Light Microscopy–

An IntroductionAn Introduction

„Anatomy“ of the light microscope (simplified)

Eye lens

Intermediate image planeIntermediate image plane

Specimen

Object lens

Condenser

Light source

Upright Scopep g p

Epi-illuminationSource

BrightfieldSource

Image from Nikonpromotional materials

Source

Inverted Microscope

BrightfieldSource

Epi-illuminationSource

Image from Nikonpromotional materials

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

A

AP A

P

Microinjection of fluorescent tracer dyes into each eye

Retinal ganglion cells in higher vertebrates

The spectrum of visible light

Spectrum of electromagnetic radiation

Spectrum of visible light

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

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)

1000mm

Magnification vs Resolution

1000mm

35 mm slide

M = 1000 mm35 mm = 28x

No limit to magnification

1000mm

Magnification vs Resolution

1000mm

35 mm slide

M = 1000 mm35 mm = 28x

no limit to magnification, but resolution is limited

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

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

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

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

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)

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

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

Brightfield microscopy

Brightfield illumination:

used for fixed, stained specimens or other types of samples with high natural absorption of visible light

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

Aequorea victoria

GFP (Green Fluorescent Protein), YFP, CFP…

Fluorescence microscope

Filters

neutral density filterh t filt

coloured filters

short pass filter

long pass filterband pass filter g pband pass filter

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)

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!

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, …

FP-tagged proteins are introduced into cells via transfection

Other transfection methods:

- DNA/Ca2+ phosphate co-precipitationp p p p

- viruses

Applications: BiFC (bimolecular fluorescent complementation)pp ( p )

Applications: Immunoistochimica – Immunohistochemistry