Confocal Microscopy Principles

Confocal Microscopy 

Dr. Serge Arnaudeau 

Bioimaging Core Facility 

Geneva

Light source 

Wide-field microscope 

in focus 

Sample 

(object plane) 

objective 

• only one plane in focus 

• but all the planes contribute to the image 

Viewing plane 

(image plane)

Light source 

in focus 

Sample 


The pinhole 

objective 

Photons passing through the pinhole are coming 

exclusively from the focal point of the objective 

pinhole 

Viewing plane 

(image plane)

Depth of field depends on pinhole size 

in focus 

objective 

small pinhole 

large pinhole 

small pinhole most of the photons coming from out of 

focus planes are rejected and do not contribute to the image

Light 

source 

Confocal microscope principle 

pinhole 

Transmissive design 

• “conjugate focal planes” 

objective 

Sample 


• illumination and detection of the same focal point 

• need to displace the sample in x and y to construct an image 

x 

y 

objective 

pinhole 

detector

Absorb high 

energy photons 

What is fluorescence? 

Excited state 

Emit lower 

energy photon 

Ground state 

In one-photon excitation λ ex < λ em (Stoke’s shift)

How does a fluorescence microscope 

Emission filter 

Dichroic filter 

objective 

work? 

Excitation filter 

sample 

Light source

Epitaxial confocal microscope for 

• use of the same objective for illumination 

and detection 

• use of a laser source to avoid the use of a 

pinhole in illumination 

fluorescence 

• 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 

photomultiplier tube 

pinhole 

dichroic mirror 

objective 

Focal point 

Laser Source

Advantage of fluorescence confocal 

• 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 

microscopy 

A section of mouse intestine 

imaged with both confocal and 

non-confocal microscopy

How big is a Laser Scanning Confocal 

Laser module 

405, 458, 477, 488, 

514, 561, 633 nm 

System electronic rack 

Microscope ? 

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-field microscope : entire depth of the specimen over 

a wide area is illuminated 

• Confocal microscope : the sample is scanned with a finely 

focused spot of illumination centered in the focal plane

Beam scanning 

Majority of laser scanning microscopes : single beam 

scanning 

Laser spot 

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 

Only confocal microscopes which use acousto-optic 

deflectors can scan at speeds of 30 frames/s

Photomultiplier Tubes (PMT) 

Photocathode 

Window 

Incident light 

Side on design 

Anode 

Dynodes 

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 dark current of a 

photomultiplier tube are determined by the composition of the photocathode 

Quantum Efficiency (%) 

100 

10 

1 

0.1 

0.01 

100 200 300 400 500 600 700 800 900 1000 

Wavelength (nm) 

Gray levels (8bits) 

255 

128 

0 

600 V 

0 V 

800 V 

gain 

50 V offset 

Low quantum efficiency and low dynamic range but very fast response time

Scanning speed influences image 

quality 

pixel dwell time 3.2 µs pixel dwell time 25.6 µs 

Better signal to noise ratio with low scanning speeds but 

samples are more exposed to the laser beam 

2 µm 

Muntjac cells – Alexa 555 anti OX Phos complex V inh prot

Scans averaging reduces noise 

Average of 2 scans Average of 8 scans 

But greatly reduce the frame rate 

10 µm 

Muntjac cells – Alexa 488 phalloidin

Airy disk and Resolution 

Due to diffraction, the image of a point source of light in the focal 

plane is not a point it’s actually an Airy diffraction pattern 

Airy diffraction pattern 

Airy disk 

The resolving power of an objective determines the size of the 

Airy diffraction pattern formed


The radius of the Airy disk is given by : 

α 

r (Airy) = 0.61 λ exc /NA (obj) 

with NA (obj) = n sinα 

n = medium refractive index 

α = objective angular aperture

intensity 


Rayleigh criterion for lateral resolution : 

the center of one airy disk falls on the first minimum of the 

other airy disk 

contrast 

resolved Rayleigh criterion unresolved

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

Confocal fluorescence : 


pointwise illumination + pointwise detection 

narrower Point Spread Function / widefield microscopy 

Axial PSF intensity profiles 

widefield confocal 

Increase in lateral resolution 

r lateral = 0.4 λ exc / NA 

confocal lateral resolution > widefield lateral resolution


Confocal PSF 

Axial resolution : 

r axial = 1.4 λ exc n/ NA 2 

The PSF is elongated in the axial direction 

Axial resolution of an objective is worse than 

its lateral resolution 

λ exc = excitation wavelength 

n = medium refractive index 

NA = objective’s numerical aperture 

For an oil immersion objective with 1.4 NA using the 488 nm laser line 

r lateral = 0.4 x 488/1.4 = 139 nm (in theory for very 

r axial = 1.4 x 488 x 1.515/(1.4) 2 = 528 nm small pinhole size)

Resolution depends on pinhole size 

Pinhole : 1 AU 

(optical slice ~ 0.8 µm) 

Pinhole : 0.5 AU 

(optical slice ~ 0.5 µm) 

Better Z discrimination with small pinhole size but needs 

strong signals 

10 µm 

Muntjac cells – Alexa 488 phalloidin

Z 

Optical sectionning 

Y 

X 

5 

4 

3 

2 

1 

z 

y 

x 

5 

4 

3 

2 

1 

3D reconstruction

Sampling in confocal microscopy 

Voxel on the sample 

z 

x 

y 

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 pixelization effect even 

with very high zoom 

y 

x 

Pixel on the image


260 nm/pixel 

16 nm/pixel 

512x512 zoom 1 

512x512 zoom 16 

20 µm 

1 µm 

130 nm/pixel 

33 nm/pixel 

512x512 zoom 2 

512x512 zoom 8 

10 µm 

2 µm 

65 nm/pixel 

512x512 zoom 4 

5 µm 

Muntjac cells 

Alexa 488 phalloidin 

Alexa 555 anti OXPhos complex V inh prot 

TO PRO-3


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 

Nyquist theorem : 

to reconstruct a sine wave : f sampling = 2 x f wave 

In imaging, frequency = spatial frequency 

f sampling = 2.3 x f highest (to compensate low-pass filtering)


The highest frequency to be sampled in the CLSM is imposed 

by the optical system : 

f highest = 1/resolution 

To fulfill the Nyquist criterion : 

undersampling > 

f sampling = 2.3/r lateral 

Pixel size ~ r lateral /2.3 > oversampling


Critical sampling distances @ 500 nm 

(for pinhole = 1 AU values by 50%)

Ideal emission separation 

Red emission filter 

Dichroic 

beamsplitter 

λ τ 

PMT 1 

λ τ 

λ τ 

PMT 2 

Green emission filter

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 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, 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

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 the 

core and the ring acquired 

with the spectral detector 

(Meta) 

Image serie of the bead at 

different wavelengths

Spectral separation 

Fluorescence separation 

after software unmixing 

Selection of the 

different fluorescences 

(core and ring)

FRAP 

Fluorescence Recovery After Photobleaching 

bleach recovery 

Use of the high power of the laser to photobleach 

a defined region of the sample 

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

FRAP experiments 

FRAP-recording for 40 min (1 frame/min)

Very high control of the scanner 

by the DSP (Digital Signal 

Processor) to position the laser 

beam and choose ROI of any 

shape 

Photobleaching 

Bovine endothelial cells 

actin filaments (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)

Other beam scanning techniques 

Multiple beam scanning : the Nipkow disk 

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 Petran in 1968.

Improvement of the Nipkow disk 

principle in the YOKOGAWA scanhead 

Collector disk 

Aperture disk 

Objective 

specimen 

Dichroic 

mirror 

Laser beam 

Microlenses 

(20 000) 

Pinholes 

(20 000) 

CCD camera

Nipkow disk confocal microscope facilitate 

Cell cycle in 

Drosophila Embryo 

expressing GFP- 

Histone 

Dr. Caetano Gonzalez 

EMBL 

studies of ligth-sensitive processes

Nipkow disk confocal microscope facilitate 

Ca 2+ waves in 

cardiomyocytes 

loaded with fluo-3 

Dr. Marisa Jaconi 

Geneva 

studies of fast processes 

Image capture at 33 Hz using an intensified camera 

(Coolsnap Cascade from Photometrics)

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

Fluo-3 

Fura-red 

10 μm 

Advantage of the LSCM : 

the line scan mode 

10 μm 

[Ca 2+ ] i (nM) 

150 

75 

0 

200 ms 

Spatially restricted, but very fast (1 line/2ms) 

250 

125 

0 

[Ca 2+ ] i (nM)

Confocal Microscopy Principles

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