Components of confocal and two-photon microscopes Internal training 07/04/2016 A. GRICHINE Platform Optical microscopy Cell imaging, IAB, ISdV
Plan Confocal laser scanning microscope o o o Principle Main components Lasers Acousto-optical tunable filters Scanner Detectors Signal optimization Rapid confocal microscopy o o o Resonant scanning Line scanning (LSM7 LIVE) Spinning disk (Andromeda) Two photon imaging o o o o o o o Pulsed lasers 2P absorption properties Group velocity dispersion compensation 2P Spectral imaging Excitation fingerprinting Emission spectroscopy Second harmonic generation imaging Non-descanned detection Advanced 2P applications
Principle of confocality
Principle of confocality Pinhole diaphragm Optical system Thick fluorescent sample
Components of CLSM
Components of CLSM Detection
Lasers Light Amplification by Stimulated Emission of Radiation http://www.phy.cuhk.edu.hk/phyworld/articles/laser/laser_e.html
Lasers in confocal microscopy Types: gas (Argon, Ar-Kr, Helium-Neon), solid state (diodes, DPSS) Monochromatic, coherent Intense (high irradiance: power/surface) Low divergence Life span and cost: high
Lamp versus laser light
Shortcomings of laser light Monochromatic Intense Narrow and mono-directional Coherent Polarized
IAB Lasers He-Ne 543 nm TRITC 633 nm Cy5 Ar 458 nm CFP 488 nm GFP 514 nm YFP DPSS 405 nm DAPI 561 nm mcherry 640 nm Alexa647 Ti:Sa 690 nm 1064 nm 2P
Laser light handling AOTF for wavelength- and power- selection Δt ~ μs T > 90 % LSM 510 ZEN Linear region LA
Laser light handling Collimator for expanding & co-focusing Diffraction limited spot Back focal aperture z Collimator misaligned
Laser light handling Pinhole for optical sectioning 3D Point Spread Function (PSF) y LSM 510 y x z x For Diam > 1AU : Diameter λ exc 488 nm NA 1.4 n 1.518 R x,y 0.51 λ eee NN 178 nm z R z 0.88 λ eee n n 2 NN 2 461 nm Pinhole of the Green channel is misaligned
Laser light handling Raster scanning Onedirectional bidirectional Linear region 2x faster, but needs for phase alignement
Signal (image) quality
Digitization From analog world to digital computing Intensity Time Space 1 Frame Nyquist criterion: 1 resel 2 pixels
Spatial sampling in CLSM Zoom or ROI Correct sampling depends on: Number of pixels 360-400 488-520 543-600 633-650 (dwell time) Electronic Zoom (scanner amplitude) Objective Numerical Aperture (Niquist criterion)
Confocal detectors PMT : PhotoMultiplier Tube ~ +1000 V 8 bits 12 bits
Confocal detectors Photodiode (Avalanche photodiode APD) Si Si Si Si B Si + Si Si Si + Si Si Si Si P Si Si Si Si p n - + E ext
Confocal detectors PMT APD HyD PDE Gain Dynamic range Noise 25% (37% GaAsP) ~ 50 000 12 16 bit High, gain dependent 45% ~ 1 000 8 bit (scan speed dependent) Very low http://www.leica-microsystems.com/science-lab/sensors-for-true-confocal-scanning/
Gain and Offset These parameters are most used to fill up the dynamic range of the detector: = maximum of gray levels for the intensity quantification GAIN OFFSET Increases amplification (PMT, EMCCD) Increases the signal intensity - number of grey levels Tends the bright pixels to saturation Global effect on brightness Make visible the faint signals Limits : linearity & noise Similar to threshold Eliminsate the background noise Faint structures become black (intensity = 0) Reduces globally the brightness Limit : risk of information loss
Signal and S/N optimization 1. Pinhole(s) alignment X-Y-Z 2. Pinhole opening (signal vs. optical section) 3. Number of pixels Zoom or ROI (resolution vs. speed & intensity) 4. Scan speed (= pixel dwell time) 5. Detector gain (noise vs. dynamics) 6. Laser intensity (bleaching vs. dynamics) 7. Amplifier offset (dynamics) 8. Line averaging (noise vs. speed)
Dichroic mirror / spectral selection
Rapid confocal imaging
Confocal / 2P point raster scanning Galvanometer scanner Laser Stop point Zone of linear scan Acceleration / deceleration Electronic zoom : amplitude of the scanner
Rapid (resonant) point scanning If 100 images 512x512 per second : Dwell time is only 0.038 µs / pixel!!! http://www.microscopyu.com
Rapid line-scan (slit-scan) 100 images 512x512 per second: Dwell time 19 µs/pixel Light sheet is shaped by special cylinder optics Optical Zoom : Spatial restriction of excitation light line Confocal «pinholes» are slit shaped
Spatial resolution in line-scan system J. Biomed. Opt. 11(6), December 26, 2006
LIVE7 confocal line-scan microscope + 512 pixels parallel excitation and detection 2 line CCD detectors (QE > 85%) Rapid Z-stack with piezo Variable confocal apertures (slits) 120 images/sec @ 512x512 1010 images/sec @ 512x50 3 lasers : 405 / 488 / 561 - Faster photobleaching Anisotropic X-Y resolution Loss of X-Z resolution Limited Optical zoom (x0.5-x2) Loss of signal intensity with zoom Field heterogeneity No 633 excitation laser Difficult transmitted light imaging 30
LIVE7 : configuration in ZEN Database : DUO Scan Odd laser names (more powerful) 2 x CCD No main dichroic, but AchroGate a «line» mirror
LIVE7 optical zoom and shading Zoom 0.5 Zoom 1 Zoom 2 Relative decrease of confocal resolution in X direction
Rapid transmitted light imaging with LIVE7 Filter BP 655-750
Confocal multipoint scanning Classic Spinning Disk configuration
Andromeda SD of imic (OBFR) Objective 63x/1.46 40x/1.4 Resolution xy 0.32 µm 0.4 µm Resolution z 0.68 µm 1 µm XY Z
Synchronisation of Spinning Disk & CCD 12 images / revolution 5000 rpm EMCCD ~ 30 images/s Pinhole size ~ 32 µm http://zeiss-campus.magnet.fsu.edu/tutorials/spinningdisk/synchronization/index.html
Andromeda SD of imic (OBFR)
Two photon excitation
Two photon absorption properties S 2 1. Non-linear process : S 1 S virt 10-15 s I fluo ~ Pw² 2. Limited in space: σ 2P, Pw S 0 1P 2P 3. Necessary irradiance (power density) : MW/cm² Compare to solar irradiance Earth surface : 0.14 W/cm 2 Sun surface : 5.9 kw/cm 2 4. Absorption spectrum is not equivalent to its 1P counterpart : λ 1P * 2 λ 2P
Continuous vs. pulsed lasers CW laser Pulsed laser
Pulsed excitation Pulse duration Frequency Illumination duration per second 200 fs 80 MHz 16 µs Mean Pw Surface Peak power density 1 mw 1 µm² 6.25 GW/cm²
2P spatial confinement of excitation λ exc 720 nm NA 1.4 n 1.518 R x,y 0.319 λ eee NN 0.91 R z 0.521 λ eee n n 2 NN 2 169 nm 403 nm (FWHM) Zipfel et al. Nonlinear magic: multiphoton microscopy in the biosciences. Nat Biotechnol. 2003 Nov;21(11):1369-77.
1P & 2P Absorption Spectra 100 80 60 40 20 0 488 350 400 450 500 553 100 80 60 40 20 0 350 400 450 500 550 600 976 1106 1 Göppert-Mayer (GM) = 10-50 cm -4 s/photon FITC ~ 40 GM Ti:Sa laser excitation range GFP, dsred ~ 100 GM
Group velocity dispersion (GVD) Femtosecond laser pulse Pulse propagation in glass chirp effect Wavelength, λ Time Red light travels longer optical path 4-prism optical arrangement for chirp precompensation
Ι Simultaneous emission 2P spectral imaging Single excitation egfp+yfp No time delay between channels egfp λ YFP!!! 1PAbsorption in NIR (e.g. Cy5, Alexa 647)
Spectral acquisition modes (LSM710) Simultaneous acquisition 34 channels (9.3 nm) Wavelength scan (3 nm) 400 750 nm PMT Diffraction grating Fluorescence
2P spectral imaging Excitation Fingerprinting Ι λ Applications : FRET, spectral analysis, colocalization
Second harmonic generation imaging (SHG) P ~ χ 1 E ω + χ 2 E 2 ω + χ 3 E 3 ω + cos 2 ω = 1 2 (1 + cos (2ω) E 2ω ~P 2 2ω = χ 2 E 2 ω SHG conditions : Intense coherent light Ordered medium (phase matching) Non-centrosymmetric molecules
Non-Descanned Detection (NDD) + Non ballistic photon detection - Stray light, reflections, HBO risk, detector scanning Applications : deep tissue imaging High sensitivity detection
Advanced 2P applications Photoablation Fluorescence lifetime imaging, FLIM 2.1 ns 1000 Fluorescence Decay Curves 100 1.6 ns 10 0 2 4 6 8 10 Time, ns Eq 10 DyOGen 1.1 ns DNA damage 3D photoactivation / Photoconversion