Comparison of coherent and spontaneous Raman microspectroscopies for noninvasive detection of single bacterial endospores

Abstract

Single bacterial spores were analyzed by using nonlinear Raman microspectroscopy based on coherent anti-Stokes Raman scattering (CARS). The Raman spectra were retrieved from CARS spectra and found to be in excellent agreement with conventionally collected Raman spectra. The phase retrieval method based on maximum entropy model revealed significant robustness to external noise. The direct comparison of signal amplitudes exhibited a factor of 100 stronger CARS signal, as compared with the Raman signal.

Fig. 1.

B. subtilis spores under the microscope. (Left) Regular optical microscopy. (Right) Phase-contrast microscopy. (Scale bar: 10 microns.)

Fig. 2.

Molecular energy level diagram in which fields 1 and 2 prepare the vibrational levels with a ground-state coherence ρ_bc. Field 3 then scatters off the coherence, generating the anti-Stoke field 4.

Fig. 3.

CARS (Upper) and retrieved Raman (Lower) spectra of a single bacteria spore. Shown is three-color geometry (532 nm, 1,064 nm, and broadband continuum).

Fig. 4.

CARS (Upper) and retrieved Raman (Lower) spectra of a single bacteria spore. Shown is two-color geometry (1,064 nm and broadband continuum).

Fig. 5.

A schematic diagram of the experimental CARS set-up. F indicates a set of filters rejecting all pump beams, but transmitting CARS signal. NLC indicates a 4-mm-long LBO crystal. Two delay lines are used to ensure the temporal overlap of all beams on the sample.