# Laboratory for experimental botany

## Image densitometry

Image densitometry is an image analysis method used to quantify the amount of staining in the specimen.

• we use it to measure the absolute or relative amount of DNA in plant nuclei
• specimens are either squashes of meristematic tissue or histological tissue sections
• specimens stained by the Feulgen reaction (with Schiff's reagent, which stochiometrically binds to DNA)
• absorbance of Feulgen-stained nuclei is proportional to the amount of nuclear DNA Transmittance of nuclei on a glass microscope slide

After Beer-Lambert law, the absorbance at a given wavelength is linearly related to the concentration of light-absorbing molecules in the sample. Absorbance is calculated from transmittance of the sample and it is the transmittance of nuclei that we measure with the microscope camera.

A = ecd
A = - log10(T) = - log10(I/I0)
A - absorbance, e - extinction coeffient, c - concentration of the light-absorbing molecules, d - lenght of the light path through the sample, T - transmittance, I0 - incident light intensity, I - transmitted light intensity

We are interested only in a relative amount of DNA in the nuclei. Squashed nuclei on the slide have approximately the same thickness, so the length of the light path through the sample (d) is constant, and so is the extinction coefficient. Therefore we can use optical density (OD) expressed in relative units to express the relative amount of DNA in the nuclei:

A = OD×d
A = ecd
=> OD ~ c (where OD is expressed in relative units)
A - absorbance, OD - optical density, d - lenght of the light path through the sample, e - extinction coeffient, c - concentration of the light-absorbing molecules

However, because the distribution of DNA in the nuclei is not homogenous, optical density must be calculated for the smallest possible parts of the nuclei (image pixels), and then all ODs are summed together to give IOD (integrated optical density). IOD is linearly related to the amount of DNA in the nucleus.

IOD = sum(ODi)
IOD - integrated optical density, ODi - optical density of individual pixels in the image of a nucleus Microscope Axiskop 2 MOT (Carl Zeiss) with a video CCD camera (Sony) used for image densitometry

## Measurement of cell size

• cell size is measured on plant tissue sections by outlining cell walls
• cell walls are visualized using their autofluorescence
• volume of cells is estimated from the area of the cell's transect (as a sphere or a cylinder)

## 3D reconstruction of plant tissues

• maize endosperm (Vilhar et al., 2002 Plant Physiology)
• root cap (Kladnik et al., 2004 Canadian Journal of Botany)