OPTIQUE et BIOLOGIE
Cycle ingénieur 2A – mars/mai 2019
Nathalie Westbrook Karen Perronet
Groupe Biophotonique, Institut d’Optique
Content of lecture 2
① Microscopy and fluorescence
② Basics of biology (part 2) Types of cells – Cell membrane – Signalling –
Chromosomes- Cell cycle – from DNA to proteins + Exercices
Microscopy and fluorescence (part 1)
Optical imaging using microscopy • Standard resolution: diffraction limit
• Contrast methods – Without markers:
Dark field, Phase contrast, Differential interference contrast
New developments in phase contrast microscopy – Fluorescence microscopy: markers, optical
configurations
• Superresolution techniques: beyond the diffraction limit
Typical optical configuration
Most microscope objectives form an image at infinity: the magnification specified (60x typ) is for the objective combined with a specific tube lens
Microscope objectives for biology applications are corrected for the spherical aberration due to the 170µm cover slip.
The larger the magnification, the smaller the working distance (object to first objective surface)
Objective forming an image at a finite distance (160mm typically)
Lateral resolution limited by diffraction (NA of the objective)
in incoherent illumination
Standard optical resolution
€
rmin = 1,22 * λ2NA
Axial resolution (depth of field) :
€
Δxmin ≈ 2nλ
NA2
Simulated PSF for NA=1,4
λ=500nm n=1,5
1nm 100nm 10µm 1mm ~20nm
Cell Virus Atom
Standard resolution in microscopy
Ribosome/mRNA
~0.1nm
Comparison with typical sizes of biological objects
Dark field image of diatoms
Similar to strioscopy (principle above): illumination at grazing incidence and only the light diffracted by the phase object is detected
(see figure below)
① Dark Field Light
source"
Object plane"
Fourier plane"
Dark spot"
Lens" Image plane"
Filtered image"Object
under study"
In a microscope, dark field contrast requires only a large annular diaphragm in the back focal plane of the condenser
From zeiss-campus.magnet.fsu.edu
Contrast without markers
② phase contrast
From www. microscopyu.com (Nikon)
A phase plate is placed on the image of the source (Fourier plane) so that the direct light interferes with the light diffracted by
the phase object
Light source"
Object plane"
Fourier plane"Lens" Image plane"
Phase contrast image"Object
under study"
Phase plate"
In a microscope, phase contrast requires:
- a small annular diaphragm in the back focal plane of the condenser
- a specific objective with a phase plate in its back focal plane
phase contrast image of buccal epithelial cells
Contrast without markers
③ differential interference contrast (DIC) An interferometer is built using 2 Wollaston prisms as beam splitters (similar to Mach
Zehnder interferometer with polarized waves).
The two orthogonally polarized waves pass through the object at two different
locations: the phase difference between those two locations in the object give rise
to an intensity change in the image.
From zeiss-campus.magnet.fsu.edu
Contrast without markers
DIC contrast as implemented in a microscope
(from www. olympusmicro.com)
Buccal Epithelial Cells – DIC DIC gives an impression of seing the topology of the object, however this is only a phase variation. DIC has the advantage of a much better resolution both lateral and axial, than phase contrast
(from Nikon’s site www.microscopyu.com)
New developments in quantitative phase microscopy
Contrast without markers
Developments in CCD cameras with large number of pixels and increased processing power have led to the development of quantitative phase imaging reconstructed from combined measurements of intensity and phase images. See for example: - Digital Holographic microscopy (LynceeTec, Phase holographic imaging, …) - SID4 bio camera by Phasics
Live COS-7 (fibroblast) cell imaged with the SID4 bio camera – x150, NA 1,3- white light illum. From Phasics web site
Principle of digital holographic microscopy From Phase Holographic imaging web site
Microscopy and fluorescence (part 2)
Optical imaging using microscopy • Standard resolution: diffraction limit
• Contrast methods – Without markers:
Dark field, Phase contrast, Differential interference contrast
New developments in phase contrast microscopy – Fluorescence microscopy: markers, optical
configurations
• Superresolution techniques: beyond the diffraction limit
Contrast with markers: fluorescence
Advantages: • detection on a dark background, high sensitivity • specificity (spectral selection) • Multicolor labeling, colocalisation possible • Several degrees of freedom (intensity, spectrum, polarization, lifetime,…) • Sensitivity to the environment (pH, concentration in ions, …)
Principle: Fluorescent labels on specific parts of the cell or tissue are excited by a light source (often in the UV or blue) and
the emitted light is spectrally selected (at a higher wavelength)
Comparison between interference contrast and fluorescence on the image of a neuron
B. Lounis, extrait cours Les Houches 2003
Fluorescent markers 1: organic fluorophores
Intrinsic/endogenous (FAD, NADH,…) low contrast
or extrinsic/exogenous (Cyanine, Alexa…) toxic
Many markers are available or under development to increase their brightness, reduce photobleaching and also for FUNCTIONAL imaging
Main provider: Molecular Probes (www.invitrogen.com)
Conjugation to specific biomolecules can be done using antibodies (immunolabeling)
Cy3 Cy5
400 500 600 7000
20
40
60
80
100
120
Spec
tres
d'ab
sorp
tion
et d
'ém
issi
on
λ (en nm)
Alexa 488 Alexa 594 Fluorescence
400 800 600 500 700 Wavelength (nm)
Absorption
Wavelength (nm) 400 700
Multiple organic fluorophores in a single cell
Invitrogen FluoCells slide #2: Bovine Pulmonary Artery Endothelial Cell Multiple-exposure image acquired using bandpass optical filter sets
BODIPY antibody labelling of Microtubules
DAPI labelling of DNA (Nuclei)
Texas Red-X phalloidin labelling of F-actin
Fluorescent markers 2: fluorescent proteins
Green Fluorescent Protein (GFP): Discovered on a jellyfish
Transfection in live cells
=> they produce the protein themselves through a genetic modification
VERY WELL SUITED for IN VIVO STUDIES
SPECIFIC LABELING is GUARANTEED
Mutants have been produced to cover a large spectrum:
Fluorescent markers 3: semiconductor nanocrystals (Quantum dots or QD)
CdSe core (2 à 6 nm) with a ZnS shell (0,5nm) High performance markers
• high brightness (ideal for single molecule applications) • long-term photostability • narrow emission spectrum, adjustable with size • broad absorption spectrum (ideal for multicolor detection with single excitation source)
Difficulties: • difficult to conjugate to biomolecules • blinking (pb for particle tracking) • larger than an organic fluorophore, can perturb a movement or an interaction
Qdots absorption and fluorescence spectra
Fluorescence spectra
Absorption spectra
narrow emission spectrum, adjustable with size
broad absorption spectrum (ideal for multicolor
detection with single excitation source)
Using fluorescence to monitor molecular processes
Fluorescence (usually from exogenous fluorophores) can be used to monitor molecular processes that cannot be spatially resolved :
– Change in fluorescence intensity or spectrum due to pH, calcium
ions, cell death (apoptosis) – Change in fluorescence lifetime to detect changes in the
environment (FLIM)
– Transfert of excitation from one fluorophore to another on a 1-10nm scale (FRET) to detect molecular interactions (between 2 proteins eg)
see animation: http://zeiss-campus.magnet.fsu.edu/tutorials/spectralimaging/fretbiosensors/indexflash.html
– Detection of dynamical processes using Fluorescence Correlation Spectroscopy or Single Molecule Spectroscopy
Basics of Biology (part 2)
Types of cells
The human body is made up of over 200 different types of cells
Examples:
- Epithelial cells with subgroups (mucosal cells, secretory cells, ciliated cells, ….)
- Blood cells
1. Erythrocytes (Red blood cells, RBC) - contain hemoglobin, transport O2, CO2
2. Leukocytes (white blood cells) ~ 1:1000 RBCs
- Lymphocytes (T cells - cell-mediated immunity, B cells - produce antibodies)
- Macrophages / Neutrophils - move to site of infection, digest bacteria
3. Thrombocytes (platelets) - cause blood coagulation
- Muscle cells - form muscle tissue, produce mechanical force by contraction
- Nerve cells/neurons - communication throughout body
- Sensory cells (e.g. hair cells of inner ear, taste buds, retina, etc.)
- Germ cells (haploids - containing only one copy of each pair of chromosomes),
e.g. egg cells, sperm cells
- Stem cells - cells that are not yet specialized (differentiated). Can turn into any
type of cell. Embryonic stem cells - pluripotent (not specialized), adult stem
cells (in bone marrow) - multipotent (somewhat specialized)
Cell Structure, Constituents and their Functions
Main distinctive cell groups:
Eukaryotic cells: animal/plant cells with complex structureProkaryotic 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 formreceptors, pores (channels), and enzymes to control transport and communicationwith the exterior.
The cell membrane
An example of study of cell membranes with fluorescence microscopy
From Owen et al, Biophys J 2006 (Photonics group at Imperial College)
ü Specific membrane staining dye
ü Fluorescence lifetime increases in the ordered phase (lipid rafts)
ü Temperature increase or depletion of cholesterol (using cyclodextrin) results in more disorder
The fluorescence spectrum changes with order (green) or disorder (red) From Lin et al, BiophysJ 2005 Cited as reference 4 in Owen et al, BiophysJ 2006
Signaling in cells
Trigger process, typically mediated by
receptor-ligand interactions
Interactions with membrane-bound
receptors (e.g. binding of external ligands)
leads to transmission of signals from
surface to interior.
Up-, down-regulates genes (expression of
specific proteins) - e.g. growth factors lead
to growth, proliferation, differentiation
Apoptosis (programmed cell death) can be triggered by external orinternal signals
Cancer is characterized by cells that grow uncontrollably - even in the
absence of growth factors and that do not respond to apoptosis signals
Signaling in cells
Trigger process, typically mediated by
receptor-ligand interactions
Interactions with membrane-bound
receptors (e.g. binding of external ligands)
leads to transmission of signals from
surface to interior.
Up-, down-regulates genes (expression of
specific proteins) - e.g. growth factors lead
to growth, proliferation, differentiation
Apoptosis (programmed cell death) can be triggered by external orinternal signals
Cancer is characterized by cells that grow uncontrollably - even in the
absence of growth factors and that do not respond to apoptosis signals
Apoptosis (programmed cell death) can be triggered by external or internal signals.""Cancer is characterized by cells that grow uncontrollably – even in the absence of grow factors – and that do not respond to apoptosis signals""
Different types of signaling in cells"(from L’essentiel de la biologie cellulaire,
introduction to chap 16)""
How life is encoded in cells
The DNA: - building block for every living being
-“storage”solution for hereditary information
DNAHistone11 X 5 nm
4 bases: A, T, G, Cform pairs by hydrogen bonding in
the double helix: G-C, A-TChromatin
The nucleus: DNA double helix
The genetic code is efficiently stored in the nucleus
DNA in the nucleus is tightly packaged by packaging proteins (histone), but
still easily accessible to enzymes. Protein-DNA complex is calledchromatin.
DNA is divided between chromosomes (24 different ones for humans).Chromosomes are only highly condensed and visible during cell division
(mitosis - mitotic chromosomes).
During most parts of the cell cycle they exist in long threads that cannot bedistinguished (interphase chromosomes).
Human cells: Total stretched DNA is 2 m in length, yet fits in 6 µm nucleus
The nucleus: chromosomes
Cell growth and replication - the cell cycle
8 h
(synthesis of proteins,
lipids, carbs)
6 h
(DNA synthesis)
4-5 h
(formation of
chromosomes)
1-2 h
See movie of fibroblast cell migration and division (DIC microscopy): https://www.microscopyu.com/galleries/cell-motility Movie of animal cell division (also DIC microscopy): http://www.dnatube.com/video/4153/Animal-Cell-Division-video
Replication
During cell division (mitosis)
Other processes exist in some viruses: RNA replication, reverse transcriptase (RNA to DNA)
From DNA to proteins
REPLICATION Involves DNA polymerase
TRANSCRIPTION Involves RNA polymerase
TRANSLATION Involves ribosomes
Real Time Sequencing from Single Polymerase molecules
Eid et al, Science 2009
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