C8116 Immunochemical techniques
Advanced microscopy III
Spring term 2025
Hans Gorris
Department of Biochemistry
May 6st, 2025
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Confocal microscopy
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Total internal reflection fluorescence mic. (TIRF)
Total internal reflection leads to emergence of an evanescent field
(with exponential decay of intensity):
=> reduces the excitation volume to a depth of ca. 100 nm
TIRF is suitable for investigating phenomena close to the glass slide
=> e.g. cell membranes
Intermolecular
FRET
Intramolecular
FRET
Single-molecule FRET
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Fluorescence correlation spectroscopy
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Fluorescence correlation spectroscopy
PC/Correlator
low fluorophore concentration
(~ 0.1 nM = 10-10 M)
+ very small focal volumen
(fL = 10-15 L)
=> single molecule in focal volume
Confocal optics
=> Compare to
single molecule tracking
Fluorescence correlation spectroscopy
Time
Fluorescence
Raw data
Each time when a fluorescent molecule
passes through the confocal volume,
there is a burst of light
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Data analysis: autocorrelation
90 s90 s90 s
SignalF(t)
30 s30 s30 s30 s30 s30 s30 s30 s30 s30 s30 s30 s30 s30 s30 s30 s30 s60 s60 s60 s60 s 60 s60 s 60 s60 s60 s60 s60 s60 s60 s60 s60 s60 s90 s90 s90 s90 s90 s90 s90 s90 s 90 s90 s90 s90 s
Time lag (Δτ) correlation no correlation
30 s 88 % 12 %
60 s 75 % 25 %
90 s 60 % 40 %
Describes how strongly two data
points with a given time lag
correlate
t (s)
𝐹 𝑡
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Fluorescence correlation spectroscopy
- brackets: averaging over time
- F(t): fluorescence signal at time t
- δF(t): deviation of the fluorescence signal at time t
from the average fluorescence signal
Calculation of G(τ) for the time series of the increments:
𝐺 𝜏 =
𝛿𝐹 𝑡 ∗ 𝛿 𝑡 + 𝜏
𝐹 𝑡 2
Calculation of increments:
𝛿𝐹 𝑡 = 𝐹 𝑡 − 𝐹 𝑡
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Fluorescence correlation spectroscopy
Time
Fluorescence
Raw data
Autocorrelation
function
𝜏 𝐷: diffusion time through confocal volume
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Investigating the mobilitiy of biomolecules
Proinsulin C-peptide (with fluorescent label)
focal volume: in solution on membrane
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Investigating the mobilitiy of biomolecules
tetramethylindocarbocyanine
(DIL) in cell membrane
fluorescent protein
(EGFP) in cytoplasm
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Investigating the mobilitiy & emission fluctuation
𝜏 𝑑: diffusion time through
confocal volume
𝜏 𝑓: fluctuation time in
confocal volume
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Interaction analysis
The size of a molecular complex changes the diffusion time
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Cross correlation spectroscopy
Interaction analysis with two fluorophores
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Interaction analysis with two fluorophores
Both binding partners carry a fluorescent label
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Interaction analysis with two fluorophores
with Ca2+
without Ca2+
Binding of calmodulin to CaM-dependent protein
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Cross-correlation spectroscopy: immunoassay
The size of a molecular complex changes the diffusion time
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1. High background fluorescence:
Conventional wide field microscopy => A single fluorophore molecule
cannot be detected
(ultimate detection limit)
2. Diffraction limit of light:
The image resolution was defined by Ernst Abbe (1873):
ca. 200 nm


sin2n
d =
Numerical apperture (NA)
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Limitations of fluorescence microscopy
Near-field optical microscopy (NSOM)
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Coating
Core Tip
=> Near field illumination (evanescent field)
Usually metal coating
to avoid stray light
Near-field scanning optical microscopy (NSOM)
Diffraction only occurs in far-field imaging, where spherical wave-fronts
leaving the aperture can be regarded locally as plane waves
=> “Simple“ solution: avoid diffraction in the first place
not diffraction limited
coming close
to the sample
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Near-field scanning optical microscopy (NSOM)
various operation modes – purely near-field or combining near-/far-field
excitation/emission or vice versa
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Fluorescent sample
Near-field scanning optical microscopy (NSOM)
Scanning approach
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Combination: NSOM and
shear force feedback (tuning fork):
Topography und optical information
Not diffraction limited:
d ≈ 30 nm
Near-field scanning optical microscopy (NSOM)
piezo control
tuning fork
objective
piezo control
Surface of sample
SNOM tip
glass optical fiber
collimator
argon laser
oscillator
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Near-field fluorescence image (4.5 mm by 4.5 mm) of single oxazine 720 molecules
dispersed on the surface of a poly(methylmethacrylate) film. Each subdiffraction peak
(full width at half maximum, 100 nm) comes from a single molecule (X. S. Xie, Acc.
Chem. Res. 29, 598 (1996)).
Near-field scanning optical microscopy (NSOM)
Detection of single fluorescent molecules
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Near-field scanning optical microscopy (NSOM)
Advantages:
➢ resolution ~ 20 nm in lateral (depending on tip size)
and ~ 2-5 nm in axial direction
➢ optical and topological information
Limitations:
➢ only applicable to surfaces
➢ tip may break in contact with specimen (scanning)
➢ far-field microscopy has many advantages
(except the diffraction limit)
Far-field optical microscopy
=> Using freely propagating light waves
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Optical resolution of light microscopy


sin2n
d =
Ernst Abbe: Diffraction of waves at a cleft (using a diffraction grating)
central bright
strip
light
intensity
Light from a point
source (e.g. a
fluorophore) is
diffracted by the
inner rim of the
objective and forms
an Airy disc.
The size of the Airy disc
depends on λ and NA of
the objective:Airy
disc
Airy
pattern


sin
61.0
n
d =
=> 2 points are
resolvable if the
maximum of one
Airy disc
coincides with the
first minimum of
the next Airy
pattern.
Corresponding intensity profile
FirstminimumMaximum
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Optical resolution of light microscopy
Rayleigh criterion: when are two objects visible as separate points
Source Nikon: http://www.microscopyu.com
e.g. a fluorescent molecule
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Optical resolution of light microscopy
red waves:
positive interference
green waves:
negative interference
Point spread function:
Max. axial und
lateral resolution
Diffraction limited spot: Point spread function
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Optical resolution of light microscopy
Stefan Hell
STED
William Moerner
- Detection of single
fluorescent molecules
- switchable fluorophores
Eric Betzig
- Near field microscopy
- STORM
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Nobel prize for Chemistry in 2014
(Far-field) microscopy beyond the diffraction limit
A) Single molecule localisation (2006)
e.g. STORM (STochastic Optical
Reconconstruction Microscopy)
B) Structured illumination (1994/1999)
z.B. STED (STimulated Emission Depletion)33
Microscopy beyond the diffraction limit
Using non-linear optical processes
STochastic Optical
Reconconstruction Microscopy
STORM
=> based on wide-field microscopy
(frequently in combination with TIRF)
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Single Molecule Tracking

Imaging (STORM)
=> Rather than using a highly diluted solution of fluorophores,
individual fluorophores are switched on/off in a sequential manner
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STORM microscopy
=> Maximum of the point spread function of a single fluor. molecule can be determined precisely
But: 1000-10.000 images required to put together a high-resolution image
=> Need for high computational power / appropriate „switchable“ fluorophores
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STORM microscopy
Pointillism in
modern art
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STORM microscopy
Microtubule
(diam. 25 nm)
Clathrincoated
pit
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STORM microscopy
Wide-field image STORM image
Fibrobalast
in the kidney
Enlarged
section
Microtubule
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STORM microscopy: images
Variation: PALM
(Photoactivated Localization Microscopy)
=> based on FP
Switching on by UV-Licht
Switching off by photobleaching
Yield: ~500 photons
Switching on to fluorescent state by supporting dye (e.g. Cy3)
Switching off to dark state: spontaneously
Yield: 6000 photons per activated fluorescent molecule 40
STORM microscopy
photoswitchable
fluorophores are
required:
Nn
d


sin2
=
Resolution:
2 points that can just be distinguished
by using STORM:
N: Number of photons emitted by a single
fluorophore molecule that can be detected
In praxis, resolution of 10 - 20 nm:
> factor 10!
Other factors are limiting:
e.g. antibodies have a diameter of 15 nm
Different sizes of molecules
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STORM microscopy
STimulated Emission Depletion Microscopy
STED
=> is based on Confocal Microscopy
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2. Spontaneous emission
Abs. Fluorescence
fl = 1–10 ns
3. Stimulated emission
Abs. Stimulated
Emission
t  1 ps
Light can interact with matter:
1. Absorption
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STED microscopy
EXC und STED are pulsed lasers with defined timing of pulses
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STED microscopy: instrumental setup
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Excitation spot Depletion spot Remaining spot
STED microscopy: improved lateral resolution
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STED microscopy: improved lateral resolution
Conventional confocal
microscopy
STED microscopy
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STED microscopy: improved lateral resolution
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STED microscopy: STED pulse intensity
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STED microscopy: STED pulse intensity
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STED microscopy: STED pulse intensity
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STED microscopy: STED pulse intensity
I: Intensity of the STED laser
Is: Required intensity to completely deplete the excited state
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STED microscopy: STED pulse intensity
Intensity
Intensity
I: Intensity of the STED laser
Is: Required intensity to completely deplete the excited state
In praxis, resolution of < 10 nm
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STED microscopy: STED pulse intensity
Conventional CLSM
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STED microscopy: scanning
Conventional CLSM
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STED microscopy: scanning
STED-CLSM (low power STED)
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STED microscopy: scanning
STED-CLSM (high power STED)
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STED microscopy: scanning
STED-CLSM
=> A higher resolution requires more scanning steps
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STED microscopy: scanning
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STED microscopy: images
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STED microscopy: images
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STED microscopy: images
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STED microscopy: images
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STED microscopy: images
Light Sheet Microscopy
=> based on wide-field microscopy
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=> Separate light paths for
excitation and emission light
Light sheet microscopy
=> Separate light paths for excitation and emission light
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Light sheet microscopy: planar illumination
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Intrinsic optical sectioning
=> only the image plane is illuminated
=> avoids photobleaching outside the sheet
Fast image acquisition
=> Whole image taken in a single exposure
(no scanning required, but scanning techniques also exist)
=> more than 100 full images can be taken per second
(depending on camera)
Applicable to larger biological samples
=> 3-D imaging
=> small living organisms
=> embryo development
Light sheet microscopy: advantages
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A sea horse: detection of autofluorescence for imaging
Light sheet microscopy: images
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pink: progenitor cells
green: aorta
blue: vein
Light sheet microscopy: images
Formation of lymph vessels in a mouse embryo
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red:
(1) red blood cells
(2) myocard
(heart muscle)
cyan: endocard
(inner lining of heart)
Light sheet microscopy: video
Heartbeat of
zebrafish
Expansion Microscopy
=> Blows up sample before imaging
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Expansion microscopy
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2 µm
Expansion microscopy: images
Different types of microtubles
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4x expanded
=> 60 nm resolution
Gao et al. (2019) Science 363, 245
Brain of fruit fly Drosophila:
Mapping of more than 40 million synapses in 62 hours
Expansion + light sheet microscopy: images
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Expansion + light sheet microscopy: video
Labeling of neuronal cells in the brain