light microscopy an introductionan introductionlight microscopy – an introductionan introduction...
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Light Microscopy–
An IntroductionAn Introduction
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„Anatomy“ of the light microscope (simplified)
Eye lens
Intermediate image planeIntermediate image plane
Specimen
Object lens
Condenser
Light source
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Upright Scopep g p
Epi-illuminationSource
BrightfieldSource
Image from Nikonpromotional materials
Source
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Inverted Microscope
BrightfieldSource
Epi-illuminationSource
Image from Nikonpromotional materials
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Overview: Microscopy
Light microscopy (LM)
• simple microscopes (magnifying glasses)simple microscopes (magnifying glasses)
• fluorescence microscopes
Electron microscopy (EM)
• transmission electron microscopy (TEM) TS TEM
• thin section TEM
• freeze fracture TEM
• scanning electron microscopy (SEM)TS TEM
SEM
FF TEM
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A
AP A
P
Microinjection of fluorescent tracer dyes into each eye
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Retinal ganglion cells in higher vertebrates
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The spectrum of visible light
Spectrum of electromagnetic radiation
Spectrum of visible light
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Absorption and colour
Colour seenColour(s) absorbed from white light
blue
red
red
blue
green
green
yellow
greenredblue
blue
g
yellow
magenta
red
blue
greenwhite light(all colours)
cyan
black
red
redblue green
white
redblue green grey
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Magnification (M)
Objects can generally be focused no closer than 25 cm from the eye
= normal viewing distance for 1x magnification
Children: up to 12 cm ⇒ 2x magnification compared to adults
Magnification of a microscope:
Mmicroscope = Mobjective x Meyepieces
e.g. 63x objective, 10x eyepieces ⇒ 630x magnification
ibl b t ifi ti t→ possible subsequent magnification steps
(e.g. digital image processing)
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1000mm
Magnification vs Resolution
1000mm
35 mm slide
M = 1000 mm35 mm = 28x
No limit to magnification
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1000mm
Magnification vs Resolution
1000mm
35 mm slide
M = 1000 mm35 mm = 28x
no limit to magnification, but resolution is limited
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Resolution
shortest distance between two points that can still be distinguished as separateshortest distance between two points that can still be distinguished as separate
point sources of light from a specimen appear as Airy diffraction patterns
Resolution depends on:
• physical parameters p y p
• "user" parameters
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Resolution depends on (physical parameters):
• Correct alignment of the microscope optical system Wavelength Resolution• Correct alignment of the microscope optical system
• Wavelength of light (λ) → see EM
(nm) (μm)
360 0.19
400 0.21
450 0.24
NA = 0.95
• Numerical aperture (NA) of objective (& condenser)
generally: r ( ) = 500 µm r (LM) = 0 25 μm
500 0.26
550 0.29
600 0.32
650 0.34generally: rmax.(eye) 500 µm rmax.(LM) 0.25 μm (= 2,000 x magnification)
700 0.37
Objective Type
Plan Achromat Plan Fluorite Plan Apochromat
M ifi ti R l ti R l ti R l ti
r = λ/2NAMagnificati
on N.A. Resolution(µm) N.A. Resolution
(µm) N.A. Resolution(µm)
4x 0.10 2.75 0.13 2.12 0.20 1.375
10x 0.25 1.10 0.30 0.92 0.45 0.61
20x 0.40 0.69 0.50 0.55 0.75 0.37
40x 0.65 0.42 0.75 0.37 0.95 0.29
60x 0.75 0.37 0.85 0.32 0.95 0.29
100x 1.25 0.22 1.30 0.21 1.40 0.20
N.A. = Numerical Aperture
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Numerical aperture (NA)
measure of an objective’s ability to gather light & resolve detail at a fixed object distancemeasure of an objective s ability to gather light & resolve detail at a fixed object distance
a
NA = n . sinα
sinα = a/b
a
b
a
bαsinα = a/b
α
n: refractive index of the medium between the objective front lens and the specimen
imaging medium: air ⇒ n = 1
n: refractive index of the medium between the objective front lens and the specimen
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Numerical aperture (NA)
measure of an objective’s ability to gather light & resolve detail at a fixed object distancemeasure of an objective s ability to gather light & resolve detail at a fixed object distance
nair = 1.0
α
noil = 1 511.51
glass coverslip
air objectives: generally NA ≤ 0.95 (limited by min. focal length of objective)
⇒ higher NA obtainable by increasing refractive index (n) of medium between specimen & objective front lens
air: n = 1.0 water: n = 1.33glycerine: n = 1.47immersion oil: n = 1.51 (= of glass)
NA = n . sinα
numerical aperture of an objective is also dependent, to a certain degree, upon the amount of correction for optical aberration
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Resolution depends on ("user" parameters):
• clean objective & specimen e.g. no fingerprints, remnants of buffer, dust, no water in immersion oil, no oil on air objectives etc.
• good illumination improper illumination may lower resolution
• low specimen contrast lowers resolution→ contrast enhancing in the specimen or in the microscope (e.g. phase contrast) may increase resolution
• correct thickness of coverslip (typically 0.17 mm; indicated on the objective)
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Darkfield microscopy
contrasting method to visualize fine structural featurescontrasting method to visualize fine structural features
specimen viewed against dark background
condenser optics
specimen
objective
annular stop (in condenser)
"brightfield"
"darkfield" brightfield darkfield
wt Drosophilacuticula
cuticula of bicoid mutant
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Brightfield vs Phase Contrast microscopy
Phase contrast:Phase contrast:
employs optical mechanism to translate minute variations in phase into corresponding changes in amplitude
that can be visualized as differences in image contrast
hi h t t i f t t i⇒ high-contrast images of transparent specimens
(e.g. living cells, microorganisms, thin tissue slices; no fixation & staining needed)
Required: Special objectives and special condensers
Also: differential interference contrast (DIC) used to obtain higher contrast images of low contrast specimen
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Brightfield microscopy
Brightfield illumination:
used for fixed, stained specimens or other types of samples with high natural absorption of visible light
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Fluorescence
fluorophore absorbs photon (λ1) and re-emits photon (λ2)
λ2 > λ1 ; usually Δt < 1 μs
higher energy & vibration states
lower singlet excited state
bsor
ptio
n (λ
1) emission (λ
ab
λ2)
gound state
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Aequorea victoria
GFP (Green Fluorescent Protein), YFP, CFP…
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Fluorescence microscope
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Filters
neutral density filterh t filt
coloured filters
short pass filter
long pass filterband pass filter g pband pass filter
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Essentials for successful fluorescence microscopy
• high intensity excitation• high intensity excitation
• appropriate excitation & emission filters
• high quality objectives (high NA, high light transmission)
• minimal autofluorescence in specimen (e.g. no glutaraldehyde fix.)
• use immersion oil without autofluorescence (normal oil autofluoresces ⇒ haze over sample)
• antifade reagents (special ones for LSM)
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Fluorescent proteins allow labelling of proteins in living cells
P t i b d t t d b tib d t i i b t l i fi d llProteins can be detected by antibody staining, but only in fixed cells
⇒ not possible to visualise protein movement / dynamics in living cells
IMPORTANT:
Excitation & emission spectra can overlap
⇒ Signal from one fluorophore mistaken for that from another
Example: What looks like "co-localisation" of two proteins is actually bleed-through.
Choose combinations of fluorophores carefully!
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There are numerous fluorescent proteins with different properties
onE
xcita
tion
Em
issi
o
⇒ wide variety of excitation & emission spectra available for different applications and fluorophore combinations
Also: destabilised GFP, BiFC, …
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FP-tagged proteins are introduced into cells via transfection
Other transfection methods:
- DNA/Ca2+ phosphate co-precipitationp p p p
- viruses
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Applications: BiFC (bimolecular fluorescent complementation)pp ( p )
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Applications: Immunoistochimica – Immunohistochemistry