Hemoglobin nanocrystals were analyzed with tip-enhanced Raman scattering (TERS), surface-enhanced resonance Raman scattering (SERRS) and conventional resonance Raman scattering (RRS) using 532 nm excitation. The extremely high spatial resolution of TERS enables selective enhancement of heme, protein, and amino acid bands from the crystal surface not observed in the SERRS or RRS spectra. Two bands appearing at 1378 and 1355 cm(-1) assigned to the ferric and ferrous oxidation state marker bands, respectively, were observed in both TERS and SERRS spectra but not in the RRS spectrum of the bulk sample. The results indicate that nanoscale oxidation changes are occurring at the hemoglobin crystal surface. These changes could be explained by oxygen exchange at the crystal surface and demonstrate the potential of the TERS technique to obtain structural information not possible with conventional Raman microscopy.
Tip-enhanced Raman scattering (TERS) spectroscopy is a promising technique for nanoscale chemical analysis. However, there are several challenges preventing widespread application of this technology, including reproducible fabrication of efficient TERS probes. These problems reflect a lack of clear understanding of the origins of, and the parameters influencing TERS. It is believed that the coating characteristics at the apex of the tip have a major effect on the near-field optical enhancement and thus the TERS activity of a metalized probe. Here we show that the aspect ratio of the tip can play a significant role in the efficiency of TERS probes. We argue that the electrostatic field arising from the lightning-rod effect has a substantial role in the observed TERS effect. This argument is supported by 'edge-enhanced Raman scattering' which is shown for a noble metal film. Furthermore, it is reported that an associated tip-surface-enhanced Raman scattering effect can be achieved by using a TERS-inactive metalized probe on a surface-enhanced Raman spectroscopy-inactive roughened surface. This observation can be explained by an interparticle enhancement of the electromagnetic field.
The effects of fixation and dehydration procedures on heme environment inside human erythrocytes were examined using resonance Raman spectroscopy. The resonance Raman spectroscopic data along with far-infrared spectra show that hemoglobin in air-dried red blood cells and those fixed in the precipitating fixatives, methanol and acetone, changes to hemichrome, a low-spin component in which the sixth coordination site of the iron is occupied by the imidazole group of the distal histidine. The cross-linking fixatives, formaldehyde and glutaraldehyde, maintain hemoglobin in its native conformational state (oxyhemoglobin) as evidenced by the appearance of the oxygen ligand marker bands along with the skeletal sensitive modes. However, polymerization with cross-linking fixatives increases the auto-oxidation kinetics of hemoglobin and gradually oxidizes it to the met state. Moreover, the dehydration of a red blood cell under N 2 results in a change of oxyhemoglobin to a six coordinate low-spin Fe II derivative of hemoglobin, hemochrome.
The effects of fixation and dehydration on the distribution of heme-based molecules inside red blood cells and the structural integrity of the cells have been investigated using Raman mapping and AFM topographic imaging. A strong correlation was observed between the thickness of the cells as determined from AFM images and the intensity of the characteristic heme bands in the Raman maps, demonstrating that heme compounds are relatively evenly distributed inside dried and fixed cells in the majority of cases. The exception occurred when cells were dried in phosphate buffered saline, where more hemichrome appears close to the periphery of the cell despite the AFM image showing a plateau like topography. Using neat formaldehyde solution as a fixative is inadequate for a complete structural preservation and results in diffusion of hemoglobin into the surrounding area. However, a mixture of formaldehyde (3%) and glutaraldehyde (0.1%) in buffer was found to be sufficient to retain the structural integrity of cells with minimal autofluorescence. This protocol was also suitable for red blood cells infected with Plasmodium falciparum parasites, and preserved the characteristic knob-like structures on the infected red blood cell surface.
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