Optical Techniques for Diagnosis
Introduction:
Light can react with tissue in different ways and its response can yield information about the state of tissue with respect to its physiology as well as pathology. The light used to probe tissue does so in a non-intrusive manner and typically uses very low levels of light unlike in therapeutic applications. The use of fiber optics allows light to probe tissue in a minimally invasive manner. Since tissue response is virtually instantaneously, the results are obtained in real-time and the use of data processing techniques and computers allows for automated detection of disease. These properties have resulted in a variety of applications where light and optical modalities have been used for tissue diagnosis.
References:
1. Lakowicz, Joseph. Principles of Fluorescence Spectroscopy. NY, Plenum Press
Spectroscopy
Tissue response to light may be measured in different ways; imaging or spectroscopy. Spectroscopy is the measurement of light intensity as a function of wavelength at high spectral resolution but without much spatial information. Multi-fiber spectroscopic probes can provide some spatial information. Conventional imaging is the technique used when spatial information is needed. Imaging, however, provides limited spectral information. Spectral imaging is a promising new method by which light spectrum at every picture element i.e. pixel, of a two-dimensional image is measured. The images acquired using such a method consists of a cube of information Ix,y (l), which contains the full spectrum at each pixel position (x, y). Thus spectral imaging combines the features of spectroscopy and imaging to provide spatial as well as spectral information.
When a photon is incident on a molecule, it may be transmitted, absorbed or scattered. Different techniques arise from these different light-tissue interactions. These techniques include;
Absorption spectroscopy - In this technique, absorption spectra are studied where the absorption of light as a function of wavelength is measured. For biologic materials, near-infrared to infrared wavelength are used which represent transitions between vibrational energy levels. e.g. diagnosis of breast cancer
Reflectance Spectroscopy - In this technique, the diffuse reflectance (elastic scattering) is measured as a function of wavelength. Diffuse reflectance is an elastic scattering phenomenon and yields a measure of the optical properties of the tissue. This technique is particularly useful in accounting for blood absorption in tissue.
Fluorescence Spectroscopy - In this technique, the fluorescence intensity is measured as a function of wavelength. Fluorescence results following transitions between electronic energy levels.
Raman Spectroscopy - Classically speaking, when the energy of the incident photon is unaltered after collision with a molecule, the scattered photon has the same frequency as the incident photon. This is known as Rayleigh or elastic scattering. When energy is transferred either from the molecule to the photon or vice versa, the scattered photon has less or more than the energy of the incident photon. This is known as inelastic or Raman scattering. A Raman spectrum is a plot of scattered intensity as a function of the energy difference between the incident and scattered photons. Raman signals are usually weak and require powerful sources and sensitive detectors. Typically, Raman peaks are spectrally narrow (a few wavenumbers; a wavenumber = 1/l), and in many cases can be associated with the vibration of a particular chemical bond in a molecule.
Principles of Fluorescence
To explain the phenomenon of fluorescence, we need to consider the molecular structure of matter. As seen before, molecules possess rotational, vibrational and electronic behavior. The absorption and emission of light by a molecule can be illustrated by the Jablonski diagram where the ground, first and second electronically excited states are depicted by S0, S1 and S2, respectively [Fig 1]. Within each of these states, the vibrational energy levels are represented by a number in parenthesis - e.g. S0(0), S0(1)…
At room temperature, molecules primarily reside in the ground state. When stimulators such as photons are absorbed (each photon has energy hna where h is the Planck's constant and na is the frequency at which absorption occurs) molecules occupying the lowest vibrational level of the ground state S0(0) are excited to the higher electronic excited levels (S1,S2).
Jablonski diagram showing the vibrational (v) and electronic (S & T) energy levels and processes that can take place with the absorption of light.
Following absorption, several processes occur. In most organic compounds, the molecules will rapidly relax to S1(0) by heat generation - referred to as Internal Conversion. (One known exception is azulene, which emits fluorescence from S2 as well). This occurs virtually instantaneously; the time scale of this event is 10-14 - 10-12 s. From S1(0), the molecule can relax to the ground state by three processes;
(1) They can internally convert (by heat generation) to any of the S0 states.
(2) They can emit fluorescence.
(3) They can emit phosphorescence.
In fluorescence, the emitted photons have an energy hnf equal to the energy difference between the excited and ground states.
hnf = ES1(0) - ES0(0)
The average time a molecule remains in the excited state before emitting fluorescence (fluorescence lifetimes) is 10^-10 - 10^-8 s.
Molecules in the S1 state can also undergo conversion to the first triplet state T1. Emission from T1 is termed as phosphorescence. The conversion from S1 to T1 is referred to as Intersystem Crossing. The transition from T1 to S0 is classically forbidden and the rate of such emission is slow. Phosphorescence lifetimes are in the order 10-3 - 100 s. Note that we will not be discussing phosphorescence anymore beyond the above discussion.
Fluorescence Spectroscopy
The fluorescence emitted can be mathematically represented as follows;
For a dilute solution with only 1 fluorophore (defined as a molecule/compound that exhibits fluorescence) the fluorescence intensity is a function of both the excitation and the emission wavelength and can be written as;
The fluorescence quantum yield Q is a measure of the proportion of molecules emitting fluorescence as compared to the total number of molecules excited.
Fluorescence intensity may be measured as a function of the excitation or emission wavelength, the two variables in the above equation.
1. The excitation spectrum depicts the fluorescence intensity as a function of the excitation wavelength for a fixed emission wavelength.
Since Q(lem) is a constant, the excitation spectrum looks like the absorption spectrum for that fluorophore.
2. The emission spectrum depicts the fluorescence intensity as a function of the emission wavelength for a fixed excitation wavelength.
Since the light absorption term is a constant, the emission spectrum represents the quantum yield for that fluorophore.
3. Excitation emission matrices (EEMs) are assembled from a series of fluorescence emission spectra collected at sequential excitation wavelengths.
For lambdaexc1, measure I vs lambdaem
lambdaexc2, measure I vs lambdaem and so on.
This data results in a matrix where the first row consists of the excitation wavelengths, the first column consists of the emission wavelength and the rest of the matrix contains the fluorescence intensities as shown below
EEMs form surface plots that are usually presented as contour plots where each contour line connects points of equal fluorescence intensity (similar to isobar and isothermal charts).
Emission spectra may vary depending on the chemistry of the fluorophore and the environment. The position of the fluorescence maxima is dependent on the environment and the dynamics of the fluorophore. A turbid sample such as tissue contains not only multiple fluorophores which may or may not interact with each other but also contain scatterers and absorbers. Thus an EEM of such a material, shows the fluorescence excitation emission maximum of each fluorophore present altered from its pure state by its environment. Any scatterers present typically manifest themselves in a broad band attenuation of the collected spectrum. The presence of an absorber is indicated by valleys at both excitation and emission wavelengths at which it absorbs. Thus, fluorescence gives a measure of the composition as well as environment of the sample of interest.
Properties of Fluorescence
1) Stokes' Shift
In general, the emission wavelength is observed to be longer than the excitation wavelength. This can be explained by the conservation of energy. Molecules excited into higher electronic and vibrational levels quickly relax into the lowest vibrational level of S1. Thus
This shift in frequency (and wavelength) is called Stokes' shift. In practice, these means that the emission wavelength is always longer than the excitation wavelength.
2) Emission spectrum is independent of the excitation wavelength.
In an emission spectrum, since the fluorescence intensity is only dependent on the quantum yield, the shape of the emission spectrum is independent of the incident frequency, because emission only occurs from the lowest vibrational state of the lowest electronically excited state i.e. S0(0).
3) Mirror Image Rule
The absorption spectra (excitation spectra) are indicative of the vibrational levels of the excited states whereas the emission spectra indicates those of ground state. Since generally excitation does not alter these energy levels (with some exceptions), the emission spectrum appears as a mirror image of the excitation spectrum. This symmetry is due to the same vibrational levels being involved in absorption and emission.
4) Fluorescence Lifetimes and Quantum Yield
G is the rate of emission of photons by fluorescence
k is the rate of internal conversion (IC)
Quantum yield, Q is defined as the proportion of photons emitted by fluorescence as compared to the photons absorbed.
When G >>> k, i.e. fluorescence is the dominant phenomenon, then Q = 1. However, by the Stokes' shift where some of the energy absorbed is always lost by heat when decaying to S1, Q cannot be 1. Therefore Q is always less than 1. Fluorescence dyes such as Rhodamine and Fluorescein have high quantum yields (~ 0.8) and thus exhibit bright fluorescence.
Fluorescence lifetime tf, is defined as the average time that a molecule spends in S1 before decaying to the ground state. It can be written as,
What fluoresces?
Molecules that exhibit fluorescence are called fluorophores. Fluorescence is exhibited primarily by organic molecules with lots of delocalized electrons i.e. molecules with lots of conjugated bonds. For example, aromatic compounds with lots of benzene rings are known to exhibit fluorescence.
Natural biological fluorophores
There are numerous biological fluorophores that exist in tissue that exhibit intrinsic fluorescence.
1) Proteins: The most common amino acid that exhibits fluorescence is typtophan. Its excitation emission maximum occurs at (290, 330 nm). Its fluorescence lifetime is 1 - 6 ns. Most proteins exhibit the peak associated with tryptophan in their EEM. Another amino acid that exhibits fluorescence is tyrosine. Its excitation emission maximum occurs at (230, 300 nm). However, although tyrosine fluoresces in solution, its fluorescence is very weak in proteins.
2) Nucleic Acids: Nucleic acids do not in general, exhibit fluorescence. One known exception is Yeast-tRNA.
3) Co-factors: Some metabolic products exhibit fluorescence. NADH has an excitation emission maximum at (340, 450 nm). Its fluorescence lifetime is 0.5 ns. However, interestingly, NAD+ does not fluoresce.
4) Flavins: Flavins such flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) exhibits fluorescence at (450, 515 nm).
Extrinsic fluorophores
The natural fluorescence properties of biological molecules are often weak and need to be enhanced. To improve the signal to noise of the fluorescence signal extrinsic fluorophores are often added to display improved spectra. Some commonly used dyes include fluorescein, rhodamine isocyanates and isothiocyanates, often used to label proteins.
Instrumentation
The typical system used to measure fluorescence is often referred to as a spectrofluorometer. The basic system used to measure fluorescence consists of;
1. Light Source
This can be a laser or a white light source.
2. Wavelength Selector (Excitation)
A laser typically provides only 1 or a limited number of wavelengths. When multiple wavelengths are required e.g. when building an EEM, a device that disperses white light as a function of wavelength and can be tuned to select specific wavelength is used. This device may be a monochromator or in a simpler case it may consist of a series of bandpass filters in a filter wheel. Most current monochromators use diffraction gratings rather than prisms for wavelength selection. At the excitation leg, a monochromator with high efficiency in the ultraviolet region is typically chosen.
3. Light delivery and Collection device:
This might consist of a series of optics and a physical sample chamber or a fiber optic probe with coupling optics designed for optimal delivery and collection of light. The geometry of this part of the system is a key issue when studying the measured signals.
4. Wavelength Selection (Emission)
The wavelength selector at the emission end may be a spectrograph or monochromator. While a monochromator allows only one wavelength of light to exit at a given time, a spectrograph allows all the wavelengths dispersed along one axis (typically the x-axis) to exit.
5. Detector
When using a monochromator, a photomultiplier tube is used as a detector. PMTs are selective and have the highest efficiency for detection i.e. are most sensitive. PMT consists of a photocathode which ejects electrons when struck with photons. The generation efficiency of photoelectrons is proportional to the incident wavelength. The electrons then go through a series of dynodes that amplify the signal. It is a single detector and can detect the fluorescence from a single area and needs the monochromator to serially select the wavelength to build a spectrum. However, current technology has yielded the use of charge coupled device cameras (CCDs). CCDs consist of a two-dimensional array of detectors that when coupled with a spectrograph allows the simultaneous detection of all wavelengths along the x-axis while providing limited spatial information along the y-axis.
Diagnostic Application of Fluorescence Spectroscopy
A variety of biological molecules contain naturally occurring or endogenous fluorophores. Some of the best known fluorophores include the aromatic amino acids; tryptophan, tyrosine and phenylalanine, the cofactor; reduced nicotinamide adenine dinucleotide (NADH), vitamin A, flavins (flavin adenine dinucleotide (FAD)), riboflavins (flavin mononucleotide (FMN)) and porphyrins.
Although a complete understanding of the quantitative information contained within a tissue fluorescence spectrum has not been achieved, many groups have attempted to use fluorescence spectroscopy for automated, fast and non-invasive characterization of disease. It has been established that the fluorescence spectra and relaxation times of normal and diseased tissues differ. This difference may be attributed to the change in the environment surrounding the fluorophore and their quantity. Auto and dye induced fluorescence have shown promise in recognizing atherosclerosis and various types of cancer. Exogenous dyes, such as hematoporphyrin derivative (HpD), were used in early studies to enhance the fluorescence of neoplastic tissue. However, analysis using autofluorescence eliminates any likelihood of toxic reactions due to the injection of an external agent. Auto-fluorescence spectra of normal, precancerous and cancerous tissues have been measured from several organ sites, both in vitro and in vivo. These include normal and diseased tissues from the human bronchus, lung, breast, cervix, brain, and gastrointestinal tract.