C8116 Immunoaffinity techniques
Advanced microscopy II
Spring term 2024
Hans Gorris
Department of Biochemistry
May 7th, 2024 1
Fredriksson (2002): Protein detection using proximity-dependent DNA ligation assays. Nat. Biotechnol. 20: 473-477
Proximity ligation
2
Homodimeric PDGF-BB
Proximity probe
A1
Proximity probe
A2
Aptamer specific
for PDGF-B
Aptamer specific
for PDGF-B
primer primer
connector oligonucelotide
probe for detection
of PCR products
F: fluorescent dye fluorescein
Q: quencher TAMRA
Preparation of recombinant proteins
Structure of glutathione beads
GST pulldown assay
4
Y2H: Protein fragment complementation assay
5
Þ remember:
“smart reporters”
Light microscopy: Upright microscope
6
Imaging light path of an optical microscope
7
intermediate
image
eye pieceOptical tube
length t
object
objective
back
focal plane
We obtain a real image (upside down) if object is placed:
(A) in focal plane (pobj = f): parallel rays emerge after lens; i.e. image is not focused
(B) between simple and double focal length (f < pobj < 2f): magnified image
(C) in double focal length (pobj = 2f): image has the same size as object
(D) beyond double focal length (pobj > 2f): demagnified image
Conjugated planes
8
1
2
3
image
plane
optical axis
(1) parallel ray
(2) central ray
(3) focal ray
object
plane
(pobj)
f2x f f’
conjugated planes
We obtain a real image (upside down) if object is placed:
(A) in focal plane (pobj = f): parallel rays emerge after lens; i.e. image is not focused
(B) between simple and double focal length (f < pobj < 2f): magnified image
(C) in double focal length (pobj = 2f): image has the same size as object
(D) beyond double focal length (pobj > 2f): demagnified image
Conjugated planes
9
1
2
3
image
plane
optical axis
(1) parallel ray
(2) central ray
(3) focal ray
object
plane
(pobj)
f f’
conjugated planes
focal
plane
back focal
plane
We obtain a real image (upside down) if object is placed:
(A) in focal plane (pobj = f): parallel rays emerge after lens; i.e. image of light bulb is not focused
(B) between simple and double focal length (f < pobj < 2f): magnified image
(C) in double focal length (pobj = 2f): image has the same size as object
(D) beyond double focal length (pobj > 2f): demagnified image
Conjugated planes
10
image
plane
object
plane
(pobj)
f f’
conjugated planes
Parallel rays
back focal
plane
Conjugated planes
11
image
plane
object
plane
(pobj)
f f’
conjugated planes
Parallel rays
back focal
plane
We must distinguish between:
(1) Imaging light path
(2) Illumination light path
=> First implemented in microscopy in 1893
by August Köhler (Zeiss company)
Conjugate planes in an optical microscope
12
=> Köhler
Illumination
in an upright
microscope
f’
f
As the light source is not
focused at the level of the
specimen, the light at
specimen level is essentially
grainless and extended, and
does not suffer deterioration
from dust and imperfections
on the glass surfaces of the
condenser.
Optical defects in lens systems (1)
13
Optical defects in lens systems (2)
14
4 color
correction
3 color
correction
2 color
correction
Objective
15
Objektive descriptions
Objective
16
n=1 n=1,5
n=1,5
NA up to 0.9 NA up to 1.49
=> Immersion liquid reduces the refractive index mismatch
Refraction at the interface
of glass (cover glass of
the sample) and air
Objective: refractive index mismatch
object
object
oilair
cover glas
17
cover glas
It is not the magnification but rather the numerical aperture (NA) of the objective
that determines the quality of on image.
NA = n × sinα
n: refractive index
α: acceptance angle
of the objective
Width of the acceptance cone
=> How much light can be focused?
High NA improves
1. Resolution
2. Brightness (also contrast)
Objective: numerical aperture
18
Wave-optical explanation: Diffraction of rays at a cleft
Requirement for an objective with a wide acceptance cone (NA)
to focus diffracted light efficiently
=> high-resolution objective
Diffraction increases with wavelength!
Optical resolution of light microscopy
19
Resolution is diffraction limited!
Possibilities to attain a higher resolution?
=> d » l / 2 » 200 nm
a
l
sin2n
d =
Optical resolution of light microscopy
Wave-optical explanation: Diffraction of rays at a cleft
central bright
strip
light
intensity
20
Bright-field microscopy
21
Light from the condenser passes through sample (transmission mode), is
attenuated by absorbing materials and collected by the objective
Total magnification (Mtot) = Mobjective x Meyepiece
• but there is a fundamental limit of resolution depending only on the objective:
λ/(2n*sinα) – note: M does not appear in this equation!
with λ: wavelength of light
n: refractive index
α: half of acceptance cone
• higher magnifications are called empty magnification
• The objective forms an image in the the intermediate image plane that
contains all information on the specimen accessible by the microscope! Any
further image magnification by eyepiece or camera lenses only changes the
size for easier observation or to fit the camera chip, but does not add any
information.
=> The resolution and brightness/contrast of an objective are essential
Dark-field microscopy
22
Dark-field microscopy prevents non-diffracted light from entering the objective.
Only light rays diffracted by the specimen are collected by the objective. Thus,
a bright image appears against a dark background, resulting in a much better
image contrast compared to bright-field microscopy.
=> Enables observation of living cells/organisms.
In biology, dark-field microscopy has been replaced by improved techniques,
but it has recently reemerged for the analysis of strongly light scattering
(plasmonic) nanomaterials.
Amphipod crustacean (25x magnification)
Condenser
should have
larger NA
than objective
Dark-field and phase contrast microscopy
23
Source: https://toutestquantique.fr/en/dark-field-and-phase-contrast/
Phase contrast microscopy
Improved cellular contrast by shifting the phase of light
=> in the phase ring, light is retarded (or advanced) by ¼ wavelength (Δφ = 90°)
24
phase ring=> Frits
Zernike 1930
Phase contrast microscopy
25
phase ring Δφ = 90°
Interference:
converts a phase
difference into an
amplitude difference
(=> visible by eye)
diffracted light
(from specimen)
non-diffracted light
(from light source)
Phase contrast image
=> Phase contrast microscopy enables label-free detection of living cells
26
Phase contrast microscopy
Bright-field image
Fluorescence microscopy
27
Epifluorescence microscopy
28
Setup of epifluorescence microscope
29
30
Comparision of microscopy in the life sciences
Setup of epifluorescence microscope
31
A) GFP-coupled pallidin
=> binds to actin
B) AlexaFluor546-phalloidin
=> binds to F-actin
C) Cy5-coupled antibody
=> binds to cell-substrateadhesion
protein
(immune fluorescence)
D) Overlay of three fluorescence
signals
3-fold fluorescence labeling of keratinocyte è detection in 3 color channels
Fluorescence microscopy
=> Sensitivity through dark background
32
natural fluorophores
Try, NADH, FADH2 UV excitation
GFP, EGFP, EYFP etc. Excitation with UV or visible light
fluorescent labels
labeling of cell components that are non-fluorescent by themselves
proteins (directly or via antibodies): FITC, TRITC, Cy-3, Cy-5
DNA, RNA: ethidium bromide, DAPI
lipids: DPH, Pyrenyl-PC
low molecular weight ions: Fluorescein (pH), Fura-2 (Ca2+)
problems: reasons: consequences:
- autofluorescence using short- high
- light scattering wavelength light background
- photobleaching strong excitation short imaging
- cytotoxicity intensities times
- labeling non-specific binding artifacts
Fluorescent dyes
33
cyclic Ser65-Tyr66-Gly67
Originally isolated from jellyfish.
=> Enormous importance via
recombinant expression!
(Nobel Prize in 2008)
Green fluorescent protein (GFP)
34
structural gen EGFPpromoter
Enhanced Green Fluorescent Protein
day light
under UV light
GFP and its derivatives
35
ECFP - marker protein for endoplasmic reticulum
EYFP - marker for Golgi
GFP chimera
36
Subcategories of fluorescence microscopy
37
Fluorescence microscopy
Wide-field
Epi TIRF
Light
sheet
STORM
Confocal
Multi-photon
STED
Super-
resolution
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
=> Both problems have been solved over the last 30 years
a
l
sin2n
d =
Fluorescence microscopy: limitations
38
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
=> Both problems have been solved over the last 30 years
Fluorescence microscopy: limitations
39
State of the art microscope systems and cameras/photomultipliers are sensitive
enough to visualize single fluorescent molecules
=> A single fluorophore can emit up to 1.000.000 photons before it photobleaches
Problem: Background signal
(Autofluorescence / Rayleigh scattering / Raman scattering)
Background can be reduced by reducing the excitation volume
- Confocal microscopy / Multi-photon microscopy
(ellipsoid excitation volume of ca. 1 x 1.5 µm = 10-15 L = 1 Femtoliter)
=> contains 1 molecule of fluorophore but also 1010 solvent molecules
- Total internal reflection microscopy (TIRF)
(planar excitation volume of ca. 100 nm depth, evanescent field)
Avoiding autofluorescence and light scattering
40
Photomultiplier
Laser
PinholeImage plane
Aperture
Lens
Beam splitter
Objective lens
Object
Focus too high
Focus too low
Focal plane
Focal plane + Pinhole
are optically conjugated!
Confocal laser scanning microscopy (CLSM)
41
A pinhole in the image plane rejects the
light coming from outside the focal plane.
The pinhole size is a trade-off between
good rejecting ability and sufficient light
throughput (typically ~ 30 – 150 µm)
Confocal laser scanning microscopy (CLSM)
42
The pinhole restricts the observed volume of the sample to a single point (the size of which is
restricted by the pinhole size). Excitation by a collimated beam (point source optically conjugated
to the pinhole) focused to a diffraction limited spot
wide field confocal
whole image at once
dichroic
image is scanned point by pointCCD
PMT
MPD
…
Confocal vs. wide-field microscopy
43
Wide-field
Confocal
Elimination of out-of-focus light improves contrast and, thus, resolution
confocal aperture open
no depth of field (hloupka pole)
confocal apterture (optimal)
depth of field, z-resolution
Confocal microscopy: improved lateral resolution
Preparation of „optical sections“ through thick samples: z resolution
From the optical sections:
Calculation of side views
3D-reconstruction
Confocal microscopy
Confocal microscopy
46
Focusing only in one plane ® axial sectioning of the sample to ~ µm slices
Summary of confocal microscopy
47
Advantages
• improved contrast
• optical sectioning (z stacks)
• multiple fluorescence measurements can be performed in individual points
(e.g. lifetime, spectra, fluorescence correlation spectroscopy)
Limitations
• more expensive and complicated setup
• slower than wide-field imaging
• longer imaging time needed
=> more photobleaching
Multi-photon microscopy
(2p-, 3p-, 4p-microscopy)
48
49
Two-photon microscopy
50
Two-photon microscopy
51
Two-photon microscopy: Axial resolution
laser pulse
focal plane
the required photon density for
2-photon excitation is
established only in the focal
plane
Ø no out-of focus fluorescence
Ø no pinhole needed
photon
non-excited
dye molecule
2p-excited
dye molecule
conventional 1p-excitation 2p-excitation
x
y
z
Illumination spot
Comparison of emission profiles 1p vs 2p
52
Two-photon microscopy
x
y
z
Illumination spot
53
Two-photon microscopy
54
Summary of two-photon microscopy
Advantages
• improved axial resolution
• reduced bleaching out of focus
• higher light collection efficiency (no pinhole)
• higher depth of light penetration (» 5 x)
• broader excitation spectra:
simultaneous excitation of more dyes
Limitations
• more expensive and complicated
instrumental setup: pulsed (femtosecond)
solid state lasers required for extremely high
excitation powers (100 kW)
• higher bleaching in the focus
• broader excitation spectra:
decreased selectivity of excitation
• scanning technique is slower
(=> confocal microscopy)
55
From two- to multi-photon microscopy
56
Sequential absorption of 2 or more photons via long-lived transition states
=> More time for absorbing a further photon
The process is ca. 1 million times more efficient than 2-photon excitation
=> a continuous wave (CW) laser diode can be used
Sequential absorption of two or more photons
57
Labeling of cancer cells:
Upconversion microscopy
Wide-field microscopy
Upconversion microscopy
Farka Z, Mickert MJ, Mikušová Z, Hlaváček A, Bouchalová P, Xu W, Bouchal P, Skládal, P, Gorris HH (2020)
Nanoscale 12, 8303
Þ Small differences in protein expression levels can be dected.
Excellen signal to
background (S/B) ratio
58
Total Internal Reflection Fluorescence microscopy
(TIRF)
59
Snell‘s law:
n1 sinα = n2 sinα‘
Holds until reaching the critical angle (θ), then: Total internal reflection
d: depth of evanescent field
λ: wavelength of light
θ: critical angle
60
Total internal reflection fluorescence mic. (TIRF)
61
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
62
Total internal reflection fluorescence mic. (TIRF)
Cytoplasm
green: Staining of actin
red: Soluble dye rhodamine
Vesicle budding by endocytosis
63
Total internal reflection fluorescence mic. (TIRF)
http://microscopy.duke.edu/gallery.html
How fast are photobleached fluorophores replaced by diffusion (D)?
Lipid bilayer adsorbed
to solid surface
- mobile lipids Lipid
monolayer adsorbed
to immobilized alkyl chains
- immobile lipids -
I0
IB
I0
IB
D can be determined by
fitting the recovery curve
with a model accounting
for the size and shape of
the bleached area.
64
Fluorescence recovery after photobleaching
Single molecule fluorescence microscopy
(frequently in combination with TIRF)
65
Conditions:
• Fluorescent probes with high quantum yield / low bleaching rates
further problems can be avoided by the choice of buffer systems:
e.g. „blinking“ by transition into a triplet state or oxidation by O2
• Wide field epifluorescence microscopy with TIRF => very low background
• Wide field microscopy provides a much better time resolution compared to scanning
techniques (video rates = up to 100 images / sec)
=> Each fluorophor molecule
is visible as a diffraction limited spot.
66
Single-molecule fluorescence microscopy
67
Single-molecule fluorescence microscopy
=> Single Particle Tracking (SPT)
68
Single-molecule fluorescence microscopy
Analysis of trajectories: Random Walk
=> Diffusion coefficients of single molecules (single molecules vs. ensemble)
Diffusion coefficient (D) given by
Stokes Einstein equation:
69
Single-molecule fluorescence microscopy
Trajectory of a single molecule temporal sequence: violet, blue, green, yellow
Dynamics of
biological membranes
on a molecular level
70
Single-molecule fluorescence microscopy
Microdomains in plasma membrane
„Lipid Rafts“
71
Single-molecule fluorescence microscopy
=> Fluorophores in membrane can be well excited in evanescent field
72
Single-molecule fluorescence microscopy
Fluorescence resonance energy transfer (FRET)
microscopy
73
Analyzing protein-protein interactions by FRET
74
𝐸! =
1
1 +
𝑅"#
𝑅$
%
R0: ET = 50%
FRET: Experimental setup
75
pulsed laser
beam
splitter
microscopepinhole
donor
filter
detector 1
acceptor
filter
detector 2
time-resolved
single photon
counting
color beam
splitter
Intramolecular
FRET
Intermolecular
FRET
Single-molecule FRET
76
- Protein-protein interactions are investigated in their natural environment
- Fusion with fluorescent proteins (e.g. GFP) are used
- The location of the interaction can be determined (=> super-resolution microscopy)
- Real-time imaging
- Heterogeneous and dynamic biological processes can be observed
Requires dedicated equipment:
Þ Strong background reduction (autofluorescence): confocal microscopy or TIRF
Þ Sensitive cameras or avalanche photodiodes
Þ Reduction of photobleaching (GFP is not very photostable)
Single-molecule FRET in vivo
77