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The chemical and structural properties of biomolecules determine their interactions, and thus their functions, in a wide variety of biochemical processes. Innovative imaging methods have been developed to characterise biomolecular structures down to the angstrom level. However, acquiring vibrational absorption spectra at the single molecule level, a benchmark for bulk sample characterization, has remained elusive. Here, we introduce off-resonance, low power and short pulse infrared nanospectroscopy (ORS-nanoIR) to allow the acquisition of infrared absorption spectra and chemical maps at the single molecule level, at high throughput on a second timescale and with a high signal-to-noise ratio (~10-20). This high sensitivity enables the accurate determination of the secondary structure of single protein molecules with over a million-fold lower mass than conventional bulk vibrational spectroscopy. These results pave the way to probe directly the chemical and structural properties of individual biomolecules, as well as their interactions, in a broad range of chemical and biological systems.

Fatty infiltration of the liver is a common finding in alcoholic patients, but the mechanisms responsible for the accumulation of hepatic fat are not clear (2). In the present investigation, an attempt was made to clarify this relationship by studying the effect of ethanol on fatty acid metabolism in rat liver in vitro (3). MATERIALS AND METHODSSeventy-one male Sprague-Dawley rats weighing from 250 to 400 g were maintained on Purina Laboratory Chow, except for a 24 hour period immediately preceding the experiment. During this period, they were given 36.5 g glucose per kg together with 7.5 g ethanol per kg or, in the experiment shown in Table III, 50 g per kg fructose, administered by means of 5 gastric tube feedings of equal volume. Approximately 3 hours after the last gastric feeding, the animals were decapitated. The liver was quickly removed, and slices were prepared in the cold with a Stadie-Riggs slicer (4). The C14-labeled 1 and unlabeled substrates were dissolved in 5 ml isotonic KrebsRinger bicarbonate buffer, pH 7.4 (6), and the final concentration of the substrates was expressed as millimoles per liter. and the radioactivity counted in a Packard liquid scintillation counter. Randomized liver slices (approximately 0.5 g) were suspended in the incubation mixture in the main compartment of incubation flasks (8). The flasks were sealed with serum caps, gassed five minutes with a mixture of 95 per cent 02 and 5 per cent CO2, and then incubated for 3 hours at 37.50 C in a Dubnoff shaking water bath. Rat epididymal fat pads (0.5 to 1.0 g) were incubated in the same manner. At the end of the incubation, 0.2 ml of 10 N sulfuric acid was injected with a needle through the serum cap into the main compartment, and 1 ml of alkaline hyamine (9) into the center well. The, flasks were then shaken for 30 minutes in an ice bath to trap the evolving CO2 in the hyamine. The hyamine-C"4O. solution' was transferred into a volumetric flask and made up to 5 ml with toluene; 2 ml of this solution was mixed with 16 ml of toluene containing 0.4 per cent DPO and 0.005 per cent POPOP (9), and the radioactivity was measured in a Packard liquid scintillation counter. To calculate the disintegrations per minute (dpm), the obtained counts were multiplied by the efficiency of the counter which was determined separately for each experiment with an internal C"4 standard. C 40-2 was expressed in dpm per gram of wet tissue or per milligram of tissue nitrogen. The incubated tissue from each flask was homogenized in the incubation mixture, and an aliquot of this homogenate was used for determination of total nitrogen by the micro-Kjeldahl method (10). The rest of the homogenate was transferred to a screw-capped bottle, to which 0.4 ml of 90 per cent (wt/vol) potassium hydroxide per ml of homogenate was added. This mixture was saponified by autoclaving for 1 hour at 120°C. After cooling, ethanol was added to obtain a final concentration of 50 per cent. The mixture was vigorously shaken with 20 ml petroleum ether for 10 minutes to remove nonsa...

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