMed]

22. B. Bhaduri, H. Pham, M. Mir, and G. Popescu, “Diffraction phase microscopy with white light,” Opt. Lett. **37**(6), 1094–1096 (2012). [CrossRef] [PubMed]

23. F. Dubois and C. Yourassowsky, “Full off-axis red-green-blue digital holographic microscope with LED illumination,” Opt. Lett. **37**(12), 2190–2192 (2012). [CrossRef] [PubMed]

24. R. Chmelík and Z. Harna, “Parallel-mode confocal microscope,” Opt. Eng. **38**(10), 1635–1639 (1999). [CrossRef]

24. R. Chmelík and Z. Harna, “Parallel-mode confocal microscope,” Opt. Eng. **38**(10), 1635–1639 (1999). [CrossRef]

25. R. Chmelík, “Three-dimensional scalar imaging in high-aperture low-coherence interference and holographic microscopes,” J. Mod. Opt. **53**(18), 2673–2689 (2006). [CrossRef]

3. H. Janečková, P. Veselý, and R. Chmelík, “Proving tumour cells by acute nutritional/energy deprivation as a survival threat: a task for microscopy,” Anticancer Res. **29**(6), 2339–2345 (2009). [PubMed]

26. H. Janečková, P. Kolman, P. Veselý, and R. Chmelík, “Digital holographic microscope with low spatial and temporal coherence of illumination,” Proc. SPIE **7000**, 70002E (2008). [CrossRef]

6. L. Lovicar, L. Kvasnica, and R. Chmelík, “Surface observation and measurement by means of digital holographic microscope with arbitrary degree of coherence,” Proc. SPIE **7141**, 71411S (2008). [CrossRef]

7. L. Lovicar, J. Komrska, and R. Chmelík, “Quantitative-phase-contrast imaging of a two-level surface described as a 2D linear filtering process,” Opt. Express **18**(20), 20585–20594 (2010). [CrossRef] [PubMed]

27. R. Chmelík and Z. Harna, “Surface profilometry by a parallel–mode confocal microscope,” Opt. Eng. **41**(4), 744–745 (2002). [CrossRef]

6. L. Lovicar, L. Kvasnica, and R. Chmelík, “Surface observation and measurement by means of digital holographic microscope with arbitrary degree of coherence,” Proc. SPIE **7141**, 71411S (2008). [CrossRef]

28. P. Kolman and R. Chmelík, “Coherence-controlled holographic microscope,” Opt. Express **18**(21), 21990–22003 (2010). [CrossRef] [PubMed]

29. M. Lošťák, P. Kolman, Z. Dostál, and R. Chmelík, “Diffuse light imaging with a coherence controlled holographic microscope,” Proc. SPIE **7746**, 77461N (2010). [CrossRef]

28. P. Kolman and R. Chmelík, “Coherence-controlled holographic microscope,” Opt. Express **18**(21), 21990–22003 (2010). [CrossRef] [PubMed]

28. P. Kolman and R. Chmelík, “Coherence-controlled holographic microscope,” Opt. Express **18**(21), 21990–22003 (2010). [CrossRef] [PubMed]

3. H. Janečková, P. Veselý, and R. Chmelík, “Proving tumour cells by acute nutritional/energy deprivation as a survival threat: a task for microscopy,” Anticancer Res. **29**(6), 2339–2345 (2009). [PubMed]

26. H. Janečková, P. Kolman, P. Veselý, and R. Chmelík, “Digital holographic microscope with low spatial and temporal coherence of illumination,” Proc. SPIE **7000**, 70002E (2008). [CrossRef]

30. E. N. Leith and J. Upatnieks, “Holography with achromatic-fringe systems,” J. Opt. Soc. Am. **57**(8), 975–980 (1967). [CrossRef]

31. T. Slabý, M. Antoš, Z. Dostál, P. Kolman, and R. Chmelík, “Coherence-controlled holographic microscope,” Proc. SPIE **7746**, 77461R (2010). [CrossRef]

## 2. Optical setup and principles of operation

_{1}, C

_{2}), infinity-corrected objectives (O

_{1}, O

_{2}) and tube lenses (TL

_{1}TL

_{2}). The essential component of the CCHM setup is the reflection diffraction grating (DG), which is placed in the reference arm of the interferometer and imaged into the output plane (OP) as proposed by Leith [30

30. E. N. Leith and J. Upatnieks, “Holography with achromatic-fringe systems,” J. Opt. Soc. Am. **57**(8), 975–980 (1967). [CrossRef]

_{1}, OL

_{2}). Since only the + 1st order of the diffraction grating is used for imaging (other diffraction orders are eliminated by spatial filtering in focal plane of output lens OL

_{2}), the image of the grating is not formed directly by the reference beam in the output plane. However, when the object beam and the reference beam recombine in the output plane, the interference fringe pattern appears, which corresponds to the diffraction grating grooves‘ image as it would be formed directly by 0th and + 1st order of the diffraction grating. Thus the spatial frequency of interference fringes

*f*in the output plane – i.e. the carrier frequency – equals to the spatial frequency of diffraction grating grooves

_{C}*f*reduced by output lenses’ magnification

_{G}*m*

_{OL}_{2}is spectrally dispersed with respect to the dispersive power of the diffraction grating so that the longer is the wavelength of light, the further the image of the source is placed from the reference arm axis. Let trace the axial ray which comes from the source, passes through the reference arm and hits the grating. When considering the + 1st diffraction order of the grating, the incident ray is diffracted by the grating at an angle

*α*according to the grating equation sin(

*α*) =

*f*, where

_{G}λ*λ*is the wavelength of light. The diffracted ray then passes through the output lens OL

_{2}and enters the output plane at an angle

*β*. The relation between

*α*and

*β*is given by sin(

*β*) = sin(

*α*)/

*m*. In the object arm of the interferometer, the light is reflected by mirror M

_{OL}_{2}and passes through the output lens OL

_{1}normally since there is no diffractive element in the path. The light is not spectrally dispersed in this arm. Thus rays of different wavelengths emitted from corresponding points of tertiary images of the source in both interferometer arms recombine in the output plane under different angles

*β*. This is caused by the dispersive power of the diffraction grating and gives rise to interference fringes parallel with grooves of the diffraction grating and of a spatial carrier frequency

*f*which is constant for all wavelengths – i.e. the interferometer is achromatic. If a specimen (Sp) is observed, an image plane off-axis hologram with the spatial carrier frequency

_{C}*f*is formed in the output plane.

_{C}*β*for all available wavelengths is crucial for achromaticity of the interferometer. When any misalignment

*θ*is introduced to the output angle

*β*, the interferometer produces interference fringes of slightly different carrier frequencies at different wavelengths. The higher values of

*θ*give rise to higher values of

*f*and vice versa. Also the positive values of

_{C}*θ*give rise to higher values of

*f*at shorter wavelengths while the negative values of

_{C}*θ*give rise to higher values of

*f*at longer wavelengths. This behavior significantly influences achromaticity of the interferometer and consequently the contrast of the interference fringes pattern in the recorded hologram. Therefore the output angle

_{C}*β*has to be properly aligned.

_{2}is shifted along the optical axis with the use of piezo-positioner. Second piezo-positioner is used to translate the reference objective O

_{2}perpendicularly to the optical axis to align precisely images from both interferometer arms formed in the output plane.

_{2}, BS

_{3}). Also a reflected-light setup can be easily achieved by introducing illumination beams into the infinity space between objectives and tube lenses. In the same way, multimodality can be achieved by implementing other imaging or micromanipulation techniques to provide combined imaging [32

32. N. Pavillon, A. Benke, D. Boss, C. Moratal, J. Kühn, P. Jourdain, C. Depeursinge, P. J. Magistretti, and P. Marquet, “Cell morphology and intracellular ionic homeostasis explored with a multimodal approach combining epifluorescence and digital holographic microscopy,” J Biophotonics **3**(7), 432–436 (2010). [CrossRef] [PubMed]

34. E. Shaffer, N. Pavillon, and C. Depeursinge, “Single-shot, simultaneous incoherent and holographic microscopy,” J. Microsc. **245**(1), 49–62 (2012). [CrossRef] [PubMed]

## 3. Incoherence of the light source

*d*of the mutual intensity function. From the formula for this function [35, p. 511] it can be computed that

_{w}*λ*is central wavelength and

_{0}*γ*is angular radius of the tertiary image of the light source as viewed from the output plane. According to [35, p. 319 ] the CL can be calculated as

*λ*is FWHM of the spectral function. For parameters of the real setup (

_{0}*γ*≈0.0063 rad,

*λ*≈570 nm and Δ

_{0}*λ*≈150 nm) we obtained values of CW

_{0}*d*≈63 µm (calculated in the output plane) and CL

_{w}*d*≈2.2 µm. However, these values are only approximate and do not reflect the increase of coherence in tertiary images of the light source introduced by the imaging process when the light source is imaged by the optical system to focal planes of the output lenses.

_{l}_{2}) in a direction perpendicular to the optical axis. Thus the image formed by the reference arm in the output plane was shifted with respect to the image formed by the object arm. The reference objective was translated in two directions – perpendicular to diffraction grating grooves (

*x*axis) and parallel with diffraction grating grooves (

*y*axis). The average values of the reconstructed amplitude were then computed in area of 5 px × 5 px within the central part of the image and the normalized values were plotted versus the lateral shift

*d*

_{OP}of the image formed by the reference arm in the output plane (Fig. 2(a)). The CW was estimated as the FWHM of the mutual intensity function, giving

*d*≈91 µm and

_{w,x}*d*≈76 µm for the two directions respectively.

_{w,y}_{2}. In this way the optical path difference (OPD) between the two interferometer arms was varied. The averaged and normalized amplitude values versus the OPD value

*d*

_{OPD}were plotted and the CL was estimated as the FWHM of the obtained mutual coherence function giving

*d*≈4 µm (Fig. 2(b)).

_{l}*d*

_{OP}(in

*x*axis) and

*d*

_{OPD}, which is probably the reason for higher measured values of

*d*and

_{w,x}*d*. This effect together with the presence of secondary maxima of the spectrum (caused by the reflection diffraction grating) may explain also the side-lobes of the curve in the Fig. 2(b).

_{l}## 4. Lateral resolution

*o*(

*x*,

*y*) and

*r*(

*x*,

*y*) respectively, where

*r*(

*x*,

*y*) =

*r*(

_{0}*x*,

*y*) exp(-i2π

*f*) and

_{C}x*r*(

_{0}*x*,

*y*) is the complex amplitude distribution of reference wave expressed in the plane perpendicular to propagation direction. Then the intensity distribution of the hologram which is generated in the output plane by interference of the two waves is given bywhere asterisk denotes the complex conjugate operator and

*x*,

*y*are coordinates defined in the output plane. The first two terms in second row of Eq. (2) correspond to the intensities of object and reference waves, respectively. In the spatial frequency spectrum of hologram these terms create so-called zero-order term,

*or** is the image term and

*o**

*r*is its complex conjugate, i.e. the twin image. Both the terms

*or** and

*o**

*r*can be used for reconstruction of the object amplitude and phase (see section 7). In Fig. 3 the spatial-frequency spectrum support (areas of non-zero values) of a hologram is depicted with all the terms of Eq. (2) for CCHM in comparison with a typical DHM setup. The form of the supports is explained in the following text. For purposes of the following paragraphs it should be noted that the Fourier transform of the terms |

*o*|

^{2}and |

*r*|

^{2}is the autocorrelation of the Fourier transform of

*o*and

*r*, respectively, and the Fourier transform of the term

*or** is the convolution of the Fourier transform of

*o*and

*r**(and analogically for the term

*o*r*).

*o*is given by numerical aperture

*NA*of the objective, total magnification

_{O}*m*(between the output plane and the object plane of objectives) and wavelength of light

*λ*as

*o*|

^{2}in the spatial frequency spectrum of a hologram is a circle of radius 2

*a*, whereLet us consider now two extreme cases of illumination: fully spatially coherent and fully spatially incoherent.

*r*is nearly a two-dimensional Dirac distribution. Thus the highest frequency produced by the terms

_{0}*or*and

_{0}**o*r*in the spatial frequency spectrum is given by

_{0}*r*is given by

_{0}*or*and

_{0}**o*r*in the spatial frequency spectrum is given by

_{0}*a*, where(marked by solid line in Fig. 3). It can be seen that the lateral resolution limit achievable by the CCHM with spatially incoherent illumination is fully comparable to conventional optical microscopes and it is half of the value for coherent illumination, the mode used in most current DHMs. Thus the lateral resolution limit of CCHM corresponds to incoherent imaging process [25

25. R. Chmelík, “Three-dimensional scalar imaging in high-aperture low-coherence interference and holographic microscopes,” J. Mod. Opt. **53**(18), 2673–2689 (2006). [CrossRef]

*λ*= 650 nm, 10 nm FWHM) and highly spatially coherent (HeNe laser,

*λ*= 633 nm). The spatially coherent illumination was provided to allow comparison of results obtained by CCHM in incoherent mode with those for a typical DHM (simulated by CCHM in coherent mode). The circles in Fig. 4 show the expected diameters of spectral supports of zero-order term and image terms corresponding to Eq. (5) and Eq. (4). Although one have to be cautious when directly comparing the diameters because of slightly different wavelengths used (650 nm vs. 633 nm), the experimental results show a good agreement with the theoretical assumptions. Also one can notice the different shapes of spatial frequency transmission profiles in the image terms, where the profile is approximately triangular for spatially incoherent illumination and rectangular for spatially coherent illumination. This is an important fact to understand the difference in the achievable lateral resolution between spatially incoherent and spatially coherent illumination. While spatially coherent illumination will provide good contrast even at the maximum transmitted spatial frequency, the spatially incoherent illumination will provide contrast approaching zero at its maximum transmitted spatial frequency. However, the maximum transmitted spatial frequency for spatially incoherent illumination will be double that of spatially coherent illumination.

## 5. Spatial frequency of the diffraction grating

*or**and

*o*r*from the zero-order term |

*o*|

^{2}+ |

*r*|

^{2}is required in the spatial frequency spectrum as it is depicted in Fig. 3. No overlap of these terms is allowed. It can be seen from Fig. 3 that the carrier frequency is then given as

*f*≥ 3

_{C}^{DHM}*a*in the case of DHM and

*f*≥ 4

_{C}^{CCHM}*a*in the case of CCHM. The need of higher carrier frequency in the case of CCHM is the consequence of higher lateral resolution. This influences negatively the available field of view (FOV) as it will be discussed in the next section. The total magnification

*m*between the output plane and the object plane of objectives O

_{1}, O

_{2}is given as

*m*=

*m*, where

_{O}m_{OL}*m*is magnification of objectives and

_{O}*m*is magnification of output lenses. The condition for carrier frequency in the output plane of the CCHM is thus given by

_{OL}*f*= 150 mm

_{G}^{−1}, which is designed for λ = 650 nm and

*NA*/

_{O}*m*≤ 0.025 ratio. When a shorter wavelength or objectives with higher

_{O}*NA*/

_{O}*m*ratio are to be used, then higher values of

_{O}*f*are required to avoid an overlap of the sideband terms and the zero-order term in the spatial frequency spectrum (e.g.

_{G}*f*= 222 mm

_{G}^{−1}is required for λ = 450 nm and

*NA*/

_{O}*m*= 0.025).

_{O}## 6. Output lens and the field of view

*m*of output lenses is dependent on the maximum spatial frequency

_{OL}*f*

_{OP,}_{max}present in the hologram at the output plane that has to be resolved and recorded digitally. Taking into account the rotation of the detector by 45° around the optical axis with respect to interference fringes, this frequency can be derived asSince the sampling rate should be at least 2.3 times higher [36] a condition for the spatial frequency

*f*(pixel density) of a CCD detector is given bywhich in terms of Eq. (7) and Eq. (8) leads toFor

_{CCD}*f*= 150 mm

_{G}^{−1}and a camera pixel size of 6.45 μm Eq. (10) gives

*m*≥ 2.7. The higher is

_{OL}*f*, the finer is the interference structure in the output plane and the larger magnification is thus needed to resolve the fringes by a detector and consequently the smaller is the field of view. Therefore it is convenient to keep

_{G}*f*as low as possible. When compared to typical DHM, the resulting FOV is smaller in the case of CCHM due to higher lateral resolution (FOV dimensions are approximately 1.5 × smaller). However, CCHM offers a better (lower) resolution/FOV ratio (when preservation of

_{G}*NA*/

_{O}*m*ratio is assumed). This means that if objective lenses with higher

_{O}*NA*(and corresponding

_{O}*m*) would be used in DHM providing comparable resolution to CCHM, it would result in smaller FOV dimensions in the case of DHM. From another view, if objective lenses with lower magnification

_{O}*m*(and corresponding

_{O}*NA*) would be used in CCHM to provide comparable FOV as in DHM, it would result in worse achievable lateral resolution in the case of DHM. Also there is a difference in achievable extremes: objective lenses with highest available

_{O}*NA*or lowest available magnification

_{O}*m*. With the highest available

_{O}*NA*objective lens the CCHM is able to provide better resolution when compared to DHM with the same lens, while with the lowest available magnification lens the DHM is able to provide larger FOV when compared to CCHM with the same lens. To extend the available FOV a larger detector with increased number of pixels can be used. There are also several methods enabling suppression of the zero order term in the frequency spectrum to improve the available bandwidth for the sideband terms (e.g [37

_{O}37. N. Pavillon, C. S. Seelamantula, J. Kühn, M. Unser, and C. Depeursinge, “Suppression of the zero-order term in off-axis digital holography through nonlinear filtering,” Appl. Opt. **48**(34), H186–H195 (2009). [CrossRef] [PubMed]

_{2}. The lateral resolution has to be sufficient across the whole FOV to transfer all the spatial frequencies produced by objectives into the output plane. The accessibility of the back focal plane is important to enable elimination of all diffraction grating orders except the imaging order (

*l*= + 1) by spatial filtering.

## 7. Image processing

24. R. Chmelík and Z. Harna, “Parallel-mode confocal microscope,” Opt. Eng. **38**(10), 1635–1639 (1999). [CrossRef]

38. T. Kreis, “Digital holographic interference-phase measurement using the Fourier-transform method,” J. Opt. Soc. Am. A **3**(6), 847–855 (1986). [CrossRef]

*f*and the size of the window corresponds to the maximum image term spatial frequency

_{C}*f*

_{max,}

_{or}_{*}. The frequency origin is translated to the center of the window and the spectrum is multiplied by an apodization function. Finally, the image complex amplitude is computed using the 2D inverse FFT and the image amplitude and phase are derived from the complex amplitude as modulus and argument, respectively.

## 8. Phase measurement accuracy and precision

39. J. Kühn, F. Charrière, T. Colomb, E. Cuche, F. Montfort, Y. Emery, P. Marquet, and C. Depeursinge, “Axial sub-nanometer accuracy in digital holographic microscopy,” Meas. Sci. Technol. **19**(7), 074007 (2008). [CrossRef]

*σ*was computed for each pixel of the blank reconstructed phase image throughout the 15 s long sequence of 140 captured images (no averaging used). In this way maps of temporal standard deviations of reconstructed phase were calculated providing information on the precision achieved in any particular pixel of the reconstructed image (Fig. 5). The measurement was performed with 14-bit camera 1376 pixels × 1038 pixels under two different degrees of temporal coherence of illumination – halogen lamp filtered with interference filter (

*λ*= 550 nm, 10 nm FWHM) and unfiltered (white light). Examples of central parts of captured holograms are shown in Fig. 5(a, b). With filtered light the interference fringes utilize 62% of dynamic range of the detector, while with white light the interference fringes utilize 28%. This gives 2.2 × lower contrast for white light when compared to filtered light. The obtained values of temporal standard deviations are in range of 0.002-0.006 rad for filtered light with mode of

*σ*in the case of white-light illumination are probably caused by imperfect alignment of the output angle

*β*, which influences achromaticity of the interferometer (see section 2) and decreases contrast of interference fringes. Higher values of

*σ*at the edges of FOV are caused by slight decrease of interference fringes’ contrast in these areas, which is a consequence of spatially incoherent illumination [28

**18**(21), 21990–22003 (2010). [CrossRef] [PubMed]

*n*= 0.5, one can obtain temporal standard deviation values converted to a real height:

## 9. Coherence gating

**18**(21), 21990–22003 (2010). [CrossRef] [PubMed]

6. L. Lovicar, L. Kvasnica, and R. Chmelík, “Surface observation and measurement by means of digital holographic microscope with arbitrary degree of coherence,” Proc. SPIE **7141**, 71411S (2008). [CrossRef]

**38**(10), 1635–1639 (1999). [CrossRef]

25. R. Chmelík, “Three-dimensional scalar imaging in high-aperture low-coherence interference and holographic microscopes,” J. Mod. Opt. **53**(18), 2673–2689 (2006). [CrossRef]

## 10. Influence of spatial and temporal coherence on the imaging properties

**53**(18), 2673–2689 (2006). [CrossRef]

27. R. Chmelík and Z. Harna, “Surface profilometry by a parallel–mode confocal microscope,” Opt. Eng. **41**(4), 744–745 (2002). [CrossRef]

**18**(21), 21990–22003 (2010). [CrossRef] [PubMed]

40. R. Chmelík, “Holographic confocal microscopy,” Proc. SPIE **4356**, 118–123 (2001). [CrossRef]

41. E. N. Leith and G. J. Swanson, “Recording of phase-amplitude images,” Appl. Opt. **20**(17), 3081–3084 (1981). [CrossRef] [PubMed]

**7141**, 71411S (2008). [CrossRef]

## 11. Conclusions

**18**(21), 21990–22003 (2010). [CrossRef] [PubMed]

**29**(6), 2339–2345 (2009). [PubMed]

26. H. Janečková, P. Kolman, P. Veselý, and R. Chmelík, “Digital holographic microscope with low spatial and temporal coherence of illumination,” Proc. SPIE **7000**, 70002E (2008). [CrossRef]

**7141**, 71411S (2008). [CrossRef]

7. L. Lovicar, J. Komrska, and R. Chmelík, “Quantitative-phase-contrast imaging of a two-level surface described as a 2D linear filtering process,” Opt. Express **18**(20), 20585–20594 (2010). [CrossRef] [PubMed]

27. R. Chmelík and Z. Harna, “Surface profilometry by a parallel–mode confocal microscope,” Opt. Eng. **41**(4), 744–745 (2002). [CrossRef]

**7141**, 71411S (2008). [CrossRef]

## Acknowledgments

## References and links

1. | R. Barer, “Interference microscopy and mass determination,” Nature |

2. | H. G. Davies and M. H. F. Wilkins, “Interference microscopy and mass determination,” Nature |

3. | H. Janečková, P. Veselý, and R. Chmelík, “Proving tumour cells by acute nutritional/energy deprivation as a survival threat: a task for microscopy,” Anticancer Res. |

4. | F. Dubois, C. Yourassowsky, O. Monnom, J. C. Legros, O. Debeir, P. Van Ham, R. Kiss, and C. Decaestecker, “Digital holographic microscopy for the three-dimensional dynamic analysis of in vitro cancer cell migration,” J. Biomed. Opt. |

5. | E. Cuche, F. Bevilacqua, and C. Depeursinge, “Digital holography for quantitative phase-contrast imaging,” Opt. Lett. |

6. | L. Lovicar, L. Kvasnica, and R. Chmelík, “Surface observation and measurement by means of digital holographic microscope with arbitrary degree of coherence,” Proc. SPIE |

7. | L. Lovicar, J. Komrska, and R. Chmelík, “Quantitative-phase-contrast imaging of a two-level surface described as a 2D linear filtering process,” Opt. Express |

8. | T. Colomb, N. Pavillon, J. Kühn, E. Cuche, C. Depeursinge, and Y. Emery, “Extended depth-of-focus by digital holographic microscopy,” Opt. Lett. |

9. | F. Dubois, L. Joannes, and J.-C. Legros, “Improved three-dimensional imaging with a digital holography microscope with a source of partial spatial coherence,” Appl. Opt. |

10. | P. Klysubun and G. Indebetouw, “A posteriori processing of spatiotemporal digital microholograms,” J. Opt. Soc. Am. A |

11. | G. Indebetouw and P. Klysubun, “Optical sectioning with low coherence spatio-temporal holography,” Opt. Commun. |

12. | G. Indebetouw and P. Klysubun, “Imaging through scattering media with depth resolution by use of low-coherence gating in spatiotemporal digital holography,” Opt. Lett. |

13. | E. N. Leith, W.-C. Chien, K. D. Mills, B. D. Athey, and D. S. Dilworth, “Optical sectioning by holographic coherence imaging: a generalized analysis,” J. Opt. Soc. Am. A |

14. | M.-K. Kim, “Tomographic three-dimensional imaging of a biological specimen using wavelength-scanning digital interference holography,” Opt. Express |

15. | P. Massatsch, F. Charrière, E. Cuche, P. Marquet, and C. D. Depeursinge, “Time-domain optical coherence tomography with digital holographic microscopy,” Appl. Opt. |

16. | W. Choi, C. Fang-Yen, K. Badizadegan, S. Oh, N. Lue, R. R. Dasari, and M. S. Feld, “Tomographic phase microscopy,” Nat. Methods |

17. | T. Zhang and I. Yamaguchi, “Three-dimensional microscopy with phase-shifting digital holography,” Opt. Lett. |

18. | L. Xu, J. M. Miao, and A. Asundi, “Properties of digital holography based on in-line conﬁguration,” Opt. Eng. |

19. | D. Carl, B. Kemper, G. Wernicke, and G. von Bally, “Parameter-optimized digital holographic microscope for high-resolution living-cell analysis,” Appl. Opt. |

20. | D. Shin, M. Daneshpanah, A. Anand, and B. Javidi, “Optofluidic system for three-dimensional sensing and identification of micro-organisms with digital holographic microscopy,” Opt. Lett. |

21. | P. Girshovitz and N. T. Shaked, “Generalized cell morphological parameters based on interferometric phase microscopy and their application to cell life cycle characterization,” Biomed. Opt. Express |

22. | B. Bhaduri, H. Pham, M. Mir, and G. Popescu, “Diffraction phase microscopy with white light,” Opt. Lett. |

23. | F. Dubois and C. Yourassowsky, “Full off-axis red-green-blue digital holographic microscope with LED illumination,” Opt. Lett. |

24. | R. Chmelík and Z. Harna, “Parallel-mode confocal microscope,” Opt. Eng. |

25. | R. Chmelík, “Three-dimensional scalar imaging in high-aperture low-coherence interference and holographic microscopes,” J. Mod. Opt. |

26. | H. Janečková, P. Kolman, P. Veselý, and R. Chmelík, “Digital holographic microscope with low spatial and temporal coherence of illumination,” Proc. SPIE |

27. | R. Chmelík and Z. Harna, “Surface profilometry by a parallel–mode confocal microscope,” Opt. Eng. |

28. | P. Kolman and R. Chmelík, “Coherence-controlled holographic microscope,” Opt. Express |

29. | M. Lošťák, P. Kolman, Z. Dostál, and R. Chmelík, “Diffuse light imaging with a coherence controlled holographic microscope,” Proc. SPIE |

30. | E. N. Leith and J. Upatnieks, “Holography with achromatic-fringe systems,” J. Opt. Soc. Am. |

31. | T. Slabý, M. Antoš, Z. Dostál, P. Kolman, and R. Chmelík, “Coherence-controlled holographic microscope,” Proc. SPIE |

32. | N. Pavillon, A. Benke, D. Boss, C. Moratal, J. Kühn, P. Jourdain, C. Depeursinge, P. J. Magistretti, and P. Marquet, “Cell morphology and intracellular ionic homeostasis explored with a multimodal approach combining epifluorescence and digital holographic microscopy,” J Biophotonics |

33. | B. Kemper, P. Langehanenberg, A. Höink, G. von Bally, F. Wottowah, S. Schinkinger, J. Guck, J. Käs, I. Bredebusch, J. Schnekenburger, and K. Schütze, “Monitoring of laser micromanipulated optically trapped cells by digital holographic microscopy,” J Biophotonics |

34. | E. Shaffer, N. Pavillon, and C. Depeursinge, “Single-shot, simultaneous incoherent and holographic microscopy,” J. Microsc. |

35. | M. Born and E. Wolf, |

36. | J. B. Pawley, |

37. | N. Pavillon, C. S. Seelamantula, J. Kühn, M. Unser, and C. Depeursinge, “Suppression of the zero-order term in off-axis digital holography through nonlinear filtering,” Appl. Opt. |

38. | T. Kreis, “Digital holographic interference-phase measurement using the Fourier-transform method,” J. Opt. Soc. Am. A |

39. | J. Kühn, F. Charrière, T. Colomb, E. Cuche, F. Montfort, Y. Emery, P. Marquet, and C. Depeursinge, “Axial sub-nanometer accuracy in digital holographic microscopy,” Meas. Sci. Technol. |

40. | R. Chmelík, “Holographic confocal microscopy,” Proc. SPIE |

41. | E. N. Leith and G. J. Swanson, “Recording of phase-amplitude images,” Appl. Opt. |

42. | R. Chmelík, P. Kolman, T. Slabý, M. Antoš, and Z. Dostál, “Interferometric system with spatial carrier frequency capable of imaging in polychromatic radiation,” patent EP2378244B1 (July 4, 2012). |

**OCIS Codes**

(090.0090) Holography : Holography

(110.4980) Imaging systems : Partial coherence in imaging

(120.5050) Instrumentation, measurement, and metrology : Phase measurement

(170.1790) Medical optics and biotechnology : Confocal microscopy

(180.3170) Microscopy : Interference microscopy

(110.0113) Imaging systems : Imaging through turbid media

**ToC Category:**

Microscopy

**History**

Original Manuscript: December 5, 2012

Revised Manuscript: February 3, 2013

Manuscript Accepted: May 2, 2013

Published: June 13, 2013

**Virtual Issues**

Vol. 8, Iss. 7 *Virtual Journal for Biomedical Optics*

**Citation**

Tomáš Slabý, Pavel Kolman, Zbyněk Dostál, Martin Antoš, Martin Lošťák, and Radim Chmelík, "Off-axis setup taking full advantage of incoherent illumination in coherence-controlled holographic microscope," Opt. Express **21**, 14747-14762 (2013)

http://www.opticsinfobase.org/vjbo/abstract.cfm?URI=oe-21-12-14747

Sort: Year | Journal | Reset

### References

- R. Barer, “Interference microscopy and mass determination,” Nature169(4296), 366–367 (1952). [CrossRef] [PubMed]
- H. G. Davies and M. H. F. Wilkins, “Interference microscopy and mass determination,” Nature169(4300), 541 (1952). [CrossRef] [PubMed]
- H. Janečková, P. Veselý, and R. Chmelík, “Proving tumour cells by acute nutritional/energy deprivation as a survival threat: a task for microscopy,” Anticancer Res.29(6), 2339–2345 (2009). [PubMed]
- F. Dubois, C. Yourassowsky, O. Monnom, J. C. Legros, O. Debeir, P. Van Ham, R. Kiss, and C. Decaestecker, “Digital holographic microscopy for the three-dimensional dynamic analysis of in vitro cancer cell migration,” J. Biomed. Opt.11(5), 054032 (2006). [CrossRef] [PubMed]
- E. Cuche, F. Bevilacqua, and C. Depeursinge, “Digital holography for quantitative phase-contrast imaging,” Opt. Lett.24(5), 291–293 (1999). [CrossRef] [PubMed]
- L. Lovicar, L. Kvasnica, and R. Chmelík, “Surface observation and measurement by means of digital holographic microscope with arbitrary degree of coherence,” Proc. SPIE7141, 71411S (2008). [CrossRef]
- L. Lovicar, J. Komrska, and R. Chmelík, “Quantitative-phase-contrast imaging of a two-level surface described as a 2D linear filtering process,” Opt. Express18(20), 20585–20594 (2010). [CrossRef] [PubMed]
- T. Colomb, N. Pavillon, J. Kühn, E. Cuche, C. Depeursinge, and Y. Emery, “Extended depth-of-focus by digital holographic microscopy,” Opt. Lett.35(11), 1840–1842 (2010). [CrossRef] [PubMed]
- F. Dubois, L. Joannes, and J.-C. Legros, “Improved three-dimensional imaging with a digital holography microscope with a source of partial spatial coherence,” Appl. Opt.38(34), 7085–7094 (1999). [CrossRef] [PubMed]
- P. Klysubun and G. Indebetouw, “A posteriori processing of spatiotemporal digital microholograms,” J. Opt. Soc. Am. A18(2), 326–331 (2001). [CrossRef] [PubMed]
- G. Indebetouw and P. Klysubun, “Optical sectioning with low coherence spatio-temporal holography,” Opt. Commun.172(1-6), 25–29 (1999). [CrossRef]
- G. Indebetouw and P. Klysubun, “Imaging through scattering media with depth resolution by use of low-coherence gating in spatiotemporal digital holography,” Opt. Lett.25(4), 212–214 (2000). [CrossRef] [PubMed]
- E. N. Leith, W.-C. Chien, K. D. Mills, B. D. Athey, and D. S. Dilworth, “Optical sectioning by holographic coherence imaging: a generalized analysis,” J. Opt. Soc. Am. A20(2), 380–387 (2003). [CrossRef] [PubMed]
- M.-K. Kim, “Tomographic three-dimensional imaging of a biological specimen using wavelength-scanning digital interference holography,” Opt. Express7(9), 305–310 (2000). [CrossRef] [PubMed]
- P. Massatsch, F. Charrière, E. Cuche, P. Marquet, and C. D. Depeursinge, “Time-domain optical coherence tomography with digital holographic microscopy,” Appl. Opt.44(10), 1806–1812 (2005). [CrossRef] [PubMed]
- W. Choi, C. Fang-Yen, K. Badizadegan, S. Oh, N. Lue, R. R. Dasari, and M. S. Feld, “Tomographic phase microscopy,” Nat. Methods4(9), 717–719 (2007). [CrossRef] [PubMed]
- T. Zhang and I. Yamaguchi, “Three-dimensional microscopy with phase-shifting digital holography,” Opt. Lett.23(15), 1221–1223 (1998). [CrossRef] [PubMed]
- L. Xu, J. M. Miao, and A. Asundi, “Properties of digital holography based on in-line conﬁguration,” Opt. Eng.39(12), 3214–3219 (2000). [CrossRef]
- D. Carl, B. Kemper, G. Wernicke, and G. von Bally, “Parameter-optimized digital holographic microscope for high-resolution living-cell analysis,” Appl. Opt.43(36), 6536–6544 (2004). [CrossRef] [PubMed]
- D. Shin, M. Daneshpanah, A. Anand, and B. Javidi, “Optofluidic system for three-dimensional sensing and identification of micro-organisms with digital holographic microscopy,” Opt. Lett.35(23), 4066–4068 (2010). [CrossRef] [PubMed]
- P. Girshovitz and N. T. Shaked, “Generalized cell morphological parameters based on interferometric phase microscopy and their application to cell life cycle characterization,” Biomed. Opt. Express3(8), 1757–1773 (2012). [CrossRef] [PubMed]
- B. Bhaduri, H. Pham, M. Mir, and G. Popescu, “Diffraction phase microscopy with white light,” Opt. Lett.37(6), 1094–1096 (2012). [CrossRef] [PubMed]
- F. Dubois and C. Yourassowsky, “Full off-axis red-green-blue digital holographic microscope with LED illumination,” Opt. Lett.37(12), 2190–2192 (2012). [CrossRef] [PubMed]
- R. Chmelík and Z. Harna, “Parallel-mode confocal microscope,” Opt. Eng.38(10), 1635–1639 (1999). [CrossRef]
- R. Chmelík, “Three-dimensional scalar imaging in high-aperture low-coherence interference and holographic microscopes,” J. Mod. Opt.53(18), 2673–2689 (2006). [CrossRef]
- H. Janečková, P. Kolman, P. Veselý, and R. Chmelík, “Digital holographic microscope with low spatial and temporal coherence of illumination,” Proc. SPIE7000, 70002E (2008). [CrossRef]
- R. Chmelík and Z. Harna, “Surface profilometry by a parallel–mode confocal microscope,” Opt. Eng.41(4), 744–745 (2002). [CrossRef]
- P. Kolman and R. Chmelík, “Coherence-controlled holographic microscope,” Opt. Express18(21), 21990–22003 (2010). [CrossRef] [PubMed]
- M. Lošťák, P. Kolman, Z. Dostál, and R. Chmelík, “Diffuse light imaging with a coherence controlled holographic microscope,” Proc. SPIE7746, 77461N (2010). [CrossRef]
- E. N. Leith and J. Upatnieks, “Holography with achromatic-fringe systems,” J. Opt. Soc. Am.57(8), 975–980 (1967). [CrossRef]
- T. Slabý, M. Antoš, Z. Dostál, P. Kolman, and R. Chmelík, “Coherence-controlled holographic microscope,” Proc. SPIE7746, 77461R (2010). [CrossRef]
- N. Pavillon, A. Benke, D. Boss, C. Moratal, J. Kühn, P. Jourdain, C. Depeursinge, P. J. Magistretti, and P. Marquet, “Cell morphology and intracellular ionic homeostasis explored with a multimodal approach combining epifluorescence and digital holographic microscopy,” J Biophotonics3(7), 432–436 (2010). [CrossRef] [PubMed]
- B. Kemper, P. Langehanenberg, A. Höink, G. von Bally, F. Wottowah, S. Schinkinger, J. Guck, J. Käs, I. Bredebusch, J. Schnekenburger, and K. Schütze, “Monitoring of laser micromanipulated optically trapped cells by digital holographic microscopy,” J Biophotonics3(7), 425–431 (2010). [CrossRef] [PubMed]
- E. Shaffer, N. Pavillon, and C. Depeursinge, “Single-shot, simultaneous incoherent and holographic microscopy,” J. Microsc.245(1), 49–62 (2012). [CrossRef] [PubMed]
- M. Born and E. Wolf, Principles of Optics, 6th edition (Pergamon Press, 1986).
- J. B. Pawley, Handbook of Biological Confocal Microscopy (Springer, 2006), pp. 65, Chap. 4.
- N. Pavillon, C. S. Seelamantula, J. Kühn, M. Unser, and C. Depeursinge, “Suppression of the zero-order term in off-axis digital holography through nonlinear filtering,” Appl. Opt.48(34), H186–H195 (2009). [CrossRef] [PubMed]
- T. Kreis, “Digital holographic interference-phase measurement using the Fourier-transform method,” J. Opt. Soc. Am. A3(6), 847–855 (1986). [CrossRef]
- J. Kühn, F. Charrière, T. Colomb, E. Cuche, F. Montfort, Y. Emery, P. Marquet, and C. Depeursinge, “Axial sub-nanometer accuracy in digital holographic microscopy,” Meas. Sci. Technol.19(7), 074007 (2008). [CrossRef]
- R. Chmelík, “Holographic confocal microscopy,” Proc. SPIE4356, 118–123 (2001). [CrossRef]
- E. N. Leith and G. J. Swanson, “Recording of phase-amplitude images,” Appl. Opt.20(17), 3081–3084 (1981). [CrossRef] [PubMed]
- R. Chmelík, P. Kolman, T. Slabý, M. Antoš, and Z. Dostál, “Interferometric system with spatial carrier frequency capable of imaging in polychromatic radiation,” patent EP2378244B1 (July 4, 2012).

## Cited By |
Alert me when this paper is cited |

OSA is able to provide readers links to articles that cite this paper by participating in CrossRef's Cited-By Linking service. CrossRef includes content from more than 3000 publishers and societies. In addition to listing OSA journal articles that cite this paper, citing articles from other participating publishers will also be listed.

« Previous Article | Next Article »

OSA is a member of CrossRef.