3-Dimensional imaging of collagen using second harmonic generation
Introduction
Almost as soon as the first laser had been built, Franken et al. (1961) demonstrated that shining pulses of deep red ruby laser light through a quartz crystal produced ultraviolet light, the second harmonic of the original light. A recent paper by Gauderon et al. (2001) summarises the process of second harmonic generation (SHG). Briefly, as electromagnetic radiation propagates through matter, the electric field (E) exerts forces on the sample’s internal charge distribution. The consequent redistribution of charge generates an additional field component. The resultant dipole moment per unit volume is referred to as the electric polarization (P), and can be expressed as a sum of linear and nonlinear terms. The nonlinear components only become significant at very high light intensities. The primary nonlinear effect is a polarization of second order in the electric field and is given by (Yariv, 1967)where subscripts denote cartesian components and superscripts the relevant frequencies. χijk2ω is a (3×3×3) third-rank tensor, termed the second-order nonlinear optical susceptibility, whose elements sum to zero for material with inversion symmetry (Gauderon et al., 2001). This means that the ability to generate second harmonics is peculiar to molecules which are not centro-symmetric. Second harmonic generation will also take place at interfaces where there is a huge difference in refractive index, such as metal surfaces.
Collagen has a highly crystalline triple-helix structure which is not centro-symmetric, and several reports have noted that it is a very effective “upconverter” of light by second harmonic generation (Georgiou et al., 2000; Roth and Freund, 1981). However to date, even though scanning second harmonic microscopy was demonstrated 23 years ago (Gannaway and Sheppard, 1978), most second harmonic imaging of collagen has been restricted to low-resolution images. In the biomedical sciences, second harmonic generation has been used to amplify the effect of potential-sensitive styryl dyes (Bouevitch et al., 1993), which has recently been extended into the microscopic realm (Campagnola et al., 2001), though the latter paper also includes an image at approximately 1-μm resolution of collagen in a fish scale. This aspect was further pursued in a very recent paper (Campagnola et al., 2002) which has examined second harmonic imaging of a wide range of structural proteins, specially collagen, at resolutions of around 1 μm.
Mertz and Moreau have explored membrane imaging using dyes which produce both SHG and two-photon fluorescent signals (Mertz and Moreaux, 2001; Moreaux et al., 2001), and have shown that SHG microscopy has the potential to offer very high resolution. Gauderon and Sheppard (2000) have calculated the weak-object transfer function for a scanning harmonic microscope and showed that it in fact offered a degree of superresolution. The expected resolution, while not as good as that of a microscope operating at the harmonic frequency, can be superior to that of a similar microscope operating at the fundamental frequency, and can be improved further by using a confocal pinhole. These findings encouraged us to believe that in fact collagen could be imaged at the maximum resolution of an optical microscope.
Using a microscope that we designed to be optimised for second harmonic detection (Cox et al., 2002) we found that we could detect the second harmonic signal from collagen with much higher resolution and sensitivity than had been reported in previous studies, typically using excitation levels lower than required for exciting two-photon fluorescence. We present here evidence that this novel approach can (a) discriminate between type I and type III collagen, hitherto only possible by chemical analysis, and (b) identify collagen fibres in situations where conventional staining techniques do not reveal them. This is to the best of our knowledge the first report of microscopic collagen imaging using SHG which has demonstrated true diffraction-limited resolution.
Section snippets
Materials and methods
Sample preparation. Human endometrium samples came from a databank of specimens kept by the Department of Obstetrics and Gynaecology at the University of Sydney (Manconi et al., 2001). Samples were either 50-μm-thick cryostat sections of unfixed tissue or 30- to 100-μm microtome sections of formalin-fixed paraffin-embedded material examined without further staining. Mouse skin samples were prepared by conventional histological techniques, sectioned at 10-μm thickness, and stained with Masson’s
Results and discussion
Characterising the signal. Our initial experiments were carried out as part of a long-term study of the three-dimensional tissue architecture in human endometrium (Manconi et al., 2001). Using titanium-sapphire laser excitation, with pulsewidths around 200 fs FWHM, we were able to obtain excellent bright, high-resolution images of collagen in both conventional histological sections and cryostat sections. Fig. 1a shows the collagen surrounding a mucus gland in human endometrium, in a cryosection
Conclusions
Since SHG, like two-photon excited fluorescence (TPF), is a nonlinear process depending on the square of the incident light intensity, it is depth selective without the requirement for confocal optics. In practical terms SHG, TPF, and confocal microscopy can be regarded as equivalent in terms of optical sectioning, since in each case the depth discrimination depends upon the quadrature of the optical point-spread function. However SHG is a coherent process whereas TPF and confocal fluorescence
Acknowledgements
We thank Colin Sheppard, Régis Gauderon, Paul Xu, and Phil Lukins for introducing us to second harmonic microscopy, and for much valuable discussion about the physical principles involved. We are equally indebted to Teresa Dibbayawan and David Sillence for information about collagen, Margot Hosie for the trichrome-stained sections of skin, Xin Wang for preparing liver sections, and Pilar Garin-Chesa and Wolfgang Rettig for the F19 antibody. M.D.G. is supported by a grant from the National
References (26)
- et al.
Probing membrane potential with non-linear optics
Biophys. J.
(1993) - et al.
Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues
Biophys. J.
(2002) - et al.
Laser-scanning coherent anti-Stokes Raman scattering (CARS) microscopy and applications to cell biology
Biophys. J.
(2002) - et al.
Optimization of second-harmonic generation microscopy
Micron
(2001) - et al.
Second and third optical harmonic generation in type I collagen, by nanosecond laser irradiation, over a broad spectral region
Opt. Commun.
(2000) - et al.
Computer-generated three-dimensional reconstruction of uterine histological parallel serial sections displaying microvascular and glandular structures in human endometrium
Micron
(2001) Xenografts for tendon and ligament repair
Biomaterials
(1994)- et al.
Coherent scattering in multi-harmonic microscopy
Biophys. J.
(2001) - et al.
Farbstoff analytische Untersuchungen zum polarisationsmikropischen Nachweis von Kollagen mit Solaminrot 4B (Teil II)
Acta Histochem.
(1991) - et al.
Extracellular matrix degradation and the role of hepatic stellate cells
Semin. Liver Dis.
(2001)
Principles of Optics
Peacock’s Elementary Microtechnique
Second-harmonic imaging of living cells
J. Biomed. Opt.
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