Rejection of Fluorescence Background in Resonance and Spontaneous Raman Microspectroscopy

Summary

We discuss the construction and operation of a complex nonlinear optical system that uses ultrafast all-optical switching to isolate Raman from fluorescence signals. Using this system we are able to successfully separate Raman and fluorescence signals utilizing pulse energies and average powers that remain biologically safe.

Abstract

Raman spectroscopy is often plagued by a strong fluorescent background, particularly for biological samples. If a sample is excited with a train of ultrafast pulses, a system that can temporally separate spectrally overlapping signals on a picosecond timescale can isolate promptly arriving Raman scattered light from late-arriving fluorescence light. Here we discuss the construction and operation of a complex nonlinear optical system that uses all-optical switching in the form of a low-power optical Kerr gate to isolate Raman and fluorescence signals. A single 808 nm laser with 2.4 W of average power and 80 MHz repetition rate is split, with approximately 200 mW of 808 nm light being converted to < 5 mW of 404 nm light sent to the sample to excite Raman scattering. The remaining unconverted 808 nm light is then sent to a nonlinear medium where it acts as the pump for the all-optical shutter. The shutter opens and closes in 800 fs with a peak efficiency of approximately 5%. Using this system we are able to successfully separate Raman and fluorescence signals at an 80 MHz repetition rate using pulse energies and average powers that remain biologically safe. Because the system has no spare capacity in terms of optical power, we detail several design and alignment considerations that aid in maximizing the throughput of the system. We also discuss our protocol for obtaining the spatial and temporal overlap of the signal and pump beams within the Kerr medium, as well as a detailed protocol for spectral acquisition. Finally, we report a few representative results of Raman spectra obtained in the presence of strong fluorescence using our time-gating system.

Protocol

1. Some care must be taken in preparing and placing a Raman sample within this system.

1. Because the system typically makes use of very high numerical aperture objectives with very short working distances, the samples are placed on a coverslip. Biological samples are typically placed on a No. 1 thickness coverslip mounted in an Attofluor cell chamber (Invitrogen, Carlsbad, CA).
2. Liquid samples, particularly those toxic to humans, are placed in a small glass bottle with a coverslip cemented to the opening by means of a silicone epoxy. The bottle is then inverted for measurement.
3. Because our system depends on precise ti....

Discussion

The field of biomedical Raman spectroscopy has seen increasing interest over the past several years as a result of its demonstrated potential for solving several difficult challenges in biological diagnostics. For example, Raman spectra have been shown to have diagnostic value in cancer detection ^{3, 4, 5, 6}. Raman spectroscopy has also been used in bacterial quantitation ^{7, 8} and bacterial drug response ⁹. It has also found application in a broad range of other biomedical applications ran.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Lenses	Thorlabs Inc.	Various	All lenses coated to have maximum transmission losses of 1% each
Tunable Ti:Sapph laser	Coherent Inc.	Chameleon	30 nJ, 200 fs, 80 MHz
40X oil immersion objective	Olympus Corporation	UApo/340	NA = 1.35
Inverted microscope	Olympus Corporation	IX-71	Modified to remove all lenses in side port
Half wave plate	Thorlabs Inc.	AHWP05M-600
Glan-Thompson polarizer	Thorlabs Inc.	GTH10M	∼10% transmission loss
Spectrometer	Princeton Instruments/Acton	SP2300i
CCD	Princeton Instruments/Acton	Pixis 100B
Mathmatical software	Mathworks	MATLAB	version 2008a
Faraday isolator	EOT	BB8-5I
Piezo-electric mirror	Newport Corp.	AG-M100
BBO crystal	CASIX	custom	1 mm thickness
Bandpass filter 1	Andover	008FC14	808 ± 0.4 nm
Dichroic mirror	Semrock	FF662-FDI01	band edge at 662 nm
Long-pass filter	Semrock	BLP01-405R	band edge at 417 nm
Bandpass filter 2	Semrock	FF02-447/60	417-447 nm
CS2	Sigma-Aldrich	335266	99% purity
Coumarin 30	Sigma-Aldrich	546127	99% purity
Immersion oil	Cargill Labs	16242	Type DF