Abstract

A theoretical and experimental study of the THG signal from a reference interface in confocal microscope allows precise analysis of beam propagation and optimization of the focusing objectives.

© 2004 Optical Society of America

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References

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    [CrossRef] [PubMed]
  2. W. Denk, �??Fluctuation analysis of motor protein movement and single enzyme kinetics,�?? Proc. Natl. Acad. Sci. U.S.A. 91, 6629 (1994)
    [CrossRef] [PubMed]
  3. K. Svoboda et al., �??In vivo dentitric calcium dynamics in neocortical pyramidal neurons,�?? Nature (London) 385, 161-165(1997)
    [CrossRef]
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    [CrossRef]
  5. P. J. Campagnola et al., �??High resolution non-linear optical microscopy of living cells by second harmonic generation,�?? Biophys. J. 77, 3341-3349 (1999)
    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef]
  8. Y. Barad et al., �??Nonlinear scanning laser microscopy by third-harmonic generation,�?? Appl. Phys. Lett. 70, 922-924 (1997)
    [CrossRef]
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    [CrossRef]
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    [CrossRef]
  12. E.O. Potma, �??Intracellular Molecular Diffusion Probed with Nonlinear Optical Microscopy,�?? thesis University of Groningen (2001)
  13. F. Cannone, G. Chirico, G. Baldini, A. Diaspro, �??Measurement of the laser pulse width on the microscope objective plane by modulated autocorrelation method,�?? J. Microscopy 210, No. 2 (2003)
    [CrossRef]
  14. R. W. Boyd, �??Nonlinear optics, Academic Press,�?? San Diego (1992) Chapter 1 and 2
  15. T. Y. Tsang, �??Optical third-harmonic generation at interfaces,�?? Phys. Rew. A 52, n° 35 (1995)
    [CrossRef]
  16. J.J. Stamnes, �??Waves in Focal Regions,�?? IOP, Bristol (1986)

Appl. Phys. Lett. (1)

Y. Barad et al., �??Nonlinear scanning laser microscopy by third-harmonic generation,�?? Appl. Phys. Lett. 70, 922-924 (1997)
[CrossRef]

Biophys. J. (1)

P. J. Campagnola et al., �??High resolution non-linear optical microscopy of living cells by second harmonic generation,�?? Biophys. J. 77, 3341-3349 (1999)
[CrossRef] [PubMed]

J. Microscopy (1)

F. Cannone, G. Chirico, G. Baldini, A. Diaspro, �??Measurement of the laser pulse width on the microscope objective plane by modulated autocorrelation method,�?? J. Microscopy 210, No. 2 (2003)
[CrossRef]

J. Microsc. (1)

M. Müller et al., �??3D microscopy of transparent objects using third-harmonic generation,�?? J. Microsc. 191, 266-274 (1998)
[CrossRef] [PubMed]

Nature (1)

K. Svoboda et al., �??In vivo dentitric calcium dynamics in neocortical pyramidal neurons,�?? Nature (London) 385, 161-165(1997)
[CrossRef]

Opt. Commun. (1)

M. D. Duncan, �??Molecular discrimination and contrast enhancement using a scanning coherent anti-Stokes Raman microscope,�?? Opt. Commun. 50, 307-312 (1984)
[CrossRef]

Opt. Lett. (2)

Phys. Rev. A (1)

M. Kempe and W. Rudolph, �??Femtosecond pulses in the focal region of lenses,�?? Phys. Rev. A 48, n°6 (1993)
[CrossRef]

Phys. Rew. A (1)

T. Y. Tsang, �??Optical third-harmonic generation at interfaces,�?? Phys. Rew. A 52, n° 35 (1995)
[CrossRef]

Proc. Natl. Acad. Sci. (1)

W. Denk, �??Fluctuation analysis of motor protein movement and single enzyme kinetics,�?? Proc. Natl. Acad. Sci. U.S.A. 91, 6629 (1994)
[CrossRef] [PubMed]

Rev. Sci. Instrum. (1)

J. Squier, M. Müller, �??High resolution non linear microscopy: A review of sources and methods for achieving optimal imaging,�?? Rev. Sci. Instrum. 72, n° 7 (2001)
[CrossRef]

Science (1)

W. Denk, J. H. Strickler, and W.W. Webb, �??Two-photon laser scanning fluorescence microscopy,�?? Science 248, 73-76 (1990)
[CrossRef] [PubMed]

Other (3)

J.J. Stamnes, �??Waves in Focal Regions,�?? IOP, Bristol (1986)

E.O. Potma, �??Intracellular Molecular Diffusion Probed with Nonlinear Optical Microscopy,�?? thesis University of Groningen (2001)

R. W. Boyd, �??Nonlinear optics, Academic Press,�?? San Diego (1992) Chapter 1 and 2

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Figures (7)

Fig. 1.
Fig. 1.

Picture of the laser beam propagating on the sample. z0 is the entrance face of the coverslip and z1 is the output face. The beam waist is moved along the z axis with a piezoelectric activator mounted on the microscope objective

Fig.2.
Fig.2.

Theoretical normalized third-harmonic intensity plot along the z axis for one (x, y) point. The beam spot is displaced on the sample with an optical scanner.

Fig. 3.
Fig. 3.

Optical setup for THG microscopy experiment. A Zeiss Axiovert 200 M microscope is modified to collect correctly the third-harmonic signal. Two lasers are respectively used: a synchronously pumped OPO providing 150 fs pulse duration at 1.5μm, with a repetition rate of 80 MHz and an average power of 350 mW and then a T-pulse laser providing 200 fs pulse duration at 1.03 μm, with a repetition rate of 50 MHz and an average power of 1.1 W. The THG light is collected by a specific condenser and after filtering of the IR wavelength, it’s recorded by a photomultiplier tube. The current generated by the PMT is amplified and collected. The computer synchronizes both the scanning process and data collection.

Fig. 4.
Fig. 4.

Experimental third-harmonic intensity plot (in nA) along fundamental laser beam power (in mW) in log scale

Fig 5.
Fig 5.

Lag of the position of the third-harmonic intensity with the centred position for λ =1.5 μm (A) and for λ = 1.03 μm (B)

Fig. 6.
Fig. 6.

FWHM of the third-harmonic intensity in each (x, y) point for λ =1.5 μm (A) and for λ = 1.03 μm (B).

Fig. 7.
Fig. 7.

logarithmic ratio (Pω / τp) in each (x, y) point for λ =1.5 μm (A) and for λ = 1.03 μm (B).

Equations (20)

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E ω = 4 P ω T π c ε ω τ p
E 3 ω = A 1 W 2 E ω 3 J
with A = i χ 3 8 λ 3
J = z 0 z 1 exp ( i Δ k z ) ( 1 + 2 iz / b ) 2 dz
I 3 ω E 3 ω E 3 ω * = B 1 W 4 I ω 3 J 2 with B = A A *
I ω E ω E ω * = D P ω τ p with D = 4 T πc ε 0
I 3 ω = K 1 W 4 P ω 3 τ p 3 J 2 with K = BD 3
FWHM ( J z 0 2 ) = Δz = 0.7927 b + 0.2178
FWHM ( I 3 ω ) = 1,5854 π λ w 0 2 + 0.2178
J 2 = 426.929 451.397 b + 162.216 b 2 24.3781 b 3 + 1.40895 b 4
I 3 ω norm = I 3 ω W L 4 J L 2 = K P ω 3 τ p 3
ln ( I 3 ω norm I 3 ω norm-centre ) = ln ( ( K P ω 3 τ p 3 ) ( K P ω 3 τ p 3 ) centre )
P ω τ p = ( P ω τ p ) centre + Δ ( P ω τ p )
1 3 ln ( I 3 ω norm I 3 ω norm-centre ) = Δ ( P ω τ p ) ( P ω τ p ) centre
w 0 = 0.518 λ NA
J = 1 4 ( b + 2 iz ) ( e bΔk 2 ( 2 i b 2 e bΔk 2 + iΔkz i b 2 e bΔk 2 ( 2 bΔk e bΔk 2 E i [ bΔk 2 ] ) + 2 b e bΔk 2 z ( 2 bΔk e bΔk 2 E i [ bΔk 2 ] ) i b 3 Δk Ei [ bΔk 2 + i Δk z ] + 2 b 2 Δk z Ei [ bΔk 2 + i Δk z ] ) )
Ei ( z ) = z e t t dt
J | z 0 = 1 4 ( b + 2 iz ) ( e bΔk 2 ( 2 i b 2 e bΔk 2 + iΔkz i b 3 Δk f ( z ) + 2 b 2 Δk z f ( z ) + 1 b + 2 i z 0 ( i b 3 ( 2 e 1 2 Δk ( b + 2 i z 0 ) + Δk ( b + 2 i z 0 ) f ( z 0 ) ) ) 1 b + 2 i z 0 ( 2 b 2 z ( 2 e 1 2 Δk ( b + 2 i z 0 ) + Δk ( b + 2 i z 0 ) f ( z 0 ) ) ) ) )
f ( u ) = Ei [ b Δk 2 + i Δk u ] for u < 0
f ( u ) = Ei [ b Δk 2 + i Δk u ] + 2 i π for u 0

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