Spectroscopy — Light and Diagnostic Imaging

Spectroscopy is the study of how matter interacts with electromagnetic radiation. Different types of radiation interact with matter in characteristic ways, forming the basis of an enormous range of analytical and medical techniques. The electromagnetic spectrum (from lowest to highest energy): Radio waves → Microwaves → Infrared → Visible light → Ultraviolet → X-rays → Gamma rays All electromagnetic radiation: - Travels at the speed of light (c = 3×10⁸ m/s) in vacuum - Exhibits wave-particle duality - Carries energy E = hf = hc/λ where h = Planck's constant (6.626×10⁻³⁴ J·s), f = frequency (Hz), λ = wavelength (m) - Higher frequency = shorter wavelength = higher energy Energy levels in atoms and molecules: Electrons exist in discrete energy levels. When a photon of exactly the right energy is absorbed, an electron jumps to a higher level (excitation). When it falls back, a photon is emitted. This quantisation means each element absorbs and emits characteristic wavelengths — its spectral fingerprint. Beer-Lambert Law: When light passes through a solution, the absorbance A = εcl, where ε = molar absorptivity, c = concentration, l = path length. Absorbance is proportional to concentration. This is the basis of spectrophotometry — measuring the concentration of coloured substances in solution (including haemoglobin, enzyme substrates, drugs).

Ultraviolet (200–400 nm) and visible (400–700 nm) light is absorbed by molecules with π bonds and lone pairs (chromophores). The absorbed wavelength depends on the electronic structure. Principle: Light of wavelength λ passes through a sample. The detector measures transmitted intensity I. Absorbance A = log(I₀/I). A plot of A vs λ gives an absorption spectrum — a characteristic curve for each molecule. Applications in medicine and biology: Pulse oximetry: Haemoglobin (Hb) and oxyhaemoglobin (HbO₂) absorb differently at 660 nm (red) and 940 nm (infrared). Pulse oximeters shine LEDs at both wavelengths through a fingertip. The ratio of absorbances gives oxygen saturation (SpO₂). Normal SpO₂ ≥ 95%. This non-invasive continuous monitoring is fundamental in anaesthesia and intensive care. Blood tests: Colorimetric assays measure metabolites by reaction with a reagent to produce a coloured compound. Glucose oxidase/peroxidase assay (glucose), Jaffe reaction (creatinine), biuret reaction (total protein). The absorbance is read at a specific wavelength and concentration calculated using Beer-Lambert Law. Bilirubin: Bilirubin (yellow-orange) absorbs at 450 nm. Jaundice (yellow skin and sclera) occurs when bilirubin > 35–50 μmol/L. Phototherapy (blue-green light, 420–490 nm) is used for neonatal jaundice — light isomerises bilirubin to a water-soluble form that can be excreted without conjugation. Haemoglobin measurement: Cyanmethaemoglobin method: blood is lysed and haemoglobin converted to cyanmethaemoglobin, which absorbs at 540 nm. Concentration calculated from absorbance. Reference: males 130–170 g/L, females 120–150 g/L.

Infrared (IR) Spectroscopy: Infrared radiation (wavelength 2.5–25 μm, wavenumber 400–4000 cm⁻¹) is absorbed by molecular vibrations — stretching and bending of covalent bonds. Each functional group vibrates at characteristic frequencies. Key IR absorptions (functional groups): O-H stretch: broad 2500–3300 cm⁻¹ (carboxylic acids), sharp 3200–3600 cm⁻¹ (alcohols) N-H stretch: 3300–3500 cm⁻¹ C=O stretch: 1700–1750 cm⁻¹ (very strong, characteristic) C-H stretch: 2850–3100 cm⁻¹ C≡N: ~2200 cm⁻¹ Medical application — breath analysis: CO₂ absorbs strongly at 4.26 μm (2349 cm⁻¹). Capnography (end-tidal CO₂ monitoring) uses infrared spectroscopy to measure CO₂ in expired breath — essential for confirming correct intubation and monitoring respiratory status. Nuclear Magnetic Resonance (NMR) Spectroscopy: NMR exploits the magnetic properties of certain nuclei (¹H, ¹³C, ³¹P). In a magnetic field, nuclei align with or against the field. Radio frequency pulses disturb this alignment. As nuclei relax back, they emit signals — the chemical shift reveals the chemical environment of each nucleus. ¹H NMR (proton NMR): different types of protons (CH₃, CH₂, aromatic, OH) resonate at different frequencies. The spectrum provides structural information for identifying compounds — essential in pharmaceutical development and quality control. MRI (Magnetic Resonance Imaging): MRI is clinical NMR applied to the body. Water protons (¹H) in different tissues have different relaxation times (T1, T2). Radio frequency pulses excite protons; spatial gradients localise the signal. Produces exquisite soft-tissue images without ionising radiation. T1-weighted images highlight fat (white); T2-weighted images highlight water/fluid (white). Gadolinium contrast agents shorten T1 relaxation, enhancing vascular structures.

X-ray Spectroscopy and Imaging: X-rays (wavelength 0.01–10 nm, energy 0.1–100 keV) are produced when high-energy electrons decelerate in a metal target (bremsstrahlung) or when inner-shell electrons are displaced (characteristic X-rays). Radiography: X-rays are differentially absorbed by tissues — bone (calcium, high atomic number) absorbs strongly (appears white); soft tissue absorbs less (appears grey); air absorbs minimally (appears black). Used for: fracture detection, pneumonia, pulmonary oedema, foreign bodies. CT scanning (Computed Tomography): A narrow X-ray beam rotates around the patient; detectors measure transmitted intensity from many angles. Computer reconstruction produces cross-sectional images with much greater density resolution than plain radiography. Hounsfield units (HU) quantify X-ray attenuation: water = 0 HU, air = −1000 HU, bone = 400–1000 HU, blood ~45 HU, fat −100 to −50 HU. X-ray Crystallography: X-rays diffract off crystal lattice planes (Bragg diffraction). The diffraction pattern reveals the three-dimensional arrangement of atoms. Used to determine protein and DNA structures — the X-ray diffraction image of DNA by Rosalind Franklin and Raymond Gosling was crucial in Watson and Crick's discovery of the double helix in 1953. Mass Spectrometry (MS): Molecules are ionised, then separated by mass-to-charge ratio (m/z) in a magnetic or electric field. The mass spectrum shows the relative abundance of ions at each m/z value. The molecular ion (M⁺) gives the molecular mass. Fragmentation patterns help identify structure. Clinical applications: Toxicology screening: urine or blood drug testing using GC-MS (gas chromatography-mass spectrometry) or LC-MS/MS (liquid chromatography-tandem MS). Highly sensitive and specific. Neonatal screening: tandem MS screens for 30+ metabolic disorders (PKU, MCAD, galactosaemia) from a heel-prick blood spot. Pharmacokinetics: measuring drug and metabolite concentrations in plasma.

Understanding the physical principles behind each imaging modality helps clinicians select the appropriate investigation: Plain X-ray: Best for: bone, lungs, foreign bodies, large abdominal gas patterns Limitations: poor soft-tissue contrast, 2D projection (structures overlap) Radiation: low (chest X-ray ~0.02 mSv) Cost: low, widely available CT scan: Best for: trauma (head, chest, abdomen), vascular (CT angiography), oncology staging, detailed bone anatomy Limitations: significant radiation, renal toxicity of iodinated contrast Radiation: moderate–high (CT abdomen ~8 mSv, equivalent to ~3 years background) Speed: very fast (seconds) MRI: Best for: soft tissue detail, brain, spine, musculoskeletal, cardiac, liver, prostate Limitations: no ionising radiation but expensive, noisy, slow, contraindicated with some metal implants No radiation Speed: minutes (slower than CT) Ultrasound: Best for: abdominal organs (gallstones, kidneys, liver), obstetrics, cardiac (echocardiography), vascular (Doppler), soft tissue masses, guided procedures Limitations: operator-dependent, poor penetration through bone and gas, limited field of view No radiation, no contrast agent needed Real-time imaging Nuclear medicine (SPECT, PET): Best for: functional imaging — bone metastases, cardiac perfusion, thyroid, renal function, cancer staging Limitations: radiation, poor spatial resolution (anatomy), requires radiopharmaceutical Radiation: moderate (bone scan ~3 mSv, FDG-PET ~7 mSv) Often combined with CT (SPECT-CT, PET-CT) for anatomical correlation.