OPTIQUE et BIOLOGIE. Cycle ingénieur 2A mars/mai Nathalie Westbrook Karen Perronet Groupe Biophotonique, Institut d Optique

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1 OPTIQUE et BIOLOGIE Cycle ingénieur 2A mars/mai 2017 Nathalie Westbrook Karen Perronet Groupe Biophotonique, Institut d Optique

2 Outline of the course (18h) 14 & 21 / 03 / 2017: Molecular and Cellular Biology Overview of optical properties and methods for biology and medicine Fluorescence microscopy (6h, NW) 28 / 03 / 2017: Molecular Biology techniques: gel electrophoresis, recombinant DNA technology, cell culture,... (2h, KP) Fluorescent proteins and their applications (1h, KP) MOOC Nano : Nanobiology week 29/03 -> 04/04 : REGISTER on FUN 25 / 04/ 2017: Optical tweezers and applications in biology - (3h, NW) 02 / 05 / 2017: Atomic force microscopy (1h, KP) Flow cytometry (1h, KP) Sequencing (1h, KP) 09/ 05 / 2017: Biomedical applications (3h, NW) 19/ 05 / 2017 : WRITTEN EXAM (2h, NW + KP) Problem sessions or discussions on publications on subjects related to the lecture: 45mn to 1h at the end of each lecture

3 Ressources Libres savoirs page du cours 2A Opt&Bio 1) Tester vos connaissances sur la cellule avec le quiz sur site Sagascience du CNRS 2) Visionner la vidéo Inner Life of the Cell de Biovisions et essayer de comprendre ce qui est décrit 3) Voir le site du prix Nobel pour les textes dits «popular» sur les prix de Chimie ) Lire l article «New Optics sheds light on biology» dans EMBO reports 2011 (disponible dans onglet biblio)

4 1 Basics of biology Content of lecture 1 Structure of the prokaryotic and eukaryotic cells To be continued in lecture 2 2 Optical properties of biological material absorption, scattering, fluorescence

5 1 Basics of Biology Several of these slides are extracted from a course «introduction to biophotonics» by Thomas Huser from UC David and Lawrence Livermore National Laboratory It refers largely to the book «Molecular Biology of the Cell» by Alberts et al, of which a simplified version exists at the IOGS library «L essentiel de la biologie cellulaire» (in french translation) Similar content can be found in the chapter 3 «basics of biology» in the book «Biophotonics» also available at the IOGS library

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9 Differences between prokaryotic and eukaryotic cells The prokaryotic cell has: - No nucleus - DNA directly in the cytosol - No internal organelles with membranes It is also smaller (about 1 micron)

10 Fluorescence images of eukaryotic cells Bovine Pulmonary Artery Endothelial Cell Blue: nucleus (DNA) Green: microtubules Red: Actin filaments Deer Skin Fibroblast cells Yellow: nucleus Green: Actin Red: Mitochondria

11 Cell Structure, Constituents and their Functions Main distinctive cell groups: Eukaryotic cells: animal/plant cells with complex structure Prokaryotic cells: bacterial cells (~1 µm size) - single-cell organisms Plasma membrane Semipermeable barrier defining the outline of the cell. Made from a double layer (bilayer) of phospholipids. Cholesterol molecules provide rigidity of the otherwise fluid bilayer. Contains proteins that can be anchored to the interior and form receptors, pores (channels), and enzymes to control transport and communication with the exterior.

12 Cell Structure, Constituents and their Functions - II Cytoplasm Everything in the interior of cells, except for the nucleus. Mixture of fluid containing salts, sugars, lipids, vitamins, nucleotides, amino acids, RNA, and proteins. Cytoskeleton Protein filaments connected to organelles to provide cell shape, transport, strength, and locomotion. Nucleus Largest organelle. Contains chromosomal DNA tightly packaged b y the DNA binding protein histone to form nucleosomes.the nucleus is separated from the cytoplasm by an inner and an outer membrane containing pores. Inside the nucleus are nucleoli that contain mostly RNA and proteins and produce ribosomes. Important factory for proteins. Nucleosome (discovered 1974, high-res structure solved 1997)

13 Cell Structure, Constituents and their Functions - III The Mitochondrion Mitochondria = The cell s power plant (intake of oxygen and harness energy ( food molecules) production of ATP ( powers cell activities) Smooth outer membrane Mitochondria have their own - circular DNA Molecule - ribosomes Convoluted inner membranes Three dimensional structure - transfer RNA Cross section, as seen in the electron microscope Schematic eucaryotic cell with Mitochondrion (orange = miochondrial DNA and ribosomes) The convoluted inner membrane houses the proteins that generate ATP from the oxidation of food molecules.

14 Cell Structure, Constituents and their Functions - IV Endoplasmic Reticulum (ER) Sheets, sacs, and tubes of membranes enclosing intracellular space called lumen. Close to nucleus. Site of attachment of ribosomes. Transitions to smooth ER, which is the site of synthesis of lipids and sugars. Golgi apparatus Stacked, flattened sacs of membranes, which ship proteins to other organelles by use of vesicles. Lysosomes Single membrane vesicles ( µm diameter) containing enzymes in an acidic environment. Sites for cellular digestion. Peroxisomes Membrane-bound vesicles ( µm diameter) containing oxidative enzymes that generate and destroy hydrogen peroxide. Chloroplasts Plant cell organelle. Site of photosynthesis (light energy - ATP).

15 2 Optical properties of biological material Ø Absorption from the UV to the IR Ø Scattering Ø Fluorescence

16 Absorption of biological material In the UV: damage to the skin, absorption of nucleic acids at 260nm, of amino acids at 280nm, of collagen, elastin, NADH in the nm range In the visible: low absorption in general (microscopic imaging), except for hemoglobin, melanine, rhodopsin (retina) and chloroplasts (in plant cells) In the near IR: «optical window», best penetration through tissue, used for in-depth imaging In the IR: water absorption, Raman spectroscopy to identify molecular content

17 Absorption Absorption spectroscopy spectroscopy is a routinely used in technique the UV in the biosciences Absorption spectroscopy is widely used in bio sciences to determine concentrations by measuring optical density (OD): Samples in 1 cm path cells quartz/glass; for <250 nm purge with N 2 Proteins: Tyr/Trypt/Phe in nm range # max at ~ 280 nm DNA # max at ~ 260 nm

18 Main sources for light loss in biological materials: absorption and light scattering Absorption in tissue in the visible and near IR 10 Tissue absorption Absorption spectra of hemoglobin Water absorption and oxyhemoglobin Hemoglobin Water 1 Absorption.1.01 OPTICAL WINDOW Wavelength 1200 Figure courtesy Dr. Stavros Demos, LLNL Optical window used for in-depth imaging (best penetration) 1400 Used to measure blood oxygenation (eg to monitor brain activity) omlc.ogi.edu/spectra/wate#6e1cb

19 Spectroscopy in the IR corresponding to molecular vibrations: Raman spectroscopy and imaging Surface Enhanced Raman Spectroscopy : example taken from the Tripp Laboratory, Univ of Georgia, Athens (USA) "! Rapid detection of trace levels of viruses with a high degree of sensitivity and specificity (no need for viral manipulation and DNA amplification) "! Metallic nanostructures enhance the weak Raman signal

20 Scattering in tissue Scattering in tissues Structures << wavelength of light: Rayleigh scattering Structure ~ wavelength: Mie scattering

21 Consequences of scattering Imaging is limited to thin samples, unless ballistic photons can be isolated Scattering decreases when wavelength increases + absorption increases in the IR due to water => Penetration depth is best in the near infrared (2 to 6 mm at 1 µm)

22 Fluorescence The property of absorbing light of short wavelength and emitting light of a longer wavelength Natural fluorescence from marine jellyfish Aequorea Victoria (from which derives the Green Fluorescent Protein GFP)

23 Excitation / Emission spectroscopy of native fluorophores in tissue (autofluorescence) Absorption absorption max. (nm) émission max. (nm) origine tryptophane protéines collagène tissu conjonctif élastine tissu conjonctif NADH chaîne respiratoire Emission flavines chaîne respiratoire Information about the biochemical composition of tissue can be obtained by analyzing its absorption and fluorescence spectra

24 Example of NADH fluorescence detection to test photosensitizer efficiency for photodynamic therapy In blue: fluorescence of NADH When a photosensitizer is injected in the cell and irradiated with light (image D), the NADH fluorescence decreases, which is a signal for cell death. Without the photosensitizer (B), the cell remains alive.