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Confocal MicroscopyDavid 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 microscopePinholeOptical SectioningSpinning Disk ConfocalPhoton Multiplier TubeCCD

Confocal principles: Scan speedOptical resolutionPinhole adjustmentDigitisation: sampling as opposed to imagingxy sampling: pixel size and zoom choicesPhotomultiplier tubes, noise, digitisation of intensityMultichannel imaging, crosstalkColour Look Up Tables

Recap of principal factors affecting image qualityImaging Thick SpecimensMultiphoton Microscopy

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What to get out of this lectureHave 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)

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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

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CLSM microscope

antivibration table

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Confocal principle

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The Pinhole

z

x

y

x

Conjugate plane

y

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Pinhole

The Pinhole

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Optical sectioning

1 m

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Laser scanning confocal microscopeLaser scanningPhotomultiplier tube

Computer

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Laser scanning confocal microscope

Laser scanning

xz scanning

xy

z

xy

z

xy

xy

z

z seriessingle section

xy scanning

x

y

x

z

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Confocal Principles

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Scan speed: t resolutionOn 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 phototoxicitylimits temporal resolution

Increasing scan speed- has opposite effect but often results in poor image qualityNote: Some types of confocal specifically optimised for fast scanning. Eg spinning disk, line scanner and resonant scanner

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Pinhole adjustment

Airy disc

0.5 Maximum optical sectioning and resolution. Discard much in-focus light

1 xy resolution approaches that of conventional microscopy, but still retain good rejection of out-of-focus information. 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

xy

z

xy

z

Open pinhole

Close pinhole

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Confocal Pinhole

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210 nm

60 nm

z = 0 z = 2 z = 4

z = 6 z = 8

Fluotar 20x/0.5Zoom = 3Pinhole = 0.7

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210 nm

60 nm

z = 0 z = 2 z = 4

z = 6 z = 8

Fluotar 20x/0.5Zoom = 3Pinhole = 3.0

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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.

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Sampling

Scanning involves digitisation in x, y, z, intensity, and 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)

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Pixel choices

512x5121024x10242048x2048

More pixels—smoother looking image - more xy informationmore light exposure of specimenlarger file sizeslower imaging (less temporal resolution)

—250 kbyte (1 channel)—3 Mbyte (3 channel)

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Digitisation can lose information

Correct choice of pixel size can minimise this

intensity

scan line

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Pixel undersampling

Specimen

Large pixels

Small pixels, lucky

alignmentSmall pixels, unlucky

alignmentVery small

pixels

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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 mRequired 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…

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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

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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

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Nyquist sampling and z seriesWhat 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.

xy

z

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Over- and undersampling

Oversampling (pixels small compared with optical resolution)

Image smoother and withstands manipulation better

Specimen needlessly exposed to laser lightImage area needlessly restrictedFile size needlessly large

Undersampling (pixels large compared with optical resolution)

Degraded spatial resolutionPhotobleaching reducedImage 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.

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Digitisation of PMT voltage

3 bit8 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

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NoiseNoise: any variability in measurement that is not due to

signal changesS/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 averagingNoise is reduced (dynamic range increased) with square root of number of framesSample exposure to light is increased

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High gain

1 scan 16 scans

Apo 63x lens

Laser 488nm 10%PMT 1000V

Laser 488nm 80%PMT 800V

Medium gain

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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 processing2. Probably helps to smooth out noise

problems (e.g. capture in 12 bit and save in 8 bit)

Quantitation/physiology

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Multi-channel imaging

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Multi-channel imagingUse a fluorochrome combinationMultiple laser lines and PMTsComplicated filter sets needed to separate lightAlternatives: AOTF, AOBS, spectrophotometric detection

Dichroic mirrors or AOBS

488, 568

<550

>550

PMT1

PMT2

bleedthrough

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Crosstalk

FITCTRITC

FITC = fluorescein isothiocyanateTRITC = tetramethyl rhodamine isothiocyanate

Usually overlap of emission spectra from L to R

Green channel Red channel

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Crosstalk

How to reduce:

Use better separated fluorochromese.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

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Preventing cross-talk

FITCTRITC

FITCTexas Red

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4 principal factors for image quality

Spatial resolutionUltimately 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 resolutionUltimately set by detector, but limited by digitisation

and low photon sampling. Aim to fill whole dynamic range with image information.

Signal-to-noise ratioDegree of visibility of image over background noise,

given variability in system.

Temporal resolutionDepends 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.

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Colour Look UpTables

Which colours to use?

You’re not restricted to the ‘true’ colour of the fluorochrome

Colour look-up table

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Colour LUT

Grey is bestGrey is bestRed is really Red is really badbad

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Imaging Thick Specimens

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The Problem

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Defocus

1µm

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Background & ScatteringConfocal Widefield

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Aberrations

Green

Red

Blue

Axial Chromatic Aberration

RedGreenBlue

Lateral Chromatic Aberration

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Chromatic

Aberrations

No-Aberration Green-Red Chromatic Aberration

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AberrationsSpherical Aberration 20-40µm

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75 um

XY

100 um

nls GFP: ex 476; em 530+/-15

Spherical Aberration on a confocalXZ

100 um

coverslip0 um

Aberrations

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2-Photon

S

S*

S

S*

1-photon absorption

Fluorescence

2-photon absorption

Fluorescence

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2-Photon

Single Photon

2 photon

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2-Photon

Image Brad Amos MRC CambridgeFrom:- Piston DW Trends Cell Biol (1999) 9: 66

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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.

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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.

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Multi Photon3 photon

• Use of near-infrared wavelengths (down to 720 nanometers) 3 photon excitation extend the fluorescence imaging range into the deep ultraviolet.

ExampleSingle, 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

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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 ConfocalSpecimen 10-30µm Thick

Fast eventLaser Scanning Confocal

Multiphoton

NoYes

Deconvolution

Spinning Disk Confocal

Resonant Scanner Specimen > 30µm Thick

Fast eventNoYesMultiphoton with

Resonant ScannerMultiphoton

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END