Joerg Bewersdorf Department of Cell Biology

U Maine

Fluorescence Microscopy

10/17/2011

• Overview • A Basic Light Microscope • Contrast in Light Microscopy • Fluorescence Microscopy • Image Artifacts

Outline

Zeiss Transmission Electron Microscope

Robert Hooke Micrographia, 1665

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Olympus Confocal Laser Scanning Microscope

Microscopy Techniques

light

el

ectr

on

widefield scanning

othe

rs

scanning probe/tip

conventional microscopy

confocal laser scanning

microscopy (CLSM)

transmission electron

microscopy (TEM)

scanning transmission

electron microscopy (STEM)

scanning electron

microscopy (SEM)

scanning near-field optical

microscopy (SNOM)

electron tomography

atomic force microscopy (AFM)

ion conductance microscopy

scanning acoustic microscopy

…

scanning Helium

microscopy

Size scales and resolution

synaptic vesicle (Takamori et al., Cell 2006)

myoglobin (Wikipedia)

amino acid (Wikipedia)

Helium atom (Wikipedia)

mitochondrion (Wikimedia Commons)

animal cell (Wikipedia)

0.1 nm

1 nm

10 nm

100 nm

1 µm

10 µm

100 µm

super-resolution

conventional EM

naked eye

diffraction limit

Elements of an Imaging System (here: Microscope)

illumination source

sample detector beam path

beam path

sun lamp halogen mercury vapor metal halide

light emitting diode (LED) laser electron gun

eye film camera CCD EM-CCD CMOS

point detector photomultiplier tube (PMT) avalanche photodiode

Contrast: absorption reflection/scattering phase fluorescence polarization birefringence …

Wave-Particle Duality Light is a Wave Light is a Particle

fluorescence

excitation

Light particle: „Photon“ with energy E = hc/λ

Wavelength λ

Airy Pattern

Diffraction

Refraction

λ

3 eV 2 eV energy

Visible spectrum

500 nm 600 nm 400 nm 700 nm

infrared ultraviolet

A Basic Light Microscope

On September 17, 1683, Leeuwenhoek wrote to the Royal Society about his observations on the plaque between his own teeth, "a little white matter, which is as thick as if 'twere batter." He repeated these observations […] on two old men who had never cleaned their teeth in their lives. […] In the mouth of one of the old men, Leeuwenhoek found "an unbelievably great company of living animalcules, swimming more nimbly than any I had ever seen up to this time. The biggest sort. . . bent their body into curves in going forwards. . . Moreover, the other animalcules were in such enormous numbers, that all the water. . . seemed to be alive.“ http://www.ucmp.berkeley.edu/history/leeuwenhoek.html

http://www.brianjford.com/wav-spf.htm http://www.cartage.org.lb/en/themes/sciences/physics/optics/OpticalInstruments/Microscope/GlassSphere/GlassSphere.htm

Exploring the Unknown: Leeuwenhoek and his Microscope

red

bloo

d ce

lls

tiny lens sample mount

How does the light get from here to there?

Solution 1:

Step 1: A Single Lens

focal length f

angle α distance d

2 * d

Step 2: Another Lens

same angle α

2 * f

Building a Microscope

focal length f

fObj = f

Magnification:

M = (2 * d) / d = 2

= fTL/ fObj ratio of the focal lengths

sample image (camera)

Remarks:

• The image is upside down (can be easily compensated)

• Needs a light source (not shown here)

• For looking through the microscope with your eyes, additional lenses (“eye pieces” or oculars) are used to take the lenses in your eyes into account.

• This schematic represents the “infinity-corrected” beam path of modern microscopes

objective tube lens

fTL = 2 * f

d

2 * d

objective back

aperture

Attributes of a Microscope

Enhancing Image Quality

Problems of first microscopes: • Chromatic aberrations: different images for different colors • Small and/or distorted field of view • Limited resolution Additional problems of fluorescence microscopes (due to dim signal): • high background from autofluorescence (lenses, media, etc.) • stray light • non-ideal color filters

Breakthrough for the first three problems:

© Whipple Museum

Flea imaged through an 18th century microscope

Ernst Abbe (1872)

A Modern Widefield Microscope Beam path of an inverted microscope 1 Intermediate image plane/phototube 2 Eyepiece 3 Intermediate image plane/front port 4 Intermediate image plane/base port 5 Switching beam path between base port/front port/vis. observation 6 Side port prisms 7 Tube lens 8 Analyzer 9 Reflector cube 10 Field diaphragm 11 Aperture stop 12 Filter slider 13 mercury vapor (HBO) or metal halide lamp 14 halogen lamp 15 Field diaphragm 16 Polarizer 17 Aperture stop 18 Condenser 19 Objective

1 2

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8 *

Contrast in Light Microscopy

Type Principle Live cells Fixed cells

Bright field Absorption; stains required No Yes

Phase contrast Refractive index differences Yes Yes

DIC* Gradient of refractive index Yes Yes

Fluorescence Emission by fluorescent molecule Yes Yes

Dark field Light scattering (Yes) (Yes)

Polarization Birefringence Yes Yes

*Differential interference contrast

Contrast Mechanisms in Light Microscopy

standard brightfield (with closed

condenser aperture)

Phase Contrast and DIC

phase contrast

phase contrast DIC

HeLa cells

Phase Contrast

Differential Interference

Contrast (DIC)

all images: www.olympusmicro.com

Fluorescence Microscopy

• Imaging of live specimen possible

• Fast imaging (~ 1 sec / image) observation of activities in living cells

• Fluorescence microscopy allows

• Highly specific staining via antibodies

• Multi-color stainings e.g. colocalization studies

• Green Fluorescent Proteins (GFP): inherent highly specific dyes

Why Fluorescence Microscopy?

S 0

S 1

Absorption

Fluorescence λexcitation

λemission

Ener

gy

Jablonski diagram of a fluorescent molecule

Fluorescence

+ + - -

- -

- -

-

- -

- + +

+ + -

-

-

-

- -

- -

-

- - - + +

Franck-Condon principle

Organic Dyes e.g. Fluorescein isothiocyanate (FITC)

Fluorescent Probes

Fluorescent Proteins Green Fluorescent Protein (GFP)

inward facing side chains of Ser65, Tyr66 and Gly67

lead to fluorophore formation

Quantum Dots

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tatio

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ndpa

ss fi

lter

emis

sion

ban

dpas

s fil

ter

500 nm 600 nm 400 nm

emission spectrum

excitation spectrum

Green Fluorescent

Protein

camera

Fluorescence Microscopy

probes.invitrogen.com/resources/spectraviewer/

S 0

S 1

Absorption

Fluorescence λexcitation

λemission

Ener

gy

Cell stained with fluorescent-phalloidin, which binds actin filaments

Phase Contrast vs. Fluorescence Microscopy

A Modern Widefield Microscope Beam path of an inverted microscope 1 Intermediate image plane/phototube 2 Eyepiece 3 Intermediate image plane/front port 4 Intermediate image plane/base port 5 Switching beam path between base port/front port/vis. observation 6 Side port prisms 7 Tube lens 8 Analyzer 9 Reflector cube 10 Field diaphragm 11 Aperture stop 12 Filter slider 13 mercury vapor (HBO) or metal halide lamp 14 halogen lamp 15 Field diaphragm 16 Polarizer 17 Aperture stop 18 Condenser 19 Objective

1 2

3

4

5

6 7

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12

13 19

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Image Artifacts

Every step in the image generation process is prone to saturation effects which create artifacts Fluorescence excitation: - too high laser intensities in laser scanning microscopy

lead to excitation saturation which broadens the apparent focus size

Light detection process: - saturation of detector capacity (overflow) - negative offsets can accidently eliminate low signals Image processing: - „photoshopping“ such as excessive use of contrast and

brightness setting creates similar effects Print or data conversion process: - appearance often changes during conversion from RGB to

CMYK

Saturation

One or more stainings appear in the wrong channels. errors in colocalization measurements. use dyes which are spectrally better separated optimize your filters, laser powers and PMT voltages use sequential scanning use „spectral unmixing“ algorithms

Crosstalk

Embedding medium and thickness of coverslip matter Use suitable objective and coverslips Adjust correction collar of objective (if exists)!

from Pawley 2006

Refractive Index induced Spherical Aberrations

overlay

Different color channels appear shifted errors in colocalization measurements. Measure fluorescent bead in 2D or 3D

for calibration and correct in image post-processing

x

z

Chromatic Aberrations

What Limits Resolution?

Optical imperfections - manufacturing limitations - refractive index mismatch and heterogeneity of sample

microscopyu.com

Wikipedia

Aberrations a single lens cannot focus light perfectly for large angles, several colors and over a large fields of view combine multiple lenses

Diffraction fundamental limit