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Multiphoton Microscopy

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Multiphoton Microscopy Living up to Life Multiphoton Microscopy 1 Jablonski Diagram: Living up to Life Nonlinear Optical Microscopy F.- Helmchen, W. Denk, Deep tissue two-photon microscopy, Nat. Methods 2, 932-940 2 Typical Samples – Living up to Life Small Dimensions & Highly Scattering • Somata 10-30 µm • Dendrites 1-5 µm • Spines ~0.5 µm • Axons 1-2 µm ls ~ 50-100 µm (@ 630 nm) ls ~ 200 µm (@ 800 nm) T. Nevian Institute of Physiology University of Bern, Switzerland F.- Helmchen, W. Denk Deep tissue two-photon microscopy. Nat. Methods 2, 932-940 3 Why Multiphoton microscopy? Living up to Life • Today main challenge: To go deeper into samples for improved studies of cells, organs or tissues, live animals Less photodamage, i.e. less bleaching and phototoxicity • Why is it possible? Due to the reduced absorption and scattering of the excitation light 4 The depth limit Living up to Life • Achievable depth: ~ 300 – 600 µm • Maximum imaging depth depends on: – Available laser power – Scattering mean-free-path – Tissue properties • Density properties • Microvasculature organization • Cell-body arrangement • Collagen / myelin content – Specimen age – Collection efficiency Acute mouse brain sections containing YFP neurons,maximum projection, Z stack: 233 m Courtesy: Dr Feng Zhang, Deisseroth laboratory, Stanford University, USA Page 5 What is Two‐Photon Microscopy? Living up to Life A 3-dimensional imaging technique in which 2 photons are used to excite fluorescence emission exciting photon emitted photon S1 Simultaneous absorption of 2 longer wavelength photons to excite a fluorochrome, emission as with 1-photon S0 1-photonexcitation 2-photon excitation 6 2-photon: excitation probability Living up to Life - importance of high NA na: probability of excitation : excitation cross section 2 Pavg: average power incident light 2 2 (peak power) Pavg NA na 2 : pulsewidth f hc : repetition rate NA: Numerical aperture h: Planck‘s constant c: Speed of light : Wavelength MP excitation is favoured when we have: • Molecules with large cross-section • High peak power • High-NA objective lenses 7 Two photon microscopy Living up to Life fluorescence yield –non‐linear process 2 Fl = fluorescence photons/sec Pavg P = average laser power Fl = pulse width fs/psec T f f = laser repetition rate Efficiency of excitation increases with the square of the laser power Living up to Life What is Two‐Photon Microscopy ? 1-Photon 2-Photon label is excited only at the focus of the beam where probability of 2P event is highest No out-of-focus-fluorescence: - No need of confocal aperture - Dye bleaching and photo toxicity limited to the plane of focus 9 Living up to Life Confocal vs. Multiphoton microscopy Two-photon optical probe interacts with the sample only in the focus region. continuous Pulse 2-photone and 1-photon excitation at the same time In dye solution 10 Optical resolution: PSF - Confocal vs. 2-Photon Living up to Life Resolution in 2-Photon Microscopy is ~ 2x worse compared to Confocal Microscopy Confocal Lateral Axial NA=1,4 Wavelength resolution resolution (nm) n= 1,51 (x-y, µm) (x-z, µm) Confocal 488 0,16 0,52 2-Photon 2-photon 900 0,29 0,97 Calculated PSFs taken from „Confocal and Two Photon Microscopy“, ed. Alberto Diaspro, 2002 11 Optical resolution of Two Photons imaging Living up to Life Geometry of MP illumination spot confocal microscopy 2P microscopy Page 12 Optical resolution: Conventional vs. Confocal Living up to Life Conventional Microscopy Confocal Microscopy . Wavelength of light . Pinhole size . Numerical aperture . Geometry of the probing beam spot 0,46 1,4 n 0,61 r r rxy xy z 2 NA NA NA NA = nsinα n = 1 for air n = 1.518 for oil immersion Page 13 Optical resolution: Confocal vs. Two photons Living up to Life Confocal Microscopy: 0,46 1,4 n rxy rz NA NA2 Example: NA=1,4 Wavelengt Lateral Axial resolution resolution h (nm) n= 1,51 (x-y, µm) (x-z, µm) Confocal 488 0,16 0,52 2-photon 900 0,29 0,97 In two photon microscopy, lower resolution due to longer wavelength Page 14 TPE volumes: wavelength & NA dependence Living up to Life Rubart , M., Two-Photon Microscopy of Cells and Tissue, Circ. Res. 2004;95;1154-1166 Page 15 Comparison of penetration: UV –IR Living up to Life (internal detectors) Eye of zebrafish larvae (stained with DAPI) Image size (xz): 125 m x 125 m - Objective: 63x 1.2 Water - Detection range: 400nm – 500nm Exc.: UV PMT: 300V Exc.: IR PMT: 360V 16 Living up to Life 2-Photon excitation probability 2-Photon excitation is a very very rare event! In bright day light a good one- or two-photon absorber absorbs in a 1-photon process: once a second in a 2-photon process: every 10 million years Solution: Use of laser sources – focused beam The probability of a molecule to absorb 2 photons simultaneously is expressed as the 2-photon cross section 17 Living up to Life Multiphoton Microscope setup 18 CW and Pulsed Lasers Living up to Life P CW 10W Mean Power 1W t P Pulsed 100kW Pulse repetition rate f t ( 80 MHz) in range of 12.5nsec psec/fsec fluorescence life time 10 -8 s few nsec 80MHz Short Pulse Advantage Fluorescence proportional to Pulse duration 1/pulse width x repetition rate 2-3nsecs (width) 10 –13 s (fs/ps) 19 Continuous lasers vs. pulsed lasers Living up to Life Pulsed mode Continuous mode Two photon excitation in Lucifer yellow at 750nm wavelength (same power) Courtesy: Magendie institute, PICIN, P. Legros, Bordeaux, France 3 conditions necessary for 2P excitation - High intensity Hence in focal point - High cross section Not always double 1P - Short pulses: Pico or Femto second laser Page 20 Typical Tuning Curve IR Laser Living up to Life Coherent Chameleon Vision II Spectra Physics MaiTai DeepSee 21 Ultra Short Pulses Living up to Life 80 MHz repetition rate FWHM 100 fs – time FWHM 10 – 15 nm Short pulses of monochromatic light have colors! - Spectral components Nat. Biotech. Vol 21 No 11, 2003, Zipfel et al. Nonlinear magic: multiphoton microscopy in the biosciences 22 Continuous or pulsed laser Living up to Life Notion of fluorescence emission rate and cross section One photon of fluorescence is generated by 2 incident photons With continuous laser the fluorescence emission rate (f) is proportional to the square of intensity f : photons/seconde ² : Intensity of laser f = ½ ² : cross section in GM (Göppert-Mayer) 1 GM= 10-50 cm4/photons The two photon cross section is the probability of a molecule to absorb 2 photons simultaneously (cross section) is dependent on the wavelength and in general between 1 and 100 GM GFP = 10 GM Qdots = 104 –105 GM Page 23 Continuous or pulsed laser Living up to Life With pulsed laser, the fluorescence emision rate is described as follows: t : Pulse duration (s) F : Frequency (Hz) 1 fm = ½ (t.F)- m² fm : Average fluorescence emission rate m² : Average intensity t. : Intensity per pulse (peak power) : cross section in GM (Göppert-Mayer) Because m = t.F. Examples t = 10-13 s F = 108 Hz (t.F)-1 = 105 To compare (t.F)-1 = 1 for continuous laser So for equal power we have 105 time more excitation of fluorophore with pulsed laser Page 24 Living up to Life Group Delay Dispersion (GDD) Electrical field Positive Dispersion GDD Intensity (Intensity)2 = 2PE efficiency in 100 fs @ 690nm ~700 fs @ 800nm ~500 fs @ 1020nm ~300 fs Amended, from Pawley, Chapter 28 Denk et al., 25 Multi-Photon Excitation in Laser Scanning Microscopy Living up to Life Principle of Precompensation blue red Laser Microscop Sample e 100 fs 400 fs +GDD Group Delay Dispersion: GDD red blue Laser Pre-Chirp Mircroscope Sample 100 fs 400 fs 100 fs -GDD +GDD 26 Performance comparison Living up to Life Dispersion Compensation –On / Off Pre-Chirp Off Pre-Chirp On Objective lens: 20 x 1,0 Sample: Brain Slice, GFP @ 920 nm Mean intensity approx. 2,5 x higher Chameleon Vision 27 Living up to Life MP‐imaging for highly scattered tissue Confocal Microscopy: • pinhole aperture rejects the out of focus fluorescence but also scattered light difficult to image highly light scattering tissue like thick brain slices Multi-photon Microscopy: • no confocal pinhole required because all fluorescent light originates from the focal spot; detectors can be placed as close as possible to the specimen (NDDs) • enables also scattered photons to be collected • much higher photon collection efficiency compared to confocal microscopy Large area detector 28 Living up to Life Detection Path • Descanned pathway can be used – but clipping at pinhole • Strategy: collect as many photons as possible → i.e. if descanned detection: open pinhole completely! • Non-Descanned-Detection: – Large-area detectors (predominantly PMTs) – epi-detection – trans-collection – high-NA Condensor, prefferably oil (!) 29 Living up to Life Photon Collection Efficiency ‐ Internal vs. NDDs Mouse brain slice: ~ 20 µm (center plane) Detection range: 500 – 550 nm PMT: 950 V Objective: 20 x 1.0 W internal RLD TLD Mean intensity: 20 52 58 30 Living up to Life Fluorochromes: TPE ‐ Overview 1 Bestvater et el. Two-photon fluorescence absorption and emission spectra of dyes relevant for cell imaging Journal of Microscopy, Vol. 208, Pt 2 November 2002, pp. 108–115 31 Living up to Life TPE cross‐sections of various fluorochromes Bestvater et el. Two-photon fluorescence absorption and emission spectra of dyes relevant for cell imaging Journal of Microscopy, Vol. 208, Pt 2 November 2002, pp. 108–115 32 Living up to Life Fluorochromes: TPE ‐ Overview 2 Svoboda & Ryohei Principles of Two-Photon Excitation Microscopy and its Applications to Neuroscience Neuron 50, 823 – 839, June 15, 2006 33 Living up to Life TP cross‐section of standard FPs Blab et al., 2001 Two-Photon Excitation Cross-Sections of the Autofluorescent Proteins.
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