Assessment of tissue volume
The volume of the disector is calculated by multiplying its (x; y; z) dimensions. If the entire space of the disector is fully filled with the examined tissue, the disector volume is equal to the tissue volume and the number of autophagosomes is directly related to the disector volume:
E. g., if the disector (x; y; z) dimensions are equal to (50; 50; 7.5) µm in the meristematic zone, the volume of the disector is:
If only a part of the examined tissue lies within the disector, the volume of such an intersection is counted by combination of the Cavalieri principle and the point-counting method.
Assessment of tissue volume by the Cavalieri principle and point counting
The volume of the intersection of the examined tissue and the disector is estimated by counting the number of regularly distributed points in selected Z-slides [6].
(i) After autophagosomes have been counted in step 20, the opened Z-stack with apllied disector must not be closed. Subsequent point-counting procedure is performed on it.
(ii) A 2D grid is generated and superimposed over the opened image using the Grid tool in Fiji: Analyze>Tools>Grid. The parametr "Area per point" is set to 300 µm2 in the dialog box.
(iii) The points of the grid superimposed over the examinated tissue within the disector are counted using e.g. Multi poins selection tool. The points of the grid that do not lie in the tissue are excluded. Points are counted in individual slides within the disector but it is not necessary to use all the slides. Counting points at every third slide (i. e. with 1.5 µm distance between two neighboring slides where the points are counted) should provide a sufficiently accurate estimate of the volume. At least 200 points in total should be counted to get a good estimate. The position of the first slide where the points are counted is randomly selected from the first to the third slide within the disector.
(iv) The estimate of the volume of the examined tissue within the disector is calculated as [6]:
estV=T⋅a⋅∑j=1nPj [estV]=µm3
where estV is the estimator of volume in µm3, T is the distance between two neighboring optical sections where the points have been counted, a is the area in µm2 represented by one testing point of the testing grid, and Pj is the number of testing points hitting the j-th (j = 1, 2, …, n) optical section. If the above indicated values are set, then:
Note: The required time is estimated for analysis of two variants, each comprising ca. 15 - 20 roots.