Raman Spectroscopy of Optically Trapped Single Biological Micro-Particles

Abstract

The combination of optical trapping with Raman spectroscopy provides a powerful method for the study, characterization, and identification of biological micro-particles. In essence, optical trapping helps to overcome the limitation imposed by the relative inefficiency of the Raman scattering process. This allows Raman spectroscopy to be applied to individual biological particles in air and in liquid, providing the potential for particle identification with high specificity, longitudinal studies of changes in particle composition, and characterization of the heterogeneity of individual particles in a population. In this review, we introduce the techniques used to integrate Raman spectroscopy with optical trapping in order to study individual biological particles in liquid and air. We then provide an overview of some of the most promising applications of this technique, highlighting the unique types of measurements enabled by the combination of Raman spectroscopy with optical trapping. Finally, we present a brief discussion of future research directions in the field.

Keywords: Raman spectroscopy; bioaerosols; optical trapping.

Figure 1

(a) The radiative pressure force, which is the dominant force experienced by non-absorbing particles, results from the transfer of momentum from photons scattered by a particle. The radiative pressure force can be divided into a scattering force, which tends to push the particle along the direction of light propagation, and a gradient force, which tends to pull the particle toward the highest intensity region. The gradient force enables trapping in a focused laser beam; (b) The photophoretic force, which is the dominant force experienced by strongly absorbing particles, results from the transfer of heat to surrounding gas molecules from a non-uniformly heated and/or non-uniformly heat-emitting particle.

Figure 2

A 4.7 μm diameter microsphere trapped inside a vacuum chamber by a counter-propagating dual-beam optical tweezer. The wavelength of the trapping beams is 1064 nm; A weak green (532 nm) laser is used for illumination. Inset is a counter-propagating dual-beam optical trap in air based on radiative pressure forces. With kind permission from Springer Science and Business Media [9].

Figure 3

An example of a photophoretic trap. The particle is trapped in the low intensity region between two counter-propagating Laguerre-Gaussian vortex beams [21] (Fair Use according to OSA).

Figure 4

(a) A 100 µm polymer sphere is trapped between two fibers; (b) A HL60 cell is trapped in a microfluidic channel between two fibers. The particle is stopped in flow while the Raman spectrum is recorded and then released [41] (Fair Use according to OSA).

Figure 5

(a) Schematic of the optical trapping apparatus used to trap both transparent and absorbing airborne particles of arbitrary morphology using a single shaped hollow laser beam. The aspheric lens forms a hollow conical focus within a glass chamber where airborne particles are trapped; (b) Calculated intensity profile near the focal spot plotted on a log-scale; (c) Image of the conical focal region produced inside the chamber obtained by introducing Johnson Smut Grass Spores and recording a long exposure time image; (d) Image of a spore trapped in air near the focal point [44] (Fair Use according to OSA).

Figure 6

Raman spectra of trapped yeast cells revealing distinct spectra depending on whether the yeast cells are alive or dead [54] (Fair Use according to OSA).

Figure 7

One of the earliest typical LTRS experimental schematics for (a) the near-infrared Raman trapping system; and (b) the optical arrangement for the sample cell [52] (With permission from ACS publications).

Figure 8

(a) Schematic of a multifoci-scan confocal Raman imaging system; (b) Lateral; and (c) axial intensity profiles of the Raman band at 1001 cm⁻¹ of a 100 nm diameter polystyrene bead [57] (With permission from AIP Publishing LLC).

Figure 9

(a) A red blood cell is stretched using optical tweezers while the Raman spectra is monitored to gauge the cell oxygenation level; (b) The Raman spectra of the stretched (bottom curve) and un-stretched (top curve) blood cell. The shaded regions highlight Raman bands which were most affected by mechanical stretching [65] (With permission from Elsevier).

Figure 10

(a) Raman spectra from 10 normal cells and 10 cells exposed to oxidative stress. The variations between each spectra illustrate the cell-to-cell variation. Nonetheless, despite broadly similar Raman spectra; a PCA shown in (b) clearly differentiates between the stressed and unstressed cells [68] (With permission from Elsevier).

Figure 11

(a) LTRS analysis of red blood cells exposed to Ag nanoparticles revealed a change in the relative intensity of the 1211 and 1224 cm⁻¹ lines, indicating a change in the methane C-H deformation region of the cell; (b) A PCA provided further insight into the temporal evolution of blood cells exposed to Ag nanoparticles [69] (Reprinted under the Creative Commons Attribution License).

Figure 12

(a) The temporal response of yeast cells to oxidative stress is characterized via LTRS; (b) Raman lines associated with varying chemical bonds within the yeast cell are monitored over time. While the bonds associated with the Raman line at 1651 cm⁻¹ and 1441 cm⁻¹ are diminished, the bonds associated with the line at 1300 cm⁻¹ (among others) are unaffected [70] (With permission of John Wiley & Sons).

Figure 13

The Raman line (a) associated with the calcium dipicolinate biomarker is monitored during the spore germination process along with the intensity of elastic scattered light; (b) for varying particles. The individual particles show different germination times, indicated by the rapid decrease in the Raman scattering line at 1017 cm⁻¹ (a); but the germination process is consistently correlated with an increase in the elastic scattering of the cell (b) [61]. (With permission from ACS publications).

Figure 14

Raman spectra and microscope images of trapped particles of either (a,b) Bacillus cereus spores; (c,d) polystyrene microspheres; (e,f) glass microspheres. The LTRS system used the unique Raman spectra to rapidly identify the particle type [73] (With permission from ACS publications).

Figure 15

(a) Raman spectra recorded from leukemic T lymphocytes exposed to varying doses of a chemotherapy drug over time; (b) Principal component analysis revealed three stages in the apoptosis process induced by the drug [8] (Fair Use according to OSA).

Figure 16

(a) LTRS studies on absorbing bioaerosols rely on a photophoretic trap to hold a particle in the low intensity region formed between two counter-propagating hollow beams; (b) Photograph of an experimental photophoretic LTRS system in which an aerosol delivery nozzle introduces particles into the photophoretic trap for on-line particle characterization [23]. (With permission from Elsevier).

Figure 17

Raman spectra of (a) dormant; and (b) germinated B. subtilis spores; (c) subtraction of curve b from curve a; (d) The Ca-DPA; and (e) DPA Raman spectra [96] (With permission from American Society for Microbiology).