Localized IR spectroscopy of hemoglobin

Abstract

IR absorption spectroscopy of hemoglobin was performed using an IR optical parametric oscillator laser and a commercial atomic force microscope. This experimental approach enables detection of protein samples with a resolution that is much higher than that of standard IR spectroscopy. Presented here are AFM based IR absorption spectra and images of micron sized hemoglobin features. IntroductionAbsorption spectroscopy is a widely applied technique for chemical characterisation. This method is able to detect both luminescent and non-luminescent materials and provide chemical specific information. An extensively used form of absorption spectroscopy is infrared absorption (IR) spectroscopy. This measures specific frequencies in the infrared region of the electromagnetic spectrum at which constituent parts of molecules corresponding to specific types of molecular bonds vibrate. This makes possible structural elucidation and compound identification of materials. As a consequence, IR absorption is extensively used as an analytical tool.

Nanoscale Mid-Infrared Imaging of Phase Separation in a Drug–Polymer Blend

“…Nanoscale mid‐IR spectroscopy has the potential to address this gap because it is ideally suited for the characterization of organic small molecules and polymers, providing both the high chemical information content of mid‐IR analysis and a high spatial (nanometer) resolution. The latter is achieved by using an AFM probe for detecting the photothermal expansion resulting from mid‐IR absorption, hence overcoming limitations in spatial resolution of current (mid‐IR and other) chemical imaging techniques originating from the diffraction limit of light 10–27. Currently, application of the technique has been mostly used for analyzing biological samples (e.g., various types of cells10–12,15,18), hemoglobin,24 the localization of energy storage polymers21 and viruses18,14 in bacteria and organometallic compounds in breast cancer cells,25 polymeric samples,8,10,16,19,22,27 and quantum dots and inorganics 13,17,26.…”

Nanoscale spectroscopy and imaging of hemoglobin

“…Previously we demonstrated single point IR absorption spectra for proteins [4]. In this letter, we demonstrate full IR absorption imaging at sub diffraction resolution of both hemoglobin aggregates and ledges with varying incident excitation radiation.…”

“…These changes in the relative amount of different bond excitations by the light arise from small sub-micron changes in the protein conformation [4]. For the region shown, the absorption arises from N-H and C-H vibrations.…”

“…However, emergent techniques allow for the utilization of a contact mode AFM to detect thermally induced dilation of a sample surface (e.g., [3,4,5]). Light incident on a sample tuned to a suitable absorption band, such as aliphatic C-H stretching for hemoglobin, will be partially absorbed, generating internal heat.…”

Nanoscale infrared absorption imaging permits non-destructive intracellular photosensitizer localization for subcellular uptake analysis

“…[22][23][24][25][26][27][28] Briefly, when incident pulses were tuned to a vibrational resonance of the cells or TBOGNPs the infrared light absorbed was proportional to the materials' absorption coefficients at the excitation wavelength, causing thermal heating which relaxed mechanically via expansion of the surface. 23 This deformation of the surface causes deflection of the mechanical probe in contact mode, which relaxes back according to a one end fixed-cantilever solution to the EulerBernoulli equation.…”