Confocal Microscopy
David Kelly November 2013
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
Handbook of Biological Confocal Microscopy. Ed. J. Pawley, Plenum Press
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Fundamentals of Light Microscopy and Electronic Imaging. D. B. Murphy, Wiley-Liss Inc.
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)
2
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
4
CLSM microscope
antivibration table
5
Confocal principle
6
The
Pinhole
Conjugate plane
y
z
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x
y
x
The
Pinhole
Pinhole
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Optical sectioning
1 mm
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confocal
Laser scanning
microscope
Photomultiplier tube
Computer
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Laser scanning
confocal
microscope
xy scanning
y
z
x
y
x
y
x
single section
z series
z
z
z
x
y
x
y
x
xz scanning
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Confocal Principles
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Scan speed: t resolution
On modern confocals this is measured in Hz
usually from 1-1400Hz
Decreasing scan speedmore 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
13
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
Open pinhole
z
z
x
y
Close pinhole
x
y
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Confocal Pinhole
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z=0
z=2
z=6
z=8
z=4
210 nm
60 nm
Fluotar 20x/0.5
Zoom = 3
Pinhole = 0.7
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z=0
z=2
z=6
z=8
z=4
210 nm
60 nm
Fluotar 20x/0.5
Zoom = 3
Pinhole = 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
pixels
(voxels)
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Pixel choices
512x512
1024x1024
2048x2048
—250 kbyte (1 channel)
—3 Mbyte (3 channel)
More pixels—
smoother looking image - more xy information
more light exposure of specimen
larger file size
slower imaging (less temporal resolution)
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Digitisation can lose information
Correct choice of
pixel size can
minimise this
intensity
scan line
21
Pixel undersampling
Specimen
Large pixels
Small pixels, lucky alignment
Small pixels, unlucky alignment
Very 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 mm
Required pixel size = 0.075 mm
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…
23
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 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 mm z resolution, optimum
z stepping is 0.1-0.2 mm (assuming optimum
pinhole size, etc).
In practice, this is often too many for a very thick
specimen. 0.5-1 mm is often fine. Especially if
pinhole opened.
z
x
y
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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 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]!”
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ie. Open the pinhole.
Digitisation of PMT voltage
Voltage is sampled at regular intervals and
converted into a digital pixel intensity value
by the analogue-digital converter (ADC)
12 bit
1 bit: 2 levels (black + white)
7
3 bit
8 levels of brightness
Level
3 bit
0
x
(Eye is a 6 bit device (~50 levels of brightness))
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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
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1 scan
16 scans
Medium gain
Laser 488nm 80%
PMT 800V
High gain
Laser 488nm 10%
PMT 1000V
Apo 63x lens
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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 processing
2. 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 imaging
Use a fluorochrome combination
Multiple laser lines and PMTs
Complicated filter sets needed to separate light
Alternatives: AOTF, AOBS, spectrophotometric detection
488, 568
>550
Dichroic mirrors
or
AOBS
PMT2
<550
bleedthrough
PMT1
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Crosstalk
FITC = fluorescein isothiocyanate
TRITC = tetramethyl rhodamine isothiocyanate
FITC
TRITC
Green channel
Red channel
Usually overlap of emission spectra from L to R
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Crosstalk
How to test:
Turn off laser line for the ‘LH’ fluorochrome
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
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Preventing cross-talk
FITC
TRITC
FITC
Texas Red
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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: superresolution (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.
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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
39
Colour LUT
Grey is best
Red is really bad
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Imaging Thick Specimens
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The Problem
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Defocus
1µm
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Background & Scattering
Confocal
Widefield
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Aberrations
Blue Green
Red
Red
Green
Blue
Axial Chromatic
Aberration
Lateral Chromatic
Aberration
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Aberrations
Chromatic
No-Aberration
Green-Red Chromatic Aberration
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Aberrations
Spherical Aberration
20-40µm
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Aberrations
nls GFP: ex 476; em 530+/-15
Spherical Aberration on a confocal
75 um
XY
XZ
coverslip
0 um
100 um
100 um
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2-Photon
Fluorescence
S
1-photon absorption
S
Fluorescence
2-photon absorption
S*
S*
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2-Photon
Single Photon
2 photon
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2-Photon
From:- Piston DW Trends Cell Biol (1999) 9: 66
Image Brad Amos MRC Cambridge
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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 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 greenishyellow light centered at 580nm.
Three-photon excites with near-infrared
light at 840nm
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Sample
Sample Mounting
Slides etc
Petri dishes, plates etc
Upright Scope
Cells, yeast etc
Epi-Fluorescence
Spinning Disk
Confocal
Resonant Scanner
Deconvolution
Yes
Multiphoton with
Resonant Scanner
Fast event
Epi-Fluorescence
No
Structured Illumination
Laser Scanning Confocal
Specimen 10-30µm Thick
Yes
Spinning Disk
Confocal
Resonant Scanner
Inverted Scope
Fast event
No
Laser Scanning Confocal
Multiphoton
Specimen > 30µm Thick
Yes
Fast event
No
Multiphoton
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END
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