## Digital confocal microscope |

Optics Express, Vol. 20, Issue 20, pp. 22720-22727 (2012)

http://dx.doi.org/10.1364/OE.20.022720

Acrobat PDF (2501 KB)

### Abstract

We demonstrate experimentally a scanning confocal microscopy technique based on digital holography. The method relies on digital holographic recording of the scanned spot. The data collected in this way contains all the necessary information to digitally produce three-dimensional images. Several methods to treat the data are presented. Examples of reflection and transmission images of epithelial cells and mouse brain tissue are shown.

© 2012 OSA

## 1. Introduction

3. Y. C. Liu and A. S. Chiang, “High-resolution confocal imaging and three-dimensional rendering,” Methods **30**(1), 86–93 (2003). [CrossRef] [PubMed]

4. S. J. Tseng, Y. H. Lee, Z. H. Chen, H. H. Lin, C. Y. Lin, and S. C. Tang, “Integration of optical clearing and optical sectioning microscopy for three-dimensional imaging of natural biomaterial scaffolds in thin sections,” J. Biomed. Opt. **14**(4), 044004 (2009). [CrossRef] [PubMed]

5. M. J. Booth, “Adaptive optics in microscopy,” Philos. Transact. A Math. Phys. Eng. Sci. **365**(1861), 2829–2843 (2007). [CrossRef] [PubMed]

6. J. M. Girkin, S. Poland, and A. J. Wright, “Adaptive optics for deeper imaging of biological samples,” Curr. Opin. Biotechnol. **20**(1), 106–110 (2009). [CrossRef] [PubMed]

7. F. Charrière, A. Marian, F. Montfort, J. Kuehn, T. Colomb, E. Cuche, P. Marquet, and C. Depeursinge, “Cell refractive index tomography by digital holographic microscopy,” Opt. Lett. **31**(2), 178–180 (2006). [CrossRef] [PubMed]

8. K. Dillon and Y. Fainman, “Computational confocal tomography for simultaneous reconstruction of objects, occlusions, and aberrations,” Appl. Opt. **49**(13), 2529–2538 (2010). [CrossRef]

9. N. Lue, W. Choi, K. Badizadegan, R. R. Dasari, M. S. Feld, and G. Popescu, “Confocal diffraction phase microscopy of live cells,” Opt. Lett. **33**(18), 2074–2076 (2008). [CrossRef] [PubMed]

10. G. Barbastathis, M. Balberg, and D. J. Brady, “Confocal microscopy with a volume holographic filter,” Opt. Lett. **24**(12), 811–813 (1999). [CrossRef] [PubMed]

11. C. Yang and J. Mertz, “Transmission confocal laser scanning microscopy with a virtual pinhole based on nonlinear detection,” Opt. Lett. **28**(4), 224–226 (2003). [CrossRef] [PubMed]

## 2. Method

12. E. Cuche, P. Marquet, and C. Depeursinge, “Spatial filtering for zero-order and twin-image elimination in digital off-axis holography,” Appl. Opt. **39**(23), 4070–4075 (2000). [CrossRef] [PubMed]

*r*and

*s*respectively, the intensity of the hologram captured on the CCD is given by

*I*= |

_{h}*r*+

*s*|

^{2}. The reference intensity |

*r*|

^{2}and incidence angle are known and the reference can be expressed as

*r*= |

*r*|exp[

*i*(

*κ*+

_{x}x*κ*y)] exp[

_{y}*ik*(

*x*

^{2}+

*y*

^{2}/(2

*R*)], where

*κ*and

_{x}*κ*are the horizontal and vertical spatial frequencies of the fringes. The last term accounts for any remnant curvature of the reference wavefront of radius

_{y}*R*. The signal wave

*s*is collinear to the optical axis. We calculate FFT{

*r I*} = FFT{

_{h}*r*|

*r*|

^{2}+

*r*|

*s*|

^{2}+

*r*

^{2}

*s** + |

*r*|

^{2}

*s*} and we digitally filter out the DC term (

*r*|

*r*|

^{2}+

*r*|

*s*|

^{2}) and the conjugate image

*r*

^{2}

*s**. We then filter the signal |

*r*|

^{2}

*s*with a circular aperture, which acts as a virtual pinhole. If the reference is flat and wide enough, it can be considered constant and FFT{|

*r*|

^{2}

*s*} ≈|

*r*|

^{2}FFT{

*s*}, where the approximation is due to any spatial variations in

*r*. The confocal signal

*I*is the intensity integrated within the pinhole:

_{conf}*D*= 1.22

_{opt}*λ*/

*N.A.*is the optimal diameter of the pinhole in physical units,

*N*is the width and height of the image, Δ

*x*is the size of the camera pixels,

*N.A.*= 1.4 is the numerical aperture of the imaging objective and

*f*= 1.8mm is its focal length. For the camera in reflection, we have

*N*= 512 and Δ

*x*= 8μm, which gives

*N*= 512 and Δ

*x*= 9.3μm, which gives

*N*larger), it is possible to increase the resolution in the Fourier space and smoothen the image, but this requires a longer time to process.

*r*s*, we need to multiply it by

*r*and then take the FFT. However, there is, in the experiment, some uncertainty about the exact phase of

*r*(both in angle and sphericity). Moreover, the signal term itself can have some remnant phase curvature due to aberrations and/or misalignment. Therefore, the first step in the procedure is to iteratively modify

*r*to compensate for angle and sphericity until a focussed spot is obtained at the expected position in the digital reconstruction. We used a custom digital holography software that displays the extracted phase in real-time to visually adjust the off-axis reference angle, which amounts to bring the Fourier transform of the beam to the center of the Fourier plane where the virtual pinhole lies. This alignment procedure is performed while the beam passes next to the sample in a zone free from scattering. The aberrations that may be due to the cover slips when we scan the sample are thus also present during alignment. However, it is true that the focus may move slightly when we scan over several tens of microns. This issue is solved by pre-recording the reference angles needed to keep the focus in the center of the Fourier plane as a function of the scan position. Once this operation is complete, the optimal angle of incidence and the curvature of the reference are recorded and applied for the virtual confocal measurement.

*P*where the correlation reaches its maximum along the Z direction and we center the virtual pinhole on the centroid of the focus spot. The plotted value is the integrated intensity that passes through the pinhole. An improved image of the sample can also be produced by plotting the maximum value of the Gaussian correlation in plane

_{opt}*P*for each scanned point.

_{opt}## 3. Sample preparation

## 4. Results

## 5. Discussion

*N*pixels within the pinhole, we need to perform the following summations over the pixels in the image:where

_{p}*F*is the image. The arrays cos(

_{xy}*k*+

_{x}x*k*) and sin(

_{y}y*k*+

_{x}x*k*) can be calculated in advance and stored in the memory. After the summation, we add the intensities of the pixels in the pinhole, which requires 4

_{y}y*N*-1 operations (

_{p}*N*squaring operations for both the real and imaginary parts and 2

_{p}*N*−1 additions of all the squares). For a square image of size

_{p}*N*by

*N*, the total number of operation is

*N*= 3

_{op}*N*

_{p}N^{2}+ 4

*N*−1. Note that for

_{p}*N*< 25, this is smaller than the number of operations for a FFT which in the order of 2

_{p}*N*(34/9

*N*log

_{2}

*N*) [13

13. S. G. Johnson and M. Frigo, “A modified split-radix FFT with fewer arithmetic operations,” IEEE Trans. Signal Process. **55**(1), 111–119 (2007). [CrossRef]

*N*= 4,

_{p}*N*= 480 and

*N*= 2,764,815. Computation speed on graphic processing units is now in the range of 10

_{op}^{12}floating point operations per second. Since the operations required can be made completely parallel, this allows us to process 36,000 frames per second. Among the fastest commercially available devices, some high-end cameras achieve 25,000fps for 512x512 pixel images. This corresponds to 12 scanned slices per second for a standard VGA resolution of 640x480 pixels. To achieve such a speed we would have to scan the laser focus rather than the sample. This adds complication since we have to account for the change in angle of the signal beam. Fortunately, in the digital approach, the angles can be digitally recorded during an initial calibration phase and used in subsequent imaging.

## 6. Conclusion

14. D. J. Brady, K. Choi, D. L. Marks, R. Horisaki, and S. Lim, “Compressive holography,” Opt. Express **17**(15), 13040–13049 (2009). [CrossRef] [PubMed]

## References and links

1. | M. Minsky, “Microscopy apparatus,” US Patent 3,013,467 (1961). |

2. | C. Sheppard and D. Shotton, |

3. | Y. C. Liu and A. S. Chiang, “High-resolution confocal imaging and three-dimensional rendering,” Methods |

4. | S. J. Tseng, Y. H. Lee, Z. H. Chen, H. H. Lin, C. Y. Lin, and S. C. Tang, “Integration of optical clearing and optical sectioning microscopy for three-dimensional imaging of natural biomaterial scaffolds in thin sections,” J. Biomed. Opt. |

5. | M. J. Booth, “Adaptive optics in microscopy,” Philos. Transact. A Math. Phys. Eng. Sci. |

6. | J. M. Girkin, S. Poland, and A. J. Wright, “Adaptive optics for deeper imaging of biological samples,” Curr. Opin. Biotechnol. |

7. | F. Charrière, A. Marian, F. Montfort, J. Kuehn, T. Colomb, E. Cuche, P. Marquet, and C. Depeursinge, “Cell refractive index tomography by digital holographic microscopy,” Opt. Lett. |

8. | K. Dillon and Y. Fainman, “Computational confocal tomography for simultaneous reconstruction of objects, occlusions, and aberrations,” Appl. Opt. |

9. | N. Lue, W. Choi, K. Badizadegan, R. R. Dasari, M. S. Feld, and G. Popescu, “Confocal diffraction phase microscopy of live cells,” Opt. Lett. |

10. | G. Barbastathis, M. Balberg, and D. J. Brady, “Confocal microscopy with a volume holographic filter,” Opt. Lett. |

11. | C. Yang and J. Mertz, “Transmission confocal laser scanning microscopy with a virtual pinhole based on nonlinear detection,” Opt. Lett. |

12. | E. Cuche, P. Marquet, and C. Depeursinge, “Spatial filtering for zero-order and twin-image elimination in digital off-axis holography,” Appl. Opt. |

13. | S. G. Johnson and M. Frigo, “A modified split-radix FFT with fewer arithmetic operations,” IEEE Trans. Signal Process. |

14. | D. J. Brady, K. Choi, D. L. Marks, R. Horisaki, and S. Lim, “Compressive holography,” Opt. Express |

**OCIS Codes**

(180.1790) Microscopy : Confocal microscopy

(090.1995) Holography : Digital holography

**ToC Category:**

Microscopy

**History**

Original Manuscript: July 6, 2012

Revised Manuscript: September 7, 2012

Manuscript Accepted: September 15, 2012

Published: September 19, 2012

**Virtual Issues**

Vol. 7, Iss. 11 *Virtual Journal for Biomedical Optics*

**Citation**

Alexandre S. Goy and Demetri Psaltis, "Digital confocal microscope," Opt. Express **20**, 22720-22727 (2012)

http://www.opticsinfobase.org/vjbo/abstract.cfm?URI=oe-20-20-22720

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### References

- M. Minsky, “Microscopy apparatus,” US Patent 3,013,467 (1961).
- C. Sheppard and D. Shotton, Confocal Laser Scanning Microscopy (Oxford, BIOS Scientific Publishers 1997).
- Y. C. Liu and A. S. Chiang, “High-resolution confocal imaging and three-dimensional rendering,” Methods30(1), 86–93 (2003). [CrossRef] [PubMed]
- S. J. Tseng, Y. H. Lee, Z. H. Chen, H. H. Lin, C. Y. Lin, and S. C. Tang, “Integration of optical clearing and optical sectioning microscopy for three-dimensional imaging of natural biomaterial scaffolds in thin sections,” J. Biomed. Opt.14(4), 044004 (2009). [CrossRef] [PubMed]
- M. J. Booth, “Adaptive optics in microscopy,” Philos. Transact. A Math. Phys. Eng. Sci.365(1861), 2829–2843 (2007). [CrossRef] [PubMed]
- J. M. Girkin, S. Poland, and A. J. Wright, “Adaptive optics for deeper imaging of biological samples,” Curr. Opin. Biotechnol.20(1), 106–110 (2009). [CrossRef] [PubMed]
- F. Charrière, A. Marian, F. Montfort, J. Kuehn, T. Colomb, E. Cuche, P. Marquet, and C. Depeursinge, “Cell refractive index tomography by digital holographic microscopy,” Opt. Lett.31(2), 178–180 (2006). [CrossRef] [PubMed]
- K. Dillon and Y. Fainman, “Computational confocal tomography for simultaneous reconstruction of objects, occlusions, and aberrations,” Appl. Opt.49(13), 2529–2538 (2010). [CrossRef]
- N. Lue, W. Choi, K. Badizadegan, R. R. Dasari, M. S. Feld, and G. Popescu, “Confocal diffraction phase microscopy of live cells,” Opt. Lett.33(18), 2074–2076 (2008). [CrossRef] [PubMed]
- G. Barbastathis, M. Balberg, and D. J. Brady, “Confocal microscopy with a volume holographic filter,” Opt. Lett.24(12), 811–813 (1999). [CrossRef] [PubMed]
- C. Yang and J. Mertz, “Transmission confocal laser scanning microscopy with a virtual pinhole based on nonlinear detection,” Opt. Lett.28(4), 224–226 (2003). [CrossRef] [PubMed]
- E. Cuche, P. Marquet, and C. Depeursinge, “Spatial filtering for zero-order and twin-image elimination in digital off-axis holography,” Appl. Opt.39(23), 4070–4075 (2000). [CrossRef] [PubMed]
- S. G. Johnson and M. Frigo, “A modified split-radix FFT with fewer arithmetic operations,” IEEE Trans. Signal Process.55(1), 111–119 (2007). [CrossRef]
- D. J. Brady, K. Choi, D. L. Marks, R. Horisaki, and S. Lim, “Compressive holography,” Opt. Express17(15), 13040–13049 (2009). [CrossRef] [PubMed]

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