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