Stimulated Raman scattering (SRS) microscopy is used to generate structural and chemical three-dimensional images of native skin. We employed SRS microscopy to investigate the microanatomical features of skin and penetration of topically applied materials. Image depth stacks are collected at distinct wavelengths corresponding to vibrational modes of proteins, lipids, and water in the skin. We observed that corneocytes in stratum corneum are grouped together in clusters, 100 to 250 μm in diameter, separated by 10- to 25-μm-wide microanatomical skin-folds called canyons. These canyons occasionally extend down to depths comparable to that of the dermal–epidermal junction below the flat surface regions in porcine and human skin. SRS imaging shows the distribution of chemical species within cell clusters and canyons. Water is predominately located within the cell clusters, and its concentration rapidly increases at the transition from stratum corneum to viable epidermis. Canyons do not contain detectable levels of water and are rich in lipid material. Oleic acid-d34 applied to the skin surface lines the canyons down to a depth of 50 μm below the surface of the skin. This observation could have implications on the evaluation of penetration profiles of bioactive materials measured using traditional methods, such as tape-stripping.
We demonstrate a fiber-based two-color source of picosecond pulses for coherent Raman scattering (CRS) microscopy.
An Yb-doped fiber laser combined with a divided-pulse amplifier produce up to 3 W of power tunable from 1030 nm to
1040 nm. A normal dispersion photonic crystal fiber is used to blue-shift the pulses through seeded four-wave mixing.
Pulses with up to 150 mW of average power are produced, tunable between 770 nm and 800 nm. Imaging of animal
tissue and cells is demonstrated.
We report a fiber-based high-power picosecond laser system for coherent Raman microscopy (CRM). This source
generates 3-ps pulses with 6 W average power at 1030 nm.
Frequency-doubling yields more than 2 W of green light,
which can be used to pump a commercial optical parametric oscillator to produce the pump and Stokes beams for CRM.
The design and performance of the laser are described, along with an application to CARS imaging.
Near-IR ultrafast pulse laser and confocal microscope are combined to create a multiphoton multichannel non-linear imaging technique, which allows in situ 3-D characterization of nonfluorescent nanoparticles in biological systems. We observed intense CARS signals generated from various metal oxides due to their high third-order nonlinear susceptibilities (Chi(3)), which do not depend on the vibrational resonance but on the electronic resonance. We show that fine and ultrafine particles of metal oxides in alveolar macrophage cells may be imaged in vitro using CARS and multiphoton fluorescence microscopy with highest optical resolution for extended periods without photobleaching effects. The advantage of the epi-detection over the forward detection for imaging sub-micron particles has been investigated.
Gary Holtom, Brian Thrall, Thomas Weber, Leyun Zhu, Derek Hopkins, Christopher Parkinson, Steven Colson, John Price, Beek Yoke Chin, Augustine Choi, Terence Risby
Virtually all laser based microscopy imaging methods involve a single laser, with ultrafast lasers emerging as the enabling tool for a variety of methods. Two-photon fluorescence is a high sensitivity method with selectivity depending on a chromophore that is either added or produced by genetic engineering. While there are fundamental advantages over white light or other fluorescence microscopies, there are unavoidable limitations such as bleaching, photoinduced damage to the cell, and the inability to label some major constituents of the cell, particularly the abundant species. Raman imaging affords chemical selectivity but application is limited due particularly to its low sensitivity and unavoidable fluorescence background. Adding a second laser beam, shifted from the first laser by a molecular vibrational frequency, increases the detected Raman signal by many orders of magnitude and in addition shifts the detected signal to the high energy (blue) side of both lasers, removing fluorescence artifacts. Signal levels sufficient to acquire high signal-to-noise ratio images of 200 by 200 pixels in one minute requires sub-nanojoule pulse energy. A convenient, tunable source of the Stokes-shifted beam is provided by an Optical Parametric Amplifier (OPA), which requires an amplified laser. 250-kHz sources have ample energy and in addition keep the average sample power on the order of 0.1 mW, a level that even sensitive biological systems tolerate at the focal spot diameter of 0.3 micrometers . Long-term viability of mammalian cells has been demonstrated during dozens of scans in a single plane. Two-photon fluorescence provides a useful complimentary data channel that is acquired simultaneously with the Raman image. Several dyes and green fluorescence protein have been used for this purpose. Interpretation of images, acquiring three dimensional images, and identification of cellular features are ongoing activities.
Femtosecond optical parametric oscillators (OPO), synchronously pumped by Ti:Sapphire lasers, operating in the near infrared (IR) region are an important light source now under active development. We report the results of our Ti:Sapphire synchronously pumped noncritically phase matched femtosecond OPOs that are based upon several crystals from the KTP family. The newly developed nonlinear crystal niobium doped KTP (Nb:KTP) has a greater birefringence than undoped KTP and is shown to extend the wavelength farther into the mid-IR. We report the first operation of a femtosecond OPO utilizing the solid-solution grown crystal Nb:KTP. Additionally, we show that CTA is very useful in mid-IR angle tuned OPOs.
Near-field fluorescence microscopy combines nanometer resolution with the ability to conduct optical measurements with high sensitivity. To illustrate the potential of the near-field technique, we summarize recent results from our laboratory which include the fluorescence imaging of single dye molecules and single proteins and picosecond fluorescent lifetime measurements on intact photosynthetic membranes, all done at room temperature with nanometric spatial resolution.
Lumazine protein is a 21200 Da protein containing a single tryptophan residue and a non-covalently bound, highly fluorescent ligand, 6,7-dimethyl-8-ribityllumazine. Visser et al. have reported that excitation into the region of the absorption of the tryptophan residue, around 300 nm, produces a distinct rise in fluorescence emission at 475 nm from the lumazine ligand. They analyzed a rise rate of around 1 ns and attributed this to energy transfer between the tryptophan donor and the lumazine as acceptor. This present report re-investigates this phenomenon using a ten times higher resolution (FWHM = 23 ps) . The fluorescence rise is found to be more complex and can only approximately be fitted by a sum of two exponential processes, with rise times of 0.02 and 0.6 ns. For the fluorescence of the tryptophan measured at 340 nm, no rise is detected but the decay is similarly much more complex than previously recognised with data taken at lower resolution. Global analysis of three 340 nm decay curves taken with time windows of 1.2, 4.8, and 55.6 ps/channel, results in about 5 exponential components being required for a satisfactory fit to the fluorescence decay.
The goal of time resolved fluorescence experiments is to determine the validity of a model for population (kinetics) or motion (decay of anisotropy), and to extract a suitable parameter set which quantitatively describes the sample. Ideally, this analysis requires no adjustments of an ad-hoc nature in order to obtain a good fit, and the time-resolved residuals will be uniformly random. Any problems at this point lead to questions about the model being used, either with respect to its correctness in functional form, or in the exact values recovered, and it may become difficult to extract useful information about the sample. Reaching the ideal situation, and confirming that there are no experimental problems, requires considerable care. All aspects of the hardware are examined, starting with the laser light source and sample illumination optics. Collection optics for the fluorescence are analyzed, along with polarization components. Electronics checks and optimizations are described, showing the effects of characteristic problems. Finally, apparent artifacts due to numerical analysis are shown. While the examples are given for time-correlated single-photon counting, many of the optics related problems have similar consequences in the frequency domain. Some of the experimental problems have implications for the design of multiple channel detection schemes.
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