Nonlinear interferometric vibrational imaging

#The ability to measure the three-dimensional structure of biological tissues is important, but common methods used to leave out basic information about the molecular composition and metabolic behavior of the tissue imaged. This molecular composition and metabolic behavior information may yield valuable scientific data on the behavior of biological systems, and would be of great clinical diagnostic value for finding diseases such as cancer. Much of the focus of biological and medical imaging today is to gain information about composition.

#Functional magnetic resonance imaging (FMRI) utilizes contrast agents specific to particular molecular species or metabolic processes to provide specific information about the three-dimensional (3-D) location of these species or processes. This method is quite versatile, and poses relatively little risk to the patient, but is limited in practice to a resolution over 100 microns. Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) utilizes a radiolabeled metabolic molecule to provide 3-D measurements of utilization of these molecules by sensing their gamma-ray emissions. Unfortunately, these methods have been cost prohibitive as diagnostic tools, and expose the patient to ionizing radiation. Fluorescence microscopy labels relevant biological structures and processes with a fluorescent compound which can be measured by conventional microscopy or confocal microscopy. The two-photon variant uses ultrafast pulses so that emission and excitation frequencies can be clearly separated, and also utilizes the additional selectivity that the intensity-sensitive nature of two-photon excitation provides. While some biological structures produce natural fluorescence, in general externally introduced fluorescence markers must be used, which can interfere with biological processes and are often toxic.

#Molecules frequently have molecular resonance frequencies that are due to the electromagnetic attractions of atoms in the molecule. These frequencies are those of molecular vibrations, molecular rotational motions, the excitation of electrons to higher energy states, and occasionally finer structures such as hyperfine interactions and optical-magnetic properties. These properties are present without the introduction of any external contrast molecule. These frequencies are usually in the mid-infrared, corresponding to photons of 1.5-50 microns of wavelength. Unfortunately, they cannot be directly excited by electromagnetic radiation of the same frequency because when they are in tissue, the surrounding water absorbs almost all of these frequencies. The range of wavelengths that the tissue is relatively transparent to is 0.6-1.5 microns. Therefore multiphoton nonlinear processes need to be employed to probe these resonances. The photons to stimulate and record the processes are typically in a region where the tissue is not absorbing, so that they can reach the tissue feature and be measured from the feature.

#FIG. 1. Coherent Anti-Stokes Raman Scattering and Coherent Stokes Raman Scattering energy-level diagrams.

#FIG. 2. Basic block diagram of NIVI

#FIG. 3. Example laser configurations are shown that produce two pulses of frequencies ω1 and ω2 overlapped. We assume that the delays have been set correctly to overlap them.

#Nonlinear interferometric vibrational imaging (NIVI) is a method used to measure the three-dimensional distribution of molecular species in various samples (biological or otherwise). Its basic operation is to stimulate the excitation of molecular bonds with particular resonance frequencies, and then use these excitations to produce radiation distinct from the excitation that can be measured. The physical process of excitation and stimulation of radiation is called Coherent Anti-Stokes Raman Scattering (CARS). Unlike previous methods that use CARS in microscopy to probe for the presence of molecular species, NIVI utilizes a heterodyne approach where a reference signal is separately generated and interferomerically compared to the signal received from the sample, allowing the signal to be formed into an image in the same way as OCT. In this way, additional information can be inferred from the emitted radiation such as the distance to the sample and phase information that yields additional structure of the molecular bonds. It also has other advantages in sensitivity and the ability to screen out background radiation that is not produced by the sample. It also can allow more flexibility in the choice of laser illumination source, because the coherent detection process does not rely on photon frequency alone to discriminate emitted radiation.

#The term “scattered photon” means photons scattered by a sample, which include linearly scattered photons and non-linearly scattered photons, such as CARS and CSRS photons.

#The term “image” means data produced by receipt of electromagnetic radiation, which may or may not be formed into a picture viewable by the human eye. This includes images produced directly onto a medium such as film or video.