Confocal Microscopy

Dr. Serge Arnaudeau Bioimaging Core Facility Geneva

Wide-field microscope
in focus

Light source

Sample (object plane)

objective

Viewing plane (image plane)

only one plane in focus but all the planes contribute to the image

The pinhole
in focus pinhole Light source

Sample (object plane)

objective

Viewing plane (image plane)

Photons passing through the pinhole are coming exclusively from the focal point of the objective

Depth of field depends on pinhole size

small pinhole in focus objective

large pinhole

small pinhole most of the photons coming from out of focus planes are rejected and do not contribute to the image

Confocal microscope principle

x pinhole Light source detector Transmissive design Sample objective (object plane) y pinhole

objective

conjugate focal planes illumination and detection of the same focal point need to displace the sample in x and y to construct an image

What is fluorescence?
Excited state

Absorb high energy photons

Emit lower energy photon Ground state

In one-photon excitation ex < em (Stokes shift)

How does a fluorescence microscope work?

Emission filter

Light source Dichroic filter

objective

Excitation filter

sample

Epitaxial confocal microscope for fluorescence

photomultiplier tube barrier filter pinhole

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
objective dichroic mirror

Laser Source

use of a stepping motor in the Z direction to make optical slices in the sample

Focal point

Advantage of fluorescence 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
A section of mouse intestine imaged with both confocal and non-confocal microscopy

How big is a Laser Scanning Confocal Microscope ?

Laser module 405, 458, 477, 488, 514, 561, 633 nm Scanning head

System electronic rack

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 )

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)

Anode Photocathode Window

Incident light

Dynodes

Side on design

Gain varies with the voltage across the dynodes and the total number of dynodes With typically 9 dynodes, gain of 4x106 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
100

Quantum Efficiency (%)

255
10

600 V

800 V

Gray levels (8bits)

gain

128

0.1

0
0.01 100 200 300 400 500 600 700 800 900 1000

0V

50 V

offset

Wavelength (nm)

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

Scanning speed influences image quality

Muntjac cells Alexa 555 anti OX Phos complex V inh prot

2 m

pixel dwell time 3.2 s

pixel dwell time 25.6 s

Better signal to noise ratio with low scanning speeds but samples are more exposed to the laser beam

Scans averaging reduces noise

Muntjac cells Alexa 488 phalloidin
10 m

Average of 2 scans

Average of 8 scans

But greatly reduce the frame rate

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
Airy diffraction pattern

Airy disk The resolving power of an objective determines the size of the Airy diffraction pattern formed

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

Rayleigh criterion for lateral resolution : the center of one airy disk falls on the first minimum of the other airy disk
intensity

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

Pinhole and Resolution

Confocal fluorescence : pointwise illumination + pointwise detection narrower Point Spread Function / widefield microscopy
Axial PSF intensity profiles

Increase in lateral resolution

rlateral = 0.4 exc / NA

widefield confocal

confocal lateral resolution > widefield lateral resolution

Pinhole and Resolution

Confocal PSF

The PSF is elongated in the axial direction Axial resolution of an objective is worse than its lateral resolution

Axial resolution : raxial = 1.4 exc n / NA2

exc = excitation wavelength n = medium refractive index NA = objectives numerical aperture

For an oil immersion objective with 1.4 NA using the 488 nm laser line rlateral = 0.4 x 488/1.4 = 139 nm raxial = 1.4 x 488 x 1.515/(1.4)2 = 528 nm (in theory for very small pinhole size)

Resolution depends on pinhole size

Muntjac cells Alexa 488 phalloidin
10 m

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

Optical sectionning
X Y

5 4
5 4 3 2 1

y 1 x 3D reconstruction

Sampling in confocal microscopy

x y 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 pixelization effect even with very high zoom

x y

Voxel on the sample

Sampling in confocal microscopy

512x512 zoom 1
260 nm/pixel

512x512 zoom 2
130 nm/pixel

512x512 zoom 4
65 nm/pixel 20 m 10 m

512x512 zoom 8 512x512 zoom 16

16 nm/pixel 5 m 33 nm/pixel

2 m 1 m

Muntjac cells Alexa 488 phalloidin Alexa 555 anti OXPhos complex V inh prot TO PRO-3

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 Nyquist theorem : to reconstruct a sine wave : fsampling = 2 x fwave In imaging, frequency = spatial frequency fsampling = 2.3 x fhighest
(to compensate low-pass filtering)

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

undersampling > Pixel size ~ rlateral/2.3 > oversampling

Sampling in confocal microscopy

Critical sampling distances @ 500 nm (for pinhole = 1 AU values by 50%)

Ideal emission separation

PMT 1

Red emission filter

Dichroic beamsplitter

PMT 2

Green emission filter

Crosstalk problems
Most of the time there is some overlapping between fluorophores emission spectra
Example of FITC and TRITC

If the fluorescence signals are not taken sequentially : some of the green fluorescence is assigned to the red channel

Using 488 nm and 543 nm lines : 22% overlap

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
Selection of the different fluorescences (core and ring)

Fluorescence separation after software unmixing

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)

Photobleaching
Bovine endothelial cells actin filaments (BODIPY FL), mitochondria (MitoTracker Red); some mitochondria are marked for photobleaching

Very high control of the scanner by the DSP (Digital Signal Processor) to position the laser beam and choose ROI of any shape

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

Laser beam Collector disk Microlenses (20 000) CCD camera

Aperture disk Dichroic mirror

Pinholes (20 000)

Objective

specimen

Nipkow disk confocal microscope facilitate studies of ligth-sensitive processes

Cell cycle in Drosophila Embryo expressing GFPHistone Dr. Caetano Gonzalez EMBL

Nipkow disk confocal microscope facilitate studies of fast processes

Ca2+ waves in cardiomyocytes loaded with fluo-3 Dr. Marisa Jaconi Geneva

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

Advantage of the LSCM : the line scan mode

Fluo-3 [Ca2+]i (nM) 250 10 m 125 0 Fura-red [Ca2+]i (nM) 10 m 150 75 0 200 ms

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