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1
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
5
CLSM microscope
antivibration table
6
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
34
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
37
Preventing cross-talk
FITCTRITC
FITCTexas Red
38
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
40
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
44
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
49
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
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
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
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