Post on 20-Dec-2015
Introduction to fluorescence microscopy
• Fluorescence• Widefield Fluorescence
microscopes• Filters and Dichroics• Objectives and abberation• CCD cameras
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
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
Microscope for widefield epifluorescence
• Mercury Lamp• Dichroic Reflector• Objective• Sample Stage• DIC analyser• Dichroic Reflector• CCD camera
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
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
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
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