Direct Comparison of Hyperspectral Stimulated Raman Scattering and Coherent Anti-Stokes Raman Scattering Microscopy for Chemical Imaging

Summary

This paper directly compares the resolution, sensitivity, and imaging contrasts of stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) integrated into the same microscope platform. The results show that CARS has a better spatial resolution, SRS gives better contrasts and spectral resolution, and both methods have similar sensitivity.

Abstract

Stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) microscopy are the most widely used coherent Raman scattering imaging technologies. Hyperspectral SRS and CARS imaging offer Raman spectral information at every pixel, which enables better separation of different chemical compositions. Although both techniques require two excitation lasers, their signal detection schemes and spectral properties are quite different. The goal of this protocol is to perform both hyperspectral SRS and CARS imaging on a single platform and compare the two microscopy techniques for imaging different biological samples. The spectral focusing method is employed to acquire spectral information using femtosecond lasers. By using standard chemical samples, the sensitivity, spatial resolution, and spectral resolution of SRS and CARS in the same excitation conditions (i.e., power at the sample, pixel dwell time, objective lens, pulse energy) are compared. The imaging contrasts of CARS and SRS for biological samples are juxtaposed and compared. The direct comparison of CARS and SRS performances would allow for optimal selection of the modality for chemical imaging.

Introduction

The Raman scattering phenomenon was first observed in 1928 by C. V. Raman¹. When an incident photon is interacting with a sample, an inelastic scattering event can spontaneously occur, in which the energy change of the photon matches a vibrational transition of the analyzed chemical species. This process does not require the use of a chemical tag, making it a versatile, label-free tool for chemical analysis while minimizing sample perturbation. Despite its advantages, spontaneous Raman scattering suffers from a low scattering cross-section (typically 10¹¹ lower than the infrared [IR] absorption cross-section), w....

Protocol

1. Instrumental setup for hyperspectral CRS imaging

NOTE: The generation of CRS signal requires the use of high-power (i.e., class 3B or class 4) lasers. Safety protocols must be addressed and proper personal protective equipment (PPE) must be worn at all times when working at such high peak powers. Consult proper documentation before experimentation. This protocol focuses on designing the beam path, chirping the femtosecond pulses, and optimizing imaging conditions. A general optical layout of this hyperspectral CRS microscope is shown in Figure 1. The configuration shown here is one of many exi....

Results

Comparisons of the spectral resolution
Figure 2 compares the spectral resolution of hyperspectral SRS (Figure 2A) and CARS (Figure 2B) microscopy using a DMSO sample. For the SRS spectrum, two Lorentzian functions (see protocol step 2.3) were applied to fit the spectrum, and a resolution of 14.6 cm^-1 was obtained using the 2,913 cm^-1 peak. For CARS, a two-peak-fitting function with a Gaus.......

Discussion

The protocol presented here describes the construction of a multimodal CRS microscope and the direct comparison between CARS and SRS imaging. For the microscope construction, the critical steps are spatial and temporal beam overlapping and beam size optimization. It is recommended to use a standard sample such as DMSO before the biological imaging for optimizing SNR and calibrating Raman shifts. Direct comparison between CARS and SRS images reveals that CARS has a better spatial resolution, while SRS gives better spectra.......

Materials

Name	Company	Catalog Number	Comments
2D galvo scanner set	Thorlabs	GVS002
Acousto-optic modulator	Isomet	M1205-P80L-0.5
AOM driver	Isomet	532B-2
Data acquisition card	National Instruments	PCle 6363	Custom ordered filter (980 sp)
Delay stage	Zaber	X-LSM050A
Deuterium oxide	Millipore Sigma	151882-100G
Dichroic mirror for beam combination	Thorlabs	DMLP1000
Dichroic mirror for signal separation	Semrock	FF776-Di01-25x36
DMSO	MiliporeSigma	200-664-3
MIA PaCa 2 Cells	ATCC	CRL-1420
Femtosecond laser system	Spectral Physics	InSightX3+
Filter for CARS	Chroma	AT655/30m
Filter for SRS	Chroma	ET980sp
Function generator	Rigol	DG1022Z
Glass rods	Lattice Electro Optics	SF-57
Half-wave plate	Newport	10RP02-51; 10RP02-46
LabVIEW 2020	National Instruments		This is the image acquisition software
Lock-in amplifier	Zurich Instrument	HF2LI
Microscope housing	Olympus	BX51W1
Objective lens	Olympus	UPLSAPO60XW
Origin Pro 2019b	OriginLab Corporation		This is the spectral fitting software
Oscilloscope	Tektronix	TBS2204B
Photodiode	Hamamatsu	S3994-01
PMT detector	Hamamatsu	H7422P-40
PMT voltage amplifier	Advanced Research Instrument Corp.	PMT4V3
Polarizing beamsplitter cube	Thorlabs	PBS255
Terminal block	National Instruments	BNC-2110