K.U.LEUVEN Johan Hofkens Satoshi Habuchi Laboratory of Photochemistry and Spectroscopy Katholieke...

K.U.LEUVEN

Johan HofkensSatoshi Habuchi

Laboratory of Photochemistry and SpectroscopyKatholieke Universiteit Leuven - Belgium

Theories and methods to study molecular interactions :

microscopy

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

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

Microscope Optical Components

Light source

Collector lens

Condenser

Objective

Eyepiece

Field Diaphragm

Aperture Diaphragm

Objective back focal plane

Intermediate image plane

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


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).


Magnification of a lens

baf

111

a

b

h

hM =

′=

Lens formula Magnification


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


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


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.

Resolution of Optical Microscopy


Diffraction

Light rays bend around edges – new wavefronts are generated at sharp edges


Interference

Constructive interference Destructive interference


Airy pattern formation

Condenser

Objective

Aperture Diaphragm

Intermediate image plane

Direct light

Diffracted light

Objective back focal plane


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


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


NA and resolution

NA = 0.10 NA = 0.18 NA = 0.36

R = 0.61λ / NA

High NA : better resolution


NA and resolution

Resolution and Numerical Apertureby Objective Type

λ = 550 nm


Wavelength and resolutionR = 0.61λ / NA

wavelength = 400 nm wavelength = 550 nm wavelength = 700 nm

Short wavelength : better resolution


Wavelength and resolution

Resolution versus Wavelength

NA = 0.95


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


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

Image Brightness

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

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.


Köhler illumination (image-forming light rays)


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.


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

Fluorescence Microscopy


Fluorescence


Epi fluorescence and transmitted light microscopy

Transmitted light microscopy Epi 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.


Light source


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)


Filters


Detector

Area detector Point detector


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

Confocal Microscopy

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.

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

Confocal Microscopy

Confocal microscope scanning system

Confocal Microscopy

Confocal images

Application for Studies of molecular Interactions

Amine-Reactive Probes Thiol-Reactive Probes

Fluorescent Probes

General fluorescent probes

target

probe

+

Fluorescent Probes

Organelle-specific fluorescent probes

Mitochondrion-Selective Probes

Fluorescent Probes

Genetically encoded fluorescent probes

Green fluorescent protein (GFP)

target GFPcDNA

vector

expression

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+


Cameleon protein

Energy transfer efficiency Ca2+ concentration


Ca2+ propagation in cells

HeLa cells are stimulated with histamine


Ca2+ propagation in cells

Nucleocytoplasmic Shuttling of MAPK


MAPK cascades

The Mitogen-activated Protein Kinase (MAPK) Cascades


Fused protein

MAPK

Dronpa

MAPK-Dronpa is initially distributed throughout the cytosol and nucleus.

Dronpa : GFP-like fluorescent protein

COS7 cells


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.


Monitoring the nuclear import and export of MAPK

C ►N

N ►C


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.

K.U.LEUVEN Johan Hofkens Satoshi Habuchi Laboratory of Photochemistry and Spectroscopy Katholieke...

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