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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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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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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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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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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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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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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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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 >

S li i f l i


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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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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



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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)

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