Label-free microscopy enables the possibility of measuring biological samples noninvasively and purely based on endogenous contrast. In particular, quantitative phase microscopy (QPM) can provide signals proportional to the intracellular refractive index with high-throughput. To improve the specificity of these measurements, we coupled QPM with Raman spectroscopy, another label-free modality that provides a signal related to the molecular content of the sample. We then developed a hybrid imaging approach where imaging is restricted to QPM to maintain a high-throughput despite the inherent slow acquisition time of Raman signals, while ensuring that the measured spectrum is representative of the whole cellular content.
This approach provides signals for both the morphology, related to the phenotype, and the intracellular molecular content at single-cell level, that we employed to study cell populations under different stimuli. In particular, we studied macrophage cells and their response to a simulated bacterial infection upon exposure to lipopolysaccharide, and show how this approach is able to noninvasively detect the activation state at single-cell level by coupling it with multivariate analysis and machine learning algorithms.
A pulsed (4.4 ns pulse length) frequency doubled Nd:YAG laser, operating at 10 Hz, was used to generate Raman
scattering from samples at a distance of 12 m. The scattered light was collected by a 6 inch telescope and the Raman
spectrum recorded using an Acton SP-2750 spectrograph coupled to a gated ICCD detector. To extend the potential
applications further, employing a spatial offset between the point where the laser hit the sample and the focus of the
telescope on the sample, enabled collection of Raman photons that were predominantly generated inside the sample and
not from its surface. This is especially effective when the content of concealed objects should be analysed. Raman
spectra of H2O2 in a 1.5 mm thick, fluorescent HDPE plastic bottle were recorded at a distance of 12 m. From the
recorded spectra it was possible to determine the H2O2 concentration in the concentration range from 2-30%. Stand-off
Raman spectra of eleven potentially dangerous chemicals (commercial and improvised explosives) were recorded at a
distance of 100 m.
We present our work on stand-off Raman detection of explosives and related compounds. Our system employs 532 or
355 nm laser excitation wavelengths, operating at 10 Hz with a 4.4 ns pulse length and variable pulse energy (maximum
180 mJ/pulse at 532 nm and 120 mJ/pulse at 355 nm). The Raman scattered light is collected by a co-axially aligned 6"
telescope and then transferred via a fiber optic cable and spectrograph to a fast gating iCCD camera capable of gating at
500 ps. We present results including the effect of different excitation wavelengths, showing that 355 nm excitation gives
rise to significantly stronger stand-off Raman signals compared to that of 532 nm. We also show the effect of appropriate
detector gating widths for discrimination of ambient light and the reduction of high background signals in the obtained
Raman spectra. Our system can be used to identify explosives and their precursors in both bulk and trace forms such as
RDX and PETN in the low mg range and TNT in the 700 μg range at a distance of 20 m, as well as detection of a 1% or
greater H2O2 solution at a distance of 6.3 m.