C8116 Immunoaffinity techniques
Advanced microscopy I
Spring term 2025
Hans Gorris
Department of Biochemistry
April 15th, 2025 1
Preparation of recombinant proteins
Structure of glutathione beads
GST pulldown assay
3
Y2H: Protein fragment complementation assay
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 remember:
“smart reporters”
Convex glass (spherical)
main plane
Brennpunkt
Optische Achse
Collective lens
optical axis
focal length
focal point
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focal plane
Collective lens
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1
2
3
image
optical axis
(1) parallel ray
(2) central ray
(3) focal ray
object
f2x f f’
Object placed between focal point and lens (pobj < f)
=> Diverging rays after lens, i.e. image cannot be focused
Collective lens
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Collective lens
Magnified virtual image behind object (loupe).
virtual
image
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Object placed between focal point and lens (pobj < f)
=> Diverging rays after lens, i.e. image cannot be focused
Visual angel
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up to 200-fold magnification
Antonie van Leeuwenhoek (1632-1723)
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Combination of two collective lenses
Mtotal = Mobjective x Meye piece
Light path of combined microscope
virtual
image
object
objective
lens
eye piece
real
intermediate
image
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Imaging light path of an optical microscope
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“upright“
microscope
intermediate
Image
eye pieceOptical tube
length t
object
objective
back
focal plane
Setup of (historical) combined microscope
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Light microscopy: Upright microscope
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Modern microscopes are infinity corrected
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finite optical
system
infinity-corrected
optical system
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
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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
17
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
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image
plane
object
plane
(pobj)
f f’
conjugated planes
Parallel rays
back focal
plane
Conjugated planes
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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
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=> 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)
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Optical defects in lens systems (2)
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4 color
correction
3 color
correction
2 color
correction
Objective
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Objektive descriptions
Objective
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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
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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
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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
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Resolution is diffraction limited
Possibilities to attain a higher resolution?
=> d   / 2  200 nm


sin2n
d =
Optical resolution of light microscopy
Wave-optical explanation: Diffraction of rays at a cleft
central bright
strip
light
intensity
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Bright-field microscopy
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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
cell sample
examples:
- Gram-staining (bacteria)
- Stained tissues (histology)
but: fixing/staining kills cells
stained cells => higher contrast
Standard (bright field) microscopy
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=> poor contrast because cells are
70% water
26% macromlecules (proteins,
nucleic acids, and polysacharides)
+ smaller amounts of others
Bright-field vs. Dark-field microscopy
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objective
condenser
illumination
light
Condenser should
have larger NA
than the objective
Dark-field microscopy
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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
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°)
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phase ring=> Frits
Zernike 1930
Phase contrast microscopy
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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
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Phase contrast microscopy
Bright-field image
Dark-field and phase contrast microscopy
36Source: https://toutestquantique.fr/en/dark-field-and-phase-contrast/
Fluorescence microscopy
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Epifluorescence microscopy
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Setup of epifluorescence microscope
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Setup of epifluorescence microscope
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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 ➔ detection in 3 color channels
Fluorescence microscopy
=> Sensitivity through dark background
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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
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cyclic Ser65-Tyr66-Gly67
Originally isolated from jellyfish.
=> Enormous importance via
recombinant expression!
(Nobel Prize in 2008)
Green fluorescent protein (GFP)
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structural gen EGFPpromoter
Enhanced Green Fluorescent Protein
day light
under UV light
GFP and its derivatives
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ECFP - marker protein for endoplasmic reticulum
EYFP - marker for Golgi
GFP chimera
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46
Comparision of microscopy in the life sciences
Subcategories of fluorescence microscopy
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Fluorescence microscopy
Wide-field
Epi TIRF
Light
sheet
STORM
Confocal
Multi-photon
STED
Super-
resolution