Introduction to Tissue Imaging

The main goal of tissue imaging is to diagnose diseases or physiological states in the human body. The electromagnetic spectrum provides a diverse set of tools for probing, manipulating, and interacting with tissue. For example, X-ray and magnetic resonance imaging (MRI) are used ubiquitously in clinical diagnostic procedures. Optical imaging has gradually found its way into clinical applications over the last couple of decades. The advantages of optical imaging are that it does not use ionizing radiation unlike X-rays, and that it has high spatial and temporal resolution unlike magnetic resonance. The main disadvantage of optical imaging is that tissue in general is highly scattering and absorbing. The scattering/absorption coefficient is defined as the inverse of the mean free path (the average distance a photon travels between consecutive scattering/absorption events).

Optical imaging methods can be broadly divided into two categories: linear and nonlinear imaging - based on the dependence of the signals on the incident light intensity. Most imaging methods are implemented in the linear regime due to the low cost and ease of use associated with operation. Some of the most common methods include: fluorescence, absorption, and reflectance. The problem with linear imaging is that depth resolved information is difficult to achieve due to light scattering. Nonlinear imaging can partly circumvent this problem since the detected signals depend nonlinearly on the incident intensity and as a result are only generated at the focus of the light beam. This strong localization allows 3D optical sectioning in images based on a nonlinear contrast. Transmitted light or back scattered light can be collected, knowing it must have originated in the voxel being interrogated. This works even in heavily scattering samples, such as tissue. Another advantage of nonlinear imaging methods is that they encompass two-photon processes whereby lower energy photons (in the near IR spectral range) may be used to probe the sample thereby taking advantage of the therapeutic window shown in Figure 1. Some nonlinear imaging methods include two-photon fluorescence (TPF), two-photon absorption (TPA) and self-phase modulation (SPM).

Two-photon fluorescence (TPF) is an important nonlinear optical effect. It relies on the quasi-simultaneous absorption of two photons (of either the same or different energies) by a molecule (i.e. the fluorophore). During the absorption process, the electronic distribution of the molecule changes and as a result it is transferred to an excited state. Radiative and nonradiative relaxation processes ultimately thermalize the absorbed energy. It is the radiative relaxation processes that give rise to TPF. During this relaxation process, radiationless relaxation in the vibrational levels of the fluorophore occur prior to the emission of a photon and as a result the energy of the emitted photon is lower compared to the sum of the energy of the absorbed photons. The fluorescent photons are easily isolated by filtering the light signals. Two-photon fluorescence microscopy is one of the most important applications of TPF in biomedical imaging. The diagnostic value of the TPF process lies in the fact that different parts of the tissue will light up depending on the localization of the fluorophore. This type of imaging has been very useful for studying and characterizing different tissues although one of its fundamental limitations is that it typically requires the use of exogenous fluorophores that need to be introduced to the sample. There is always the question of toxicity that ultimately hinders the application of these methods to humans in vivo. As a result, there is a need to develop microscopies that only depend on the intrinsic properties and contents of cells. As will be discussed, TPA and SPM are promising processes that are capable of providing nonlinear contrast and only depend on the endogenous content of cells.

TPA occurs when a molecule absorbs two photons simultaneously. Because two photons are involved, this process does not scale linearly with intensity, but with the intensity squared. SPM is also a nonlinear optical effect. A pulse of light will induce a varying refractive index in the medium due to the optical Kerr effect. This variation in the refractive index will produce phase shifts to different parts of the spectral components of the light pulse. It also depends nonlinearly on the input light intensity. The strength of the TPA and SPM signals depend on the properties and content of the tissue being interrogated. For example, SPM coefficients vary greatly when comparing different solutions and as a result SPM may be a very useful probe of the heterogeneous environment found in cells.

Beyond TPF, TPA and SPM, there are other nonlinear processes. Two photons of different wavelengths may be simultaneously absorbed by molecules in a sample. This process is known as sum frequency absorption (SFA). If absorption of one photon accesses a discrete energy level, excited state absorption (ESA) can take place. This process can be accessed in pump-probe experiments since the first (pump) pulse accesses higher energy levels that are subsequently probed using the second (probe) pulse. The time delay between the pulses allows for the determination of the relevant timescales associated with excited state relaxation.

Nonlinear contrast is extremely useful in imaging tissue since it has the potential to enhance images and access new information. The Warren lab is currently investigating TPA in hemoglobin and melanin. Techniques for differentiating between oxy- and deoxyhemoglobin as well as eumelanin and pheomelanin by SFA and ESA are being developed. Images based on either of these molecules would be useful in diagnosing skin cancer, for which no reliable diagnostic short of biopsy exists. The Warren lab is also interested in functional imaging whereby the activation of neuronal circuits is being investigated with SPM microscopy.