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Confocal Laser Scanning Microscopy (CLSM) and its application to fossil dinoflagellate cysts


3. Image acquisition

A point light source is imaged on the object plane. Emitted fluorescence light or reflected light is directed to a photomultiplier through the detector pinhole. The point is displayed on a computer screen as a pixel.
By scanning the object point by point, line by line, very thin, sharp, blur-free optical sections are recorded. A series of optical sections forms an image stack.


Types of images
(Example Willeidinium baiocassinum FEIST-BURKHARDT 1995):

Willeidinium baiocassinum FEIST-BURKHARDT 1995
(specimen embedded in ventral orientation)

By scanning the object point by point, line by line, very thin, sharp, blur-free optical sections are recorded.
This is one single optical section (number 17) out of a series of 44 optical sections through the 22.4 Ám thick specimen.

Willeidinium baiocassinum FEIST-BURKHARDT 1995
(specimen embedded in ventral orientation)

Series of optical sections.
The 22.4 Ám thick specimen is scanned from up to down in 44 optical sections with a distance between the optical sections of 0.51 Ám.
All 44 optical sections are illustrated here in an image gallery.

Willeidinium baiocassinum FEIST-BURKHARDT 1995
(specimen embedded in ventral orientation)

Extended focus image.
By projection of all optical sections of a specimen (the image stack) into one plane a so-called extended focus image is generated. This image displays a high depth of focus which is comparable to that of SEM micrographs.
Number of projected optical sections: 44.
Thickness of specimen: 22.4 Ám.

Willeidinium baiocassinum FEIST-BURKHARDT 1995
(specimen embedded in ventral orientation)

Axial optical sectioning of the object is possible in two ways.
Either by
- direct acquisition of x/z- or y/z-optical cross sections, or
- calculation of vertical optical sections using the normal x/y-image stack (as shown here).

Willeidinium baiocassinum FEIST-BURKHARDT 1995
(specimen embedded in ventral orientation)

Rotation and volume rendering.
Rotation of the image stack and projection into one plane allows viewing the object from different angles.

Willeidinium baiocassinum FEIST-BURKHARDT 1995
(specimen embedded in ventral orientation)

Stereoscopic imaging.
The combination of a left and a right image either as
- stereo pairs or
- red/green anaglyphs
allows easy visualisation of 3D-structures!

Please use red/green glasses for stereoscopic viewing of this red/green anaglyph.