Chemically resolved imaging of biological cells and thin films by infrared scanning near-field optical microscopy.

The infrared (IR) absorption of a biological system can potentially report on fundamentally important microchemical properties. For example, molecular IR profiles are known to change during increases in metabolic flux, protein phosphorylation, or proteolytic cleavage. However, practical implementation of intracellular IR imaging has been problematic because the diffraction limit of conventional infrared microscopy results in low spatial resolution. We have overcome this limitation by using an IR spectroscopic version of scanning near-field optical microscopy (SNOM), in conjunction with a tunable free-electron laser source. The results presented here clearly reveal different chemical constituents in thin films and biological cells. The space distribution of specific chemical species was obtained by taking SNOM images at IR wavelengths (lambda) corresponding to stretch absorption bands of common biochemical bonds, such as the amide bond. In our SNOM implementation, this chemical sensitivity is combined with a lateral resolution of 0.1 micro m ( approximately lambda/70), well below the diffraction limit of standard infrared microscopy. The potential applications of this approach touch virtually every aspect of the life sciences and medical research, as well as problems in materials science, chemistry, physics, and environmental research.

Time-resolved, light scattering measurements of cartilage and cornea denaturation due to free electron laser radiation.

Light scattering is used to monitor the dynamics and energy thresholds of laser-induced structural alterations in biopolymers due to irradiation by a free electron laser (FEL) in the infrared (IR) wavelength range 2.2 to 8.5 microm. Attenuated total reflectance (ATR) Fourier-transform IR (FTIR) spectroscopy is used to examine infrared tissue absorption spectra before and after irradiation. Light scattering by bovine and porcine cartilage and cornea samples is measured in real time during FEL irradiation using a 650-nm diode laser and a diode photoarray with time resolution of 10 ms. The data on the time dependence of light scattering in the tissue are modeled to estimate the approximate values of kinetic parameters for denaturation as functions of laser wavelength and radiant exposure. We found that the denaturation threshold is slightly lower for cornea than for cartilage, and both depend on laser wavelength. An inverse correlation between denaturation thresholds and the absorption spectrum of the tissue is observed for many wavelengths; however, for wavelengths near 3 and 6 microm, the denaturation threshold does not exhibit the inverse correlation, instead being governed by heating kinetics of tissue. It is shown that light scattering is useful for measuring the denaturation thresholds and dynamics for different biotissues, except where the initial absorptivity is very high.