Abstract

In this paper, we present a method to determine, for the first time, the ratio of forward-propagating second harmonic (SHG) signal to back-propagating SHG signal (F/B) in vivo on the surface of intact tissue samples without any biopsy or tissue sectioning, using only epidetection (i.e., via a single objective lens). This method has the additional benefit of using the confocal detection apparatus already contained within common commercially available two-photon laser-scanning microscopes, and hence can allow the measurement of the SHG F/B ratio in vivo with minimal purchase of new equipment.

© 2010 OSA

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  1. R. M. Williams, W. R. Zipfel, and W. W. Webb, “Interpreting second-harmonic generation images of collagen I fibrils,” Biophys. J. 88(2), 1377–1386 (2005).
    [CrossRef]
  2. R. Lacomb, O. Nadiarnykh, and P. J. Campagnola, “Quantitative second harmonic generation imaging of the diseased state osteogenesis imperfecta: experiment and simulation,” Biophys. J. 94(11), 4504–4514 (2008).
    [CrossRef] [PubMed]
  3. O. Nadiarnykh, R. B. Lacomb, P. J. Campagnola, and W. A. Mohler, “Coherent and incoherent SHG in fibrillar cellulose matrices,” Opt. Express 15(6), 3348–3360 (2007).
    [CrossRef] [PubMed]
  4. X. Han, R. M. Burke, M. L. Zettel, P. Tang, and E. B. Brown, “Second harmonic properties of tumor collagen: determining the structural relationship between reactive stroma and healthy stroma,” Opt. Express 16(3), 1846–1859 (2008).
    [CrossRef] [PubMed]
  5. A. C. Kwan, D. A. Dombeck, and W. W. Webb, “Polarized microtubule arrays in apical dendrites and axons,” Proc. Natl. Acad. Sci. U.S.A. 105(32), 11370–11375 (2008).
    [CrossRef] [PubMed]
  6. S. V. Plotnikov, A. C. Millard, P. J. Campagnola, and W. A. Mohler, “Characterization of the myosin-based source for second-harmonic generation from muscle sarcomeres,” Biophys. J. 90(2), 693–703 (2006).
    [CrossRef]
  7. O. Nadiarnykh, R. B. LaComb, M. A. Brewer, and P. J. Campagnola, “Alterations of the extracellular matrix in ovarian cancer studied by second harmonic generation imaging microscopy,” BMC Cancer 10(1), 94 (2010).
    [CrossRef] [PubMed]
  8. F. Légaré, C. Pfeffer, and B. R. Olsen, “The role of backscattering in SHG tissue imaging,” Biophys. J. 93(4), 1312–1320 (2007).
    [CrossRef] [PubMed]
  9. L. Wang and S. L. Jacques, “Hybrid model of Monte Carlo simulation and diffusion theory for light reflectance by turbid media,” J. Opt. Soc. Am. A 10(8), 1746–1752 (1993).
    [CrossRef]
  10. W. F. Cheong, S. A. Prahl, and A. J. Welch, “A review of the optical properties of biological tissues,” IEEE J. Quantum Electron. 26(12), 2166–2185 (1990).
    [CrossRef]
  11. T. J. Farrell, M. S. Patterson, and B. Wilson, “A diffusion theory model of spatially resolved, steady-state diffuse reflectance for the noninvasive determination of tissue optical properties in vivo,” Med. Phys. 19(4), 879–888 (1992).
    [CrossRef] [PubMed]
  12. W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
    [CrossRef] [PubMed]

2010 (1)

O. Nadiarnykh, R. B. LaComb, M. A. Brewer, and P. J. Campagnola, “Alterations of the extracellular matrix in ovarian cancer studied by second harmonic generation imaging microscopy,” BMC Cancer 10(1), 94 (2010).
[CrossRef] [PubMed]

2008 (3)

R. Lacomb, O. Nadiarnykh, and P. J. Campagnola, “Quantitative second harmonic generation imaging of the diseased state osteogenesis imperfecta: experiment and simulation,” Biophys. J. 94(11), 4504–4514 (2008).
[CrossRef] [PubMed]

A. C. Kwan, D. A. Dombeck, and W. W. Webb, “Polarized microtubule arrays in apical dendrites and axons,” Proc. Natl. Acad. Sci. U.S.A. 105(32), 11370–11375 (2008).
[CrossRef] [PubMed]

X. Han, R. M. Burke, M. L. Zettel, P. Tang, and E. B. Brown, “Second harmonic properties of tumor collagen: determining the structural relationship between reactive stroma and healthy stroma,” Opt. Express 16(3), 1846–1859 (2008).
[CrossRef] [PubMed]

2007 (2)

2006 (1)

S. V. Plotnikov, A. C. Millard, P. J. Campagnola, and W. A. Mohler, “Characterization of the myosin-based source for second-harmonic generation from muscle sarcomeres,” Biophys. J. 90(2), 693–703 (2006).
[CrossRef]

2005 (1)

R. M. Williams, W. R. Zipfel, and W. W. Webb, “Interpreting second-harmonic generation images of collagen I fibrils,” Biophys. J. 88(2), 1377–1386 (2005).
[CrossRef]

2003 (1)

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

1993 (1)

1992 (1)

T. J. Farrell, M. S. Patterson, and B. Wilson, “A diffusion theory model of spatially resolved, steady-state diffuse reflectance for the noninvasive determination of tissue optical properties in vivo,” Med. Phys. 19(4), 879–888 (1992).
[CrossRef] [PubMed]

1990 (1)

W. F. Cheong, S. A. Prahl, and A. J. Welch, “A review of the optical properties of biological tissues,” IEEE J. Quantum Electron. 26(12), 2166–2185 (1990).
[CrossRef]

Brewer, M. A.

O. Nadiarnykh, R. B. LaComb, M. A. Brewer, and P. J. Campagnola, “Alterations of the extracellular matrix in ovarian cancer studied by second harmonic generation imaging microscopy,” BMC Cancer 10(1), 94 (2010).
[CrossRef] [PubMed]

Brown, E. B.

Burke, R. M.

Campagnola, P. J.

O. Nadiarnykh, R. B. LaComb, M. A. Brewer, and P. J. Campagnola, “Alterations of the extracellular matrix in ovarian cancer studied by second harmonic generation imaging microscopy,” BMC Cancer 10(1), 94 (2010).
[CrossRef] [PubMed]

R. Lacomb, O. Nadiarnykh, and P. J. Campagnola, “Quantitative second harmonic generation imaging of the diseased state osteogenesis imperfecta: experiment and simulation,” Biophys. J. 94(11), 4504–4514 (2008).
[CrossRef] [PubMed]

O. Nadiarnykh, R. B. Lacomb, P. J. Campagnola, and W. A. Mohler, “Coherent and incoherent SHG in fibrillar cellulose matrices,” Opt. Express 15(6), 3348–3360 (2007).
[CrossRef] [PubMed]

S. V. Plotnikov, A. C. Millard, P. J. Campagnola, and W. A. Mohler, “Characterization of the myosin-based source for second-harmonic generation from muscle sarcomeres,” Biophys. J. 90(2), 693–703 (2006).
[CrossRef]

Cheong, W. F.

W. F. Cheong, S. A. Prahl, and A. J. Welch, “A review of the optical properties of biological tissues,” IEEE J. Quantum Electron. 26(12), 2166–2185 (1990).
[CrossRef]

Dombeck, D. A.

A. C. Kwan, D. A. Dombeck, and W. W. Webb, “Polarized microtubule arrays in apical dendrites and axons,” Proc. Natl. Acad. Sci. U.S.A. 105(32), 11370–11375 (2008).
[CrossRef] [PubMed]

Farrell, T. J.

T. J. Farrell, M. S. Patterson, and B. Wilson, “A diffusion theory model of spatially resolved, steady-state diffuse reflectance for the noninvasive determination of tissue optical properties in vivo,” Med. Phys. 19(4), 879–888 (1992).
[CrossRef] [PubMed]

Han, X.

Jacques, S. L.

Kwan, A. C.

A. C. Kwan, D. A. Dombeck, and W. W. Webb, “Polarized microtubule arrays in apical dendrites and axons,” Proc. Natl. Acad. Sci. U.S.A. 105(32), 11370–11375 (2008).
[CrossRef] [PubMed]

Lacomb, R.

R. Lacomb, O. Nadiarnykh, and P. J. Campagnola, “Quantitative second harmonic generation imaging of the diseased state osteogenesis imperfecta: experiment and simulation,” Biophys. J. 94(11), 4504–4514 (2008).
[CrossRef] [PubMed]

LaComb, R. B.

O. Nadiarnykh, R. B. LaComb, M. A. Brewer, and P. J. Campagnola, “Alterations of the extracellular matrix in ovarian cancer studied by second harmonic generation imaging microscopy,” BMC Cancer 10(1), 94 (2010).
[CrossRef] [PubMed]

O. Nadiarnykh, R. B. Lacomb, P. J. Campagnola, and W. A. Mohler, “Coherent and incoherent SHG in fibrillar cellulose matrices,” Opt. Express 15(6), 3348–3360 (2007).
[CrossRef] [PubMed]

Légaré, F.

F. Légaré, C. Pfeffer, and B. R. Olsen, “The role of backscattering in SHG tissue imaging,” Biophys. J. 93(4), 1312–1320 (2007).
[CrossRef] [PubMed]

Millard, A. C.

S. V. Plotnikov, A. C. Millard, P. J. Campagnola, and W. A. Mohler, “Characterization of the myosin-based source for second-harmonic generation from muscle sarcomeres,” Biophys. J. 90(2), 693–703 (2006).
[CrossRef]

Mohler, W. A.

O. Nadiarnykh, R. B. Lacomb, P. J. Campagnola, and W. A. Mohler, “Coherent and incoherent SHG in fibrillar cellulose matrices,” Opt. Express 15(6), 3348–3360 (2007).
[CrossRef] [PubMed]

S. V. Plotnikov, A. C. Millard, P. J. Campagnola, and W. A. Mohler, “Characterization of the myosin-based source for second-harmonic generation from muscle sarcomeres,” Biophys. J. 90(2), 693–703 (2006).
[CrossRef]

Nadiarnykh, O.

O. Nadiarnykh, R. B. LaComb, M. A. Brewer, and P. J. Campagnola, “Alterations of the extracellular matrix in ovarian cancer studied by second harmonic generation imaging microscopy,” BMC Cancer 10(1), 94 (2010).
[CrossRef] [PubMed]

R. Lacomb, O. Nadiarnykh, and P. J. Campagnola, “Quantitative second harmonic generation imaging of the diseased state osteogenesis imperfecta: experiment and simulation,” Biophys. J. 94(11), 4504–4514 (2008).
[CrossRef] [PubMed]

O. Nadiarnykh, R. B. Lacomb, P. J. Campagnola, and W. A. Mohler, “Coherent and incoherent SHG in fibrillar cellulose matrices,” Opt. Express 15(6), 3348–3360 (2007).
[CrossRef] [PubMed]

Olsen, B. R.

F. Légaré, C. Pfeffer, and B. R. Olsen, “The role of backscattering in SHG tissue imaging,” Biophys. J. 93(4), 1312–1320 (2007).
[CrossRef] [PubMed]

Patterson, M. S.

T. J. Farrell, M. S. Patterson, and B. Wilson, “A diffusion theory model of spatially resolved, steady-state diffuse reflectance for the noninvasive determination of tissue optical properties in vivo,” Med. Phys. 19(4), 879–888 (1992).
[CrossRef] [PubMed]

Pfeffer, C.

F. Légaré, C. Pfeffer, and B. R. Olsen, “The role of backscattering in SHG tissue imaging,” Biophys. J. 93(4), 1312–1320 (2007).
[CrossRef] [PubMed]

Plotnikov, S. V.

S. V. Plotnikov, A. C. Millard, P. J. Campagnola, and W. A. Mohler, “Characterization of the myosin-based source for second-harmonic generation from muscle sarcomeres,” Biophys. J. 90(2), 693–703 (2006).
[CrossRef]

Prahl, S. A.

W. F. Cheong, S. A. Prahl, and A. J. Welch, “A review of the optical properties of biological tissues,” IEEE J. Quantum Electron. 26(12), 2166–2185 (1990).
[CrossRef]

Tang, P.

Wang, L.

Webb, W. W.

A. C. Kwan, D. A. Dombeck, and W. W. Webb, “Polarized microtubule arrays in apical dendrites and axons,” Proc. Natl. Acad. Sci. U.S.A. 105(32), 11370–11375 (2008).
[CrossRef] [PubMed]

R. M. Williams, W. R. Zipfel, and W. W. Webb, “Interpreting second-harmonic generation images of collagen I fibrils,” Biophys. J. 88(2), 1377–1386 (2005).
[CrossRef]

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

Welch, A. J.

W. F. Cheong, S. A. Prahl, and A. J. Welch, “A review of the optical properties of biological tissues,” IEEE J. Quantum Electron. 26(12), 2166–2185 (1990).
[CrossRef]

Williams, R. M.

R. M. Williams, W. R. Zipfel, and W. W. Webb, “Interpreting second-harmonic generation images of collagen I fibrils,” Biophys. J. 88(2), 1377–1386 (2005).
[CrossRef]

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

Wilson, B.

T. J. Farrell, M. S. Patterson, and B. Wilson, “A diffusion theory model of spatially resolved, steady-state diffuse reflectance for the noninvasive determination of tissue optical properties in vivo,” Med. Phys. 19(4), 879–888 (1992).
[CrossRef] [PubMed]

Zettel, M. L.

Zipfel, W. R.

R. M. Williams, W. R. Zipfel, and W. W. Webb, “Interpreting second-harmonic generation images of collagen I fibrils,” Biophys. J. 88(2), 1377–1386 (2005).
[CrossRef]

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

Biophys. J. (4)

R. M. Williams, W. R. Zipfel, and W. W. Webb, “Interpreting second-harmonic generation images of collagen I fibrils,” Biophys. J. 88(2), 1377–1386 (2005).
[CrossRef]

R. Lacomb, O. Nadiarnykh, and P. J. Campagnola, “Quantitative second harmonic generation imaging of the diseased state osteogenesis imperfecta: experiment and simulation,” Biophys. J. 94(11), 4504–4514 (2008).
[CrossRef] [PubMed]

S. V. Plotnikov, A. C. Millard, P. J. Campagnola, and W. A. Mohler, “Characterization of the myosin-based source for second-harmonic generation from muscle sarcomeres,” Biophys. J. 90(2), 693–703 (2006).
[CrossRef]

F. Légaré, C. Pfeffer, and B. R. Olsen, “The role of backscattering in SHG tissue imaging,” Biophys. J. 93(4), 1312–1320 (2007).
[CrossRef] [PubMed]

BMC Cancer (1)

O. Nadiarnykh, R. B. LaComb, M. A. Brewer, and P. J. Campagnola, “Alterations of the extracellular matrix in ovarian cancer studied by second harmonic generation imaging microscopy,” BMC Cancer 10(1), 94 (2010).
[CrossRef] [PubMed]

IEEE J. Quantum Electron. (1)

W. F. Cheong, S. A. Prahl, and A. J. Welch, “A review of the optical properties of biological tissues,” IEEE J. Quantum Electron. 26(12), 2166–2185 (1990).
[CrossRef]

J. Opt. Soc. Am. A (1)

Med. Phys. (1)

T. J. Farrell, M. S. Patterson, and B. Wilson, “A diffusion theory model of spatially resolved, steady-state diffuse reflectance for the noninvasive determination of tissue optical properties in vivo,” Med. Phys. 19(4), 879–888 (1992).
[CrossRef] [PubMed]

Nat. Biotechnol. (1)

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

Opt. Express (2)

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

A. C. Kwan, D. A. Dombeck, and W. W. Webb, “Polarized microtubule arrays in apical dendrites and axons,” Proc. Natl. Acad. Sci. U.S.A. 105(32), 11370–11375 (2008).
[CrossRef] [PubMed]

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Figures (10)

Fig. 1
Fig. 1

Experimental setup for in vivo measurement of tumor collagen SHG F/B ratio.

Fig. 2
Fig. 2

Back scattering of forward-propagating SHG and propagation of direct backward propagating SHG

Fig. 3
Fig. 3

Monte Carlo simulation of forward propagating and subsequently backscattered SHG photons which reach the object plane.(a) Steady state radial distribution of the backscattered SHG photons over a large range of radial position (3mm) (b) The same distribution over a short radial range (50um)

Fig. 4
Fig. 4

Relative SHG intensity vs. relative pinhole size curves at different collagen SHG F/B ratios, based upon Eq. (4). The relative SHG intensities equal 1 at a pinhole size of r = Rmax = 28.

Fig. 5
Fig. 5

Configuration of objective, sample and scatterer holder

Fig. 6
Fig. 6

Determination of SHG spot size. (a) CCD captured image of SHG focal spot after the tube lens (b) SHG intensity along the straight line that goes through the center of SHG spot. The red straight line in (a) is the line along which SHG intensities are measured. The red curve in (b) is the Gaussian fit to the intensities we measured in (a)

Fig. 7
Fig. 7

In vivo SHG images of rat tail collagen fibers in an intact rat tail. Images 1-5 are SHG images of the same ROI when the size of the pinhole varied from 60um, 100um, 150um, 200um, to 7000um, respectively. The bright spots are blue fluorescent polystyrene beads for calibration. Images are 600 um across.

Fig. 8
Fig. 8

Epidetected total collagen SHG intensity (blue triangles), or total bead TPEF intensity (blue squares) vs. pinhole size, fit to the model given by Eq. (4) (red lines). The horizontal axis is pinhole size in units of backward propagating SHG spot size on the pinhole plane i.e. fraction of ω. The vertical axis is normalized SHG intensity. Note that the fitting curves identically equal 1 when the pinhole size reaches R = 28, whose data point is not shown, and that the intensity information for R = 28 provides the normalization value and hence is included in the overall fit.

Fig. 9
Fig. 9

Monte Carlo simulation of forward propagating SHG that is subsequently backscattered and reaches the object plane, with varying µs. Steady state radial distribution of the backscattered SHG photons when (a) µs = 100 cm−1 (b) µs = 150 cm−1 (c) µs = 200 cm−1 (d) µs = 250 cm−1 (e) µs = 300 cm−1 (f) µs = 350 cm−1 (g) µs = 400 cm−1 (h) µs = 450 cm−1 (i) µs = 500 cm−1. In each case µa = 0.7 cm−1

Fig. 10
Fig. 10

Monte Carlo simulation of forward propagating SHG that is subsequently backscattered and reaches the object plane, with varying µa. (a) Steady state radial distribution of the backscattered SHG photons when (a) µa = 0.05 cm−1 (b) µa = 0.1 cm−1 (c) µa = 1 cm−1 (d) µa = 2 cm−1 (e) µa = 3 cm−1 (f) µa = 4 cm−1 (g) µa = 5 cm−1 (h) µa = 6 cm−1 (i) µa = 7 cm−1. In all three cases µs = 500 cm−1

Tables (2)

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Table 1 Results of in vivo measured rat tail collagen SHG F/B ratio

Tables Icon

Table 2 Results of in vitro measured rat tail collagen SHG F/B ratio

Equations (5)

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ISHG(r)=Bexp[2(rω)2]+FC,
ISHG(r)=B[exp[2(rω)2]+FBC],
Ipixel02πdθ0R{exp[2(rω)2]+(FB)C}rdr,
Irel(R)=02πdθ0R{exp[2(rω)2]+(FB)C}rdr02πdθ0Rmax{exp[2(rω)2]+(FB)C}rdr,
measuredcollagenSHGF/BRatiomeasuredbeadsTPEFF/BRatio=realcollagenSHGF/BRatiorealbeadsTPEFF/BRatio.

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