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

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References

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2010

2009

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

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]

2008

2007

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]

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

2006

2003

2000

1999

Badizadegan, K.

Balberg, M.

Barbastathis, G.

Booth, M. J.

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

Brady, D. J.

Charrière, F.

Chen, Z. H.

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]

Chiang, A. S.

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

Choi, K.

Choi, W.

Colomb, T.

Cuche, E.

Dasari, R. R.

Depeursinge, C.

Dillon, K.

Fainman, Y.

Feld, M. S.

Frigo, M.

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]

Girkin, J. M.

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]

Horisaki, R.

Johnson, S. G.

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]

Kuehn, J.

Lee, Y. H.

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]

Lim, S.

Lin, C. Y.

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]

Lin, H. H.

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]

Liu, Y. C.

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

Lue, N.

Marian, A.

Marks, D. L.

Marquet, P.

Mertz, J.

Montfort, F.

Poland, S.

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]

Popescu, G.

Tang, S. C.

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]

Tseng, S. J.

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]

Wright, A. J.

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]

Yang, C.

Appl. Opt.

Curr. Opin. Biotechnol.

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]

IEEE Trans. Signal Process.

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]

J. Biomed. Opt.

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]

Methods

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

Opt. Express

Opt. Lett.

Philos. Transact. A Math. Phys. Eng. Sci.

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

Other

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

C. Sheppard and D. Shotton, Confocal Laser Scanning Microscopy (Oxford, BIOS Scientific Publishers 1997).

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Figures (6)

Fig. 1
Fig. 1

(a) Optical set-up for digital holographic confocal microscopy. A reference beam Rt (resp. Rr) is extracted for the transmission (resp. reflection) hologram. The sample, sealed between two cover slips, is placed between two microscope objectives (MO1 and 2) and scanned using a 3D translation stage. The back focal plane of objective MO1 (resp. MO2) is imaged on camera CCDt (resp. CCDr) using two relay lenses L1 and L2 (resp. L3 and L4) in a 4f configuration. The optical Fourier transform of the focus is produced on the camera in transmission (resp. reflection). The reference beam is introduced off-axis using a second beam splitter. (b) Modified set-up for conventional confocal imaging with a pinhole of 50µm in front of the detector.

Fig. 2
Fig. 2

Digital hologram filtering with the virtual pinhole. (a) Schematic of the virtual confocal principle: the field on the camera is the Fourier transform of the focus in the sample. The field is discretized by the CCD array as suggested by the drawing. This data is Fourier transformed in the computer to simulate the lens that would focus the light through the pinhole. (b) Transmission digital confocal image of an epithelial cell obtained by using a fixed pinhole. (c) Graph showing the motion of the image of the focus in the XY plane due to aberration as the sample is scanned along line A-B on picture (b). The circle represents the pinhole. (d) Corrected image obtained by tracking the focus in three dimension. (e) Corrected image obtained by performing the correlation between the measured focus and an ideal Gaussian beam.

Fig. 3
Fig. 3

Mouse brain tissue images. (a) 500μm by 500μm XY section 20μm deep in the tissue, without pinhole filtering. (b) 100μm by 100μm XY section close to the center of image (a), without the pinhole. (c) XZ section, Z coordinate ranging from 0 to 40μm in depth. The trace of the section in the XY plane is shown by the dashed line on image (b). (c) (d) and (e) Images corresponding to (a), (b) and (c) respectively, obtained with pinhole filtering. The scanning step for all sections is 1μm.

Fig. 4
Fig. 4

(a) to (c) Three successive XY sections in a human epithelial cell. The sections are spaced by 5μm in Z. The images have been obtained in reflection using the digital confocal microscope.

Fig. 5
Fig. 5

(a) to (c) Images of the same cell as in Fig. 4, obtained with digital confocal in transmission without dynamic pinhole placement. The sections are spaced by 5μm in Z. (d) to (f) Transmission images obtained with dynamic placement of the virtual pinhole. (g) to (i) Transmission images obtained using the Gaussian correlation metric.

Fig. 6
Fig. 6

(a) Reflection digital confocal XZ section with fixed pinhole of the cell shown in Fig. 4 and 5. The two horizontal lines correspond to the interfaces between the coverslips and the water solution. (b) Transmission digital confocal image with fixed virtual pinhole. (c) Transmission confocal section obtained by tracking the focus in three dimensions with the virtual pinhole. (d) and (e) Reflection and transmission sections respectively without virtual pinhole. (f) Transmission section obtained using the Gaussian correlation.

Equations (3)

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I conf = k x , k y pinhole | FFT{s } k x , k y | 2
D ˜ opt = NΔx λf D opt =1.22 NΔx N.A.f
p= x,y F xy cos( k x x+ k y y)+i x,y F xy sin( k x x+ k y y)

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