Laser Scanning Confocal Microscopy Practical session - PTBIOP-Homepage
Transcript of Laser Scanning Confocal Microscopy Practical session - PTBIOP-Homepage
Biology ImagingDr. Arne Seitz
Biomicroscopy II, 2009- 1 -
Laser Scanning Confocal Microscopy
Practical session
Biology ImagingDr. Arne Seitz
Biomicroscopy II, 2009- 2 -
Laser Scanning Confoal MicroscopyOutlook
• Light efficiency• Signal to Noise Ratio• Bleaching• Resolution
– Physical resolution– Nyquist Sampling– Optimal Pixel/Voxel Size
• Strategies for optimal imaging
Biology ImagingDr. Arne Seitz
Biomicroscopy II, 2009- 3 -
Light efficiencyPhotons emitted by a single fluorophore
- How many photons can one obtain from a typical fluorophore in confocal microscopy?
- How does can we influence the photon flux?
- Why is the photo flux important for (Laser scanning confocal) microscopy?
Biology ImagingDr. Arne Seitz
Biomicroscopy II, 2009- 4 -
Light efficiencyPhotons emitted by a single fluorophore
ba
a
tot kkk
SS
+=
][][ 1
ka = σ * I
kb = 1/τF τF =fluorescent lifetime
Photon output (steady state)= kb*[S1]
kbka
S0
S1
very fast
Biology ImagingDr. Arne Seitz
Biomicroscopy II, 2009- 5 -
Light efficiencyPhotons emitted by a single fluorophore
1mW @488 nm foccused to a radius of 0.25 µm (e.g. objective NA 1.25)I = 5.1 *105 W/cm2 = 1.25*1024 photons/(cm2*s)
Fluoresceine: ε = 80 000 l*mol-1*cm-1
Cross section/molecule = 3.06 * 10 -16 cm2/molecule
ka = σ * I = 3.8 * 108 s-1
kb = 1/τF
τF = 4.5 ns kb = 2.2*108 s-1
S1
kbka
S0
very fast
Biology ImagingDr. Arne Seitz
Biomicroscopy II, 2009- 6 -
Light efficiencyPhotons emitted by a single fluorophore
ba
a
tot kkk
SS
+=
][][ 1
0 2 4 6 8 100,0
0,2
0,4
0,6
0,8
1,0
0,0
0,2
0,4
0,6
0,8
1,0
S 1/S
tot
S
0/Sto
t
ka/kb
kbka
S0
S1
very fast
Biology ImagingDr. Arne Seitz
Biomicroscopy II, 2009- 7 -
Light efficiencyPhotons emitted by a single fluorophore
Photon output: Qe*kb*[S1] = 0.9 * 0.63 * 2.2*108 s-1 =1.3* 108 photons/s
512*512 pixel / s = 3.8 µs/pixel ~ 500 photons/pixel(for fluoresceine excited with 488 nm)
ka = σ * I = 3.8 * 108 s-1
kb = 1/τF
τF = 4.5 ns kb = 2.2*108 s-1
S1
kbka
S0
very fast
Biology ImagingDr. Arne Seitz
Biomicroscopy II, 2009- 8 -
Light efficiencyPhotons emitted by a single fluorophore
Single Fluorophore (Fluoresceine): 512*512 pixel / s = 3.8 µs/pixel ~ 500 photons/pixel
But losses due to- Geometry (fluorophore emitting in all directions)
-NA 1.4 40% efficiency-NA 0.3 5% efficiency
- Scattering- Optical elements
- Objective - mirrors, filters- pinhole
- Detector efficiency 10-30%
Each fluorescent molecule contributes on the order of only one photoelectron/pixel/sweep
Biology ImagingDr. Arne Seitz
Biomicroscopy II, 2009- 9 -
Signal to Noise RatioOverview
- What is-Signal-Noise-Background
-How do the above mentioned terms influence image quality
-How can we influence signal, noise and background
Biology ImagingDr. Arne Seitz
Biomicroscopy II, 2009- 10 -
Signal to Noise RatioDefinition
Signal Photons
Noise Fluctuation in the Signal (=photonflux)
Background Unwanted signal (=photons)
Biology ImagingDr. Arne Seitz
Biomicroscopy II, 2009- 11 -
11
Signal to Noise RatioMain sources of noise
Ntot – total noiseNd – detector noise Ns – signal (shot) noiseNph – number of photons
2 2 2tot s dN N N= +
• Shot noise prevails at high light level. It defines the fundamental limit of the noise.
• Detector noise prevails at low light level. • Statistics is applied to photons (electrons), but not to grey values.
phS NN =
Biology ImagingDr. Arne Seitz
Biomicroscopy II, 2009- 12 -
Signal to Noise RatioSignal-Noise-Background
Signal to Background constant
Signal to Noise increases
Contrast increases
Biology ImagingDr. Arne Seitz
Biomicroscopy II, 2009- 13 -
Signal to Noise Ratio
Dwell time: 50 µs Dwell time: 6 µs Dwell time: 1.6 µs
Biology ImagingDr. Arne Seitz
Biomicroscopy II, 2009- 14 -
Pinhole SizePinhole: 10 AU Pinhole: 5 AU Pinhole: 1 AU
Closing the pinhole increases the z-sectioning capabilities because light from out of focus planes is suppressed, but also decreases the SNR/contrast(less photons are detected even from the focal plane).
Biology ImagingDr. Arne Seitz
Biomicroscopy II, 2009- 15 -
Laser Scanning Confocal MicroscopyReduce Noise
Strategies to reduce image noise- reduce scan speed = increases pixel time- averaging
line averagingframe averagingintegrate
- increase laser intensity
Biology ImagingDr. Arne Seitz
Biomicroscopy II, 2009- 16 -
Laser Scanning Confoal MicroscopyBleaching
Excitation- Cone of light- Bleaching occurs above and below focal plane- z-sampling affects bleaching
Biology ImagingDr. Arne Seitz
Biomicroscopy II, 2009- 17 -
Laser Scanning Confoal MicroscopyBleaching mechanisms
Photon output: 1.3* 108 photons/s = 1.3* 108 cycles/s
High energy densityHigh cycle timeHigh bleaching probability
- Photochemistry of bleaching poorly understood. - Differs from fluorophor to fluorophor-Main Bleaching route: Triplet state
- Absorb another photon- Energy exchange with triplet oxygen, which becomes highly react ive singlet oxygen
S2
S0
S1τ ~ ns
T2
T1
τ ~ µs-s
Ene
rgy
Biology ImagingDr. Arne Seitz
Biomicroscopy II, 2009- 18 -
Laser Scanning Confoal MicroscopyBleaching
Parameters affecting bleaching probability- Fluorophore- Laser intensity- Pixel Dwell time- Sampling frequency
Bleaching (Photon)Noise
Biology ImagingDr. Arne Seitz
Biomicroscopy II, 2009- 19 -
Laser Scanning Confoal MicroscopyOutlook
• Resolution– Point Spread Function– Nyquist Sampling– Optical Slice thickness
Biology ImagingDr. Arne Seitz
Biomicroscopy II, 2009- 20 -
Laser Scanning Confoal MicroscopyPoint Spread Function
Biology ImagingDr. Arne Seitz
Biomicroscopy II, 2009- 21 -
Laser Scanning Confoal MicroscopyOptical Resolution limit
The optical resolution resel1 can be defined as the radius of the first dark fringe in the diffraction pattern, or half the diameter of the Airy disc.1 Robert H. Webb, Confocal optical microscopy, Rep. Prog. Phys. 59 (1996) 427-471
NAnresel λ
υλ ⋅
=⋅
⋅=
61.0sin61.0 wide-field
xy-plane
NAnresel λ
υλ ⋅
=⋅
⋅=
44.0sin44.0 confocal
xy-plane
2
5.1NA
nreselaxialλ⋅⋅
=confocalaxial
Example: λ=500 nm; n=1.518; NAObj =1.4wf xy-plane resel=217 nm conf. xy-plane resel=157 nm conf. axial resel=580 nm
Biology ImagingDr. Arne Seitz
Biomicroscopy II, 2009- 22 -
Laser Scanning Confoal MicroscopyDigital sampling
Biology ImagingDr. Arne Seitz
Biomicroscopy II, 2009- 23 -
Laser Scanning Confoal MicroscopyDigital sampling
Smallest resolvable structure must be sampeled (at least) twice.
Biology ImagingDr. Arne Seitz
Biomicroscopy II, 2009- 24 -
Laser Scanning Confoal MicroscopyOptical zooming
Biology ImagingDr. Arne Seitz
Biomicroscopy II, 2009- 25 -
Laser Scanning Confoal MicroscopyDigital zooming
Biology ImagingDr. Arne Seitz
Biomicroscopy II, 2009- 26 -
Laser Scanning Confoal MicroscopyResolution
• Physical Resolution– Parameters affecting resolution
• Wavelength• NA of objective• Refractive index
– Difference in lateral and axial resolution• Digital resolution (Sampling)
– Nyquist sampling– Optical zooming– Number of pixels
Biology ImagingDr. Arne Seitz
Biomicroscopy II, 2009- 27 -
Laser Scanning Confoal MicroscopyScanning conditions
Biology ImagingDr. Arne Seitz
Biomicroscopy II, 2009- 28 -
Laser Scanning Confoal MicroscopyScan Mode
Scan Mode– unidirectional/bidirectional– Sequential– Cross talk– Cross excitation
Biology ImagingDr. Arne Seitz
Biomicroscopy II, 2009- 29 -
Laser Scanning Confoal MicroscopyScan mode
Uni-directional
Bi-directional
Biology ImagingDr. Arne Seitz
Biomicroscopy II, 2009- 30 -
Laser Scanning Confoal MicroscopyScan mode
Resp
onse
Current / A
Galvanometer Hysteresis
Biology ImagingDr. Arne Seitz
Biomicroscopy II, 2009- 31 -
Laser Scanning Confoal MicroscopyScan mode
• Scan Mode– Galvanometric scanning mirrors
• Hysteresis– Unidirectional
• slower, more precise– Bidirectional
• Faster• Hysteresis compensation required
Biology ImagingDr. Arne Seitz
Biomicroscopy II, 2009- 32 -
Laser Scanning Confoal MicroscopyBleed-Through
Biology ImagingDr. Arne Seitz
Biomicroscopy II, 2009- 33 -
Laser Scanning Confocal MicroscopySummary
• Widefield microscopy– Detector efficiency : 60%-80%– Detector noise : 4 RMS electrons/pixel– Pixel Dwell time : 10-1000 ms– Opical sectioning : no– Backgrond supression : no
• Laser scanning confocal microscopy– Detector efficiency : 3%-12%– Detector Noise : < 0.01 RMS electrons/pixel– Pixel dwell time : 1-100 µs– Optical sectioning : yes– Background supression : yes