Introduction to Fluorescence Microscopy. Introduction to fluorescence microscopy Fluorescence Widefield Fluorescence microscopes Filters and Dichroics.

Introduction to Fluorescence Microscopy

Introduction to fluorescence microscopy

• Fluorescence• Widefield Fluorescence

microscopes• Filters and Dichroics• Objectives and abberation• CCD cameras

Natural Fluorescence

Transmittance is subtractive while fluorescence is additive

excitation

emission

Fluorescence Microscopy: basics of theory

• Absorbance spectrum limits excitation.

• Energy states limit excitation

• Molecule returns to lowest vibrational state emitting heat

• Light is emitted on return to ground state

Fluorescence spectra correlated to sise of conjugated ring

Multichannel fluorescence labelling• Direct coupling to macromolecules• Fluorescent dyes and substrates• Fluorescent fusion proteins• Fluorescent Antibodies

Ch2(Red)Texas Red anti-rabbit& Rabbit anti-Gal

Ch1(Green)UBI-GFP

Arterial edothelial cellCh1(Green) FITC TubulinCh2(Red) mitotrackerCh3(Blue) DAPI

Ch1 Ch2

Light path of a Epi-fluorescent Microscope

http://microscopy.fsu.edu/primer/techniques/fluorescence/anatomy/fluoromicroanatomy.html

Upright Scope

BrightfieldSource

Epi-illuminationSource

Image from Nikonpromotional materials

Inverted Microscope

BrightfieldSource

Epi-illuminationSource

Image from Nikonpromotional materials

Microscope for widefield epifluorescence

• Mercury Lamp• Dichroic Reflector• Objective• Sample Stage• DIC analyser• Dichroic Reflector• CCD camera

Mercury lamp spectra• http://www.mcb.arizona.edu/ipc/fret/

Standard Band Pass Filters

Transmitted LightWhite Light Source

625/50 nm BandPass Filter Texas Red

600 -650 nm Light

Standard Long Pass Filters

Transmitted LightLight Source500 nm Long Pass Filter

>520 nm Light

Transmitted LightLight Source575 nm Short Pass Filter

<575 nm Light

Standard Short Pass Filters

Optical Filters

Dichroic Filter/Mirror at 45 deg

Reflected light

Transmitted LightLight Source

510 LP dichroic Mirror

GFP Longpass Emission Set

D425/60x

470DCXR

E480LP

Dichroic for multi-colour samples

• The excitation (red/oarnge) and emission (blue) spectra for three common fluors (Alexa 488, 555 and 633)

• Requires a dichroic mirror with three bands of reflection and intervening windows of transmission

Dielectric filtercomponents

“glue”

Interference in Thin Films

• Small amounts of incident light are reflected at the interface between two material of different RI

• Thickness of the material will alter the constructive or destructive interference patterns - increasing or decreasing certain wavelengths

• Optical filters can thus be created that “interfere” with the normal transmission of light

Microscope for widefield epifluorescence

• Mercury Lamp• Dichroic Reflector• Objective• Sample Stage• DIC analyser• Dichroic Reflector• CCD camera

The objective determines the content of your image!

• The objective is critical to the efficiency of light collection (your signal)

• And determines the accuracy of the image (inaccuracy is called aberration).

• The objective determines resolution.

Objective markings

PLAN-APO-40X 1.30 N.A. Oil 160/0.22

Flat field Apochromat Magnification Numerical Tube Coverglass Factor

colouredrings

Aperture Length Thickness

- Infinity corrected

Immersion mediumOil (black ring)Water (W)Air (white ring)

A

NA=n(sin )

Light cone

(n=refractive index)

• Resolving power is directly related to numerical aperture.

• The higher the NA the greater the resolution

• Resolving power:The ability of an objective to resolve two

distinct lines very close together

NA = n sin

is 1/2 the angular aperture of the objective

Numerical Aperture

Limits of resolution result from interference

• Rayleigh limit for self-luminous objects

• Abbé limit for illuminated objecs

Microscope Objectives

Images from http://micro.magnet.fsu.edu/index.html

Microscope Objectives

SpecimenCoverslip

Oil

MicroscopeObjective

Stage

60x 1.4 NAPlanApo

Refractive IndexCellsWater 1.333glycerol 1.466Glass 1.52Zeiss Oil 1.515Diamond 2.42

Refractive Index

Objective

n=1.52

n = 1.52

n = 1.52

Specimen

Coverslip

Oil

n=1.33

n = 1.52

n = 1.0

n = 1.5

Water

n=1.52

Air

Spherical Aberration

• Peripheral ray focus is shorter than more central (paraxial) ray focus.

• Compromise is ‘Circle of Least Confusion’

Monochromatic Aberrations - Coma

Coma is when a streaking radial distortion occurs for object points away from the optical axis. It should be noted that most coma is experienced “off axis” and therefore, should be less of a problem in confocal systems.

1

23

Images reproduced from:

http://micro.magnet.fsu.edu/

Monochromatic Aberrations - Astigmatism

If a perfectly symmetrical image field is moved off axis, it becomes either radially or tangentially elongated.

Images reproduced from:

http://micro.magnet.fsu.edu/

Microscope for widefield epifluorescence

• Mercury Lamp• Dichroic Reflector• Objective• Sample Stage• DIC analyser• Dichroic Reflector• CCD camera

The CCD Camera

• Extended red sensitivity• 1344(H) X1024(V) pixels• 8.3 frames per second

(to 45 fps with 8X8 bin)

Scanning the CCD chip

Scanning the CCD chip

Scanning the CCD chip

http://www.dta.it/basic.htm

Binning 2X2 16fps4X4 28fps8x8 45fps

Full well capacity (18000 electrons)Bloom

Readout noise (6 electrons)

Dynamic Range (6/18000 = 3000)

Electron Multiplying Charge Coupled Device (EMCCD)

• Impact ionization leads to secondary electrons and amplification before readout

• Amplification can be as much as 1000x

• Readout noise is excluded but shot noise is amplified

Fluorescence Microscopy in the

Sussex Centre for Advanced Microscopy

Roger Phillips

Biols 2C9,10,11

Ext 7585

[email protected]