Raman Spectroscopy of Spores using LTRS

We acquired Raman spectra from spores using our custom-built LTRS instrument. The instrument is built around an inverted microscope (IX71, Olympus) [44 , 60 (link)]. We used a Gaussian laser beam operating at 785 nm (Cobolt 08-NLD) that is coupled into the microscope using a dichroic shortpass mirror with a cut-off wavelength of 650 nm (DMSP650, Thorlabs). Imaging and focusing of the beam were achieved by a 60 $\times$ water immersion objective (UPlanSApo60xWIR, Olympus) with a numerical aperture of 1.2 and a working distance of 0.28 mm. The same laser was used for Raman light excitation. In general, we operated the laser at a fixed output power of 100 mW corresponding to a power of about 60 mW in the sample (total energy of 1.2 J when exposed for 20 seconds). This power chosen was well below those previously recorded to damage spores [61 (link), 62 (link)].
We collected the backscattered light by the microscope objective and passed it through a notch filter (NF785-33, Thorlabs) to reduce the Rayleigh scattered laser line. Further, to increase the signal-to-noise ratio, we mounted a 150 $\mathrm{\mu }$ m diameter pinhole in the focal point of the telescope. The filtered light was coupled into our spectrometer (Model 207, McPherson) through a 150 $\mathrm{\mu }$ m wide entrance slit where a 600 grooves/mm holographic grating disperses the light [63 (link)]. The Raman spectrum was then captured using a Peltier cooled CCD detector (Newton 920N-BR-DDXW-RECR, Andor) operated at -95 ${}^{\circ }$ C. Our system has a Raman wavenumber spectral resolution of < 3 cm ${}^{-1}$ and accuracy of $\sim$ 3 cm ${}^{-1}$ .