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This presentation has been put together as a common effort of Urs Ziegler, Anne Greet Bittermann, Mathias Hoechli. Many pages are copied from Internet web pages or from presentations given by Leica, Zeiss and other companies. Please browse the internet to learn interactively all about optics. For questions & registration please contact www.zmb.unizh.ch .
Interactive models, movies and animations can be published on web-pages or into power point-presentations.
Digital images:
2-dimensional distribution of image Points (Pixel)
x y
Digital resolution
Detectors record a limited amount of image points (pixel number) within a xy grid. Each image point has its own grey level (dynamic range). Increasing the amount of image points as well as the number of grey levels leads to bigger image les and longer calculation times. 256 grey levels are coded by 8 bit. 256 grey levels are presented by a computer monitor. Today, detectors are pushed to discriminate 1024, 4096 or more grey levels. The human eye can discriminate about 60 gray levels (6 bit).
3D Data set
x y z
)!z )!z )!z )!z
The information within the optical sections along the z-axis can be used to reconstruct a 3-dimensional image.
4D Data set
3-D stacks recorded along the time course
x y z
)!z )!z )!z )!z )!z )!z )!z )!z
t1
t2
5D Data set
Wavelenghts adding another dimension of uorescent data. Time laps of multi-channel 3D stacks generate a 5D data set. Wavelenght information is displayed as pseudo-colors.
x y z
)!z )!z )!z )!z )!z )!z )!z )!z )!z )!z )!z )!z
Voxels
A voxel (= volume element) is the 3D-equivalent of the 2D-pixel. It is the smallest unit of a sampled volume.
The given maximal lateral (x,y) resolution of 0.2 m and the axial (z) resolution of 0.4 m of a voxel results in an elongated shape (point spread function).
Neighbours
For the calculation and visualisation are the neighbor voxels of great importance.
Image Gallery
Galleries of images are the most simple data presentation. for xyz xyt xy! ...
Projecting the structures of all sections to the ground level (Extended Focus)
Projection types
Average Projection:
Simple to very complex mathematical procedures. Summing up the grey values of all voxels with identical xy-coordinates along the z-stack, divided by the numbers of optical sections.
Background signal gets projected too and might cause noise/blur. Suppress background rst!!
x1 y1 x2 y2 x3 y3
Z3 Z2
x1
Z1
y1 x2 y2 x3 y3
Z1
Z2
Z3
Z4
Z4
Z5
Z5
Projektion
Projektion
10
Maximum intensity projection with one sided illumination and shadow. (easy3D)
x z
y z
X-Y
Y-Z
{ { { {
Computer representation of section levels in XY, XZ, YZ
X-Z
2. Surface rendering
Segmentation of z-stacks Depth encoding of voxels Shadowing
3. Animations
time course rotations zooms etc.
Volume rendering
Even if fog (background) limits the visibility, we get an idea of the structure of the trees.
Volume rendering
Volume Screen
Virtual ray
Ray Tracing
A virtual ray passing the volume accumulates the grey levels of the voxels, normalizes the summed value and presents it on the screen.
Adjustment of the grey level according to the distance between voxel and screen.
Adjustment of the grey value according to the grey value of the voxel just passed.
screen
Surface rendering
The normal to the polygon and the viewing direction include the angle " .
Threshold 68
Threshold 138
Stereo-Representation
The depth feeling can be simulated by calculating two separate slightly tilted 3D-models of the same scene as if they were viewed by the left eye and the right eye. The nal stereo pair can be observed using different techniques.
The 3D impression can be achieved squinting the eyes or using special stereo viewers (or crossing the eyes).
StereoRepresent ation II
The 2 pictures of the stereo pair are colored in red & green and superimposed. The 3D impression can be achieved using bicolor goggels.
Looking inside
Surface view
x-z
Mo l,stained with acridine orange - 20 optical sections 3D-representation: x-y, x-z, y-z
Looking inside
Looking inside
Animations - y through
Volume and surface rendering allow you to turn and zoom the data set. Extreme Zoom allows you to virtually enter the sample.
Measurements
i.e.: - Automated data segmentation - Particle counting - Size regognition - Distance measuerments - Filament tracking - Movement tracing " Results are visualized in the 3D model " Results are listed as numbers in Exel-sheets
Colocalisation
The relation of the intensity values from 2 channels are presented in a two dimensional histogram. In case of colocalization, the intensity clouds of both channels are overlapping. Colocalization is not an absolute fact but allways relate to voxel size and resolution.
Animations
Animations are series of single images put together into a movie. The images might be a volume view, a projection, a slice, a time point. The animation is done by just playing the sequential data set, or by rotating 3D models or volume representations, by zoom-in & ythrough motions, changing of surfaces and transparencies, etc.
Today#s computer allow to calculate and represent animated sequences reasonably fast. Movie les can be published i.e in power point or on the web. Also interactive le formats are possible.
Animation in time
t1 t2 t3 t4
Changes of a 3D-volume with time might be presented as a gallery of projection views or as a movie. Animation and stereo view facilitate the recognition of spheric relations in this context.
Deconvolution
What is to be gained? Increase in resolution x, y, z Noise is reduced The image formation process is optimized (astigmatism, point spread function, ...)
Wideeld uorescent data can be improved a lot by deconvolution. Confocal data show less z-distortions, less out-of-focus blur,... -> deconvolution shows only very little effect.
Convolution - Theory
Deconvolution procedure
measured
Measure object of known size, but smaller than the resolution of the microscope (i.e. 100nm uorescent beads) Compare the microscope image with the ideal/theoretical representation of the object. Determine the difference of the measured and the real object. Correct unknown objects with the determined difference.
real
Deconvolution effect