Abstract

In this paper, we present a 3-D temporal focusing microscope based on an electrically tunable lens (ETL) and a femtosecond regenerative laser amplifier. The focus-tunable lens provides a fast and compact way to perform non-mechanical z-scanning and resolves the blurry image issue compared with GVD-based z-scanning methods. The optical performance of the temporal focusing system, including z-scanning characteristics, the associated the magnification variation, and the lateral and axial resolution, has been studied and characterized using calibrated Rhodamine-6G thin film sample, fluorescent beads, and pollen samples. Lastly, we demonstrate the optical cross-sectioning and z-scanning capability with an in vivo experiment, where Ca2+ imaging of neurons in GaCamp6 labeled zebrafish was performed.

© 2015 Optical Society of America

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  1. W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
    [Crossref] [PubMed]
  2. E. J. Botcherby, C. W. Smith, M. M. Kohl, D. Débarre, M. J. Booth, R. Juškaitis, O. Paulsen, and T. Wilson, “Aberration-free three-dimensional multiphoton imaging of neuronal activity at kHz rates,” Proc. Natl. Acad. Sci. U.S.A. 109(8), 2919–2924 (2012).
    [Crossref] [PubMed]
  3. G. Y. Fan, H. Fujisaki, A. Miyawaki, R. K. Tsay, R. Y. Tsien, and M. H. Ellisman, “Video-rate scanning two-photon excitation fluorescence microscopy and ratio imaging with cameleons,” Biophys. J. 76(5), 2412–2420 (1999).
    [Crossref] [PubMed]
  4. Q. T. Nguyen, N. Callamaras, C. Hsieh, and I. Parker, “Construction of a two-photon microscope for video-rate Ca2+ imaging,” Cell Calcium 30(6), 383–393 (2001).
    [Crossref] [PubMed]
  5. K. H. Kim, C. Buehler, and P. T. C. So, “High-speed, two-photon scanning microscope,” Appl. Opt. 38(28), 6004–6009 (1999).
    [Crossref] [PubMed]
  6. A. H. Buist, M. Muller, J. Squier, and G. J. Brakenhoff, “Real-time two-photon absorption microscopy using multipoint excitation,” J. Microsc. 192(2), 217–226 (1998).
    [Crossref]
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    [Crossref] [PubMed]
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    [Crossref] [PubMed]
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    [Crossref] [PubMed]
  10. T. Nielsen, M. Fricke, D. Hellweg, and P. Andresen, “High efficiency beam splitter for multifocal multiphoton microscopy,” J. Microsc. 201(3), 368–376 (2001).
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    [Crossref] [PubMed]
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  16. A. Straub, M. E. Durst, and C. Xu, “High speed multiphoton axial scanning through an optical fiber in a remotely scanned temporal focusing setup,” Biomed. Opt. Express 2(1), 80–88 (2011).
    [Crossref] [PubMed]
  17. B. F. Grewe, F. F. Voigt, M. van ’t Hoff, and F. Helmchen, “Fast two-layer two-photon imaging of neuronal cell populations using an electrically tunable lens,” Biomed. Opt. Express 2(7), 2035–2046 (2011).
    [Crossref] [PubMed]
  18. F. O. Fahrbach, F. F. Voigt, B. Schmid, F. Helmchen, and J. Huisken, “Rapid 3D light-sheet microscopy with a tunable lens,” Opt. Express 21(18), 21010–21026 (2013).
    [Crossref] [PubMed]
  19. E. J. O. Hamel, B. F. Grewe, J. G. Parker, and M. J. Schnitzer, “Cellular level brain imaging in behaving mammals: an engineering approach,” Neuron 86(1), 140–159 (2015).
    [Crossref] [PubMed]

2015 (1)

E. J. O. Hamel, B. F. Grewe, J. G. Parker, and M. J. Schnitzer, “Cellular level brain imaging in behaving mammals: an engineering approach,” Neuron 86(1), 140–159 (2015).
[Crossref] [PubMed]

2013 (1)

2012 (2)

H. Dana and S. Shoham, “Remotely scanned multiphoton temporal focusing by axial grism scanning,” Opt. Lett. 37(14), 2913–2915 (2012).
[Crossref] [PubMed]

E. J. Botcherby, C. W. Smith, M. M. Kohl, D. Débarre, M. J. Booth, R. Juškaitis, O. Paulsen, and T. Wilson, “Aberration-free three-dimensional multiphoton imaging of neuronal activity at kHz rates,” Proc. Natl. Acad. Sci. U.S.A. 109(8), 2919–2924 (2012).
[Crossref] [PubMed]

2011 (2)

2008 (1)

R. Du, K. Bi, S. Zeng, D. Li, S. Xue, and Q. Luo, “Analysis of fast axial scanning scheme using temporal focusing with acousto-optic deflectors,” J. Mod. Opt. 56, 99–102 (2008).

2006 (1)

2005 (2)

2001 (3)

S. W. Hell and V. Andresen, “Space-multiplexed multifocal nonlinear microscopy,” J. Microsc. 202(3), 457–463 (2001).
[Crossref] [PubMed]

T. Nielsen, M. Fricke, D. Hellweg, and P. Andresen, “High efficiency beam splitter for multifocal multiphoton microscopy,” J. Microsc. 201(3), 368–376 (2001).
[Crossref] [PubMed]

Q. T. Nguyen, N. Callamaras, C. Hsieh, and I. Parker, “Construction of a two-photon microscope for video-rate Ca2+ imaging,” Cell Calcium 30(6), 383–393 (2001).
[Crossref] [PubMed]

2000 (1)

1999 (2)

K. H. Kim, C. Buehler, and P. T. C. So, “High-speed, two-photon scanning microscope,” Appl. Opt. 38(28), 6004–6009 (1999).
[Crossref] [PubMed]

G. Y. Fan, H. Fujisaki, A. Miyawaki, R. K. Tsay, R. Y. Tsien, and M. H. Ellisman, “Video-rate scanning two-photon excitation fluorescence microscopy and ratio imaging with cameleons,” Biophys. J. 76(5), 2412–2420 (1999).
[Crossref] [PubMed]

1998 (2)

A. H. Buist, M. Muller, J. Squier, and G. J. Brakenhoff, “Real-time two-photon absorption microscopy using multipoint excitation,” J. Microsc. 192(2), 217–226 (1998).
[Crossref]

J. Bewersdorf, R. Pick, and S. W. Hell, “Multifocal multiphoton microscopy,” Opt. Lett. 23(9), 655–657 (1998).
[Crossref] [PubMed]

1990 (1)

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[Crossref] [PubMed]

Andresen, P.

T. Nielsen, M. Fricke, D. Hellweg, and P. Andresen, “High efficiency beam splitter for multifocal multiphoton microscopy,” J. Microsc. 201(3), 368–376 (2001).
[Crossref] [PubMed]

Andresen, V.

S. W. Hell and V. Andresen, “Space-multiplexed multifocal nonlinear microscopy,” J. Microsc. 202(3), 457–463 (2001).
[Crossref] [PubMed]

Bewersdorf, J.

Bi, K.

R. Du, K. Bi, S. Zeng, D. Li, S. Xue, and Q. Luo, “Analysis of fast axial scanning scheme using temporal focusing with acousto-optic deflectors,” J. Mod. Opt. 56, 99–102 (2008).

Booth, M. J.

E. J. Botcherby, C. W. Smith, M. M. Kohl, D. Débarre, M. J. Booth, R. Juškaitis, O. Paulsen, and T. Wilson, “Aberration-free three-dimensional multiphoton imaging of neuronal activity at kHz rates,” Proc. Natl. Acad. Sci. U.S.A. 109(8), 2919–2924 (2012).
[Crossref] [PubMed]

Botcherby, E. J.

E. J. Botcherby, C. W. Smith, M. M. Kohl, D. Débarre, M. J. Booth, R. Juškaitis, O. Paulsen, and T. Wilson, “Aberration-free three-dimensional multiphoton imaging of neuronal activity at kHz rates,” Proc. Natl. Acad. Sci. U.S.A. 109(8), 2919–2924 (2012).
[Crossref] [PubMed]

Brakenhoff, G. J.

A. H. Buist, M. Muller, J. Squier, and G. J. Brakenhoff, “Real-time two-photon absorption microscopy using multipoint excitation,” J. Microsc. 192(2), 217–226 (1998).
[Crossref]

Buehler, C.

Buist, A. H.

A. H. Buist, M. Muller, J. Squier, and G. J. Brakenhoff, “Real-time two-photon absorption microscopy using multipoint excitation,” J. Microsc. 192(2), 217–226 (1998).
[Crossref]

Callamaras, N.

Q. T. Nguyen, N. Callamaras, C. Hsieh, and I. Parker, “Construction of a two-photon microscope for video-rate Ca2+ imaging,” Cell Calcium 30(6), 383–393 (2001).
[Crossref] [PubMed]

Dana, H.

Débarre, D.

E. J. Botcherby, C. W. Smith, M. M. Kohl, D. Débarre, M. J. Booth, R. Juškaitis, O. Paulsen, and T. Wilson, “Aberration-free three-dimensional multiphoton imaging of neuronal activity at kHz rates,” Proc. Natl. Acad. Sci. U.S.A. 109(8), 2919–2924 (2012).
[Crossref] [PubMed]

Denk, W.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[Crossref] [PubMed]

Du, R.

R. Du, K. Bi, S. Zeng, D. Li, S. Xue, and Q. Luo, “Analysis of fast axial scanning scheme using temporal focusing with acousto-optic deflectors,” J. Mod. Opt. 56, 99–102 (2008).

Durst, M.

Durst, M. E.

Ellisman, M. H.

G. Y. Fan, H. Fujisaki, A. Miyawaki, R. K. Tsay, R. Y. Tsien, and M. H. Ellisman, “Video-rate scanning two-photon excitation fluorescence microscopy and ratio imaging with cameleons,” Biophys. J. 76(5), 2412–2420 (1999).
[Crossref] [PubMed]

Fahrbach, F. O.

Fan, G. Y.

G. Y. Fan, H. Fujisaki, A. Miyawaki, R. K. Tsay, R. Y. Tsien, and M. H. Ellisman, “Video-rate scanning two-photon excitation fluorescence microscopy and ratio imaging with cameleons,” Biophys. J. 76(5), 2412–2420 (1999).
[Crossref] [PubMed]

Fittinghoff, D.

Fricke, M.

T. Nielsen, M. Fricke, D. Hellweg, and P. Andresen, “High efficiency beam splitter for multifocal multiphoton microscopy,” J. Microsc. 201(3), 368–376 (2001).
[Crossref] [PubMed]

Fujisaki, H.

G. Y. Fan, H. Fujisaki, A. Miyawaki, R. K. Tsay, R. Y. Tsien, and M. H. Ellisman, “Video-rate scanning two-photon excitation fluorescence microscopy and ratio imaging with cameleons,” Biophys. J. 76(5), 2412–2420 (1999).
[Crossref] [PubMed]

Grewe, B. F.

E. J. O. Hamel, B. F. Grewe, J. G. Parker, and M. J. Schnitzer, “Cellular level brain imaging in behaving mammals: an engineering approach,” Neuron 86(1), 140–159 (2015).
[Crossref] [PubMed]

B. F. Grewe, F. F. Voigt, M. van ’t Hoff, and F. Helmchen, “Fast two-layer two-photon imaging of neuronal cell populations using an electrically tunable lens,” Biomed. Opt. Express 2(7), 2035–2046 (2011).
[Crossref] [PubMed]

Hamel, E. J. O.

E. J. O. Hamel, B. F. Grewe, J. G. Parker, and M. J. Schnitzer, “Cellular level brain imaging in behaving mammals: an engineering approach,” Neuron 86(1), 140–159 (2015).
[Crossref] [PubMed]

Hell, S. W.

S. W. Hell and V. Andresen, “Space-multiplexed multifocal nonlinear microscopy,” J. Microsc. 202(3), 457–463 (2001).
[Crossref] [PubMed]

J. Bewersdorf, R. Pick, and S. W. Hell, “Multifocal multiphoton microscopy,” Opt. Lett. 23(9), 655–657 (1998).
[Crossref] [PubMed]

Hellweg, D.

T. Nielsen, M. Fricke, D. Hellweg, and P. Andresen, “High efficiency beam splitter for multifocal multiphoton microscopy,” J. Microsc. 201(3), 368–376 (2001).
[Crossref] [PubMed]

Helmchen, F.

Hsieh, C.

Q. T. Nguyen, N. Callamaras, C. Hsieh, and I. Parker, “Construction of a two-photon microscope for video-rate Ca2+ imaging,” Cell Calcium 30(6), 383–393 (2001).
[Crossref] [PubMed]

Huisken, J.

Juškaitis, R.

E. J. Botcherby, C. W. Smith, M. M. Kohl, D. Débarre, M. J. Booth, R. Juškaitis, O. Paulsen, and T. Wilson, “Aberration-free three-dimensional multiphoton imaging of neuronal activity at kHz rates,” Proc. Natl. Acad. Sci. U.S.A. 109(8), 2919–2924 (2012).
[Crossref] [PubMed]

Kim, K. H.

Kohl, M. M.

E. J. Botcherby, C. W. Smith, M. M. Kohl, D. Débarre, M. J. Booth, R. Juškaitis, O. Paulsen, and T. Wilson, “Aberration-free three-dimensional multiphoton imaging of neuronal activity at kHz rates,” Proc. Natl. Acad. Sci. U.S.A. 109(8), 2919–2924 (2012).
[Crossref] [PubMed]

Li, D.

R. Du, K. Bi, S. Zeng, D. Li, S. Xue, and Q. Luo, “Analysis of fast axial scanning scheme using temporal focusing with acousto-optic deflectors,” J. Mod. Opt. 56, 99–102 (2008).

Luo, Q.

R. Du, K. Bi, S. Zeng, D. Li, S. Xue, and Q. Luo, “Analysis of fast axial scanning scheme using temporal focusing with acousto-optic deflectors,” J. Mod. Opt. 56, 99–102 (2008).

Miyawaki, A.

G. Y. Fan, H. Fujisaki, A. Miyawaki, R. K. Tsay, R. Y. Tsien, and M. H. Ellisman, “Video-rate scanning two-photon excitation fluorescence microscopy and ratio imaging with cameleons,” Biophys. J. 76(5), 2412–2420 (1999).
[Crossref] [PubMed]

Muller, M.

A. H. Buist, M. Muller, J. Squier, and G. J. Brakenhoff, “Real-time two-photon absorption microscopy using multipoint excitation,” J. Microsc. 192(2), 217–226 (1998).
[Crossref]

Nguyen, Q. T.

Q. T. Nguyen, N. Callamaras, C. Hsieh, and I. Parker, “Construction of a two-photon microscope for video-rate Ca2+ imaging,” Cell Calcium 30(6), 383–393 (2001).
[Crossref] [PubMed]

Nielsen, T.

T. Nielsen, M. Fricke, D. Hellweg, and P. Andresen, “High efficiency beam splitter for multifocal multiphoton microscopy,” J. Microsc. 201(3), 368–376 (2001).
[Crossref] [PubMed]

Oron, D.

Parker, I.

Q. T. Nguyen, N. Callamaras, C. Hsieh, and I. Parker, “Construction of a two-photon microscope for video-rate Ca2+ imaging,” Cell Calcium 30(6), 383–393 (2001).
[Crossref] [PubMed]

Parker, J. G.

E. J. O. Hamel, B. F. Grewe, J. G. Parker, and M. J. Schnitzer, “Cellular level brain imaging in behaving mammals: an engineering approach,” Neuron 86(1), 140–159 (2015).
[Crossref] [PubMed]

Paulsen, O.

E. J. Botcherby, C. W. Smith, M. M. Kohl, D. Débarre, M. J. Booth, R. Juškaitis, O. Paulsen, and T. Wilson, “Aberration-free three-dimensional multiphoton imaging of neuronal activity at kHz rates,” Proc. Natl. Acad. Sci. U.S.A. 109(8), 2919–2924 (2012).
[Crossref] [PubMed]

Pick, R.

Schmid, B.

Schnitzer, M. J.

E. J. O. Hamel, B. F. Grewe, J. G. Parker, and M. J. Schnitzer, “Cellular level brain imaging in behaving mammals: an engineering approach,” Neuron 86(1), 140–159 (2015).
[Crossref] [PubMed]

Shoham, S.

Silberberg, Y.

Smith, C. W.

E. J. Botcherby, C. W. Smith, M. M. Kohl, D. Débarre, M. J. Booth, R. Juškaitis, O. Paulsen, and T. Wilson, “Aberration-free three-dimensional multiphoton imaging of neuronal activity at kHz rates,” Proc. Natl. Acad. Sci. U.S.A. 109(8), 2919–2924 (2012).
[Crossref] [PubMed]

So, P. T. C.

Squier, J.

D. Fittinghoff, P. Wiseman, and J. Squier, “Widefield multiphoton and temporally decorrelated multifocal multiphoton microscopy,” Opt. Express 7(8), 273–279 (2000).
[Crossref] [PubMed]

A. H. Buist, M. Muller, J. Squier, and G. J. Brakenhoff, “Real-time two-photon absorption microscopy using multipoint excitation,” J. Microsc. 192(2), 217–226 (1998).
[Crossref]

Straub, A.

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[Crossref] [PubMed]

Tal, E.

Tsay, R. K.

G. Y. Fan, H. Fujisaki, A. Miyawaki, R. K. Tsay, R. Y. Tsien, and M. H. Ellisman, “Video-rate scanning two-photon excitation fluorescence microscopy and ratio imaging with cameleons,” Biophys. J. 76(5), 2412–2420 (1999).
[Crossref] [PubMed]

Tsien, R. Y.

G. Y. Fan, H. Fujisaki, A. Miyawaki, R. K. Tsay, R. Y. Tsien, and M. H. Ellisman, “Video-rate scanning two-photon excitation fluorescence microscopy and ratio imaging with cameleons,” Biophys. J. 76(5), 2412–2420 (1999).
[Crossref] [PubMed]

van ’t Hoff, M.

van Howe, J.

Voigt, F. F.

Webb, W. W.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[Crossref] [PubMed]

Wilson, T.

E. J. Botcherby, C. W. Smith, M. M. Kohl, D. Débarre, M. J. Booth, R. Juškaitis, O. Paulsen, and T. Wilson, “Aberration-free three-dimensional multiphoton imaging of neuronal activity at kHz rates,” Proc. Natl. Acad. Sci. U.S.A. 109(8), 2919–2924 (2012).
[Crossref] [PubMed]

Wiseman, P.

Xu, C.

Xue, S.

R. Du, K. Bi, S. Zeng, D. Li, S. Xue, and Q. Luo, “Analysis of fast axial scanning scheme using temporal focusing with acousto-optic deflectors,” J. Mod. Opt. 56, 99–102 (2008).

Zeng, S.

R. Du, K. Bi, S. Zeng, D. Li, S. Xue, and Q. Luo, “Analysis of fast axial scanning scheme using temporal focusing with acousto-optic deflectors,” J. Mod. Opt. 56, 99–102 (2008).

Zhu, G.

Zipfel, W.

Appl. Opt. (1)

Biomed. Opt. Express (2)

Biophys. J. (1)

G. Y. Fan, H. Fujisaki, A. Miyawaki, R. K. Tsay, R. Y. Tsien, and M. H. Ellisman, “Video-rate scanning two-photon excitation fluorescence microscopy and ratio imaging with cameleons,” Biophys. J. 76(5), 2412–2420 (1999).
[Crossref] [PubMed]

Cell Calcium (1)

Q. T. Nguyen, N. Callamaras, C. Hsieh, and I. Parker, “Construction of a two-photon microscope for video-rate Ca2+ imaging,” Cell Calcium 30(6), 383–393 (2001).
[Crossref] [PubMed]

J. Microsc. (3)

A. H. Buist, M. Muller, J. Squier, and G. J. Brakenhoff, “Real-time two-photon absorption microscopy using multipoint excitation,” J. Microsc. 192(2), 217–226 (1998).
[Crossref]

S. W. Hell and V. Andresen, “Space-multiplexed multifocal nonlinear microscopy,” J. Microsc. 202(3), 457–463 (2001).
[Crossref] [PubMed]

T. Nielsen, M. Fricke, D. Hellweg, and P. Andresen, “High efficiency beam splitter for multifocal multiphoton microscopy,” J. Microsc. 201(3), 368–376 (2001).
[Crossref] [PubMed]

J. Mod. Opt. (1)

R. Du, K. Bi, S. Zeng, D. Li, S. Xue, and Q. Luo, “Analysis of fast axial scanning scheme using temporal focusing with acousto-optic deflectors,” J. Mod. Opt. 56, 99–102 (2008).

Neuron (1)

E. J. O. Hamel, B. F. Grewe, J. G. Parker, and M. J. Schnitzer, “Cellular level brain imaging in behaving mammals: an engineering approach,” Neuron 86(1), 140–159 (2015).
[Crossref] [PubMed]

Opt. Express (5)

Opt. Lett. (2)

Proc. Natl. Acad. Sci. U.S.A. (1)

E. J. Botcherby, C. W. Smith, M. M. Kohl, D. Débarre, M. J. Booth, R. Juškaitis, O. Paulsen, and T. Wilson, “Aberration-free three-dimensional multiphoton imaging of neuronal activity at kHz rates,” Proc. Natl. Acad. Sci. U.S.A. 109(8), 2919–2924 (2012).
[Crossref] [PubMed]

Science (1)

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[Crossref] [PubMed]

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Figures (5)

Fig. 1
Fig. 1 Schematic of the fast 3-D temporal focusing microscope. M: mirror; PBS: polarization beam-splitter; DM: dichroic mirror; ETL: electrically tunable lens; L_c: collimating lens fc = 500mm; L_r1: first relay-lens fr1 = 300mm; L_r2: second relay-lens fr2 = 200mm; L_i: imaging lens (zoomlens, Canon);
Fig. 2
Fig. 2 Two-photon fluorescence images of pollen samples obtained at different depths: (a)-(e) images scanned by a motorized stage from 0~40 µm, and (f)-(j) images scanned by tuning the ETL. Note the slight change of magnification (or the field of view size). Scale bar = 20µm.
Fig. 3
Fig. 3 (a) Relationship between the axial shift of focal plane and the ETL current input; (b) relationship between the image magnification and the ETL current input; (c) point-spread-function (PSF) measurement along lateral and axial (z axis) directions as a function of the ETL current input; (d) illustration of the axial (focal plane) scanning, where positive values refer to a decrease in working distance and vice versa.
Fig. 4
Fig. 4 Optical model of the temporal focusing microscope. Note the optical path from the grating to P is omitted. P is the intermediate imaging plane of the grating, and BFL refers to the back focal length of the ETL and the objective lens.
Fig. 5
Fig. 5 In-vivo two-photon Ca2+ imaging of zebrafish. Left: image of a 4dpf larva zebrafish head obtained by a confocal microscope. The red circle indicates a zoom-in area imaged using the temporal focusing microscope. Right: (b) - (h) fluorescence images from the temporal focusing microscope of the 4dpf zebrafish larva brain at different depths; the z position of each image is obtained by Fig. 3(b); and the magnification is re-scaled using Fig. 3(c); scale bar = 20 µm; pixels: 256 × 256. Images were collected in the left hemisphere of the tectum opticum.

Equations (4)

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B F L = f o 1 + f o f E T L d 2
M i m a g i n g = f i f o × ( d 2 f o f E T L 1 )
F O V = D f c f r 1 M i l l u min a t i o n = D f c f r 1 1 M i m a g i n g f i f r 2
N A a × ( 1 d 2 f E T L )

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