Raman Tweezers Spectroscopy of Live, Single Red and White Blood Cells

An optical trap has been combined with a Raman spectrometer to make high-resolution measurements of Raman spectra of optically-immobilized, single, live red (RBC) and white blood cells (WBC) under physiological conditions. Tightly-focused, near infrared wavelength light (1064 nm) is utilized for trapping of single cells and 785 nm light is used for Raman excitation at low levels of incident power (few mW). Raman spectra of RBC recorded using this high-sensitivity, dual-wavelength apparatus has enabled identification of several additional lines; the hitherto-unreported lines originate purely from hemoglobin molecules. Raman spectra of single granulocytes and lymphocytes are interpreted on the basis of standard protein and nucleic acid vibrational spectroscopy data. The richness of the measured spectrum illustrates that Raman studies of live cells in suspension are more informative than conventional micro-Raman studies where the cells are chemically bound to a glass cover slip

Micro-Raman Spectroscopy of Silver Nanoparticle Induced Stress on Optically-Trapped Stem Cells

We report here results of a single-cell Raman spectroscopy study of stress effects induced by silver nanoparticles in human mesenchymal stem cells (hMSCs). A high-sensitivity, high-resolution Raman Tweezers set-up has been used to monitor nanoparticle-induced biochemical changes in optically-trapped single cells. Our micro-Raman spectroscopic study reveals that hMSCs treated with silver nanoparticles undergo oxidative stress at doping levels in excess of 2 µg/ml, with results of a statistical analysis of Raman spectra suggesting that the induced stress becomes more dominant at nanoparticle concentration levels above 3 µg/ml

Alignment and calibration of the experimental set-up by using 785 nm laser light to ensure maximum signal-to-noise ratio in Raman spectra of 3 µm polystyrene beads.

<p>Raman spectra of a polystyrene bead trapped at ∼5 mW power and Raman excitation at ∼10 mW with (a) Acquisition time: 2 s (b) Acquisition time: 10 s. The insets show the image of the polystyrene bead before and after trapping.</p

Raman spectrum of a trapped granulocyte over the wavelength range 400–1750 cm⁻¹.

<p>Laser power: ∼10 mW, acquisition time: 120 s, average of 5 accumulations. The insets show the image of a granulocyte before and after trapping.</p

Schematic representation of our Raman Tweezers set-up.

<p>BE: beam expanders, L: lenses, DM: dichroic mirrors, BF: band pass filter used to “clean up” the spectrum of light from the diode laser used for Raman excitation (see text).</p

Characterization of the two dichroic mirrors used to transmit Raman signals to the spectrometer and visible light to the CCD camera.

<p>Transmission curves (% T) of dichroic mirrors (a) DM₁ (785 nm) and (b) DM₂ (1064 nm) over a wide range of wavelengths (200–900 nm).</p

Raman spectrum of a trapped lymphocyte over the wavelength range 400–1750 cm⁻¹.

<p>Laser power: ∼10 mW, acquisition time: 120 s, average of 5 accumulations. The insets show the image of a lymphocyte before and after trapping.</p

Raman spectra of trapped granulocyte and lymphocyte over the wavelength range 400 to 1750 cm⁻¹.

<p>Laser power: ∼30 mW, acquisition time: 120 s, average of 5 accumulations.</p

Observed Raman frequencies with corresponding band assignments for RBCs and whole blood-Serum.

<p>Abbreviations: - ν & δ: In-plane modes, γ: Out -of- plane modes, sym: symmetric, asym: asymmetric, Str: stretching, p: protein, Phe: Phenylalanine.</p