Basics of confocal microscopy
1. How does the
confocal microscope work ?
1. Examples
2. Special techniques
Optical light microscopy course
26.10.2012
Kirsi Rilla
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1. How does the confocal
microscope work ??
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Brief history of confocal
microscopy
- Marvin Minsky in 1957
- First confocal images in 1973
- 1980s: commercially available
- 1990s: progress in optics and elecronics:
- more efficient lasers laserien tehokkuus
- quality of mirrors
- sensitivity of photodetectors
- development of better fluorochromes
- higher resolution of monitors and printers
- saving and processing of images
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How does the confocal microscope work ?
Pinhole is in a plane conjucated to
that of the focal plane = Confocal
- A modified light microscope with a laser
- Controlled by a computer
- ”Optical sectioning”
3D projection
Images: Olympus
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Point spread function (PSF)
PSF =light is diffracted or scattered,
source is a point but image is not
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Widefield fluorescence
microscopy
light source
lenses and filters
Detection
excitation
emission
dicroidic
mirrow
Fluorescent sample
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Widefield fluorescence
microscopy
In fluorescent microscopy the sample itself emisses light
Electrons of a fluorophore are excited by light (400-700 nm)
When electrons go back to their normal state, they emit
light with longer wavelenght than the excitation light
All parts of specimen throughout the optical path will be
excited and the fluorescence detected by a photodetector
or camera
Confocal microscope
laser
lenses, filters
Detection
excitation
emission
dicroidic
mirrow
Fluorescent sample
confocal
unit
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Confocal microscopy
In confocal microscopy laser light source is used to
excitate the fluorophore
Pinhole is used to eliminate out-of-focus information
Only the light within the focal plane can be detected,
so the image quality is much better than that of
conventional images
The light emitted by the sample is collected to digital
mode and analyzed by computer.
A three-dimensional illustration of the object can be
created by combining the ”optical sections” from
several planes.
Widefield - Confocal microscopy
Widefield
Confocal
Focal adhesions – vinculin staining
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Pinhole diameter effects
small pinhole diameter:
-> thin optical section
= high z-resolution possible
= low signal strength
big pinhole diameter:
-> thick optical section
= low z-resolution
= brighter signal
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Confocal microscopy
pinhole
Image: Spector & Coldman:
Basic methods in microscopy
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Types of confocal microscopes
Best resolution and out-of-focus suppression as well as highest
multispectral flexibility is achieved only by the classical
single point confocal system !
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1. Point scanner
- One spot of light scans the
whole specimen
-The signal is collected
to photomultiplier tube (PMT)
- Simultaneous scanning
of several fluorochromes
- High resolution
- Pinhole size adjustable
- Speed 5-154 fps
- Photobleaching/Phototoxicity
Image: Spector & Coldman:
Basic methods in microscopy
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2. Spinning-disk
- Excitation through thousands of
small holes (= pinhole).
- The whole view imaged in real time
- CCD-camera
- Resolution weak
- Pinhole size not adjustable
- Fast (1000 fps)
- Lower photobleaching and phototoxicity
Image: Spector & Coldman:
Basic methods in microscopy
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3. Slit scanner
- Light collected through
a narrow slit
- As fast (or even faster)
as spinning disk
- Photobleaching lower than
in point scanner
- Sensitive CCD-camera
- Resolution weaker
than in point-scanner
Kuvan lähde: Spector & Coldman:
Basic methods in microscopy
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Perkin Elmer Ultraview confocal
scanner (Spinning disk)
BioMater Centre
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ZEISS LSM 700 (point scanner)
Institute of Biomedicine/Anatomy
Zeiss Axio Observer inverted microscope
Zeiss LSM 700 confocal module
Zeiss XL-LSM S1 incubator with temperature and CO2 control.
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Image generation in confocal
LSM
1. Line-by-line scanning of the specimen
2. Pixel-by-pixel detection of the fluorescence emitted,
by means of photomultiplier tube (PMT)
3. Digitization of the object information contained in the electrical signal
5 things that affect the quality of
confocal images
1.Detector gain
- PMT detector gain setting amplifies fluorescence signal
- Higher values incease noise
- Optimal between 500-800
2. Amplifier offset
- Tells the PMT what is the cutoff point for black
- Do not adjust aggressively – in biology perfectly
black backgrouds do not exist
3. Pinhole size
- Opening the pinhole will reduce the resolution but will give more light
4. Scan speed
- Slower scan speed reduces noise but leads to faster photobleaching
5. Laser power
- Increasing laser power gives more signal but speds up photobleaching
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2. EXAMPLES OF
APPLICATIONS
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ZEISS LSM 700
Equipment:
- Zeiss Axio Observer inverted microscope
- Zeiss LSM 700 confocal module (point scanner)
- Zeiss XL-LSM S1 incubator
Progress (2010-2012):
Users
- more than 30 users from 10 research groups
Education
- 4 basic or advanced training sessions
- 2 courses for graduate students
Publications
- 10 published, > 5 submitted, > 5 manuscripts
Applications:
-2D and 3D imaging
- Protein kinetics
(FRAP, FRET, Photoconversion etc.)
- Live imaging of 3D cultures
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Basic 2D imaging
Salt crystals
Salt crystals
Mycoplasm
Brain tissue
Breast cancer cells Breast cancer tissue
A bird feather
Fly wing
Tissue sections –breast cancer
Immunofluorescence staining: 3 colors
(Blue = nuclei, Red = hyaluronan, Green = TSG-6 )
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Tissue samples -cartilage
Fresh, stained tissue imaging in 3D
Depth coding
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3D-Confocal microscopy
Optical sections…
…3D-image
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3D-Confocal microscopy
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3D imaging
3D epithelial culture
Cartilage tissue
Breast cancer cell
A bird feather
Tick head
Fly limb
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Invasion
Example of 3D imaging: Invasion of
cancer cells
Confocal microscopy- Autofluorescence
Examples:
Erytrocytes
(hemoglobin)
Strawberry flower
(Chlorophyll)
Skin epidermis
(keratins)
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Autofluorescence
Examples:
Tick head
(chitin)
Bird feather
(keratins)
Fly wing
(keratins)
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Confocal microscopy - GFP-techniques
- Protein consisting of 238 amino acids (27 kDa)
- Several derivatives (BFP, CFP, YFP)
- Low bleaching and phototoxicity
- Fusion proteins
Nobel prize of chemistry 2008 !
GFP
Protein
of interest
Aequorea victoria
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GFP-techniques
DsRed
Mitochondria
Lysosomes
Nucleus
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GFP-techniques
GFP-fusion protein
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GFP techniques – Double transfection
Green: GFP-HAS3
Red: Cherry-Myosin X
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Problems/limitations of GFP-techniques
GFP-CD44
- Photobleaching
- Phototoxicity
- High overexpression
- Fusion protein
function !
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Why to study live cells?
Live Cells
Fixed cells
Hyaluronan glycogalyx labeled with hyaluronan probe
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Confocal microscopy –live cell
markers
Examples
Organelle-specific markers:
- Nuclei (DRAQ5)
- Plasma membrane (FM1-43)
- Golgi (Ceramide)
- ER (ER tracker)
- Lysosomes (lysotracker)
Other markers:
- Labeled antibodies
- Probes (fHABR)
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Benefits of live cell microscopy
- No fixation artefacts
- Cell movements and shape changes
- Intracellular traffic
- Time lapse movies
3D movie:
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Benefits of live cell microscopy
- Same cell before-after treatment
- Save time and money
- No fixation, permeabilization,
staining or mounting steps
3D movie with two
colors:
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Requirements/limitations of live
cell microscopy
Conditions:
- Inverted microscope, plates!
-Temperature (36,5 – 37°C)
- pH value (7,3 -7,4)
- Evaporation of water
Limitations:
- Expensive
- Experience and knowledge
- Timetable
- Time consuming
- Artefacts and mistakes
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Live cell microscopy -problems
Problems:
- Focus drift
- Phototoxicity
- Photobleaching
Compromise between:
- Quality of images
- Temporal resolution
- Phototoxicity/bleaching
Blue = Nuclei
Green = GFP-HAS3
Red = Hyaluronan probe
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3. SPECIAL TECHNIQUES
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Super-resolution
For many years it was a source of
frustration for biologists that the
internal components of a cell were
practically invisible to them.
Researchers believed that the
wavelength of light determined a
fundamental limit to the resolution
of optical microscopes.
However, it now seems that the
wavelength of light was not such
a limiting factor after all.
Super-resolution technology allows
researchers to see details that
are difficult or impossible to image
with conventional light microscopes
— at resolutions of 100
nanometres or better.
Resolution:
Human eye
100 µm = 0,1 mm
Light microscope
200 nm = 0,2 µm
Electron microscope
0,2-2 nm
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Super-resolution techniques
● STED: Stimulated Emission Depletion
● PALM: Photo-Activated Localization
Microscopy
● STORM: Stochastic Optical Reconstruction
Microscopy
● SIM: Structured-illumination microscopy
- The diffraction of light causes the PSF (point spread function).
- All these techniques excite fluorescence only in a subresolution
spot. The detector still sees the same PSF, but we know where that
light comes from!
Two-Photon excitation microscopy
- The object is excitated with two photons at the same time, the
energy of which is half of that used in normal one-photon excitation
(and the wavelenght douple, near infrared, 700 nm – 1100 nm)
- No need for ”pinhole”
- Excitation occurs only at the plane of focus
+ Less phototoxicity
and photobleaching
+ Better contrast
+ For thick specimen
+ For live tissues
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FRAP
Fluorescense recovery after photobleaching (FRAP)
- The recovery of fluorescence after photobleaching is monitored
- Movements of lipids and proteins on the plasma membrane
- Molecular movements
- iFRAP = inverted FRAP
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FRAP (=fluorescence recovery after
photobleaching)
…To study protein kinetics in live cells
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Photoactivation and
photoconversion
- Photoactivation: an inactive chromophore is activated to
emission of fluorescence light (for example PA-GFP)
- Photoconversion: The wavelenght emitted by a chromophore
is changed after activation (for example EosFP or Dendra)
- Photoswitching: Reversible activation
(for example Dronpa)
Image: Carl Zeiss, Inc.
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Photoconversion
Photoconversion: the light emitted by a chromophore
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changes after excitation light of 405 nm.
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Examples of photoactivable
fluorescent proteins
PA-GFP, photoactivatable green fluorescent protein;
PS-CFP, photoswitchable cyan fluorescent protein;
KFPs, kindling fluorescent proteins.
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Properties of Dendra2
- Monomeric fluorescent protein (26 kDa)
- Photoconversion from green to red
- High contrast photoconversion
- Activated by violet (405 nm) and blue (488 nm) light
- Ideal for monitoring real time protein traffic
- Recommended for tracking cell, organelle, and protein
movement, and for determination of protein halflife
octocoral Dendronephthya
Movie: Carl Zeiss, Inc.
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Photoconversion
…for tracking organelle or protein movements
Photoconversion
1. Free cytosolic protein: 2. Transmembrane protein:
405 nm
405 nm
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Movie: Carl Zeiss, Inc.
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Fluorescense loss in photobleaching
(FLIP)
The disappearance of fluorescent signal is monitored
The same area of interest is repeatedly excitated (Bleaching)
Because the molecular movements, also surrounding areas
are gradually bleached
The areas not bleached /quenced are areas with restricted
molecular movements
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FRET
Fluorescence resonance energy transfer (FRET)
- Interaction between proteins
436 nm
436 nm
distance ~3-7 nm
distance > 10 nm
CFP
NO FRET
480 nm
YFP
CFP
FRET
480 nm
YFP
535nm
Fluorochrome pairs:
–”donor” and ”acceptor” (for example CFP ja YFP)
-When pairs of fluorochromes are close to each other,
- Excitation of CFP results in energy transfer for CFP to YFP
 sensitized emission from YFP
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- Dependent on the close distance between molecules
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Total Internal Reflection
Fluorescence (TIRF)
-Only molecules that are at the
TIRF surface are excited and fluoresce
- Lower bleaching
- High signal/noise ratio
- High contrast
- For surfaces like plasma membrane
- Only a thin area
- Vesicle traffic
- Membrane research
- Detection of single
molecules
1. Specimen
2. Evanescent wave range
3. Cover slip
4. Immersion oil
Kuvan lähde: Wikipedia
5. Objective
6. Emission beam (signal)
7. Excitation beam
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Preparation of confocal samples
Confocal microscopy is an expensive and time consuming technique.
Only good preparations are worth to be examined. The higher resolution
power of confocal microscopes has special demands on the sample:
For the best maintenance of structures in fixed cell cultures
- use freshly prepared buffered paraformaldehyde for fixation
- choose fluorochromes for optimal excitation and minimal
crosstalk
- use water soluble embedding media
which polymerizes and contains antibleach-agent.
- use cover glass- set ups (cover glass thickness of 0,17 mm)
- use immersion objectives (oil or glycerol immersion)
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Decisions in signal detection
Temporal Resolution
(= speed)
Triange of
frustration
Spatial
Resolution
Sensitivity
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THANK YOU!
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