Contents
Lecture 1: The basics about
fluorescence and confocal
microscopy
• Optical microscope basics
• Fluorescence microscopy
• Confocal microscope
Marica Ericson
2008-10-20
CHALMERS BIOCENTER
Advanced Technologies in Bioscience, 7,5 hp
Theme: An introduction to advanced fluorescence imaging techniques
The microscope
The microscopical range
• Greek: µικρόν (micron) = small + σκοπεῖν
(skopein) = to look or see
•
•
•
•
•
Human eye : 0.1 mm
Simple magnifier: 10 µm
Optical microscope: 1 µm (200 nm limit)
Electron microscope: 0.5 – 3 nm
Scanning Probe microscope: 0.1 – 10 nm
Image from: Wikipedia
The brightfield microscope
Object
Light source
Condenser
Robert Hook (1635 –1703)
Micrographia
Image
Objective
Monastery San Franscesco di Cortina
Photo A. Diaspro
1
How good can we get?
Diffraction
• Magnification – size
• Resolution – distinguish objects
• Contrast – resolved
wikipedia
Airy disc – diffraction from a point
source
Resolution
micro.magnet.fsu.edu
From Confocal and Two-photon microscopy,
A. Diaspro
Brightfield microscope
Brightfield microscopy
1. Eyepiece (2x, 5x or 10x).
2. Objective turret
3. Objective lens (4x, 5x, 10x,
20x, 40x, 50x or 100x).
4. Z-level control (coarse)
5. Fine z-level control
6. Stage with object holder
7. Light source
8. Condenser, with diaphragms
and filters.
9. X,Y control
• Advantages
– Simplicity
– No sample preparation, allowing viewing of
live cells.
• Limitations
– Very low contrast of most biological samples.
– Low apparent optical resolution due to the
blur of out of focus material.
wikipedia
2
Fluorescence
Fluorescence
S2
S1
E = hυ
S0
Contrast using fluorescence
The brightfield microscope
Object
Light source
Image
• Only specific structures are stained
• Staining of different states, eg. live or dead
• Specific markers, eg. pH, Ca2+, etc.
Condenser
The fluorescence microscope
Objective
Fluorophores
From Molecular Probes catalouge
Light source
Excitation filter
Condenser
Object
Image
Emission filter
Objective
3
GFP
Fluorophores
Mouse brain tissue section stained with antibodies (GFAP, Alexa
Fluor 568) and Hoechst 33342 nuclear counterstain. 12-bit digital
camera, Nikon Eclipse 80i microscope equipped , bandpass
emission fluorescence filter optical blocks.
http://nobelprize.org/
www.microscopyu.com
GFP – Green fluorescent protein
GFP – Mutants
Hadjantonakis et al. 1998
Alba, the fluorescent bunny, Eduardo Kac 2000
http://nobelprize.org/
Fluorescence microscope
Filterblock
Filterblock /
Filtercube
www.microscopyu.com
wikipedia
4
Inverted microscope
Fluorescence images
• Cell suspensions
Bovine pulmonary arthery endothelial
cells. Nuclei stain: DAPI, microtubles:
antibody bound to FITC, actin
filaments: phalloidin bound to TRITC.
Dividing human cancer cell. DNA
(blue), INCENP-protein (green),
microtubules (red).
wikipedia
Image formation in fluorecence
microscopy
wikipedia
The confocal microscope
Beamsplitter
Focal
plane
Out of focus fluorescence
Pinhole
Objective
LASER
Ar
364 nm
VIS
Ar
458, 477, 488,
496, 502, 515 nm
HeNe
543, 594, 612, 633 nm
Out of
focus
Scanning
• Intense light source confined to small beam
size
• Easily focused to diffraction limited spot
• Coherence is not required (can cause
problems)
• Different dyes have different spectral
characteristics: many laser lines are required
UV
Focal
plane
y
x
z
IR
Ti:Sapphire
5
Z-stack
3D view
www.hopkinsmedicine.org
www.hopkinsmedicine.org
Projection
Point spread function PSF
x
y
X,Z - plane
z
Wikipedia
www.hopkinsmedicine.org
Resolution
Numerical aperture, NA
How small objects
can we see?
NA = n sin θ
n = refractive index
n ≈ 1 (air)
n ≈ 1.33 (water)
n ≈ 1.56 (oil)
That is limited by:
Df ≈
θ
Objective
λ
2 NA
Ernst Abbe, 1840 - 1905
6
Resolution, NA= 0.2
Resolution, NA= 0.7
www.microscopyu.com
Resolution, NA= 1.0
www.microscopyu.com
Resolution, NA= 1.3
www.microscopyu.com
www.microscopyu.com
Sample
Components
of a confocal
microscope
Objectives
Objective
Scanning mirrors
Dichroic mirror
LASER
P
M
T
P
M
T
P
M
T
Sync.
www.microscopyu.com
7
Optical sectioning, example
Time laps, example
Motion in the Golgi sacs can be visualized in the area of
cytoplasm near the nucleus of an opossum kidney epithelial
cell.
www.microscopyu.com
Photobleaching
Photobleaching
S2
X
S1
ISC
T1
Phosphorescence
S0
Deactivation
by reaction
www.microscopyu.com
Summary
• Brightfield optical microscope
• Fluorescence microscopy
– Fluorophores
– Filters (excitation and emission)
Let’s see some
real stuff!
• Confocal microscope
– Avoid out of focus light
– Scanning
– Optical Sectioning
• Resolution
8