Principles of confocal microscopy / Department of Biology1
Fluorescence and confocal
microscopy
Basic principles and applications
Mgr. Ivana Andrejčinová
437403@mail.muni.cz
Light source: halogen lamps
- low-contrast specimens
(cell almost transparent)
- additional optical mechanisms
(Phase contrast, DIC..)
- use of dyes
(crystal violet, eosin..)
Principles of confocal microscopy / Department of Biology2
Conventional Light vs. Fluorescence Microscopy
- High-intensity light source: metal halide
lamps, light-emitting diodes (LEDs), lasers
(confocal microscopy)
- Use of fluorophores
- Better resolution
Fluorophore - chemical compound absorbing
light at one wavelength and re-emitting it at
another wavelength
Principles of confocal microscopy / Department of Biology3
Principle of fluorescence
(ray of photones)
Molecular system absorbs light
(high energy, short
wavelength)
Electrone is hit by photone energy, gets
excited and transits from the ground state to
higher energy state
Molecular system emits light
(lower energy, longer wavelenght)
After several nano-seconds (the fluorescence lifetime)
the electrone will relax back to the ground state and
release the energy
Fluorophore - chemical compound absorbing
light at one wavelength and re-emitting it at
another wavelength
Principles of confocal microscopy / Department of Biology4
Principle of fluorescence
(ray of photones)
Molecular system absorbs light
(high energy, short
wavelenght)
Electrone is hit by photone energy, gets
excited and transits from the ground state to
higher energy state
Molecular system emitts light
(lower energy, longer wavelenght)
After several nano-seconds (the fluorescence lifetime)
the electrone will relax back to the ground state and
release the energy
Fluorophore - chemical compound absorbing
light at one wavelength and re-emitting it at
another wavelength
Principles of confocal microscopy / Department of Biology5
Principle of fluorescence
(ray of photones)
Molecular system absorbs light
(high energy, short
wavelenght)
Electrone is hit by photone energy, gets
excited and transit from the ground state to
higher energy state
Molecular system emitts light
(lower energy, longer wavelenght)
After several nano-seconds (the fluorescence lifetime)
the electrone will relax back to the ground state and
release the energy
Fluorophore - chemical compound absorbing
light at one wavelength and re-emitting it at
another wavelength
Principles of confocal microscopy / Department of Biology6
Principle of fluorescence
(ray of photones)
Molecular system absorbs light
(high energy, short
wavelenght)
Electrone is hit by photone energy, gets
excited and transit from the ground state to
higher energy state
Molecular system emitts light
(lower energy, longer wavelenght)
After several nano-seconds (the fluorescence lifetime)
the electrone will relax back to the ground state and
release the energy
Principles of confocal microscopy / Department of Biology7
Widefield fluorescence microscopy
Wide spectrum of wavelenghts
Essential components:
• Excitation filter – allows only light of specific
wavelengths that excites the fluorophore to
pass through
• Dichroic mirror – reflects one type of light
and allows other type of light to pass through
• Emission filter – blocks excitation light and
transmits emission light to the
eyepiece/detector
Reflected light has shorter wavelength
Passing light has longer wavelength (carries
less energy than excitation light)
Contains fluorophore emmiting fluorescence
Principles of confocal microscopy / Department of Biology8
Widefield fluorescence microscopy
Wide spectrum of wavelenghts
Contains fluorophore emmiting fluorescence
HeLa cells DAPI GLUT1
Principles of confocal microscopy / Department of Biology9
Widefield fluorescence microscopy
HeLa cells DAPI GLUT1
Limitations:
• the whole sample is illuminated
• Fluorescence detected not only from one specific
focal plane, but also above and below it
(out of focus signal)
• Light can be scattered (liquid-filled compartments)
Background noise
• Problems when identifying specific subcellular
localization of the target
• Not suitable for 3D structure modelling
Confocal Laser Scanning vs. Widefield fluorescence
microscopy
• The entire sample
illuminated by light
source
• Image recorded by
camera at once
• Laser focused at the
focal plane
• Laser is scanned
along the sample
pixel by pixel (PMTs)
Confocal Laser Scanning microscopy (CLSM)
Additional components:
• Laser – bright source of pinpoint illumination
• 1. pinhole – focuses the beam of light
on the specific small part of the sample
• 2. pinhole – positionned in the focal plane,
selects the light coming from the targetted
point of the sample (reduces background
illumination)
two pinhole apertures
at confocal positions
Confocal Laser Scanning microscopy (CLSM)
Additional components:
• Laser – bright source of pinpoint illumination
• 1. pinhole – focuses the beam of light
on the specific small part of the sample
• 2. pinhole – positionned in the focal plane,
selects the light coming from the targetted
point of the sample (reduces background
illumination)
two pinhole apertures
at confocal positions
Laser scanning
• Motorized mirrors
(moving laser across
the sample)
Principles of confocal microscopy / Department of Biology13
Confocal Laser Scanning microscopy (CLSM)
Photomultiplier tubes (PMTs)
• collect photones and amplify
signal (creating digital signal
processed by computer)
– generated image
Scanning mirrors
• Motorized mirrors allow laser
scanning of the sample along
X- and Y-axis within the same
focal plane
Detection unit
Principles of confocal microscopy / Department of Biology14
1. acquisition of signal from each channel separately minimal
bleed through (scanning along x and y)
Confocal Laser Scanning microscopy (CLSM)
DAPI Actin YAP
cardiomyocyte
Principles of confocal microscopy / Department of Biology15
1. acquisition of signal from each channel separately minimal
bleed through (scanning along x and y)
2. software generates merged image
Confocal Laser Scanning microscopy (CLSM)
DAPI Actin YAP
cardiomyocyte
Merged
Principles of confocal microscopy / Department of Biology16
Resolution of images - widefield FM vs. CLSM
Principles of confocal microscopy / Department of Biology17
Key Advantage - Optical sectioning (CLSM)
• Clear images of thin sections within a thick sample
Principles of confocal microscopy / Department of Biology18
• Clear images of thin sections within a thick sample
Key Advantage - Optical sectioning (CLSM)
✓ reduced background compared
to widefield FM
✓ optical sectioning (0,5-1,5 um)
of up to 100 um thick sample
✓ generation 3D images
✓ examination of living and fixed
specimens (better resolution)
Principles of confocal microscopy / Department of Biology19
̶ Speed !
̶ Photobleaching of the sample
(long-lasting acquisition)
̶ Limitated number of excitation
wavelengths (availability of lasers)
̶ high cost of purchasing
Advantages and Disadvantages of CLSM
Principles of confocal microscopy / Department of Biology20
Fluorescence Microscopy Applications
1. Immunostaining of fixed samples
• Direct
• Indirect
Principles of confocal microscopy / Department of Biology21
1. Immunostaining of fixed samples
• Indirect
1. Fixation and Permeabilization
Cross-linkers (e.g. PFA) – form covalent chemical bonds between the proteins
Organic solvents (alcohol, acetone) – remove lipids, dehydration of the tissue, protein precipitation
Permeabilization reagents (Triton X-100, NP-40)
2. Blocking (e.g. BSA) – prevents unspecific binding of primary Ab
3. First Immunostaining – primary Ab recognizes and binds to the epitope of target protein
4. Second Immunostaining – secondary Ab conjugated with a fluorophore binds to the primary Ab (species-specific)
5. Nuclear staining (e.g. DAPI)
6. Mounting medium (PBS, Mowiol)
Fluorescence Microscopy Applications
Indirect immunofluorescence staining
Principles of confocal microscopy / Department of Biology22
1. Immunostaining of fixed samples
• Direct
✓ Faster
✓ Easier to handle
(rapid analysis,
standardized experiments,
useful for clinical practice)
- Validated Ab with
high-affinity (expensive)
• Indirect
✓ Flexibility in design
(combining different prim.
Ab with different sec. Ab)
✓ Signal amplification
✓ Economical procedure
- time-consuming workflow
Fluorescence Microscopy Applications
Indirect immunofluorescence staining
Principles of confocal microscopy / Department of Biology23
1. Immunostaining of fixed samples
• Direct
• Indirect
➢ Detection of several fluorophores
➢ organelle-specific localization
➢ co-localization
➢ FISH-ing for chromosomal
abnormalities
➢ 3D visualization of cells and tissue
sections (100um) (CLSM)
Fluorescence Microscopy Applications
Indirect immunofluorescence staining
Principles of confocal microscopy / Department of Biology24
2. Live cell imaging
• Genetically encoded GFP/FPs (fluorescent proteins) fused
with protein of interest
• tracking localization (organelle-specific proteins),
abundance or changes within the labelled protein
over time or in response to treatments
Fluorescence Microscopy Applications
Synaptophysin
Amyloid Precursor Protein
TDP43
Principles of confocal microscopy / Department of Biology25
2. Live cell imaging
• Genetically encoded GFP/FPs (fluorescent proteins) fused
with protein of interest
• tracking localization (organelle-specific proteins),
abundance or changes within the labelled protein
over time or in response to treatments
• Organelle-specific dyes
• MitoTracker, LysoTracker, ER Tracker...
• Fluorescent DNA intercalating agents (fixed samples as well)
• Hoechst (minor-groove binding dye, A/T-rich regions)
Fluorescence Microscopy Applications
Principles of confocal microscopy / Department of Biology26
2. Live cell imaging
• Fluorescent dye detecting changes in
• ion concentration (fluo-labelled chelators – concentration-dependent shift in spectral properties)
• voltage
• pH (phagocytosis assay)
Ca2+ levels changes
Fluorescence Microscopy Applications
Principles of confocal microscopy / Department of Biology27
2. Live cell imaging
• Fluorescent dye detecting changes in
• ion concentration (fluo-labelled chelators – concentration-dependent shift in spectral properties)
• voltage
• pH (phagocytosis assay)
Fluorescence Microscopy Applications
Principles of confocal microscopy / Department of Biology28
2. Live cell imaging
• Fluorescent dye detecting changes in
• ion concentration (fluo-labelled chelators – concentration-dependent shift in spectral properties)
• voltage
• pH (phagocytosis assay)
➢ 4D – time-lapse experiment
➢ in response to treatment
➢ cell cycle
Fluorescence Microscopy Applications
Principles of confocal microscopy / Department of Biology29
Thank you for your attention
Mgr. Ivana Andrejčinová
437403@mail.muni.cz
Principles of confocal microscopy / Department of Biology30
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https://www.esric.org/education/education-centre/principles-of-fluorescence
https://www.nexcelom.com/applications/celigo/fluorescent-assays/internalization-and-phagocytosis/
https://www.ptglab.com/news/blog/if-imaging-widefield-versus-confocal-microscopy/
https://www.leica-microsystems.com/science-lab/how-to-prepare-your-specimen-for-immunofluorescence-microscopy/
https://bitesizebio.com/33529/fluorescence-microscopy-the-magic-of-fluorophores-and-filters/
https://www.zeiss.com/content/dam/Microscopy/Downloads/Pdf/FAQs/zen2010-lsm780_basic_fcs_experiments.pdf
https://www.ucc.ie/en/media/academic/anatomy/imagingcentre/imagegallery/confocalgallery/Laser-Scanning-Confocal-
Microscopy-Introduction.pdf
Dey P. (2018) Fluorescence and Confocal Microscope: Basic Principles and Applications in Pathology. In: Basic and Advanced
Laboratory Techniques in Histopathology and Cytology. Springer, Singapore. https://doi.org/10.1007/978-981-10-8252-8_25
Chan, Jefferson; Dodani, Sheel C.; Chang, Christopher J. (2012). Reaction-based small-molecule fluorescent probes for
chemoselective bioimaging. Nature Chemistry, 4(12), 973–984. doi:10.1038/nchem.1500