Components

Four components are required for any in vivo fluorescence imaging system:

Fluorescence Contrast Agents

Fluorescence contrast agents, or probes, that highlight cells or processes of interest are one of the key components of molecular imaging. Probes used in imaging applications consist of a molecule (the biomarker), which can be targeted or non-targeted, conjugated to a fluorophore that enables visualization of the biomarker. Probes can be designed to accumulate in specific types of tissues or cells, typically through binding with a specific antibody, or to respond to specific stimuli.

Fluorophores used in molecular imaging can be either naturally occurring or artificially created. Naturally-occurring fluorophores include green fluorescence protein (GFP) and its red equivalent, RFP. GFP has mostly been used to measure human tumor development and metastasis in small-animal cancer models. It is also possible to use GFP in any application where fluorescent cell labeling is desired, such as gene expression, viral infection, and embryonic development. GFP is not known to interfere with cell growth or function.

For deep-tissue imaging, artificial fluorophores engineered with excitation and emission wavelengths in the near-infrared (NIR) region between 650 nm to 900 nm, have significant advantages over natural fluorophores because they have lower scattering properties in tissue and lower absorption in blood and water. Applications using NIR fluorophores include cancer typing, treatment monitoring, tracking of drug metabolism, and determining tumor localization,  size, and growth rate. For example, dye-peptide conjugates, such as cyanine-peptide, are fluorescent dyes that can be chemically bound to a target molecule and have the potential to target specific tumors, assess drug delivery, and monitor treatment in animal models.

The choice of a natural or artificial fluorophore in a probe is dependent on the targeted application. Factors that influence the choice of fluorophore include its availability and stability, as well as the location of the process of interest.

Reporter genes have also been used successfully in optical imaging applications. Commonly used reporter genes that induce visually identifiable characteristics usually involve fluorescent and luminescent proteins such as the enzyme luciferase, which catalyzes a reaction with luciferin to produce light. Since the expression of the reporter gene is used as a marker for successful uptake of the gene of interest, it must not be natively expressed in the cell or organism under study.

Also used for labeling metabolic processes in vivo, semiconductor quantum dots (Qdots) are nanometer-sized crystals that have been covalently linked to biomarkers such as peptides, antibodies, nucleic acids, or small molecules. Compared with organic fluorophores, Qdots have unique optical and electronic properties, including selectable fluorescence emission from visible to infrared wavelengths and large extinction coefficients across a wide spectral range. Qdots are available in single or multicolored forms. Multicolored Qdots have the same excitation wavelength, but will fluoresce at different emission wavelengths. Qdots have hydrophilic properties, so they are soluble in aqueous buffers and sensitive to pH changes. Although toxicity is a current disadvantage of using Qdots, some biocompatible and non-toxic coated nanocrystals have been used successfully for in vivo animal studies.

Excitation Light Sources

Light energy is essential to fluorescence and can be divided into two main categories:

  • Broad wavelength sources such as UV, mercury arc, and xenon arc lamps, which are used in some imaging and fluorescence spectroscopy systems.
  • Line sources with discrete wavelengths, such as lasers, which are used in other systems.

Both of these types of light sources may either be delivered in short pulses or in a continuous manner.

Filters

Selection of appropriate optical filters to define the excitation and emission wavelength band is another important issue for fluorescence detection. Detection filters should maximize transmission of signals at or around the desired fluorescence wavelength, while minimizing transmission of signals at other wavelengths, such as the excitation signal, background noise, or endogenous sources of fluorescence.

Three types of optical filters are commonly used in fluorescence imaging systems:

  • Long-pass (LP) filters, which allow wavelengths longer than the characteristic wavelength to pass.
  • Short-pass (SP) filters, which allow wavelengths shorter than the characteristic wavelength to pass.
  • Band-pass (BP) filters, which allow wavelengths in the filter bandwidth — which are centered on the characteristic wavelength — to pass.

Matching a fluorescent label with a suitable excitation source and emission filter is the key to optimal detection efficiency.

Detectors

Different types of detectors can be used in optical imaging systems. Due to their inherently slow response time, standard charge coupled device (CCD) cameras are used almost exclusively in CW fluorescence and bioluminescence systems, while time-correlated single photon counting (TCSPC) modules using either avalanche photo diodes, gated intensified CCDs, or photo-multiplier tubes are preferred for TD instruments such as the Optix system.

Standard CCD and TCSPC systems are both applicable to intensity-based optical molecular imaging. However, single-projection, standard CCD-based systems do not allow depth recovery, accurate tomographic imaging or fluorescence lifetime determination. Standard CCD cameras are also subject to read-out noise, which can limit the accuracy of low-light level measurements and thus limit the effective sensitivity of the system. These problems do not exist in properly cooled TCSPC systems, because of their fast temporal dynamics, which is typically on the sub-nanosecond scale, and large sensitivity. By design, a single photon entering a TCSPC detector is usually sufficient to generate a detectable signal.