Unbiased estimation of chloroplast number in mesophyll cells: advantage of a genuine three-dimensional approach

Abstract

Chloroplast number per cell is a frequently examined quantitative anatomical parameter, often estimated by counting chloroplast profiles in two-dimensional (2D) sections of mesophyll cells. However, a mesophyll cell is a three-dimensional (3D) structure and this has to be taken into account when quantifying its internal structure. We compared 2D and 3D approaches to chloroplast counting from different points of view: (i) in practical measurements of mesophyll cells of Norway spruce needles, (ii) in a 3D model of a mesophyll cell with chloroplasts, and (iii) using a theoretical analysis. We applied, for the first time, the stereological method of an optical disector based on counting chloroplasts in stacks of spruce needle optical cross-sections acquired by confocal laser-scanning microscopy. This estimate was compared with counting chloroplast profiles in 2D sections from the same stacks of sections. Comparing practical measurements of mesophyll cells, calculations performed in a 3D model of a cell with chloroplasts as well as a theoretical analysis showed that the 2D approach yielded biased results, while the underestimation could be up to 10-fold. We proved that the frequently used method for counting chloroplasts in a mesophyll cell by counting their profiles in 2D sections did not give correct results. We concluded that the present disector method can be efficiently used for unbiased estimation of chloroplast number per mesophyll cell. This should be the method of choice, especially in coniferous needles and leaves with mesophyll cells with lignified cell walls where maceration methods are difficult or impossible to use.

Keywords: Chloroplast counting; Norway spruce (Picea abies L. Karst.); confocal microscopy; coniferous needle structure; disector method; mesophyll; profile counting; stereology..

Fig. 1.

Systematic uniform random (SUR) sampling of positions of cross-sections along the needle. 0, tip; z, position of the first cross-section: a random integer from an interval 0–5 was chosen using a table of random numbers; this number determined the position of the first cross-section (z) in the needle (0 corresponded to 0.5mm from the tip and 1 corresponded to 1mm, up to 5, which corresponded to 3mm from the tip), T=3mm (the interval between subsequent cross-sections). (This figure is available in colour at JXBV online.)

Fig. 2.

Norway spruce needle cross-section: image acquisition, sampling, and processing. (A) Anatomical structure of a Norway spruce needle in a cross-section. Autofluorescence of chlorophyll in chloroplasts was detected in the red channel and autofluorescence of phenolics was detected in the green channel. (B) Histochemical lignin detection in cell walls (pink) of mesophyll cells using a phloroglucinol/HCl test (O’Brian and McCully, 1981). (C) Sampling frames were superimposed on a needle transverse section using the Rectangles module in Ellipse software. (D) Stacks of optical sections were acquired at the positions of rectangles in (C) with higher resolution. (E) Estimation of needle cross-sectional area by a point counting method using the Point Grid module in Ellipse software (F) Two frames showing the subsequent acquisition of series for counting mesophyll cells. (A, C–F), confocal microscopy: (B) bright-field light microscopy.

Fig. 3.

Chloroplast counting using the virtual 3D disector probe. (A) Scheme of the disector probe with chloroplasts. The disector probe is a 3D block. Chloroplasts lying within this block or intersecting its planes are counted, except those intersecting the exclusion planes. The exclusion planes in this scheme are represented by the dark planes and the transparent front plane (all bordered by a full line): the bottom KLMN (so-called look-up plane), KNRO, KLPO, and half-planes PLS and RNT. The top rectangle OPQR is called the reference plane and does not belong to the exclusion planes. Chloroplasts that lie fully inside the probe are always counted. In this scheme, eight chloroplasts are counted (ticked) and six chloroplasts are not counted (crossed). (B–D) Three optical sections of the stack of real serial optical sections acquired by confocal microscopy. Within section numbers 16–27, the disector probe was placed using the Disector module in the Ellipse software, section 16 being the reference plane of the probe (B), section 22 inside the disector probe (C), and section 27 the look-up plane of the disector probe (D). Counted particles are those within the probe not intersecting the exclusion planes: point, counted particle; cross, particle is not counted. In this example, eight chloroplasts were counted (chloroplasts numbers 3 and 10–13 are intersecting the exclusion planes). (This figure is available in colour at JXB online.)

Fig. 4.

Scheme of the hypothetic spatial mosaic of 3D disector sampling probes illustrating that particles are unambiguously sampled by only one disector sampling probe. The probes a, b, and c are located next to each other. The exclusion planes in this scheme are represented by grey planes. Particles are labelled according to the label of the probe they are counted in. In this scheme, in probe a eight chloroplasts are counted (labelled a), in probe b eight chloroplasts are counted (labelled b), and in probe c nine chloroplasts are counted (labelled c). Chloroplasts labelled by crosses are not counted by any of the three probes shown here, but they would be unambiguously sampled by adjacent hypothetical probes. (This figure is available in colour at JXB online.)

Fig. 5.

3D reconstruction and 3D model of a Norway spruce mesophyll cell. (A–D) Different optical sections of a mesophyll cell in a stack of images captured by a confocal microscope by using an objective 20×. (E) 3D reconstruction of the surface of the mesophyll cell from the confocal image stack shown in (A)–(D) using Ellipse software. (F) Face view of a model of a mesophyll cell with 210 chloroplasts made in Cortona 3D software. (G) 3D reconstruction of the mesophyll cell with chloroplasts from a stack of optical sections from confocal microscopy using Ellipse software. The positions of chloroplasts do not correspond to the in vivo state. (H) Side view of the model from (F) with six lines indicating planes of cross-sections. (I) 2D sections through the model cell shown in (F) and (H). Letters indicate the position of section planes in (H). Numbers indicate the number of chloroplast profiles in each of these sections.

Fig. 6.

Comparison of 3D- and 2D-based methods for chloroplast number estimation. The mean number of chloroplasts per mesophyll cell in Norway spruce needle estimated by 3D disector was 209.65±17.44 (mean±SE) (open column), and by profile counting in 2D (filled column) was 20.92±1.32. This result was statistically significantly different (P<0.001).