exposed to light illumination of a wavelength 
between 600 and  700nm  (therapeutical 
wavelength). Photoactivation induces a toxic 
effect in the tumor cells by producing singlet 
oxygen (,O?) and/or free radicals in the 
cytoplasm. This reaction which takes place 
mainly in the dark after light exposure, affects 
essential organelles in their function (cell 
membrane, mitochondria) and leads to tumor 
cell necrosis. 
In addition to the therapeutical application 
and in contrast to the traditional cancer 
treatment modalities photosensitizers are very 
well suited for diagnostic purposes. Laser light 
irradiation of shorter wavelength (488nm or 
514nm) induces a strong fluorescence which is 
utilized to localize and quantify the extend of 
tumor cells or tissue. 
In this paper we discuss a methodology to 
extract quantitative information about the 
distribution, density, size and locality of the 
photosensitizer accumulated in tumor cells or 
tissue from a stack of microscopical confocal 
laser scanning images. 
2. CLINICAL SETUP 
Although first treatments in PDT have been 
performed at the Department of Gynaecology 
and Obstetrics of the University Hospital in 
Zurich, this modality is at an early stage. This 
clinic has established a versatile system for both 
research and clinical applications in the field of 
PDT [2, 3, 4]. This setup allows to study 
biomedical processes during photodynamic 
activity for a broad range of conditions, 
extending from the patient down to subcellular 
structures. Investigations at the microscopic 
level will be performed in true three dimensions 
with living cells, colonies and tissue specimens 
in vitro. This is possible by a combination of 
confocal laser scanning microscopy (CLSM) 
  
which allows the non-destructive optical slicing 
of a probe, and specific hard- and software 
which has been developed at IBTZ at the 
University and ETH in Zurich. 
3. CONFOCAL LASER SCANNING 
MICROSCOPY 
In a CLSM a finely-focused laser spot is 
arranged in such a way that it coincides with the 
back-projected image of a point detector 
forming a confocal arrangement [6, 7]. The 
specimen is scanned through this confocal spot 
in three dimensions in order to generate a stack 
of image. The main benefits from this technique 
are three fold: 
- an improvement in resolution by a factor of 
up to two over conventional light micro- 
scopy. 
- a decrease in scattered light strength which 
results in an additional resolution 
improvement. 
- optical sectioning capability by rejecting 
out of focus information thus allowing 
systematic investigations of thick speci- 
mens. 
The CLSM setup used consists of a Leica 
True Confocal Scanner (TCS 4D). The resulting 
stack of images contains up to 140 planar slices 
with a minimal lateral resolution of ~170nm at 
512 by 512 pixels and a corresponding slice 
thickness of about ^600nm!. The signal strength 
is digitized into 256 grey levels. 
4. IMAGE PROCESSING SOFTWARE: 
QUASIA3D 
The CLSM technique offers a precise depth 
discrimination combined with an enhanced 
lateral resolution not attainable with 
conventional light microscopy. To be able to 
1 for an emission wavelength of 514nm. The lateral as well as the on-axis resolutions in a CLSM are dependant of 
the emission wavelength of the fluorescence dye and the variable pinhole diameter of the CLSM.. 
314 
International Archives of Photogrammetry and Remote Sensing. Vol. XXXI, Part B5. Vienna 1996 
  
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