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