Confocal Microscopy

Dr. Serge Arnaudeau

BioimagingCore Facility
Geneva
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
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
Depth of field depends on pinhole size
small pinhole most of the photons coming from out of
focus planes are rejected and do not contribute to the image
in focus
objective
small pinhole
large pinhole
Confocal microscope principle
pinhole pinhole
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
Light
source
detector
objective
Transmissive design
x
y
What is fluorescence?
Ground state
Absorb high
energy photons
Emit lower
energy photon
Excited state
In one-photon excitation
ex
<
em
(Stokesshift)
How does a fluorescence microscope
work?
Excitation filter
Emission filter
Dichroic filter
sample
objective
Light source
Epitaxial confocal microscope for
fluorescence
pinhole
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
Advantage of fluorescence confocal
microscopy
A section of mouse intestine
imaged with both confocal and
non-confocal microscopy
abilityto control depthof field
elimination or reduction of
background information away
from the focal plane
capability to collect serial optical
sections from thick specimens
How bigisa Laser Scanning Confocal
Microscope ?
System electronic rack
Laser module
405, 458, 477, 488,
514, 561, 633 nm
Scanning head
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, Alexa
488
)
514 nm (YFP)
Helium neon 543 nm gas laser (TRITC, Cy3, Alexa
546
)
561 nm DPSS laser (Texas red, Alexa
568
)
Helium neon 633 nm gas laser (TOTO3, Cy5 )
Wide-field illumination cone versus
point scanning of specimens
Wide-fieldmicroscope: entiredepthof thespecimenover
a widearea isilluminated
Confocal microscope : thesampleis scannedwitha finely
focusedspot of illumination centeredin thefocal plane
Beamscanning
Majorityof laser scanning microscopes : single beam
scanning
Laser spot
Only confocal microscopes which use acousto-optic
deflectors 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 not
fast enough to resolve
transient physiological signals
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 dynodes
With typically 9 dynodes, gain of 4x10
6
can be achieved
Photomultiplier Tubes (PMT)
The spectral response, quantum efficiency, sensitivity, and darkcurrent of a
photomultiplier tube are determined by the composition of the photocathode
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Low quantum efficiency and low dynamic range but very fast response time
Scanning speed influences image
quality
Better signal to noise ratio with low scanning speeds but
samples are more exposed to the laser beam
pixel dwell time 3.2 s pixel dwell time 25.6 s
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Scans averaging reduces noise
Average of 2 scans Average of 8 scans
But greatlyreducethe frame rate
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Airy disk and Resolution
Due to diffraction, the image of a point source of light in the focal
plane is not a point its actually an Airy diffraction pattern
The resolving power of an objective determines the size of the
Airy diffraction pattern formed
Airy disk
Airy diffraction pattern
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

Airy disk and Resolution

Rayleighcriterion for lateral resolution:
the center of one airy disk falls on the first minimum of the
other airy disk
resolved Rayleigh criterion unresolved
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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
Pinhole and Resolution
Confocal fluorescence :
pointwiseillumination + pointwisedetection
narrower Point Spread Function / widefieldmicroscopy
r
lateral
= 0.4
exc
/ NA
widefield confocal
Axial PSF intensity profiles
Increasein lateral resolution
confocal lateral resolution> widefieldlateral resolution
Pinhole and Resolution
Axial resolution:
r
axial
= 1.4
exc
n/ NA
2

exc
=excitation wavelength
n =medium refractiveindex
NA =objectivesnumerical aperture
Confocal PSF
The PSF iselongatedin the axial direction
Axial resolutionof an objective isworsethan
itslateral resolution
For an oil immersion objective with1.4 NA usingthe 488 nm laser line
r
lateral
= 0.4 x 488/1.4 = 139 nm (in theoryfor very
r
axial
= 1.4 x 488 x 1.515/(1.4)
2
=528 nm small pinholesize)
Resolutiondependson pinholesize
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 needs
strong signals
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Optical sectionning
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z
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3D reconstruction
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 by
slower movement of the laser
on a reduced area :
no pixelizationeffect even
with very high zoom
x
y
z
x
y
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 cells
Alexa 488 phalloidin
Alexa 555 anti OXPhos complex V inh prot
TO PRO-3
Sampling in confocal microscopy
What isthe zoomingfactor limit?
This islinkedto the X,Y resolutionof the optics
Samplingissufficient whenthereisenoughpixels to
separatetwoadjacent Airy disk
In imaging, frequency= spatial frequency
f
sampling
= 2.3 x f
highest
(to compensatelow-passfiltering)
Nyquist theorem:
to reconstruct a sine wave: f
sampling
= 2 x f
wave
Sampling in confocal microscopy
The highest frequency to be sampled in the CLSM is imposed
by the optical system :
f
highest
= 1/resolution
To fulfill the Nyquist criterion :
f
sampling
= 2.3/r
lateral
Pixel size ~r
lateral
/2.3 > oversampling undersampling>
Sampling in confocal microscopy
Critical sampling distances @ 500 nm
(for pinhole = 1 AU values by 50%)
Ideal emission separation
Green emission filter
Red emission filter
Dichroic
beamsplitter
PMT 2
PMT 1

Crosstalk problems
Most of the time there is some overlapping between
fluorophoresemission spectra
Example of FITC and TRITC
Using 488 nm and 543 nm lines : 22% overlap
If the fluorescence signals
are not taken sequentially :
some of the green
fluorescence is assigned to
the red channel
Crosstalk problems
To avoid bleed-through of one fluorescence in another
channel, multitrackconfigurations 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
Spectral separation
When the emission spectra of the fluorophoresare very close :
Spectral detector (like the Meta detector) allow the record of the
emission spectra of each pixel of the image
Exampleof latex beadwith
narrowfluorescences in the
coreandthering acquired
withthespectral detector
(Meta)
Image serieof thebeadat
different wavelengths
Spectral separation
Fluorescence separation
after software unmixing
Selection of the
different fluorescences
(core and ring)
FRAP
Fluorescence Recovery After Photobleaching
The recovery of fluorescence in this region indicates
any kind of movement (diffusion or transport) of
fluorescent molecules
The recovery time (half-recovery time) indicates the
speed of this mobility
Use of the high power of the laser to photobleach
a defined region of the sample
bleach recovery
FRAP experiments
FRAP-recording for 40 min (1 frame/min)
Photobleaching
Bovine endothelial cells
actinfilaments (BODIPY FL),
mitochondria (MitoTracker Red);
some mitochondria are marked
for 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 any
shape
Other beam scanning techniques
Multiple beam scanning : the Nipkowdisk
Disk rotation
One way to increase the scanning
speed is to increase the number
of scanning spots.
The spinning disk with pinholes
was introduced into a microscope
by Mojmir Petranin 1968.
Improvement of the Nipkowdisk
principle in the YOKOGAWA scanhead
specimen
Laser beam
Collector disk
CCD camera
Dichroic
mirror
Aperture disk
Microlenses
(20 000)
Pinholes
(20 000)
Objective
Nipkowdisk confocal microscope facilitate
studies of ligth-sensitive processes
Cell cycle in
Drosophila Embryo
expressing GFP-
Histone
Dr. Caetano Gonzalez
EMBL
Ca
2+
waves in
cardiomyocytes
loaded with fluo-3
Dr. Marisa J aconi
Geneva
Image capture at 33 Hz using an intensified camera
(Coolsnap Cascade from Photometrics)
Nipkowdisk confocal microscope facilitate
studies of fast processes
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 only
one 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 :
the line scan mode
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Fluo-3
Fura-red
Spatially restricted, but very fast (1 line/2ms)