VAST CAPABILITIES AND LIMITATIONS OF IN VIVO IR SPECTROSCOPY Chu Nguyen Kien
OUTLINE Introduction Background Instruments Case study Current Applications Conclusion
INTRODUCTION In Vivo (Latin within the living ) Experimentation using a whole, living organism such as animals or plants In Vitro (Latin within the glass ) Experimentation using partial, or dead organisms in a controlled setting In vivo better suited for observing overall effects of an experiment on an living subject Ex. Animal testing
INTRODUCTION Infrared Spectroscopy (IR) Technique used to study interaction between a molecule and radiation from the IR region (wavelength 700 nm 1mm) Typical IR spectrum shows absorbance as a function of wavenumber (cm -1 ) Near-IR region (790 nm 2500 nm) Molecular overtone and combination of vibrational modes of molecules
BACKGROUND Vibrations caused by molecules Oscillation of atoms or molecules formed by chemical bonds Transfer of energy from photon at given wavelength (λ) causes molecular vibrations Classical model of Hooke s law V = 1 2π k u
V = 1 2π k u Where, V is wavenumber (cm -1 ) k is force constant of bond c is speed of light (2.998 m/s) u is the reduced mass μ = m 1m 2 m 1 +m 2 m is atomic mass of atom Gives valuable information in functional group region (4000-1000cm -1 ) and fingerprint region ( <1000 cm -1 )
INSTRUMENTS Dispersive Spectrometer Radiation source, monochromator, and detector IR radiation from beam passes through sample and reference Monochromator disperses broad spectrum of IR radiation into narrow IR frequencies Detector convert analog spectral output into electrical signal, processed and converted to IR spectrum Absorption of sample produces change of IR radiation intensity
FOURIER TRANSFORM INFRARED (FTIR) SPECTROMETRY Used to obtain infrared spectrum of absorption or emission of solid, liquid, or gas. High-spectral-resolution data over wide range Components: Source black-body source Interferometer Michelson interferometer Sample holder sample Detector measures analog spectral signal Amplifier multiples signal to an acceptable intensity for A/D converter A/D converter converts analog signal to digital signal
ThermoFisher. Introduction to Fourier Transform Infrared Spectrscopy Thermo Fisher Scientific Inc.: Waltham, Massachusetts, USA, 2013
MICHELSON INTERFEROMETER Incoming light traveling in a plane hits beam splitter (halfsilvered mirror) Transmits and reflects 50% of incident light Path 1 and 2 reflected back by mirror 1 and movable mirror 2 Path 2 interferes with path 1 once transmitted back by beam splitter Difference in intensity measured
FIBRE OPTIC PROBE Collection of spectra for in vivo experimentation Fiber optics channel spectral information by internally reflecting rays of near-infrared light at angle of incidence within acceptable angle Can penetrate farther into skin than midinfrared NIRS based on relative transparency of tissue to light in Near IR region Omega, Fast Response Infrared Fibre Optic Thermometer, Omega Engineering INC. Irlam, Manchester, UK, 2003
CASE STUDY
Used Near-IR Spectroscopy (NIRS), an optical method, to measure tissue O 2 consumption and delivery Non-invasive determination of local oxygen consumption and blood flow in human skeletal muscle Travels through overlying tissue as well Adipose tissue thickness (ATT) on subcutaneous layer affects NIRS measurements Study determined influence of ATT on quantitative in vivo measurement of local mvo 2 and forearm blood flow (FBF) in large group of heavy subjects as well as influences of ATT during exercise or gender differences
METHOD 34 female, 44 male participants Average Age range (28.2 ± 12.5 years) Height (177.6 ± 9.0 cm) Weight (71.6 ± 11.4 kg) Skinfold thickness measured between the NIRS optodes using skinfold caliper, then divided by 2 to obtain ATT Oxygen-dependent absorption changes of haemoglobin (Hb) and myoglobin (Mb) Continuous-wave NIR spectrometer generating light at 905, 850, and 770 nm can differentiate oxy- and deoxy-hb/mb
METHOD Subject was seated for 15-20 minutes with ambient room temperature of approx. 21 C Right hand rested on handgrip dynamometer Pneumatic cuff placed around upper arm in order to apply venous or arterial occlusion Measured venous and arterial occlusion between 30 second measurements and 5 min recovery times Isometric handgrip exercise performed for 1 minute, and measured again Maximum voluntary contraction (MVC) force of subject determined before test
RESULTS Forearm measurments Sum of [O 2 HB] and [HHb] reflects total amount of Hb ([thb]), and changes in [thb] can be interpreted as changes in blood volume in the tissue Rate of increase in [thb] during venous occlusion Myocardial Volume Oxygen (mvo 2 ) Measured by NIRS using arterial occlusion by evaluating the rate of decrease in [Hb diff ] ([Hb diff ] = [O 2 Hb]-[HHb]) Concentrations in μm*s -1 converted to ml*min -1 *100mL -1 for both measurements
RESULTS Van Beekvelt et al., Adipose tissue thickness affects in vivo quantitative near-ir spectroscopy in human skeletal muscle, The Biochemical Society and the Medical Research Society 2001, pp 24 ATT mean value of 3.7 ± 1.9mm with a range of 1.4 8.9mm FBF had coefficient of variation during venous occlusion of 22.4% and mvo 2 for arterial occlusion was 16.2%
Van Beekvelt et al., Adipose tissue thickness affects in vivo quantitative near-ir spectroscopy in human skeletal muscle, The Biochemical Society and the Medical Research Society 2001, pp 25
CAPABILITIES AND LIMITATIONS Advantages Allows for the more accurate representation effects on living organisms Quick analysis with no sampling procedure As fibre optics and quality of materials increases, instruments can still improve vastly Limitations Difficult to create a standard Risk assessment is a priority Still developing as data is too complex and daunting Overlap of absorption contributors from various unwanted analytes
CONCLUSION In vivo IR spectroscopy allows for the further advancement of quick noninvasive studies Non-destructive technique used in bioanalytical studies Learning curve as issues still arise from complex nature of living organisms
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