1. 1 Confocal Microscopy David Kelly November 2013 Handbook of Biological Confocal Microscopy. Ed. J. Pawley, Plenum Press Fundamentals of Light Microscopy and Electronic Imaging. D. B. Murphy, Wiley-Liss Inc. Confocal design: CLSM microscope Pinhole Optical Sectioning Spinning Disk Confocal Photon Multiplier Tube CCD Confocal principles: Scan speed Optical resolution Pinhole adjustment Digitisation: sampling as opposed to imaging xy sampling: pixel size and zoom choices Photomultiplier tubes, noise, digitisation of intensity Multichannel imaging, crosstalk Colour Look Up Tables Recap of principal factors affecting image quality Imaging Thick Specimens Multiphoton Microscopy

2. 2 What to get out of this lecture Have an understanding of how a modern confocal microscope works Become familiar with the principal factors affecting image quality in the CLSM Begin to have an idea when and how to manipulate these factors for your purposes This often means knowing when and where to make compromises (e.g. light collection versus spatial resolution)

3. 3 Benefits of Confocal Microscopy Reduced blurring of the image from light scattering Increased effective resolution Improved signal to noise ratio Clear examination of thick specimens Z-axis scanning Depth perception in Z-sectioned images Magnification can be adjusted electronically

4. 4 Confocal Design

5. 5 CLSM microscope antivibration table

6. 6 Confocal principle

7. 7 The Pinhole z x y x Conjugate plane y

8. 8 Pinhole The Pinhole

9. 9 Optical sectioning 1  m

10. 10 Laser scanning Photomultiplier tube Computer

11. 11 Laser scanning xz scanning x y z x y z x y x y z z seriessingle section xy scanning x y x z

12. 12 Confocal Principles

13. 13 Scan speed: t resolution On modern confocals this is measured in Hz usually from 1-1400Hz Decreasing scan speed- more light collected (dwell time increased) more chance of photobleaching and phototoxicity limits temporal resolution Increasing scan speed- has opposite effect but often results in poor image quality Note: Some types of confocal specifically optimised for fast scanning. Eg spinning disk, line scanner and resonant scanner

14. 14 Pinhole adjustment Airy disc 0.5Maximum optical sectioning and resolution. Discard much in-focus light 1xy resolution approaches that of conventional microscopy, but still retain good rejection of out-of-focus information. Still lose some in-focus photons. >1Maximise light collected. But this mostly comes from adjacent out-of-focus planes - lose z resolution. xy resolution not badly affected x y z x y z Open pinhole Close pinhole

15. 15 Confocal Pinhole

16. 16 210 nm 60 nm z = 0 z = 2z = 4 z = 6z = 8 Fluotar 20x/0.5 Zoom = 3 Pinhole = 0.7

17. 17 210 nm 60 nm z = 0 z = 2z = 4 z = 6z = 8 Fluotar 20x/0.5 Zoom = 3 Pinhole = 3.0

18. 18 Pinhole Summary In practise, pinhole size is mainly used to control optical section thickness other than to achieve highest lateral or Z-resolution Occasionally, pinhole size can be used to adjust amount of photon received by PMT to change the signal intensity and increase SNR. In addition to the "optimal" 1 AU, Pinhole 1-3 AU is the range of choice. Bigger pinhole give you stronger signal but with the compromised confocal effects.

19. 19 Sampling Scanning involves digitisation in x, y, z, intensity, and t Resolution is affected by sampling during the digitisation process 0000000000000 00022456611000000 0000000000000 000000065120000 00000000990000 0000000700000 00650002821520 0003000000000 0000000000000 pixels (voxels)

20. 20 Pixel choices 512x512 1024x1024 2048x2048 More pixels— smoother looking image - more xy information more light exposure of specimen larger file size slower imaging (less temporal resolution) —250 kbyte (1 channel) —3 Mbyte (3 channel)

21. 21 Digitisation can lose information Correct choice of pixel size can minimise this intensity scan line

22. 22 Pixel undersampling Specimen Large pixels Small pixels, lucky alignment Small pixels, unlucky alignment Very small pixels

23. 23 Nyquist sampling (xy) Optimum pixel size for sampling the image is at least 1/2 spatial resolution 100x, 1.35 NA, 520 nm (blue-green) Spatial resolution = 0.15  m Required pixel size = 0.075  m Actual pixel size at 512x512 is usually too large (will be shown on screen or calculate from field size/pixel number) How to adjust to meet Nyquist criteria? Use higher pixel number (e.g. 1024x1024 2048x2048) or use a zoom factor…

24. 24 Zooming Using the same scanning raster, speed, illumination on a smaller area of the field of view May ideally need 2–5x zoom to satisfy the Nyquist criteria.

25. 25 Nyquist Sampling Equation i) 0.4 x wavelength/NA = Resolvable Distance ii) 2 pixels is smallest optically resolvable distance iii) Resolvable Distance/2 = smallest resolvable point

26. 26 Nyquist Sampling Example X10 Objective with 0.3 NA using GFP 0.4 x 520 = 693nm 0.3 693 = 346.6nm smallest resolvable distance 2 Scan Size = 1500µm Box Size = 1024 pixels 1500 = 1464nm 1024 1464 = 4.2 zoom for nyquist in xy 346.6 Or a box size large enough to produce a pixel size of 346.6

27. 27 Nyquist sampling and z series What distance between z steps? Optimum z step for sampling the image is 1/2 the axial resolution For high NA lens of 0.3  m z resolution, optimum z stepping is 0.1-0.2  m (assuming optimum pinhole size, etc). In practice, this is often too many for a very thick specimen. 0.5-1  m is often fine. Especially if pinhole opened. x y z

28. 28 Over- and undersampling Oversampling (pixels small compared with optical resolution) Image smoother and withstands manipulation better Specimen needlessly exposed to laser light Image area needlessly restricted File size needlessly large Undersampling (pixels large compared with optical resolution) Degraded spatial resolution Photobleaching reduced Image artefacts (blindspots, aliasing) “If you must sample below the Nyquist limit, then spoil the resolution [to match better the pixel size]!” ie. Open the pinhole.

29. 29 Digitisation of PMT voltage 3 bit 8 levels of brightness 0 7 1 bit: 2 levels (black + white) (Eye is a 6 bit device (~50 levels of brightness)) x Level Voltage is sampled at regular intervals and converted into a digital pixel intensity value by the analogue-digital converter (ADC) 12 bit 3 bit

30. 30 Noise Noise: any variability in measurement that is not due to signal changes S/N ratio determines the lower limit of the ability to distinguish true changes in the measurement (dynamic range) Photon sampling variability (shot noise): Statistical fluctuations in photons hitting PMT. Electronic noise: Variability in PMT generated current. These things are exacerbated at high gain settings Reduce noise by sampling more photons: Reducing scan rate (increasing pixel dwell time), or opening pinhole. Frame averaging Noise is reduced (dynamic range increased) with square root of number of frames Sample exposure to light is increased

31. 31 High gain 1 scan16 scans Apo 63x lens Laser 488nm 10% PMT 1000V Laser 488nm 80% PMT 800V Medium gain

32. 32 Digitisation of intensity Normally 8 bit (256 brightness levels) Extended dynamics 12 bit (4095 brightness levels) But useful dynamic range is degraded by noise Why need so many bits? 1. Spare dynamic range for exploring intensity details during image processing 2. Probably helps to smooth out noise problems (e.g. capture in 12 bit and save in 8 bit) Quantitation/physiology

33. 33 Multi-channel imaging

34. 34 Multi-channel imaging Use a fluorochrome combination Multiple laser lines and PMTs Complicated filter sets needed to separate light Alternatives: AOTF, AOBS, spectrophotometric detection Dichroic mirrors or AOBS 488, 568 <550 >550 PMT1 PMT2 bleedthrough

35. 35 Crosstalk FITC TRITC FITC = fluorescein isothiocyanate TRITC = tetramethyl rhodamine isothiocyanate Usually overlap of emission spectra from L to R Green channelRed channel

36. 36 Crosstalk How to reduce: Use better separated fluorochromes e.g. FITC + Texas Red versus FITC + TRITC Put the weak signal in the ‘LH’ channel Sequential imaging rather than simultaneous imaging How to test: Turn off laser line for the ‘LH’ fluorochrome

37. 37 Preventing cross-talk FITC TRITC FITC Texas Red

38. 38 4 principal factors for image quality Spatial resolution Ultimately set by the optics, but can be limited by digitisation (therefore affected by image size and zoom). Affected by pinhole: super- resolution (1.4x) is possible at small pinholes Intensity resolution Ultimately set by detector, but limited by digitisation and low photon sampling. Aim to fill whole dynamic range with image information. Signal-to-noise ratio Degree of visibility of image over background noise, given variability in system. Temporal resolution Depends on raster scan rate (+averaging). 512x512 at 2/s. Imaging depends on compromising between these factors, e.g. you might want to optimise resolution of light intensity at expense of spatial or temporal resolution.

39. 39 Colour Look UpTables Which colours to use? You’re not restricted to the ‘true’ colour of the fluorochrome Colour look-up table

40. 40 Colour LUT Grey is best Red is really bad

41. 41 Imaging Thick Specimens

42. 42 The Problem

43. 43 Defocus 1µm

44. 44 Background & Scattering ConfocalWidefield

45. 45 Aberrations Green Red Blue Axial Chromatic Aberration Red Green Blue Lateral Chromatic Aberration

46. 46 Chromatic Aberrations No-Aberration Green-Red Chromatic Aberration

47. 47 Aberrations Spherical Aberration 20-40µm

48. 48 75 um XY 100 um nls GFP: ex 476; em 530+/-15 Spherical Aberration on a confocal XZ 100 um coverslip 0 um Aberrations

49. 49 2-Photon S S* S 1-photon absorption Fluorescence 2-photon absorption Fluorescence

50. 50 2-Photon Single Photon 2 photon

51. 51 2-Photon Image Brad Amos MRC Cambridge From:- Piston DW Trends Cell Biol (1999) 9: 66

52. 52 Advantages of 2 Photon  Longer observation times for live cell studies  Increased fluorescence emission detection  Reduced volume of photobleaching and phototoxicity. Only the focal-plane being imaged is excited, compared to the whole sample in the case of confocal or wide-field imaging.  Reduced autofluorescence of samples  Optical sections may be obtained from deeper within a tissue that can be achieved by confocal or wide-field imaging. There are three main reasons for this: the excitation source is not attenuated by absorption by fluorochrome above the plane of focus; the longer excitation wavelengths used suffer less Raleigh scattering; and the fluorescence signal is not degraded by scattering from within the sample as it is not imaged.  All the emitted photons from multi-photon excitation can be used for imaging (in principle) therefore no confocal blocking apertures have to be used.  It is possible to excite UV fluorophores using a lens that is not corrected for UV as these wavelengths never have to pass through the lens.

53. 53 Limitations of 2-Photon  Slightly lower resolution with a given fluorochrome when compared to confocal imaging. This loss in resolution can be eliminated by the use of a confocal aperture at the expense of a loss in signal.  Thermal damage can occur in a specimen if it contains chromophores that absorb the excitation wavelengths, such as the pigment melanin.  Only works with fluorescence imaging.

54. 54 Multi Photon 3 photon Use of near-infrared wavelengths (down to 720 nanometers) 3 photon excitation extend the fluorescence imaging range into the deep ultraviolet. Example Single, dual, and triple photon excitations of tryptophan, Single photon excites at 280nm with emission of fluorescence at 348 nanometers (UV). Two-photon excites with greenish- yellow light centered at 580nm. Three-photon excites with near-infrared light at 840nm

55. 55 Sample Sample Mounting Upright ScopeInverted Scope Slides etc Petri dishes, plates etc Cells, yeast etc Fast event Epi-Fluorescence Spinning Disk Confocal Resonant Scanner Yes No Epi-Fluorescence Structured Illumination Laser Scanning Confocal Specimen 10-30µm Thick Fast event Laser Scanning Confocal Multiphoton No Yes Deconvolution Spinning Disk Confocal Resonant Scanner Specimen > 30µm Thick Fast event No Yes Multiphoton with Resonant Scanner Multiphoton

56. 56 END