What to get out of this lecture Benefits of Confocal Microscopy Confocal Design CLSM microscope Confocal principle

What to get out of this lecture Benefits of Confocal Microscopy Confocal Design CLSM microscope Confocal principle www.phwiki.com

What to get out of this lecture Benefits of Confocal Microscopy Confocal Design CLSM microscope Confocal principle

Miller, Brett, Morning Show Host/Producer has reference to this Academic Journal, PHwiki organized this Journal Confocal Microscopy David Kelly November 2013 H in addition to book of Biological Confocal Microscopy. Ed. J. Pawley, Plenum Press Fundamentals of Light Microscopy in addition to 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 in addition to 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 What to get out of this lecture Have an underst in addition to ing 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 in addition to how to manipulate these factors as long as your purposes This often means knowing when in addition to where to make compromises (e.g. light collection versus spatial resolution) 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

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Confocal Design CLSM microscope antivibration table Confocal principle

The Pinhole z x y x y Pinhole The Pinhole Optical sectioning 1 mm

Laser scanning Photomultiplier tube Computer Laser scanning xz scanning x y z x y z x y x y z z series single section xy scanning x y x z Confocal Principles

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 in addition to phototoxicity limits temporal resolution Increasing scan speed- has opposite effect but often results in poor image quality Note: Some types of confocal specifically optimised as long as fast scanning. Eg spinning disk, line scanner in addition to resonant scanner Pinhole adjustment Airy disc 0.5 Maximum optical sectioning in addition to resolution. Discard much in-focus light xy resolution approaches that of conventional microscopy, but still retain good rejection of out-of-focus in as long as mation. Still lose some in-focus photons. >1 Maximise 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 Confocal Pinhole

210 nm 60 nm z = 0 z = 2 z = 4 z = 6 z = 8 Fluotar 20x/0.5 Zoom = 3 Pinhole = 0.7 210 nm 60 nm z = 0 z = 2 z = 4 z = 6 z = 8 Fluotar 20x/0.5 Zoom = 3 Pinhole = 3.0 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 in addition to 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.

Sampling Scanning involves digitisation in x, y, z, intensity, in addition to t Resolution is affected by sampling during the digitisation process 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 22 45 66 11 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 65 12 0 0 0 0 0 0 0 0 0 0 0 0 99 0 0 0 0 0 0 0 0 0 0 0 7 0 0 0 0 0 0 0 6 5 0 0 0 2 8 21 5 2 0 0 0 0 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 pixels (voxels) Pixel choices 512×512 1024×1024 2048×2048 More pixels— smoother looking image – more xy in as long as mation more light exposure of specimen larger file size slower imaging (less temporal resolution) —250 kbyte (1 channel) —3 Mbyte (3 channel) Digitisation can lose in as long as mation Correct choice of pixel size can minimise this intensity scan line

Pixel undersampling Nyquist sampling (xy) Optimum pixel size as long as sampling the image is at least 1/2 spatial resolution 100x, 1.35 NA, 520 nm (blue-green) Spatial resolution = 0.15 mm Required pixel size = 0.075 mm Actual pixel size at 512×512 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. 1024×1024 2048×2048) or use a zoom factor 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.

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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 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 as long as nyquist in xy 346.6 Or a box size large enough to produce a pixel size of 346.6 Nyquist sampling in addition to z series What distance between z steps Optimum z step as long as sampling the image is 1/2 the axial resolution For high NA lens of 0.3 mm z resolution, optimum z stepping is 0.1-0.2 mm (assuming optimum pinhole size, etc). In practice, this is often too many as long as a very thick specimen. 0.5-1 mm is often fine. Especially if pinhole opened. x y z

Over- in addition to undersampling Oversampling (pixels small compared with optical resolution) Image smoother in addition to withst in addition to s 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. 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 in addition to converted into a digital pixel intensity value by the analogue-digital converter (ADC) 12 bit 3 bit 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

Sample Sample Mounting Upright Scope Inverted 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 END

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