A VERYbrief history of the confocal microscope 1950s

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Mary Bridges
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1 Confocal Microscopy

2 Confocal Microscopy Why do we use confocal microscopy? A brief history of the confocal microscope Advantages/disadvantages of a confocal microscope Types of confocal microscopes The parts of a confocal Illumination Lasers and other light sources Regulating the laser, the AOTF etc. Detectors PMTs Others Multi-point detectors used for confocal microscopy Imaging using the confocal Sampling and photobleaching Spectral unmixing

confocal microscopes The parts of a confocal Illumination Lasers and other light sources Regulating the

4 A VERYbrief history of the confocal microscope 1950s 1960s 1970s 1980s The basic concept of confocal microscopy was originally developed by Marvin Minsky(patented in 1957). M. David Egger and MojmirPetranfabricated a multiple-beam confocal microscope that utilized a spinning (Nipkow) disk. Egger went on to develop the first mechanically scanned confocal laser microscope, and published the first recognizable images of cells in G. Fred Brakenhoff developed a scanning confocal microscope in Tony Wilson, Brad Amos, and John White nurtured the concept and later (during the late 1980s) demonstrated the utility of confocal imaging in the examination of fluorescent biological specimens. The first commercial confocal microscopes appeared in Olympus

Egger went on to develop the first mechanically scanned confocal laser microscope, and published the first recognizable images of cells in 1973. G.

5 1987 -the Bio-RadMRC500 LSCM

6 2014 The Zeiss LSM 780

7 Comparing wide-field and confocal microscopes Leica

8 Confocal Imaging It s all about the size of your pinhole Rutgers University 2012

9 The pinhole. PMT Out of focus light rays pinhole In focus light rays objective lens focal plane

10 The pinhole. Pawley Handbook of Biological Confocal Microscopy

11 Confocal pinhole and spatial resolution 100% 0% Signal intensity Half Intensity Width BPPR 250nm 500nm 1000nm SVI

12 Confocal Imaging Olympus

13 Wide field optical sections

14 Wide field optical sections

15 PSFs 200nm 100nm Olympus

16 Pros and Cons of confocal microscopy Resolution X-Yresolution approaches diffraction limit of nm Z resolution usually nm Images become more distortedas you move away from the coverslip Sensitivity PMT (orgasp) detectors are VERY sensitive - with precise calibration can actually count photons Canvary dwell time or scan speed. Can sample a single point for extended time / average multiple samples Tightly-focused highintensity laser light can cause significant bleaching of fluorescent molecules Rapid Optical Sectioning Can re-construct a 3- dimensional imageof a sample. Does not require extensive computer post-processing Simultaneous detection of multiple labels Increasedspeed can independently set gain/intensity of each channel Only limited by number of laser lines and number of detectors Signal overlap/bleed through can be a significant problem.

Can sample a single point for extended time / average multiple samples Tightly-focused highintensity laser light can cause significant bleaching of fluorescent molecules Rapid Optical Sectioning Can

17 Limitations Resolution Objects smaller than nm CAN NOT be resolved using a traditional confocal Objectsless than resolution limit may be falsely identified as co-localized if less than 200nm apart Pinhole settings are veryimportant. Improper settings can cause significant artefacts. Laser illumination Tightly-focused highintensity laser light can cause significant bleaching of fluorescent molecules You are limited by the available laser wavelengths. Limited number of reporter molecules Signal overlap/bleed through can be a significant problem. Getting fluorescent reporters into cells can be difficult Getting fluorescent reporters into living cells can be even more difficult

Laser illumination Tightly-focused highintensity laser light can cause significant bleaching of fluorescent molecules You are limited by the available laser wavelengths.

18 The main types of confocal microscopes Laser Scanning Confocal Microscope (LSCM) Multi-photon microscope NipkowdiskConfocal Microscope (Spinning Disk) Other types of Confocal Microscopes Zeiss 700/710/780 Leica SPE/SP8 Olympus FV1000 Nikon A1,C2+ Images are created from scanning a finely focused spotof laser light across a sample. Uses an adjustable pinhole to remove out of focus light Uses a high wavelength (+650nm) pulsed laser to scan across a series of points in the sample. The laser only has sufficient power to excite flourophoreat the focal plane NO pinhole needed Usesa disk with multiple (fixed) pinholes and microlensesthat spins and illuminates and images multiple points of a sample simultaneously. Slit-scanners Resonant Scanners Live-scanners (array detectors) And many more

Uses an adjustable pinhole to remove out of focus light Uses a high wavelength (+650nm) pulsed laser to scan across a series of points in the sample.

19 Types of Confocal Microscopes Nikon

20 The Basic Confocal Microscope Cooper and Hausman Figure 1.26 Olympus

21 Yokogawa CSU practical spinning disc confocal microscopes Minsky s design revisited Zeiss

22 Demystifying the various parts of a confcal microscope Detectors PMTs The pinhole! Illumination Lasers AOTFs/AOBMs

23 Illumination

24 Lasers Continuous Wave Lasers Gas lasers (Ar, ArKr, HeCd, HeNe ) Solid state laser Laser-pumped laser (Ti-sapphire) Laser diode pumped laser (DPSS) Semiconductor lasers Ultra-fast lasers: (pulsed) multiphoton Mode-locked Ti-sapphire ( nm) Mode-locked Cr-Frosterite ( nm) Colliding Pulse Mode locked Dye Laser

27 Lasers Olympus

28 Gas Lasers

29 Solid State Lasers

30 Common lasers you will find attached to a confocal microscope. PRABHAT and RDOGAN (2010) FLUORESCENCE IMAGING: Optical filters optimize laser-based fluorescence imaging systems

31 The acousto-opticaltuneable filter (AOTF) Olympus

32 Olympus

33 Detectors PMTs and more

34 The photomultiplier tube (PMT) Hamamatsu

35 LiveWeb2 The photomultiplier tube (PMT) side-on PMT Best PMTs reach 30% efficiency... Side on PMT more sensitive (quantum efficiencies) But better electron gain in end-on PMTs due to more dynodes (14 vs 9)

36 Slide 37 LiveWeb2 LiveWeb,

37 The photomultiplier tube (PMT) The effect of Gain and Offset Olympus / Claxton et ai. Laser Scanning Confocal Microscopy

38 GaAsPs Gallium arsenide phosphide photocathode (semiconductor material) 40% Quantum Efficiency (QE)(+20%) GaAsP is used also in LED (red, organge, yellow) Maximum iameter of cathode in PMT 5mm due to manufacturing, larger diameter possible for Hybrid Photodetectors HPDs Zeiss / Leica

39 Avalanche Photodiodes (APDs) Olympus

40 Hybrid (HyD) detectors Combine aspects of PMTs and APDs Photon Vacuum Tube APD

41 A comparison of the various confocal detectors PMT PMT GaAsP APD/SPAD HyD Photon Detection Efficiency (QE) (500 nm) % Pulse noise % 60 % 60 % 5 % 3 % Dark Current #/s 15,000 15, (*) 2,500 max M #/s very low very low Area/mm Leica

42 Non-PMT based confocals.. EMCCD camera Andor

43 CCD cameras as a confocal detector Zeiss/Hamamatsu

44 CCD cameras as a confocal detector Zeiss/Hamamatsu

45 How are CCD/CMOS cameras as confocal detectors?

46 Imaging using a confocal microscope (what do all those buttons on the screen actually do?

47 Confocal microscopes allow for 3-dimensional reconstruction of an sample (as long as it is not too thick) Nikon

48 Acquisition of properly spaced Z-stacks allows 3D reconstruction

49 X-Y sampling criteria for confocals

50 Sampling and resolution

51 Resolution (a.k.a. image size) Lower resolution (256x256) FASTER! Artifactual co-localization Higher resolution (1024x1024) SLOWER! Slightly more photobleaching Diffraction limited

52 Sampling and resolution

53 Over/Under Sampling Under-sampled (Severely) Properly sampled Over-sampled

54 Z-sampling sources of aberration

55 Scan speed (a.k.a. dwell time) Higher speed (dwell time) FASTER! Slower speed SLOWER! More photo-bleaching

56 Laser power Higher power (too bright!) BRIGHTER! More photo-bleaching Lower power (just right) More photo-bleaching Can be too low

57 Gain (a.k.a. PMT sensitivity) Higher gain BRIGHTER! Artifactual co-localization Lower gain

58 Bit (greyscale) depth What does the 8, 12, 16 bit button actually do? # of pixels # of pixels # of pixels # of grey levels

PMT sensitivity (gain) set too low Laser power too low Detector

59 Setting the gain properly How to avoid clipping PMT sensitivity (gain) set appropriately Lost! PMT sensitivity (gain) set too low Laser power too low Detector range Lost! PMT sensitivity (gain) set too high Take the images correctly - you can always increase brightness later

60 Avoiding signalbleed-through Olympus

61 Avoiding signalbleed-through Olympus

62 Avoiding signalbleed-through Olympus

63 When you simply can tavoid bleedthrough Zeiss

64 Spectral Imaging and linear unmixing. Zeiss

65 Spectral Imaging and linear unmixing. Zeiss

66 Spectral Imaging and linear unmixing. Zeiss

67 Spectral Imaging and linear unmixing. Zeiss

68 The confocal conflict.. (The triangleof disappointment) Resolution Speed Brightness Slower speed Brighter image PMTs can collect more photons Why can t I just scan simultaneously? You can. as long as you take into account bleedthroughbetween the channels Higher resolution Takes longer to scan Can bleach samples Nyquist sampling (2.3) is best Brightness Why not just turn up the laser power? Sample bleaching Only get one chance to get image Saturated PMTs lose resolution FHWM separation is lost

CONFOCAL LASER SCANNING MICROSCOPY TUTORIAL

CONFOCAL LASER SCANNING MICROSCOPY TUTORIAL Robert Bagnell 2006 This tutorial covers the following CLSM topics: 1) What is the optical principal behind CLSM? 2) What is the spatial resolution in X, Y,