tical slicing 
id software 
3TZ at the 
NNING 
iser spot is 
des with the 
nt detector 
[6, 7]. The 
onfocal spot 
rate a stack 
is technique 
y a factor of 
light micro- 
ength which 
resolution 
by rejecting 
us allowing 
thick speci- 
| of a Leica 
[he resulting 
planar slices 
f ~170nm at 
onding slice 
onal strength 
TWARE: 
yrecise depth 
n enhanced 
able with 
9 be able to 
'e dependant of 
6 
  
analyze and visualize the resulting stacks of 
slice images the concept of a dedicated volume 
processing software with a strong emphasis on 
the demands of CLSM has been defined. 
QUASIA3D (QUantitative — Analytical 
System for Image Acquisition in 3D) addresses 
in particular two problems which are inherent to 
volumes generated by CLSM: 
-a high number of detected objects per 
volume which when visualized give rise to 
a large number of polygons (-5*10° and 
more) each with a small number of pixels 
(<30). 
- numerous parameters influence the imaging 
process in CLSM and lead to distortions of 
the volume. information. The most 
important problems are: 
. complex light absorption processes for 
excitation and emission 
. local sensitivity due to position 
dependant relative aperture 
» possible bleaching effects of 
photosensitizers 
. light scattering 
» low SNR due to the extremely fine 
focus volume 
5. QUANTIFICATION SCHEME IN 3D 
The main specification of QUASIA-3D for 
the quantitative approach is to deduce 
fluorophore content from the recorded 
brightness of a confocal image stack. The 
quantification process relays on the fluorometry 
equation: 
J floresence = Jicadiaion P nücropkor M icrophore 
with 
j : Emitted (observed) flux density 
floresence 
: Illumination spatial light flux 
density total 
J irradiation 
with 
Bootie Photosensitizer dye concentration 
: Photoefficiency of the dye for the 
M oropliors = p. 
specific irradiation wavelength 
If we consider for a moment a fibre fluorometer 
which records the total flux from an object in 
space we find: 
3 
— Q 2 
flux woud > 4 Tt NN) iin Y] oom | p sid 
Volume 
7-4 TT N, "7 irradiation : yarns E VA ner ire 
with Qthe space angle covered by the 
fluorometer, m,the gathering efficiency of the 
fibre tip and m the mass of all the fluorophores. 
The situation for a microscope is similar. With 
the numerical aperture NA = tan(a) 
Q= f 2m sin} dx -2 (1-cos(a) 
1 
2 
1+NA 
  
—-2Tt|1— 
  
the total recording efficiency is 
N oil n lens nu M ia 
4 4 
solvent cover 
  
n=o 
4 
Sensor (n +n f (n +n f n +n f 
oil lens oil cover solvent cover 
with othe sensitivity of the microscope's 
registration sensor, n. the refraction indices of 
the various media (immersion oil, lens, cover 
glass). 
Based on a known San the reconstruction 
for an ideal microscope would be trivial. Due to 
several distortions the CLSM observations are 
far from ideal for quantification purposes [8]. 
The list shows various influences ordered in 
International Archives of Photogrammetry and Remote Sensing. Vol. XXXI, Part B5. Vienna 1996