Coherent Raman Tissue Imaging in the Brain

Abstract

Imaging in neuroscience has been dramatically impacted by the advent of multiphoton microscopy. Multiphoton-excited fluorescence (MPF) in combination with endogenous fluorophores or labeling by fluorescent molecules has proven to be particularly powerful. However, endogenous fluorescence is limited to relatively few molecular species, and practical labeling schemes do not exist for many classes of molecules. Coherent Raman scattering (CRS) techniques, including coherent anti-Stokes Raman scattering and stimulated Raman scattering, allow imaging without the need for staining or fluorescent labeling. Such label-free imaging is desirable in biomedical research, because labeling often perturbs the function of small metabolite and drug molecules and may be too toxic to use in vivo. CRS techniques have similar imaging parameters to MPF, making use of pulsed near-infrared lasers to deliver high-sensitivity, high spatial resolution in three dimensions and rapid image acquisition. In this introduction, we will discuss the basic principles of CRS imaging, present the instrumentation requirements for high-speed CRS imaging, and show an example of imaging brain tumors and healthy tissue based on their intrinsic vibrational signatures. This discussion is intended to introduce the benefits and tradeoffs associated with different CRS techniques and show one example of the powerful capabilities of label-free chemical imaging.

Temporal Focusing Microscopy

“…This inherent mechanism for rejection of out-of-focus scattering eliminates the need for a confocal pinhole in the detection path. To date, a variety of multiphoton processes has been used for imaging, including both incoherent processes such as two-photon and three-photon fluorescence and coherent processes such as second-harmonic and third-harmonic generation and coherent Raman processes (see Principles of Multiphoton-Excitation Fluorescence Microscopy [Denk 2007]; Monitoring Membrane Potential with Second-Harmonic Generation [Wilson et al 2014]; Coherent Raman Tissue Imaging in the Brain [Saar et al 2014]). More recently, multiphoton photoactivation has become an important tool in biosciences (Nikolenko et al 2007), enabling either controlled uncaging of a biologically active molecule in a particular spot within the specimen or as a tool for switching fluorescent molecules between an emissive and a nonemissive state.…”

High-resolution in vivo optical imaging of stroke injury and repair

“…This enables identification and quantification of many molecules otherwise inaccessible by other imaging technologies. In particular, experimental setups based on coherent anti-Stokes Raman scattering microscopy may be only a few years away from in vivo applications (Camp Jr et al, 2014; Saar et al, 2014; Tu and Boppart, 2014). Super-resolution imaging brakes the optical resolution limits of Abbe's diffraction and enables nanometer-scale spatial resolution suitable for visualization of the tiny perisynaptic processes, radial glia endfeet, endothelial tight junctions, etc.…”

A journey to uncharted territory: new technical frontiers in studying tumor–stromal cell interactions

“…Label-free imaging approaches, such as Coherent anti-Stokes Raman Scattering (CARS), offers another solution to the IVM labeling problem. CARS relies on the unique vibrational properties of proteins for an imaging readout, thereby, in principle, enabling highly multiplexed protein visualization at a single cell resolution without fluorescent taggging 55,56 . Although still in its infancy in IVM applications, CARS has already demonstrated a capability to monitor subcellular events and cellular interactions with a temporal perspective 57,58 .…”

“…CARS relies on the unique vibrational properties of proteins for an imaging readout, thereby, in principle, enabling highly multiplexed protein visualization at a single cell resolution without fluorescent tagging. 55,56 Although still in its infancy in IVM applications, CARS has already demonstrated a capability to monitor subcellular events and cellular interactions with a temporal perspective. 57,58 Traditionally, IVM has been used to monitor cellular level behaviors, such as immune responses.…”