Confocal Microscopy. (Increasing contrast and resolu6on using op6cal sec6oning) Lecture 7. November 2017
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1 Confocal Microscopy (Increasing contrast and resolu6on using op6cal sec6oning) Lecture 7 November 2017
2
3 3 Flavours of Microscope Confocal Laser Scanning Problem: Out of Focus Light Spinning disc 2-Photon
4 A short History of Confocal Microscope Confocal concept patented by Marvin Minsky in 1957 Eggar and Petran developed spinning disc confocal in late 1960s Brakenhoff, Stelzer developed stage scanning confocal in late 1970 White, Amos and Wilson developed the MRC500 point scanning confocal -Marketed commercially in 1987
5 Comparison Widefield Vs Confocal Widefield Confocal Out of focus light blurs image Out of focus light is blocked
6 Principle of Confocal Microscopes Pinhole Pinhole diaphragm in the Conjugated focal plane = CONFOCAL in focus light (from the op6cal sec6on) passes through the pinhole and into the detector
7 Pinhole blocks out-of-focus light light from below the op6cal sec6on crosses infront of the pinhole and doesn t pass through the pinhole aperture
8 Pinhole blocks out-of-focus light light from above the op6cal sec6on also doesn t pass through the pinhole aperture
9 Confocal Microscopes Confocal Laser Scanning Spinning disc
10 Laser Scanning Confocal Laser Scanning Confocals are great to get preay images
11 Laser Scanning Confocal
12 Laser Light Source laser light source
13 Laser Light Source Laser Emission Spectra enables 6ghter control of fluorophores excited
14 AOTF Acousto-Op6c Tunable Filter AOTF
15 THEORY AOTF Acousto-Op6c Tunable Filter acousto-op6c effect: Acous6c wave excited within the quartz gives rise to varia6ons in the refrac6ve index The wavelength of the diffracted light is dependent on the acous6c frequency in the quartz. By tuning the frequency of the acous6c wave, the desired wavelength of the op6cal wave can be diffracted acoustoop6cally.
16 AOTF Acousto-Op6c Tunable Filter Quick On/Off of lasers Very fast changes between excita6on wavelengths
17 Galvo Scanning Mirrors Galvo Scanning Mirrors
18 Galvo Scanning Mirrors Sample excited at one point at a 6me Rela6vely slow
19 Adjustable Pinhole AOTF pinhole
20 Pinhole Op6cal Sec6oning THEORY Shorter the wavelength the thinner the op6cal sec6on Diameter of the pinhole: Smaller pinhole thinner op6cal sec6on FWHM=Full Width Half-Maximum The higher the NA. the thinner the sec6on Weak signal > open pinhole > more light but thicker sec6on
21 Op6cal sec6on Confocal enables 3D reconstruc6on
22 Confocal enables 3D reconstruc6on Adult Drosophila head (C. Rezeval Goodwin Lab)
23 Variable Detector Slit variable detector slit
24 Spectral Unmixing Defrac6on gra6ng separates wavelengths over physical area Light emiaed from fluorophore as a spectrum Variable slit lets through only certain wavelengths
25 Spectral Unmixing At each pixel: summed spectrum summed spectrum = + egfp (50%) auto-fluorescence (50%) = + egfp (75%) auto-fluorescence (25%) Match the summed spectrum with all possible summed combina6ons from a library At each pixel you therefore know the propor6on of each fluorophore present
26 Spectral Unmixing removal of autofluorescence At each pixel: Calculate the propor6on of the pixel is due to autofluorescence. Subtract the autofluorescence from the true GFP value.
27 PMT Photon Mul6plier Tube PMT detectors
28 PMT Photon Mul6plier Tube Very Low Noise Huge Signal Amplifica6on (~1x10)
29 insect autofluorescence
30 Airy-Scan technology
31 THEORY Airy-Scan technology Small Pinhole, signal loss but resolution gain..
32 THEORY Airy-Scan technology let through all the emitted light capture 0.2AU on each detector
33 THEORY Airy-Scan technology point of light scanned with 1AU standard detector
34 THEORY Airy-Scan technology point of light scanned with 0.2AU Airyscan detector >increased resolution
35 THEORY Airy-Scan technology each 0.2AU Airyscan detector provides >increased resolution
36 THEORY Airy-Scan technology each 0.2AU Airyscan detector info is reassigned and summed
37 THEORY Airy-Scan technology effective PSF is now smaller.. > increased resolution (1.4x - 1.7x)
38 kinetochores (James Banecror, Gruneberg Lab)
39 bleed-through Absorp6on spectral profiles Absorp Emission spectral profiles Excite at 477nm overlapping emission
40 minimising bleed-through Variable Slits Absorp6on spectral profiles Absorp Emission spectral profiles
41 minimising bleed-through Sequen6al Scanning Absorp6on spectral profiles Absorp Emission spectral profiles Excite at 477nm Excite at 514nm Temporal separa6on
42 minimising bleed-through Adjust detector slit widths Use sequen6al scanning
43
44 Confocal Microscopes Confocal Laser Scanning Spinning disc Both are confocals
45 Spinning Disc Confocal Great for live cell imaging Can collect many images per second
46 Yokogawa CSU-X1 Micro lens Array Nipkow Disk Nipkow Disk To CCD camera Sample
47 Yokogawa CSU-X1 Micro lens Array
48 Yokogawa Spinning Disc Confocal just a pinhole array Op6mised for cofocality and crosstalk too much light is blocked from reaching the specimen Only 4% light passes through disc
49 Yokogawa Spinning Disc Confocal micro-lens array increase the light reaching the specimen Typically 56% light passes through disc
50 Yokogawa CSU-X1 Nipkow Disk Sample
51 The Nipkow Disk Paul Nipkow, 1884 Eggar and Petran, 1967 Approx pinholes Single frame created with each 30-degree of rota6on of disc (12 frames per rota6on)
52 The Nipkow Disk Larger pinholes - brighter image, but less confocal Pinholes fixed size: Typically = 50um (op6mised for biology)
53 The Nipkow Disk Constant Baale: Smaller spacing - more light gets through, but crosstalk Pinhole Spacing Typically = 2.5um apart
54 Yokogawa
55 Cell division in brain stem cells (neuroblasts), Raff Lab
56 MT binding protein in Drosophila embryo, Raff Lab
57 Point Scanning Vs Spinning Disc Point Scanning Spinning Disc Speed Slow (secs) Fast (msecs) Sensi6vity OK OK Flexibility Good Poor Bleaching Poor Good Preay Pictures Unbeatable! Preay damn good! Preay Movies Good if process slow Unbeatable!
58 3 Flavours of Microscope Confocal Laser Scanning Problem: Out of Focus Light Spinning disc 2-Photon
59 2-photon Microscope Not a confocal for imaging deeper into thick specimens less damaging to biological samples
60 Confocal Vs 2-photon 1 Photon Excita6on 2 Photon Excita6on There is no out of focus light
61 1 Photon Excita6on THEORY high energy state lowest singlet excited state excita6on energy absorp6on energy loss fluorescence ground state
62 2 Photon Excita6on THEORY Almost simultaneous high energy state 2 nd low energy (IR~700nm) pulsed excita6on Low energy (IR ~700nm) Pulsed excita6on energy absorp6on energy loss lowest singlet excited state fluorescence ground state
63 Principle of 2-photon Microscope Near simultaneous, two photon event highly unlikely, only really possible a focal point Tightly focused excita6on
64 2-photon Microscope Pulsed excita6on laser is then scanned across the sample. Longer wavelengths are scaaered to a lesser degree than shorter ones, and penetrate deeper into the sample. In addi6on, these lower-energy photons are less likely to cause damage outside the focal volume.
65 Spindle forma6on in mouse ooctye, labelled with Hoechst, Alexa 680. M Schuh. EMBL, Heidelberg, Germany
66 3 Flavours of Microscope Confocal Laser Scanning Problem: Out of Focus Light Spinning disc 2-Photon
67 hap://
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