Basics in Confocal Microscopy

Handouts

23.2. – 26.2.2015EMBL internal course

Advanced Light Microscopy Facilityalmf@embl.de

Confocal Principle

Z

Confocal Pinhole

Dichroic beamsplitter

Objective lens

Focal plane

Photomultiplier

Point Scanning

Illumination Pinhole

Optical sectioning

Advantages+High resolution+Optical sectioning+Multiple channels

simultaneously+FRAP and photo

-activation studies possible

Disadvantages−Less light efficient bleaching

−Slower acquisition−Expensive

Arabidopsis root, transmission image overlaid with isosurface rendered GFP fluorescence indicating the beginning of cell division (reporter construct byP. Dörner, Edinburgh)

AOTFAcousto Optical Tunable Filter

0

20

40

60

80

100

120

400 450 500 550 600 650 700

wavelength (nm)

Tran

smitt

ance

%

458 476488 514 543 633405 561 594

Leica AOBS

Carl Zeiss LSM710 / 510 META

Olympus Fluoview 1000

Single beam Multiple beams Nipkow Disk

Multibeam Confocal Microscopy

Root hair cell of Arabidopsis, GFP in the Cytoplasm (T. Timmers, toulouse). Depth color coded.

Advantages+Optical sectioning (less than CLSM)+Higher framerate as CLSM+Less photobleaching and –toxicity than

CLSM

Disadvantages−Less depth descrimination than CLSM−Multichannel usually sequential

no optical zoom in

Single beam Multiple beamsNipkow Disk

Multibeam Scanning

Line Scanning

Spectral Detection

Leica SP2 AOBS Zeiss LSM META Olympus Fluoview1000

Imaging e.g. CFP and YFP simultaneous is important for some biological applications.

Using a multitude of different dyes, e.g. marking several genes on chromosomes, leads to spectral overlap which can be corrected by linear unmixing.

Emission spectra of different FPs

Advantages:+ High signal to noise ratio+ Very fast acquisition possible+ Single molecule detection+ Very good for studying vesicle-membrane

fusion events and cell adhesion

Image from TILL Photonics brochure

TIRF MicroscopyIn Total Internal Reflection (TIRF) microscopy light is coupled into the optics above a critical angle which reflects the light totally but creates an evanescent wave about 50-200 nm next to the reflecting surface (cover slip).

Disadvantages:− Only fluorescence directly at cover slip

Extremely thin depth of fieldLaser adjustment to TIRF mode visualized with fluorescein solution

Time-lapse in TIRF mode of VSVG-GFP expressing cells. Vesicle movement near the plasma membraneand fusions with the plasma membrane are observed.

Which Microscope Type To Use ?

Non-confocal

Single-beam confocal

Multi-beam confocal

z xy

Applications:Non-confocal: • fast and long-term time-lapse of flat samples• FRAP + Photoactivation with additional laser

Multibeam:• Fast and long-term time-lapse with

increased Z-resolution and less phototoxicitythan CLSM

Confocal:• Imaging for high resolution 3D rendering• Multi-channel time-lapse• FRAP + Photoactivation