Biophotonics, Tryptophan and Disease

Biophotonics, Tryptophan and Disease is a comprehensive resource on the key role of tryptophan in wide range of diseases as seen by using optics techniques. It explores the use of fluorescence spectroscopy, Raman, imaging techniques and time-resolved spectroscopy in normal and diseased tissues and shows the reader how light techniques (i.e. spectroscopy and imaging) can be used to detect, distinguish and evaluate diseases. Diseases covered include cancer, neurodegenerative diseases and other age-related diseases.

Biophotonics, Tryptophan and Disease offers a clear presentation of techniques and integrates material from different disciplines into one resource. It is a valuable reference for students and interdisciplinary researchers working on the interface between biochemistry and molecular biology, translational medicine, and biophotonics.

• Shows the key role of tryptophan in diseases
• Emphasizes how optical techniques can be potent means of assessing many diseases
• Points to new ways of understanding autism, aging, depression, cancer and neurodegenerative diseases

• Language: English
• Publisher: Academic Press
• Release date: Oct 9, 2021
• ISBN: 9780128227916

Book preview

Part I

Biophotonics to investigate tryptophan and its metabolites

Chapter 1: The physics of key biophotonic techniques

Laura A. Sordilloa; Peter P. Sordillob    a Departments of Physics and Electrical Engineering, The City College of New York, New York, NY, United States

b Department of Hematology-Oncology, Lenox Hill Hospital, New York, NY, United States

Abstract

The measurement of tryptophan, an essential amino acid and key fluorophore, can reveal important information about diseases as diverse as cancer, autism, heart disease and schizophrenia. Biophotonic techniques, such as fluorescence spectroscopy, multiphoton microscopy and resonance Raman spectroscopy, are now used widely, and have led to the creation of optical devices which can be utilized in the assessment and detection of these diseases.

Keywords

Biophotonic techniques; Fluorescence spectroscopy; Raman spectroscopy; Resonance Raman; Tryptophan; Disease; Cancer; Neurodegenerative diseases

Introduction

Measurement of tryptophan, an essential amino acid and key fluorophore, can reveal important information about diseases as diverse as cancer, autism, heart disease and schizophrenia. Biophotonic techniques, such as fluorescence spectroscopy, multiphoton microscopy and resonance Raman spectroscopy, are now used widely, and have led to the creation of optical devices which can be utilized in the assessment and detection of these diseases. When photons interact with a biological sample, molecules, which contain electric charge, can absorb the incoming photon, thus stimulating an electronic transition from the lowest (ground) energy state to a higher (excited) energy state. The allowed energy levels and bands are determined by the rules of quantum mechanics and the energy (E) of the photon is represented by Planck's equation (see Eq. 1.1)

si1_e    (1.1)

where h is Planck's constant (h is 6.63 × 10− 34 (Joules × s)), ν is the frequency, c is the speed of light and λ is the wavelength of light.

Electronic transitions diagrams, or Jablonski energy diagrams, can be utilized to describe the excited molecule in its different energy states following absorption of light (Fig. 1.1).¹ The electron, initially excited by the incoming photon (hνA), returns to the ground singlet state (S0), resulting in the emission of a photon (hνF). This process is known as fluorescence. The molecule absorbs light in approximately ~ 10− 15 s, or 1 femtosecond (fs), followed by a vibrational relaxation on the time scale of ~ 10−12 s. The lifetime of fluorescence is in approximately ~ 10− 9 s or ~ 1 nanosecond (ns). The entire process of absorption and fluorescence typically occurs in nanoseconds. Other phenomena can occur, including phosphorescence, nonradiative loss and energy transfer.

Fig. 1.1

Fig. 1.1 The Jablonski diagrams showing the absorption, fluorescence and phosphorescence processes, where the gray horizontal lines represent vibrational levels, S is the singlet state and T is the triplet state. No permission required.

The fluorescence quantum yield (ΦF), or the efficiency of the emission, of a fluorophore is defined as the ratio of the number of emitted photons over the number of absorbed photons, as described in Eq. (1.2)

si2_e    (1.2)

Tryptophan fluorescence

The fluorescence from tryptophan, an essential amino acid, in proteins has been extensively studied due to its sensitivity to its local environment. This intrinsic characteristic can be utilized to study changes in dynamics, structure, and intermolecular interactions, and can be represented by changes in its fluorescence spectral profile (i.e., intensity, lifetime, band shape and shift). These changes may be used to distinguish normal from diseased tissues.²–⁵ Abnormal tryptophan metabolism down the kynurenine pathway has been linked to cancer and neurodegenerative diseases, such as Alzheimer's and Parkinson's.⁶–¹⁰ Tryptophan and its metabolites (such as kynurenine, serotonin and kynurenic acid) have been investigated using fluorescence spectroscopy.¹¹ The ratios of tryptophan to tryptophan metabolites can shed light on the progression of neuroinflammation in many diseases. For example, van Rooijen utilized tryptophan fluorescence to obtain information on the structural features of α-synuclein oligomers.¹² Oligomeric α-synuclein has been linked to the development and progression of Parkinson's disease.⁷, ¹³–¹⁵,

Table 1.1 shows the optical properties (quantum yield, as well as absorption, fluorescence (at wavelength peak maxima (nm)) and absorptivity) of tryptophan, tyrosine and phenylalanine.¹⁶ Tryptophan has a higher quantum yield (ΦF = ~ 0.2) compared to that of other aromatic amino acids, such as tyrosine and phenylalanine. In fact, the quantum yield of tyrosine (ΦF = ~ 0.14) is nearly equivalent to tryptophan, yet the emission of tyrosine is less than tryptophan due to quenching (a result of many factors including the process of energy transfer from tyrosine to tryptophan). Due to changes in its local environment, manifested by changes in the spectral profile of tryptophan, the tryptophan fluorescence quantum yield can vary (to values as high as 0.35). Tryptophan absorbs in the ultraviolet (UV) range (at around 280 nm) and emits at ~ 340 nm. Tryptophan can also absorb at 215–220 nm. The fluorescence intensity peak maximum can also vary by ~ 40 nm.¹⁷

Table 1.1

The chemical structures of L-tryptophan, L-tyrosine and L-phenylalanine are shown in Fig. 1.2. Unlike tyrosine and phenylalanine, L-tryptophan possesses an indole ring.

Fig. 1.2

Fig. 1.2 Chemical structures of L-tryptophan, L-tyrosine and L-phenylalanine with the indole ring of tryptophan highlighted. No permission required.

In order to understand better what gives tryptophan its uniqueness, it is important to elucidate the excited-state properties of indole, the side-chain chromophore of tryptophan. Extensive studies on the fluorescence of indoles have been reported.¹⁷–²⁰ The spectral profiles of indole show two absorption regions at ~ 280 nm and around 220 nm. The ¹La and ¹Lb electronic transitions can exhibit distinct optical properties such as the degree of sensitivity to its environment. These overlapping transitions give way to complexities, which have been the focus of many investigations.

While tryptophan excites in response to UV light at ~ 280 nm, multiphoton microscopy, which uses longer wavelengths of light in the near infrared (NIR) (650–950 nm) region and is based on the non-linear relationship between light and matter, can also be utilized in the investigation of tryptophan fluorescence. NIR (from 650 to 950 nm) and short wavelength infrared (SWIR) (from 1000 to 2500 nm) light region can penetrate more deeply through tissue, revealing abnormalities hidden beneath tissue layers.²¹–²⁴ Multiphoton microscopic devices for deep tissue imaging of biomolecules, which involve an elaborate optical setup, have become more accessible due, in part, to the Ti:Sapphire laser. For instance, tryptophan can also be excited using the three-photon (3P) technique and light at a wavelength of 840 nm (three times 280 nm (or 1P)), resulting in a fluorescence at ~ 340 nm. Fig. 1.3 shows a simplified energy diagram highlighting multiphoton excitations, using three-photon (3P) at a wavelength of 840 nm, two-photon (2P) at 560 nm and one photon (1P) at 280 nm, and fluorescence at 340 nm of tryptophan.

Fig. 1.3

Fig. 1.3 Simplified energy diagram highlighting multiphoton excitations, using three-photon (3P) at a wavelength of 840 nm, two-photon (2P) at 560 nm and one photon (1P) at 280 nm, and fluorescence at 340 nm of tryptophan. No permission required.

Features of a fluorescent spectral profile from tryptophan

When a photon is released as fluorescence (the result of a fall from the excited state S1 to the ground state S0), the emission is recorded as a Gaussian-like spectral profile with a peak maximum at a specific wavelength (λem). Throughout this process, energy is lost in the form of internal conversion or vibrational relaxation of an excited-state electron to the lowest energy level within that excited state, which is the reason why the emission is always at a longer wavelength than absorption. The wavelength difference between that of the incident photon and that of the emitted photon is the Stokes shift. Emission or de-excitation can be described by Eem = hc/λem, or, in the case of absorption or excitation by Eexc = hc/λexc. Thus, we have shown that Eexc > Eem or λexc < λem.

The fluorescent intensity (I) is a result of the n excited fluorophores in the material. The spectral profile from fluorophores in tissue can be represented by a 2-D plot of the emission intensity (I) versus the wavelength of light (usually in nm). For example, the fluorescent spectral profiles, using a 280 nm excitation wavelength, from normal and cancerous human breast tissues (based on key fluorophores) are shown in Fig. 1.4.⁵ The wavelength peak maxima are located at ~ 340 nm. Due to the distinct absorption and emission wavelengths of biomolecules (or optical fingerprints), the spectral profiles can be analyzed in terms of the relative content of key biomolecules such as tryptophan and NADH. In this case, the features of the fluorescent spectral profiles from normal and cancerous tissues can be normalized using the ratio of intensity (I) peaks, most likely due to tryptophan (emission peak at ~ 340 nm) and NADH (emission peak at ~ 440 nm). The ratios si3_e revealed a higher relative content of tryptophan over other biomolecules in cancerous versus normal breast tissues. Further investigation showed that there exists a strong correlation between increased relative tryptophan content and high histological grade (a recognized, but underappreciated, predictor of prognosis in breast cancer patients).

Fig. 1.4

Fig. 1.4 Fluorescence spectral profiles from breast cancer (high grade) and breast normal tissues represented by 2D-plots of intensity (arbitary units) versus wavelength (nm). From Sordillo LA, Sordillo PP, Budansky Y, Pu Y, Alfano RR. Differences in fluorescence profiles from breast cancer tissues due to changes in relative tryptophan content via energy transfer: Tryptophan content correlates with histologic grade and tumor size but not with lymph node metastases. J Biomed Opt. 2014;19(12): 125002. https://doi.org/10.1117/1.jbo.19.12.125002.

The design of photonic devices used in the evaluation of intrinsic tryptophan fluorescence

Light sources and detectors

Lamp-based light sources such as xenon and mercury-arc lamps are common light sources for inducing UV fluorescence spectroscopy. These broadband light sources provide a spectrum of color from the UV to the NIR. A bandpass filter is typically used to isolate the UV wavelengths needed for tryptophan excitation. Now, with the advent of solid-state UV light sources such as diode-pumped solid-state UV lasers and light-emitted diodes (LEDs), the efficiency of UV light sources has drastically improved.²⁵–²⁸ Commercial LEDs in the UVC range (UVC (200–280 nm), UVB (280–315 nm) and UVA (315–400 nm)) are now available and are compact, low cost, resilient, and have minimal energy consumption. They are currently being used in a variety of applications, including surface disinfection and water purification.²⁷, ²⁸ UVC lasers, on the other hand, typically rely on frequency doubling (or second harmonic generation) to reach shorter UV wavelengths of light. This nonlinear process can be achieved using a laser with photons of frequency ω and a non-linear material known as a BBO crystal (beta barium borate). The fundamental wave of light produces a nonlinear polarization wave with double the fundamental frequency (2ω). The Ti:Sapphire and Nd:YAG laser (with wavelengths at ~ 680–1100 and 1064 nm, respectively), for instance, can also be used in the investigation of tryptophan (see Table 1.2).

Table 1.2

An optical detector (or photodetector) can be utilized for biomedical applications (as well as sensing and optical communications) and typically involves a photodiode with a high sensitivity and response to light in a certain spectral wavelength range. The electronics that enable optical detection is known as optoelectronics. A photomultiplier tube (which works based on the photoelectric effect) can be used as a light sensor for low-light measurements.²⁹, ³⁰ Time-resolved fluorescence spectroscopy (for lifetime measurements) requires an ultrafast detector such as a streak camera.³¹

Instruments used for tryptophan fluorescence

Luminescence spectrophotometers or spectrometers can measure fluorescence, phosphorescence and bioluminescence. Typically, spectrophotometers include a xenon broadband lamp light source with controllable power levels (to avoid photobleaching). Light is obtained using a computer controlled moveable grating. The incoming light at select wavelengths hits (or passes through) the sample (which may be housed in a cuvette) before being collected by the detector. Most spectrophotometers are equipped with the ability to provide either excitation, emission or synchronous scanning at wavelength ranges from 200 to 900 nm. A synchronous scan is the simultaneous scanning of both the excitation and emission, with a fixed wavelength equaled to the difference between he emission and absorption peak maxima (Stokes shift).

With the advent of smaller, more compact, optical components such as LEDs, optical fibers and miniature detectors (CCDs), there have been a great number of novel fluorescent-based devices proposed for fluorescence spectroscopy. We utilized a compact LED-based device, known as the S³-LED ratiometer unit, for the assessment and detection of relative tryptophan levels in breast cancer tissues.⁴ The unit contained multiple LEDs with wavelengths in the UV and visible range (to highlight the optical fingerprints of key biomolecules in tissue including tryptophan, NADH, collagen and flavin) coupled to optical fibers. Light from the select LED traveled through the optical fiber before reaching the sample (which was contained in a dark box), where it was absorbed and emitted by the biomolecules in the tissue. A deep UV LED at 280 nm was used to excite the breast cancer tissue samples. The emitted light from the key biomolecules, tryptophan and NADH, in the tissue samples was collected using an Ocean Optics spectrometer, then was recorded on a computer program. The unit provided a straightforward and effective way of displaying the intrinsic characteristics of human breast cancer. The authors concluded that the compact unit may also be utilized to assess cancer margins, therefore reducing the need for repeat surgeries.

Three-photon (3P) excitation can also be utilized in the investigation of tryptophan fluorescence.¹⁶, ³²–³⁴, Typically, 3P excitation at 840 nm requires the use of the ultrafast Ti:Sapphire laser (which can produce select excitation at wavelength from ~ 680 to 1100 nm with femtosecond pulses). The fluorescence is collected using a detector with sensitivity at around 340 nm. The use 3P fluorescence lifetime imaging and Förster resonance energy transfer has been reported as a technique to investigate the metabolic activity of tryptophan and NADH. Jyothikumar et al. used a Coherent Mira 900 Ti:Sapphire laser, bandpass filters, and a PMC-100-0 photomultiplier tube.³⁵ They found that this method can be utilized to monitor tryptophan quenching patterns in a variety of diseases, such as in cancer and diabetes.

Raman spectroscopy of tryptophan

Basic principles of Raman spectroscopy

Photon-matter interaction can result in elastic or inelastic scattering. Raman scattering is an inelastic scattering process. When a scattered photon loses energy in a Raman process, the transition is referred to as a Stokes transition. Similarly, when a scattered photon gains energy, it is known as an anti-Stokes transition. Elastic scattering (or Rayleigh scattering) can give information on the size of the inner structures of the material, while Raman can give Raman bands of vibrational modes from specific chemical bonds.

Resonance Raman and tryptophan

The resonance Raman (RR) effect is an enhanced Raman technique which utilizes an excitation laser at a wavelength that matches the absorption wavelength maximum from select biomolecules in the sample. Though understanding of this phenomenon is not new, it is now being implemented more frequently in the assessment of disease. This approach can highlight, without the addition of contrast agents (label-free), key biomolecules such as beta-carotene and flavin (represented by an increase in the Raman signal). Liu et al. used a confocal micro-Raman system equipped with a 532 nm laser to obtain the RR spectra from samples of plaque in the aortic tunica intimal walls of human cadavers.³⁶, ³⁷ Key Raman peaks were located at 1012, 1161 and 1517 cm− 1 and identified as key locations where the resonance effect was significant (enlargement of the Raman signal). In addition, peaks 758 and 1589 cm− 1 were associated with tryptophan.

In the 1980s and 1990s, extensive studies on UV RR spectroscopy (with UV laser light excitation) from amino acids had been reported.³⁸–⁴² The typical laser light source for UV RR of tryptophan (for select enhancement of tryptophan) was the Nd:YAG laser (1064 nm) light source. The fourth harmonic line (266 nm) of the fundamental frequency (1064 nm) was used to generate UV RR excitation of tryptophan. This method has also been used with other optical tools to produce additional excitation wavelengths, for instance, at around 220 nm (to match an absorption peak of tryptophan at 220 nm). In one study, RR frequencies of major bands of tryptophan in solution from 218 nm excitation (using the Nd:YAG laser system) revealed frequencies at 762, 884, 1016, 1241, 1361, 1555, and 1622 cm− 1.⁴¹

Due to the vast literature on UV RR spectroscopy of tryptophan, UV RR has been suggested as a powerful tool to probe protein folding.⁴³–⁴⁶ Folding of a membrane protein can result in changes in the microenvironment of tryptophan, and thus in its vibrational signature. Today, the Ti:Sapphire laser, which can be tuned to a desired excitation wavelength in a wide spectral range (usually  ~650-1100 nm), can be utilized in UV RR spectroscopy. Sanchez et al. studied UV RR spectroscopy of folded and unfolded states of an integral membrane protein using excitation light at 230 nm to enhance the Raman signal from tryptophan vibrational modes due the La − b and bb electronic transitions⁴⁶. They found that UV RR spectra revealed residue specific, structural details of a protein in the native environment of a bilayer membrane. Asamoto et al. showed that UV RR spectroscopy can be used to study membrane protein structure and dynamics. Using excitation at 228 nm (to selectively enhance tryptophan and tyrosine), several prominent L-tryptophan Raman modes were identified at 759, 878, 1340, 1362, and