Building Your Own 2-Photon Microscope: Challenges, Advantages and Limitations

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1 Building Your Own 2-Photon Microscope: Challenges, Advantages and Limitations Roberto Weigert, Ph.D. Chief, Intracellular Membrane Trafficking Section Oral and Pharngeal Cancer Branch NIDCR-NIH

2 Building Your Own 2-Photon Microscope:! Challenges, Advantages and Limitations! How did we manage to build a 2-photon microscope?! 12% Ethanol (Preferabl Italian)! put together! Caffeine (Strictl Italian Espresso) Being a pain in the neck.!

3 Building Your Own 2-Photon Microscope!! Turn Ke Sstem! Wh?! Build our own! More expensive! $400K-500K! $150K!! $150K Laser! 1) Budget! 1) Low-Start up package! 2) Maintenance! 2) Flexibilit! 3) Upgrades! Bu Confocal microscope! Convert to a 2-photon! Thorlabs! Labvision! First step (first major decision)! Which Platform?! Olmpus! 1) We copied the sstem built here! 2) Flexibilit! 3) Support!

4 Upright vs. Inverted! Upright! Flexibilit:!!!1) 2-photon!!2) Confocal!!!! All the organs! 1) Intravital imaging! 2) Live Cell imaging! Motion artifacts! 1) All the organs! Custom made holding device specificall designed for the organ of interest! w/o holding device! with holding device! Inverted! All the organs but the brain! Live Cell Imaging! Positioning and securing the organ to the coverslip!

5 Upright vs. Inverted! Upright! All the organs! Inverted! Inverted converted to upright! All the organs but the brain! Live Cell Imaging!

6 Upright vs. Inverted! 1) Optimized for visible light! 2) Increase the light path! 1) Model available with PMT on top! 3) Loss of power (5-10%)! 4) No effects on laser pulse width! 5) Requires extra stage! 6) Head can be rotated! 7) Adaptors for lenses! Emitted light (visible)! Excitation beam (IR)!

7 Upright vs. Inverted! Upright! All the organs! Upright converted to inverted!

8 Laser Laser combiner 488 nm 561 nm 633 nm UV laser 405 nm 1) Microscope Scanning unit Microscope Ti-sapphire lasers Tunable vs. single wavelength Laser High power lasers (3-4 W) Repetition rates: Hz Pulses: fs Beam diameter: 1.2 +/- 0.2 mm Tunable: nm

9 Laser output power! Ti-sapphire lasers! 1) Microscope! 2) Laser! Laser combiner!488 nm,561 nm,633 nm! UV laser 405 nm! High power lasers (3-4 W)! Scanning unit! Microscope!

10 Loss of power throughout the optics! (A)! Ti-sapphire lasers! Beam expander! (B)! Laser combiner!488 nm,561 nm,633 UV laser 405 nm! nm! High power lasers (3-4 W)! Dichroic mirrors! 1) Microscope! (C)! (D)! Scanning unit! Microscope! 2) Laser! (A) 800 nm 3520 mw (100%)! (B)! 3280 mw (93%)! (C)! 864 mw (25%)! (D)! 340 mw (10%) 60x N.A 1.2!

11 Control the power at the specimen! Ti-sapphire lasers! Beam expander! 3) Control output power! 1) Microscope! 2) Laser! Laser 488 nm,561 nm,633 combiner! UV laser 405 nm! nm! High power lasers (3-4 W)! Scanning unit! Microscope! 1) ND filters! a) Single! b) Carousel with multiple filters (8-10)! a) Manuall or software controlled! 2) ND continuous filter wheel!

12 Control the power at the specimen! Ti-sapphire lasers! Beam expander! 3) Control output power! 1) Microscope! 2) Laser! Laser 488 nm,561 nm,633 combiner! UV laser 405 nm! nm! High power lasers (3-4 W)! Scanning unit! Microscope! 1) ND filters! 2) ND continuous filter wheel! 3) AOM (Acousto-optic modulator)! 4) EOM (Electro-optic modulator)! a) Eas integration with the software! b) Size of the beam matching the aperture of the AOM! c) Significant pulse broadening (up to 600 fs)! a) Need for a pre-chirping sstem! d) Deflection of the beam! a) Not practical if different wavelengths are needed! b) Need for an automatic realignment set up (expensive)

13 (a)! Broadening of the pulse width! 4) Autocorrelator! 3) Control output power! (a)! Ti-sapphire lasers! Beam expander! Laser combiner!488 nm,561 nm,633 nm! UV laser 405 nm! Pulses: fs! (a) Measure the pulse at the source! (b)! Measure the pulse and the power at the specimen! 1) Microscope! Scanning unit! Microscope! (b)! 2) Laser!

14 Size of the laser beam! 5) Beam expander! 4) Autocorrelator! Ti-sapphire lasers! 3) Control output power! 1) Microscope! 2) Laser! Laser combiner! 488 nm,561 nm,633 nm! UV laser 405 nm! Scanning unit! Microscope! 1) Control the size of the beam! 2) Control the power at the specimen!

15 Filling the backaperture of the lens! Filling the back aperture of the lens! Underfilling! Filling! Essential for large lenses such as the 20X! Control the power at the specimen b overfilling!

16 Challenge: alignment of the beam! 5) Beam expander! 4) Autocorrelator! Ti-sapphire lasers! 3) Control output power! 1) Microscope! 2) Laser! Laser combiner! 488 nm,561 nm,633 nm! UV laser 405 nm! Scanning unit! Microscope!

17 Proper optics! 4) Autocorrelator! 5) Beam expander! Laser combiner!488 nm,561 nm,633 UV laser 405 nm! nm! 3) Control output power! 1) Microscope! 6) Optics! Ti-sapphire lasers! Scanning unit! 2) Laser! Microscope! 6) Excitation Dichroic mirror reflect above nm!

18 Laser combiner!488 nm,561 nm,633 nm! UV laser 405 nm! Non-descanned detectors! 4) Autocorrelator! 5) Beam expander! 3) Control output power! 1) Microscope! 6) Optics! Ti-sapphire lasers! Scanning unit! 2) Laser! Microscope! 7) Detectors! A) Descanned detectors! B) Non-descanned detector! Positioning!

19 Non-descanned detectors GaAS PMT- 1 GaAs detector PMT- 1 DM SDM 560 SDM 570 PMT- 3 PMT- 2 PMT- 3 BF680 SDM 500 BF 680 DM Cooled PMT from Hamamatzu R Gallium Arsenide PMT PMT- 2

20 Non-descanned detectors Objective inverter with PMT

21 5) Beam expander 4) Autocorrelator Laser combiner 488 nm,561 nm,633 nm UV laser 405 nm 3) Control output power 1) Microscope 6) Optics Ti-sapphire lasers 2) Laser Scanning unit CCD camera Microscope 7) Detectors Confocal microscop Two-photon microscop

22

23 A! Confocal! Two-photon! 0 µm! 0 µm! B! 60x Oil N.A. 1.4! z 15 µm! 30 µm! x 60x Water N.A. 1.2! z 60 µm! x 60 µm! 60 µm! x 30 µm! 120 µm! x 120 µm! Confocal! 0 µm! Two-photon! 0 µm! 25x WaterN.A. 1.05! z 45 µm! x 45 µm! z 125 µm! x 125 µm! 90 µm! x Confocal! 90 µm! 250 µm! x 250 µm!

24 D! 30x Silicon oil N.A. 1.05! z Two-photon! 50 µm! 100 µm! 0 µm! 150 µm! 300 µm! Confocal! Two-photon! 50 µm! x 150 µm! 100 µm! 300 µm!

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