Resonance Raman spectroscopy of optically trapped functional erythrocytes

Abstract

We introduce a novel setup combining a micro-Raman spectrometer with external optical tweezers, suitable for resonance Raman studies of single functional trapped cells. The system differs from earlier setups in that two separate laser beams used for trapping and Raman excitation are combined in a double-microscope configuration. This has the advantage that the wavelength and power of the trapping and probe beam can be adjusted individually to optimize the functionality of the setup and to enable the recording of resonance Raman profiles from a single trapped cell. Trapping is achieved by tightly focusing infrared (IR) diode laser radiation (830 nm) through an inverted oil-immersion objective, and resonance Raman scattering is excited by the lines of an argon:krypton ion laser. The functionality of the system is demonstrated by measurements of trapped single functional erythrocytes using different excitation lines (488.0, 514.5, and 568.2 nm) in resonance with the heme moiety and by studying spectral evolution during illumination. We found that great care has to be taken in order to avoid photodamage caused by the visible Raman excitation, whereas the IR trapping irradiation does not seem to harm the cells or alter the hemoglobin Raman spectra. Stronger photodamage is induced by Raman excitation using 488.0- and 514.5-nm irradiation, compared with excitation with the 568.2-nm line.

Hemoglobin oxygen saturation measurements using resonance Raman intravital microscopy

“…In some studies, the cells were immobilized by methanol fixation on a glass slide in air (27) and adsorbed on polylysine-coated glass surfaces (33). In addition, Raman spectra have been obtained from individual optically trapped cells by using different excitation lines (34). Those studies demonstrated the potential for Raman spectroscopy in the study of red blood cell disorders such as thalassemia, sickle cell disease, and malaria.…”

“…Previous studies on isolated red blood cells have shown that the type of glass coating and the laser irradiation had an important influence on the Raman spectra (33,34). Cells may lose their intrinsic shape as result of substrate-induced effects and photo-induced effects when Raman spectroscopy is used (32,34).…”

“…Cells may lose their intrinsic shape as result of substrate-induced effects and photo-induced effects when Raman spectroscopy is used (32,34). However, these investigations pointed out that low accumulated photon dose and proper sample preparation allowed RR spectroscopic studies of single red blood cells.…”

“…In terms of HbO 2 Sat determination, this method has the advantage that the oxygenation information is related only to the specific interaction of the photons with the Hb molecules, generating a unique Raman spectrum (6,7,12,36,40,41). The vibrational bands of heme are well known (1,5), and previous Raman investigations of Hb have been carried out on isolated and purified Hb or in isolated red blood cells (27,33,34). However, the technique has not yet been used to determine HbO 2 Sat in microvessels in vivo.…”

Raman Spectroscopy of Blood and Blood Components

“…The technique allowed much more detailed investigations of hemoglobin, and many of the early demonstrations of LTRS were performed using RBCs as samples. [54][55][56][57] As LTRS technology has matured, it has been used to refine previous studies that had experienced limitations due to sampling protocols. Dasgupta 58 These bands were consistent with hemoglobin existing in an aggregated form, confirming earlier work by researchers from the Monash Figure 3.…”