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K.U.LEUVEN
Johan HofkensSatoshi Habuchi
Laboratory of Photochemistry and SpectroscopyKatholieke Universiteit Leuven - Belgium
Theories and methods to study molecular interactions :
microscopy
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Overview
1. Microscope principle
- Lenses and geometrical optics
- Resolution of optical microscope
- Image brightness
2. Application for studies of molecular interactions
- Köhler illumination
- Fluorescence microscopy
- Confocal microscopy
- Fluorescent probes
- Ca2+ measurements in cells
- Nucleocytoplasmic shuttling of MAPK
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Why doing microscopy
Microscope is an instrument to produce magnified images of small objects.
- Magnified image
- Separate details in an image
- Make details visible to eye or camera
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Microscope Optical Components
Light source
Collector lens
Condenser
Objective
Eyepiece
Field Diaphragm
Aperture Diaphragm
Objective back focal plane
Intermediate image plane
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Lenses and Geometrical Optics
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Lenses and Geometrical Optics
Refraction of light
Direction change of a ray of light passing from one transparent medium to another with different optical density. A ray from less to more dense medium is bent perpendicular to the surface.
Snell’s Law
1
2
2
1
2
1 ==sin
sin
n
n
V
V
θ
θ
V: velocity of light in material
n: refractive index material
θ1
θ2
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Lenses and Geometrical Optics
Focal length
• Light from an object that is very far from the front of a lens will be brought to a focus at a fixed point behind the lens. This is known as the focal point (F) of the lens.
• The distance from the center of the lens to the focal plane is know as the focal length (f).
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Lenses and Geometrical Optics
Magnification of a lens
baf
111
a
b
h
hM =
′=
Lens formula Magnification
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Lenses and Geometrical Optics
Real image and virtual image
• When the distance between the object and the lens is longer than the focal length, the rays become convergent, giving the real image (inverted).
• When the distance between the object and the lens is shorter than the focal length, the rays can not be convergent, giving the virtual image (non inverted) which always appear upright to the observer.
Real image Virtual image
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Lenses and Geometrical Optics
Microscope conjugate field planes
fobj : < 10 mm
optical tube length : 160 mm
feye : few cm
Example
fobj : 8 mm, feye : 25 mm
208
160obj M
object to eye distance : 250 mm
1025
250eye M
2001020eyeobj MMM
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Lenses and Geometrical Optics
Infinity-corrected optical system
• The region between the objective and tube lens (infinity space) provides a path of parallel light rays.
• Complex optical components can be placed without loosing the performance of the microscope.
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Resolution of Optical Microscopy
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Resolution of Optical Microscopy
Diffraction
Light rays bend around edges – new wavefronts are generated at sharp edges
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Resolution of Optical Microscopy
Interference
Constructive interference Destructive interference
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Resolution of Optical Microscopy
Airy pattern formation
Condenser
Objective
Aperture Diaphragm
Intermediate image plane
Direct light
Diffracted light
Objective back focal plane
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Resolution of Optical Microscopy
Rayleigh criterion
The limit at which two Airy disks can be resolved into separate entities is often called the Rayleigh criterion.
Resolution
R = 0.61λ / NAλ : wavelength
NA : numerical aperture
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Resolution of Optical Microscopy
Numerical aperture (NA)
Numerical Aperture (NA) = n(sin μ)
n : refractive index of the imaging medium
μ : one-half angle of the angular aperture A
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Resolution of Optical Microscopy
NA and resolution
NA = 0.10 NA = 0.18 NA = 0.36
R = 0.61λ / NA
High NA : better resolution
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Resolution of Optical Microscopy
NA and resolution
Resolution and Numerical Apertureby Objective Type
λ = 550 nm
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Resolution of Optical Microscopy
Wavelength and resolutionR = 0.61λ / NA
wavelength = 400 nm wavelength = 550 nm wavelength = 700 nm
Short wavelength : better resolution
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Resolution of Optical Microscopy
Wavelength and resolution
Resolution versus Wavelength
NA = 0.95
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Resolution of Optical Microscopy
Axial resolution
The axial range, through which an objective can be focused without any appreciable change in image sharpness, is referred as the objective depth of field.
High NA : better axial resolution
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Resolution of Optical Microscopy
NA and immersion medium
Low refractive index High refractive index
NA = n(sin μ)
NA = 1.0 sin(65°) = 0.90
n = 1.00
μ = 65°
NA = 1.51 sin(65°) = 1.38
n = 1.51
μ = 65°
Low resolution High resolution
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Image Brightness
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Image Brightness
( )2MNA∝Brightness
Magnification
10x
NA
0.15
0.45
20x 0.40
0.85
40x 0.70
1.30
60x 0.80
1.40
100x 0.85
1.40
Transmitted light intensity
2.25
20.24
4.00
18.06
3.10
10.56
1.80
5.44
0.72
1.96
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Köhler Illumination
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Köhler Illumination
Köhler illumination (specimen illuminating light rays)
This technique is recommended by all
manufactures of modern laboratory
microscopes because it can produce
specimen illumination that is uniformly bright
and free from glare, allowing the user to
realize the microscope’s full potential.
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Köhler Illumination
Köhler illumination (image-forming light rays)
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Köhler Illumination
Aperture diaphragm
Aperture adjustment and proper focusing of the condenser are of critical importance in realizing the full potential of the objective. Specifically, appropriate use of the aperture diaphragm is most important in securing correct illumination, contrast, and depth of field.
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Köhler Illumination
Condenser alignment and field diaphragm opening size
Correct height of the condenser is critical to quantitative microscopy and optimum photomicrography.
The field diaphragm controls only the width of the bundle of light rays reaching the condenser – it does not affect the optical resolution, and the intensity of illumination. Proper adjustment of the field diaphragm is important for preventing glare.
Adjust the height of the condenser
Move the image of the field diaphragm to the center
Open the field diaphragm until it is just beyond the field of view
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Fluorescence Microscopy
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Fluorescence Microscopy
Fluorescence
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Fluorescence Microscopy
Epi fluorescence and transmitted light microscopy
Transmitted light microscopy Epi fluorescence microscopy
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Fluorescence Microscopy
Epi fluorescence microscopy
Advantage
• The objective, first serving as a well corrected condenser and then as the image-forming light gatherer, is always in correct alignment relative to each of these functions.
• Most of the unwanted or unused excitation light reaching the specimen travels away from the objective.
• The area being illuminated is restricted to the area being observed
• The full NA of the objective is utilizable.
• It is possible to combine with transmitted light observation.
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Fluorescence Microscopy
Light source
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Fluorescence Microscopy
FluorophoreA good fluorophore
- Large extinction coefficient ( ≈ 105 cm-1M-1)
- High fluorescence quantum yield ( > 0.8)
- Large shift of the fluorescence vs. absorption, Stokes shift ( > 40 nm)
- Low quantum yield of photobleaching ( < 10-6)
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Fluorescence Microscopy
Filters
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Fluorescence Microscopy
Detector
Area detector Point detector
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Fluorescence Microscopy
Fluorescence imagesHuman Cervical Adenocarcinoma Cells (HeLa Line)
EGFP : Green
Mito Tracker Red CMXRos : Red
• Peroxisomes
• Intracellular microtublar network
Hoechst 33342 : Blue
• DNA in the nucleus
Transformed African Green Monkey Kidney Fibroblast Cells (COS-7)
Cy3 : Red
Alexa Fluor 488 : Green
• Microtubles
• Cytoskeletal filamentous actin network
DAPI : Blue
• DNA
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Confocal Microscopy
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Confocal Microscopy
Confocal principle Excitation light passes through a pinhole aperture that is situated in a conjugate plane with a scanning point on the specimen.
As the laser is reflected by a dichromatic mirror and scanned across the specimen in a defined focal plane, secondary fluorescence emitted from points on the specimen (in the same focal plane) pass back through the dichromatic mirror and are focused as a confocal point at the detector pinhole aperture.
The significant amount of fluorescence emission that occurs at points above and below the objective focal plane is not confocal with the pinhole (termed Out-of-Focus Light Rays). Because only a small fraction of the out-of-focus fluorescence emission is delivered through the pinhole aperture, most of this extraneous light is not detected by the photomultiplier and does not contribute to the resulting image.
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Confocal Microscopy
Resolution in confocal microscopy
Rlat = 0.61λ / NA
Epi fluorescence microscopy Confocal fluorescence microscopy
Rlat = 0.43λ / NA
Axial Point Spread Function Intensity Profiles
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Confocal Microscopy
Confocal microscope scanning system
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Confocal Microscopy
Confocal images
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Application for Studies of molecular Interactions
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Amine-Reactive Probes Thiol-Reactive Probes
Fluorescent Probes
General fluorescent probes
target
probe
+
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Fluorescent Probes
Organelle-specific fluorescent probes
Mitochondrion-Selective Probes
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Fluorescent Probes
Genetically encoded fluorescent probes
Green fluorescent protein (GFP)
target GFPcDNA
vector
expression
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Ca2+ Measurements in Cells
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Ca2+ Measurements in Cells
Cameleon protein• Fluorescent indicators for measuring Ca2+ concentration.
- Energy donor : ECFP
ECFP EYFPCaM M13
440 nm 475 nm
ECFP
EYFP440 nm
530 nm
- Energy acceptor : EYFP
- Linker : calmodulin (CaM) + calmodulin-binding peptide M13 (myosin light chain kinase)
+4Ca2+
-4Ca2+
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Ca2+ Measurements in Cells
Cameleon protein
Energy transfer efficiency Ca2+ concentration
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Ca2+ Measurements in Cells
Ca2+ propagation in cells
HeLa cells are stimulated with histamine
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Ca2+ Measurements in Cells
Ca2+ propagation in cells
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Nucleocytoplasmic Shuttling of MAPK
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Nucleocytoplasmic Shuttling of MAPK
MAPK cascades
The Mitogen-activated Protein Kinase (MAPK) Cascades
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Nucleocytoplasmic Shuttling of MAPK
Fused protein
MAPK
Dronpa
MAPK-Dronpa is initially distributed throughout the cytosol and nucleus.
Dronpa : GFP-like fluorescent protein
COS7 cells
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Nucleocytoplasmic Shuttling of MAPK
Photoswitching of Dronpa
Intense excitation at 488 nm changes Dronpa to the dim state but even weak irradiation at 400 nm restores it to the bright deprotonated form.
Fluorescence could be switched on and off repeatedly.
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Nucleocytoplasmic Shuttling of MAPK
Monitoring the nuclear import and export of MAPK
C ►N
N ►C
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Nucleocytoplasmic Shuttling of MAPK
Monitoring the nuclear import and export of MAPK
Only the nuclear accumulation of MAPK is confirmed by the normal fluorescence images.
The acceleration of the bidirectional flow of MAPK across the nuclear envelope is vidualized by reversible protein highlighting.