Download Basics in Confocal Microscopy Handouts

Basics in Confocal Microscopy
Handouts
23.2. – 26.2.2015
EMBL internal course
Advanced Light Microscopy Facility
[email protected]
Confocal Principle
Photomultiplier
Confocal Pinhole
Dichroic beamsplitter
Point Scanning
Illumination Pinhole
Objective lens
Focal plane
Advantages
+ High resolution
+ Optical sectioning
+ Multiple channels
simultaneously
+ FRAP and photo
-activation studies
possible
Disadvantages
− Less light efficient
 bleaching
− Slower acquisition
− Expensive
Optical sectioning
Z
Arabidopsis root, transmission image overlaid with isosurface rendered
GFP fluorescence indicating the beginning of cell division (reporter construct by
P. Dörner, Edinburgh)
AOTF
Acousto Optical Tunable Filter
Leica AOBS
120
100
T r a n s m itta n c e %
80
60
40
20
0
400
450
500
550
600
wavelength (nm)
405
458
476488 514
543
561
594
633
650
700
Carl Zeiss LSM710 / 510 META
Olympus Fluoview 1000
Multibeam Confocal Microscopy
Single beam
Multiple beams
Nipkow Disk
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
Root hair cell of Arabidopsis, GFP in the Cytoplasm
(T. Timmers, toulouse). Depth color coded.
Multibeam Scanning
Single beam
Multiple beams
Nipkow Disk
Line Scanning
Spectral Detection
Imaging e.g. CFP and YFP simultaneous is
important for some biological applications.
Emission spectra of different FPs
Using a multitude of different dyes, e.g. marking
several genes on chromosomes, leads to
spectral overlap which can be corrected by
linear unmixing.
Leica SP2 AOBS
Zeiss LSM META
Olympus Fluoview1000
TIRF Microscopy
In 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).
Image from TILL Photonics brochure
Extremely thin depth of field
Advantages:
+ High signal to noise ratio
+ Very fast acquisition possible
+ Single molecule detection
+ Very good for studying vesicle-membrane
fusion events and cell adhesion
Laser adjustment to TIRF mode visualized
with fluorescein solution
Time-lapse in TIRF mode of VSVG-GFP expressing
cells. Vesicle movement near the plasma membrane
and fusions with the plasma membrane are observed.
Disadvantages:
− Only fluorescence directly at cover slip
Which Microscope Type To Use ?
Non-confocal
Multi-beam confocal
Single-beam
confocal
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 phototoxicity
than CLSM
Confocal:
• Imaging for high resolution 3D rendering
• Multi-channel time-lapse
• FRAP + Photoactivation
z
xy