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Confocal Microscopy
Dr. Serge Arnaudeau
Bioimaging Core Facility
Geneva
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Wide-field microscope
in focus
Viewing plane(image plane)Sample
(object plane)objective
Light source
only one plane in focus
but all the planes contribute to the image
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The pinhole
Photons passing through the pinhole are coming
exclusively from the focal point of the objective
in focus
pinhole
Viewing plane(image plane)Sample
(object plane)objective
Light source
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Depth of field depends on pinhole size
small pinhole most of the photons coming from out offocus planes are rejected and do not contribute to the image
in focusobjective
small pinhole
large pinhole
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Confocal microscope principle
pinholepinhole
conjugate focal planes
illumination and detection of the same focal point
need to displace the sample in x and y to construct an image
Sample(object plane)
objective
Lightsource
detector
objectiveTransmissive design
xy
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What is fluorescence?
Ground state
Absorb highenergy photons
Emit lowerenergy photon
Excited state
In one-photon excitation ex < em (Stokes shift)
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How does a fluorescence microscope
work?
Excitation filter
Emission filter
Dichroic filter
sample
objective
Light source
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Epitaxial confocal microscope for
fluorescencepinhole
photomultiplier tube
dichroic mirror
objective
Focal point
Laser Source
use of the same objective for illumination
and detection
use of a laser source to avoid the use of a
pinhole in illumination
use of a PMT to make photon counting for
each focal point
use of galvanometric mirrors to XY scan the
field of view
use of a stepping motor in the Z direction to
make optical slices in the sample
barrier filter
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Advantage of fluorescence confocal
microscopy
A section of mouse intestineimaged with both confocal andnon-confocal microscopy
ability to control depth of field
elimination or reduction of
background information away
from the focal plane
capability to collect serial optical
sections from thick specimens
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How big is a Laser Scanning Confocal
Microscope ?
System electronic rack
Laser module
405, 458, 477, 488,514, 561, 633 nm
Scanning head
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LASER
Light Amplification by Stimulated Emission of Radiation
High intensity
Spatial and temporal coherence
Monochromatic Focused
Lasers installed in our Laser Scanning Microscopes
405 nm Diode laser (DAPI, CFP)
Argon ion gas laser with 458 nm (CFP)
488 nm (FITC, GFP, Alexa488 )514 nm (YFP)
Helium neon 543 nm gas laser (TRITC, Cy3, Alexa546 )
561 nm DPSS laser (Texas red, Alexa568 )Helium neon 633 nm gas laser (TOTO3, Cy5 )
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Wide-field illumination cone versus
point scanning of specimens
Wide-field microscope : entire depth of the specimen over
a wide area is illuminated
Confocal microscope : the sample is scanned with a finelyfocused spot of illumination centered in the focal plane
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Beam scanning
Majority of laser scanning microscopes : single beam
scanning
Laser spot
Only confocal microscopes which use acousto-opticdeflectors can scan at speeds of 30 frames/s
To scan the specimen in a raster
pattern, the Laser Scanning
Microscope uses a pair of computer
controlled galvanometric mirrors.
The scanning speed is limited by
these mirrors.
Good image quality but notfast enough to resolve
transient physiological signals
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Photomultiplier Tubes (PMT)
Dynodes
Anode
Photocathode
Window
Incident light
Side on design
Gain varies with the voltage across the dynodes and the total number of dynodesWith typically 9 dynodes, gain of 4x106 can be achieved
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Photomultiplier Tubes (PMT)
The spectral response, quantum efficiency, sensitivity, and dark current of a
photomultiplier tube are determined by the composition of the photocathode
100
10
1
0.1
0.01
QuantumEfficie
ncy(%)
100 200 300 400 500 600 700 800 900 1000
Wavelength (nm)
Graylevels(8bits)
0
255
128
600 V
0 V
800 V
50 V offset
gain
Low quantum efficiency and low dynamic range but very fast response time
S i d i fl i
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Scanning speed influences image
quality
Better signal to noise ratio with low scanning speeds butsamples are more exposed to the laser beam
pixel dwell time 3.2 s pixel dwell time 25.6 s
2 m
Muntjac
cells
Alexa555
antiOXPhoscomplexVinhprot
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Scans averaging reduces noise
Average of 2 scans Average of 8 scans
But greatly reduce the frame rate
10 m
Muntjaccells
Alexa488phalloid
in
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Airy disk and Resolution
Due to diffraction, the image of a point source of light in the focalplane is not a point its actually an Airy diffraction pattern
The resolving power of an objective determines the size of theAiry diffraction pattern formed
Airy disk
Airy diff raction pat tern
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Airy disk and Resolution
The radius of the Airy disk is given by :
r(Airy) = 0.61 exc /NA(obj)
with NA(obj) = n sin
n = medium refractive index
= objective angular aperture
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Airy disk and Resolution
Rayleigh criterion for lateral resolution :
the center of one airy disk falls on the first minimum of the
other airy disk
resolved Rayleigh criterion unresolved
intensity contrast
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Pinhole and Resolution
Confocal pinhole size = diameter of the Airy disk (1 Airy unit)
84% of in focus light pass to the detector
Airy disk units are a convenient way to normalize confocal
pinhole size :
Pinhole size = 1 Airy unit = best signal to noise ratio
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Pinhole and Resolution
Confocal fluorescence :
pointwise illumination + pointwise detection
narrower Point Spread Function / widefield microscopy
rlateral = 0.4 exc / NA
widefield confocal
Axial PSF intensity profiles
Increase in lateral resolution
confocal lateral resolution > widefield lateral resolution
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Pinhole and Resolution
Axial resolution :
raxial = 1.4 exc n / NA2
exc = excitation wavelength
n = medium refractive index
NA = objectives numerical aperture
Confocal PSFThe PSF is elongated in the axial direction
Axial resolution of an objective is worse than
its lateral resolution
For an oil immersion objective with 1.4 NA using the 488 nm laser line
rlateral = 0.4 x 488/1.4 = 139 nm (in theory for very
raxial = 1.4 x 488 x 1.515/(1.4)2 = 528 nm small pinhole size)
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Resolution depends on pinhole size
Pinhole : 1 AU(optical slice ~ 0.8 m)
Pinhole : 0.5 AU(optical slice ~ 0.5 m)
Better Z discrimination with small pinhole size but needsstrong signals
10 m
Muntjaccells
Alexa488phalloidin
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Optical sectionning
12345
1
2
4
3
5
x
yz
X
Y
Z
3D reconstruction
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Sampling in confocal microscopy
Voxel on the sample
Pixel on the image
The image is built as the laser
moves on the sample
Zooming is produced byslower movement of the laser
on a reduced area :
no pixelization effect even
with very high zoom
x
y
z
x
y
S li i f l i
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Sampling in confocal microscopy
5 m
260 nm/pixel
65 nm/pixel
16 nm/pixel
1 m
20 m
130 nm/pixel
10 m
33 nm/pixel
2 m
512x512 zoom 1
512x512 zoom 2
512x512 zoom 4
512x512 zoom 8
512x512 zoom 16
Muntjac cellsAlexa 488 phalloidinAlexa 555 anti OXPhos complex V inh protTO PRO-3
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Sampling in confocal microscopy
What is the zooming factor limit?
This is linked to the X,Y resolution of the optics
Sampling is sufficient when there is enough pixels to
separate two adjacent Airy disk
In imaging, frequency = spatial frequency
fsampling = 2.3 x fhighest (to compensate low-pass filtering)
Nyquist theorem :
to reconstruct a sine wave : fsampling = 2 x fwave
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Sampling in confocal microscopy
The highest frequency to be sampled in the CLSM is imposed
by the optical system :
fhighest = 1/resolution
To fulfill the Nyquist criterion :
fsampling = 2.3/rlateral
Pixel size ~ rlateral/2.3 > oversamplingundersampling >
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Sampling in confocal microscopy
Critical sampling distances @ 500 nm(for pinhole = 1 AU values by 50%)
Id l i i ti
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Ideal emission separation
Green emission filter
Red emission filter
Dichroicbeamsplitter
PMT 2
PMT 1
C t lk bl
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Crosstalk problems
Most of the time there is some overlapping between
fluorophores emission spectra
Example of FITC and TRITC
Using 488 nm and 543 nm lines : 22% overlap
If the fluorescence signalsare not taken sequentially :
some of the green
fluorescence is assigned to
the red channel
C t lk bl
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Crosstalk problems
To avoid bleed-through of one fluorescence in another
channel, multitrack configurations allow sequential
acquisition of lines (or frames) by very fast switching of the
laser lines by means of AOTF
Minimize crosstalk between channels
More accurate quantification in co-localization experiments
S t l ti
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Spectral separation
When the emission spectra of the fluorophores are very close :
Spectral detector (like the Meta detector) allow the record of the
emission spectra of each pixel of the image
Example of latex bead with
narrow fluorescences in thecore and the ring acquired
with the spectral detector
(Meta)
Image serie of the bead at
different wavelengths
S t l ti
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Spectral separation
Fluorescence separation
after software unmixing
Selection of the
different fluorescences
(core and ring)
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FRAP e periments
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FRAP experiments
FRAP-recording for 40 min (1 frame/min)
Photobleaching
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Photobleaching
Bovine endothelial cells
actin filaments (BODIPY FL),
mitochondria (MitoTracker Red);
some mitochondria are markedfor photobleaching
Bleaching of marked mitochondria with pinpoint
accuracy (left)
Merged images of mitochondria before and after
photobleaching :bleached portions appeared in red(right)
Very high control of the scanner
by the DSP (Digital Signal
Processor) to position the laser
beam and choose ROI of anyshape
Other beam scanning techniques
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Other beam scanning techniques
Multiple beam scanning : the Nipkow disk
Disk rotation
One way to increase the scanning
speed is to increase the numberof scanning spots.
The spinning disk with pinholes
was introduced into a microscopeby Mojmir Petran in 1968.
Improvement of the Nipkow disk
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p p
principle in the YOKOGAWA scanhead
specimen
Laser beamCollector disk
CCD camera
Dichroicmirror
Aperture disk
Microlenses(20 000)
Pinholes(20 000)
Objective
Nipkow disk confocal microscope facilitate
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studies of ligth-sensitive processes
Cell cycle in
Drosophila Embryoexpressing GFP-
Histone
Dr. Caetano GonzalezEMBL
Nipkow disk confocal microscope facilitate
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Ca2+waves incardiomyocytes
loaded with fluo-3
Dr. Marisa J aconiGeneva
Image capture at 33 Hz using an intensified camera(Coolsnap Cascade from Photometrics)
studies of fast processes
Other beam scanning techniques
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Other beam scanning techniques
Slit scanning : a new approach in confocal microscopy
The circular laser beam is transformed
to a line which scan the sample in onlyone direction
The emitted fluorescence of that line
passed through a confocal line pinhole
This line (512 pixels) is detected by a
ultrafast line CCD detector
Scan speeds of 100 frames/s
can be achieved
Advantage of the LSCM :
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the line scan mode
[C
a2+]i
(nM)
0
75
150
200 ms
10
m
0
125
250
[Ca2+]i
(nM)
10 m
Fluo-3
Fura-red
Spatially restricted, but very fast (1 line/2ms)