The physical mechanism underlying multiphoton luminescence in gold is still the subject of debate. To obtain a better understanding of the mechanism, experiments that study the luminescence spectra of single particles are necessary. In this study, the multiphoton luminescence spectrum was measured for surrounding media of different refractive indices. The resulting spectra of single gold nanospheres with diameters in the range of a few tens of nanometers were found to
be strongly dominated by the absorption peak of the plasmon resonance. This is in agreement with the theory proposed by Boyd et al. (1986)1. According to Lorenz-Mie Theory, an increase in the refractive index of the surrounding medium results in a redshift of the plasmon resonance spectrum; a corresponding shift in the multiphoton luminescence spectrum has now been found experimentally.
Theileria annulata is an intracellular parasite that infects and transforms bovine leukocytes, inducing continuous proliferation of its host cell both in vivo and in vitro. Theileria-infected cells can easily be propagated in the laboratory and serve as a good model for laser ablation studies. Using single pulses from an amplified ultrashort pulse laser system, we developed a technique to introduce submicrometer holes in the plasma membrane of the intracellular schizont stage of Theileria annulata. This was achieved without compromising either the viability of the organisms or that of the host cell that harbors the parasite in its cytoplasm. Multiphoton microscopy was used to generate image stacks of the parasite before and after ablation. The high axial resolution allowed precise selection of the region of the membrane that was ablated. It also allowed observation of the size of the holes generated (in fixed, stained cells) and determination of the structural changes in the parasite resulting from the laser pulses (in living cells in vitro). This technique opens a new possibility for the transfection of Theileria or delivery of molecules to the schizont that may prove useful in the study of this special host-parasite relationship.
Noble metal nanoparticles are characterized by a strong peak in the scattering and absorption spectrum, termed the
plasmon resonance. Researchers have taken advantage of this to create a new label for biological molecules. A
disadvantage of techniques based on scattering and absorption is that the detected signal is at the same wavelength
as the incident light, making it more challenging to discriminate between signal and background. Gold
nanoparticles also luminesce, suggesting an alternate method for their detection. A tightly focused ultra-short
pulse laser beam can be used to achieve multiphoton excitation of the particles; the resulting luminescence
exhibits a peak in the same region of the spectrum as the plasmon resonance. Because excitation is nonlinear,
significant luminescence is only observed when the particle is in the focus, permitting localization with both high
lateral and axial resolution. The physical mechanism underlying multiphoton luminescence in gold is still the
subject of debate. Here, we present a systematic study in single gold nanospheres with diameters between 15 nm
and 100 nm using peak laser intensities between 10 and 350 GW/cm2. A scattering confocal microscope
incorporated in the setup was used to distinguish single particles from clusters. We observed that not all gold
nanospheres have a detectable multiphoton luminescence signal; however, laser intensities above an exposure-time
dependent threshold can alter such particles so that they do. In addition, we found that gold nanoparticles
exposed to laser intensities above about 150 GW/cm2 can exhibit behavior reminiscent of the bleaching and
blinking of conventional fluorophores.
Collagen possesses a strong second order nonlinear susceptibility; when it is irradiated with intense laser light, some of the reflected and transmitted light will have twice the frequency of the incident beam, a phenomenon known as second harmonic generation (SHG). Polarization modulation of an ultra-short pulse laser beam can be used to simultaneously measure collagen fiber orientation, SHG intensity, and a parameter related to the second order non-linear susceptibility. This technique has made it possible to discriminate among patterns of fibrillar orientation in many tissues. In the present study the role that organizational complexity plays in the relationship between nonlinear optical properties and collagen structure is investigated. As a component of tissues and organs, collagen’s structure and function is inextricably intertwined with that of the many other matrix components; to what extent do these noncollagenous components affect its nonlinear properties? To answer this, we investigated SHG in two different collagenous tissues, liver and cartilage; in addition we looked at the effect of progressive pathological changes in these tissues on SHG. At the other end of the spectrum, we studied collagen organized at the minimal level of complexity necessary for SHG detection: fibrils generated from solutions containing only a single type of collagen. Data obtained from these studies suggest that collagen’s strong nonlinear susceptibility, a property no other biologically significant macromolecule shares to the same degree, may serve as more than the basis of a novel imaging device for soft tissue. Collagen’s nonlinear optical properties in conjunction with its vast capacity for self-initiated conformational change--through self-assembly, site recognition, post-translational modification, and the like -make it an attractive candidate molecule for any of several demanding engineering applications, such as nanopatterning.
The effects of structural perturbation on second harmonic generation in collagen were investigated. Type I collagen fascicles obtained from rat tails were structurally modified by increasing nonenzymatic cross-linking, by thermal denaturation, by collagenase digestion, or by dehydration. Changes in polarization dependence were observed in the dehydrated samples. Surprisingly, no changes in polarization dependence were observed in highly crosslinked samples, despite significant alterations in packing structure. Complete thermal denaturation and collagenase digestion produced samples with no detectable second harmonic signal. Prior to loss of signal, no change in polarization dependence was observed in partially heated or digested collagen.
We use polarization-modulated second harmonic generation to image fiber orientation in collagen tissues, with an axial resolution of about 10 micrometers and a transverse resolution of up to 1 micrometers . A linearly polarized ultra-short pulse (200 fs) Ti:Sapphire laser beam is modulated using an electro-optic modulator and quarter-wave plate combination and focused onto a translation stage mounted sample using a microscope objective. The generated second harmonic light is collected using a photomultiplier tube and demodulated using phase sensitive detection to obtain signal intensity and fiber orientation information. In order to obtain second harmonic generation images of different types of collagen organization, we analyze several different tissues, including rat-tail tendon, mouse aorta, mouse fibrotic liver, and porcine skin. We can use our technique to image fibrotic tissue in histological sections of damaged liver and to identify burned tissue in porcine skin to a depth of a few hundred microns. Polarization-modulated second harmonic generation potentially could be a useful clinical technique for diagnosing collagen related disease or damage, especially in the skin.
The measurement of the second order nonlinear susceptibility of collagen in various biological tissues has potential applications in the detection of structural changes which are related to different pathological conditions. We investigate second harmonic generation in a rat-tail tendon, a highly organized collagen structure consisting of parallel fibers. Using an electro-optic modulator and a quarter-wave plate, we modulate the linear polarization of an ultra-short pulse laser beam that is used to measure second harmonic generation in a confocal microscopy setup. Phase-sensitive detection of the generated signal, coupled with a simple model of the collagen protein structures, allows us to measure a parameter (gamma) related to nonlinear susceptibility and to determine the relative orientation of the structures. Our preliminary results indicate that it may be possible to use this parameter to characterize the structure.
A confocal microscopy imaging system was devised to selectively detect second harmonic signals generated by biological tissues. Several types of biological tissues were examined using this imaging system, including human teeth, bovine blood vessels, and chicken skin. All these tissues generated strong second harmonic signals. There is considerable evidence that the source of these signals in tissue is collagen. Collagen, the predominant component of most tissues, is known to have second order nonlinear susceptibility. This technique may have diagnostic usefulness in pathophysiological conditions characterized by changes in collagen structure including malignant transformation of nevi, progression of diabetic complications, and abnormalities in wound healing.
A confocal microscopy imaging system was devised to selectively detect second harmonic signals generated by biological tissues. Several types of biological tissues were examined using this imaging system, including human teeth, bovine blood vessels, and chicken skin. All these tissues generated strong second harmonic signals. There is considerable evidence that the source of these signals in tissue is collagen. Collagen, the predominant component of most tissues, is known to have second order nonlinear susceptibility. This technique may have diagnostic usefulness in pathophysiological conditions characterized by changes in collagen structure including malignant transformation of nevi, progression of diabetic complications, and abnormalities in wound healing.
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