Fluorescence lifetime imaging is a rather new and effective tool that can be used to study complex biological samples, either at microscopic or macroscopic levels. The map of the fluorescence lifetime allows one to discriminate amongst different fluorophores and to achieve valuable insights into the behaviour of emitting molecules, leading to information like local pH, oxygen concentration in cells, etc. Moreover, the distribution in space of any fluorescent marker achievable with this technique can be exploited for diagnostic purposes in medicine. After a brief introduction on the motivations for applying fluorescence lifetime imaging in biology and medicine, the basic principles of this technique will be addressed. Then, the two possible implementations of fluorescence lifetime imaging (i.e. the frequency domain and the time domain methods) will be presented. For this purpose, special attention will be devoted to practical aspects of image acquisition and processing, especially for what concerns the time domain method. Then, the analysis of the state-of-the-art systems will include a brief discussion on new concepts that have recently been introduced in this research field. Finally, two interesting applications of fluorescence lifetime imaging will be presented. The former refers to skin tumour detection and has been successfully applied in a preliminary clinical trial, the latter regards DNA chips reading and has been tested only at laboratory level, yet it has produced promising results for its future implementation in commercial systems.

Different approaches for absorption and scattering spectroscopy of living tissues are discussed. In particular, a unique system for time-resolved reflectance and transmittance spectroscopy is presented, capable of acquiring in vivo absorption and scattering spectra of diffusive media between 600 and 1000 nm. A review of typical spectra obtained from a variety of tissue structures is shown, including female breast, forearm, abdomen, and forehead. A second-level analysis of the measured spectra permits an estimation of the concentrations of the key tissue absorbers, as well as of the Mie-equivalent scattering radii. Further, absorption and scattering spectra can be used to estimate the penetration depth of light in tissues as a function of wavelength, which is a crucial parameter in view of the possible application of optical in vivo molecular imaging in clinical diagnosis. Finally, an example of the applicability of the methodology to other biological media such as fruits and vegetables is shown.

The feasibility of in vivo measurements in the range of 1000 to 1100 nm and the potential benefits of operation in that wavelength range for diagnostic applications are investigated. To this purpose, an existing system for time-resolved diffuse spectroscopy is modified to enable in vivo studies to be carried out continuously from 600 to 1100 nm. The optical characterization of collagen powder is extended to 1100 nm and an accurate measurement of the absorption properties of lipid is carried out over the entire spectral range. Finally, the first in vivo absorption and scattering spectra of breast tissue are measured from 10 healthy volunteers between 600 and 1100 nm and tissue composition is evaluated in terms of blood parameters and water, lipid, and collagen content using a spectrally constrained global fitting procedure.

Applications of time-resolved photoluminescence spectroscopy (TRPL) and fluorescence lifetime imaging (FLIM) to the analysis of cultural heritage are presented. Examples range from historic wall paintings and stone sculptures to 20th century iconic design objects. A detailed description of the instrumentation developed and employed for analysis in the laboratory or in situ is given. Both instruments rely on a pulsed laser source coupled to a gated detection system, but differ in the type of information they provide. Applications of FLIM to the analysis of model samples and for the in-situ monitoring of works of art range from the analysis of organic materials and pigments in wall paintings, the detection of trace organic substances on stone sculptures, to the mapping of luminescence in late 19th century paintings. TRPL and FLIM are employed as sensors for the detection of the degradation of design objects made in plastic. Applications and avenues for future research are suggested.

The absorption spectrum of collagen powder is measured between 610 and 1040 nm by time-resolved transmittance spectroscopy. Absorption spectra of breast from healthy volunteers are then interpreted, adding collagen to the other absorbers previously considered (i.e., oxy- and deoxyhemoglobin, water, and lipids). A significant amount of collagen, depending on breast type, is estimated to be present. Adding collagen to the fitting procedure affects remarkably the estimated values of blood content and oxygenation. The quantification of collagen has potential implications for the assessment of breast density and cancer risk.

We describe a system for absorption and scattering spectroscopy of diffusive media based on time-resolved reflectance and transmittance measurements. The system is operated with mode-locked lasers tunable in the 550-1050 nm spectral range and on a detection chain based on time-correlated single-photon counting. All measurement procedures such as laser tuning and optimization, signal conditioning, data acquisition, and analysis are completely automated, permitting spectral measurements over the whole range in a few minutes. The criticalities of the system are discussed together with the strategies to compensate them. The Medphot protocol devised for the characterization of photon migration instruments was applied to assess the system performances in terms of accuracy, linearity, noise, stability, and reproducibility. Finally, an example of application of the instrument to the spectroscopy of powders is presented.

Ultraviolet-induced fluorescence spectroscopy is a commonly used technique for the characterization and identification of painting materials, such as organic binders and colorants. Its interpretation is strictly connected to both the experimental setup and an understanding of the physical and chemical interactions among materials in paint layers, which are commonly composed of a fluorescent organic binder and a pigment. When irradiated with ultraviolet radiation, the light emitted by fluorophores present in the organic binder undergoes several types of interactions, in particular scattering and absorption by neighboring pigmented particles and auto-absorption. As a result of scattering and absorption phenomena, the emission spectrum is deformed according to the physical properties of the surrounding pigmented particles. This can lead to shifts of the emission maxima and/or to the formation of apparent new emission bands. The extent of the modifications to the emission spectra, caused by auto-absorption and selective absorption phenomena, may lead to the erroneous characterization or identification of the fluorescent materials. As a consequence, the interpretation of the emission signal can be greatly compromised. A correction based on the Kubelka-Munk theory is proposed to evaluate the extent of the spectral distortion and is assessed on modern replicas of wall paintings of known composition. Although the model cannot be applied to all cases, qualitative distinctions between real and apparent emissions are achieved.

We investigate anisotropic light propagation in biological tissue in steady-state and time domains. Monte Carlo simulations performed for tissue that consists of aligned cylindrical and spherical scatterers show that steady-state and time-resolved reflectance depends strongly on the measurement direction relative to the alignment of the cylinder axis. We examine the determination of optical properties using an isotropic diffusion model and find that in the time domain, in contrast to steady-state spatially resolved reflectance measurements, the obtained absorption coefficient does not depend on the measurement direction and is close to the true value. Contrarily, the derived reduced scattering coefficient depends strongly on the measurement direction in both domains. Measurements of the steady-state and time-resolved reflectance from bovine tendon confirm the theoretical findings.