Images of Stomata using the Wavelet Spot Detection and the Watershed Transform

Segmenting High-quality Digital Images of Stomata using the Wavelet
Spot Detection and the Watershed Transform

by Duarte K. T. N., de Carvallo M. A.G., Martins P. S. (2017)

Kaue T. N. Duarte, Marco A. G. de Carvalho, Paulo S. Martins,

University of Campinas (UNICAMP), School of Technology, R. Paschoal Marmo, 1888, 13484 Limeira, Brazil


In Proceedings of the 12th International Joint Conference on Computer Vision, Imaging and Computer Graphics Theory and Applications (VISIGRAPP 2017), pages 540-547 – ISBN: 978-989-758-225-7 – DOI: 10.5220/0006168105400547 –

Figure 3: Stomata from Ugni Molinae Species in RGB to CIELab (a) RGB-channel R; (b) RGB-channel G; (c) RGB-channel
B; (d) CIELab-channel L; (e) CIELab-channel a; (f) CIELab-channel b.


Stomata are cells mostly found in plant leaves, stems and other organs. They are responsible for controlling the gas exchange process, i.e. the plant absorbs air and water vapor is released through transpiration. Therefore, stomata characteristics such as size and shape are important parameters to be taken into account. In this paper, we present a method (aiming at improved efficiency) to detect and count stomata based on the analysis of the multi-scale properties of the Wavelet, including a spot detection task working in the CIELab colorspace.

Figure 4: Stomata Segmentation Process from UgniMolinae Species (a) Channel a* ; (b) Binary spots from Wavelet Spot
Detection; (c) Morphological Gradient; (d) Open; (e) Erode; (f) Reconstruct; (g) Close; (h) Watershed lines and spots; (i)
Stomata detected. The images (a) and (c-g) had their contrast enhanced for better visualization.

We also segmented stomata images using the Watershed Transform, assigning each spot initially detected as a marker. Experiments with real and high-quality images were conducted and divided in two phases. In the first, the results were compared to both manual enumeration and another recent method existing in the literature, considering the same dataset. In the second, the segmented results were compared to a gold standard provided by a specialist using the F-Measure. The experimental results demonstrate that the proposed method results in better effectiveness for both stomata detection and segmentation.

A rapid permanent mounting medium for Bryophytes

Hoyer’s solution as a rapid permanent mounting medium for Bryophytes

by Anderson L. E. (1954)

Lewis E. Anderson, Duke University, Durham, S. Carolina


In Bryologist57(3): 242-244 –

Leaf Clearing Protocol to Observe Stomata

Figure 1. Leaf clearing and fixing. The ethanol:acetic acid solution fixes the tissue and the tissue becomes transparent.

Leaf Clearing Protocol to Observe Stomata and Other Cells on Leaf Surface

by Sharma N. (2017)

In bio-101 2017-09-05 – DOI: 0.21769/BioProtoc.2538

Figure 2. Washing the tissue. Cleared tissue (A, B) is washed in 1 N KOH (C, D). Tissue is washed in water (E, top slide) and placed in Hoyer’s solution (E, bottom slide).


In this protocol, leaves are cleared and fixed in an ethanol and acetic acid solution, and mounted in Hoyer’s solution.

Figure 3. Slide preparation. Individual leaves form a precipitate when they are placed in Hoyer’s solution.

The cleared leaves are imaged under differential interference contrast (DIC) microscope. This protocol is beneficial for studying stomata, hair cells, and other epidermal cells in plants.

Figure 4. Same leaf imaged with Brightfield or DIC filters. DIC image (B) gives better resolution than Brightfield (A) to observe different cell types. Arrows: stomata (young and mature).
Figure 5. Quantification of stomata. Number of stomata (X) and pavement cells (Y) are counted to calculate stomatal index.

The described method allows analysis of stomatal apertures with minimal leaf manipulation and usage of the same leaf for sequential measurements

Fig 1. Experimental setup for stomatal aperture measurements.(a) Schematic representation of the workflow; (b) epifluorescent microscopic picture of the Arabidopsis leaf stained with rhodamine 6G; (c) the same picture as in (b) after application of the option “sharpen” in ImageJ. Bars, 50 μm

A Rapid and Simple Method for Microscopy-Based Stomata Analyses

by Eisele J. F., Fässler F., Bürgel P. F., Chaban C. (2016)

Jochen F. Eisele, Florian Fäßler, Patrick F. Bürgel, Christina Chaban,

Department of Plant Physiology, Center for Plant Molecular Biology (ZMBP), University of Tübingen, Tübingen, Germany



Fig 3. Visualization of stomatal apertures in intact leaves and epidermis peels.(a,b) Epifluorescent images of intact leaves mounted in water (upper panel) and in 30% glycerol (lower panel). (a) Staining with 1 μM rhodamine 6G; (b) staining with 10 μM rhodamine 6G. (c) Photograph of leaf epidermis peels. (d) Confocal image of cells in the peeled epidermis; λexc = 488 nm, λem = 505–545 nm (upper panel); λexc = 561 nm, λem = 600–640 nm (middle panel); bright field (lower panel). Bars, 50 μm.


There are two major methodical approaches with which changes of status in stomatal pores are addressed: indirectly by measurement of leaf transpiration, and directly by measurement of stomatal apertures.

Application of the former method requires special equipment, whereas microscopic images are utilized for the direct measurements. Due to obscure visualization of cell boundaries in intact leaves, a certain degree of invasive leaf manipulation is often required. Our aim was to develop a protocol based on the minimization of leaf manipulation and the reduction of analysis completion time, while still producing consistent results.

We applied rhodamine 6G staining of Arabidopsis thaliana leaves for stomata visualization, which greatly simplifies the measurement of stomatal apertures. By using this staining protocol, we successfully conducted analyses of stomatal responses in Arabidopsis leaves to both closure and opening stimuli.

We performed long-term monitoring of living stomata and were able to document the same leaf before and after treatment. Moreover, we developed a protocol for rapid-fixation of epidermal peels, which enables high throughput data analysis.

The described method allows analysis of stomatal apertures with minimal leaf manipulation and usage of the same leaf for sequential measurements, and will facilitate the analysis of several lines in parallel.

A stomatal diffusion porometer

Design, calibration and field use of a stomatal diffusion porometer

by Kanemasu E. T., Thurtell G. W., Tanner C. B. (1969)

In Plant Physiol. 44: 881–885 – DOI: 10.1104/pp.44.6.881


Modifications of the design and calibration procedure of a diffusion porometer permit determinations of stomatal resistance which agree well with results obtained by leaf energy balance. The energy balance and the diffusion porometer measurements indicate that the boundary layer resistances of leaves in the field are substantially less than those predicted from heat transport formulas based on wind flow and leaf size.

Rapid technique for obtaining leaf prints for stomatal count

A rapid technique for obtaining leaf prints for stomatal count with Fevicol

by Nayeem K. A., Dalvi D. G. (1989)

Wheat Research Unit, Marathwada Agricult. Univ., Parbhani 431 402, India


In Curr. Sci. 58: 640 – 641 –


AP4 Porometer

AP4 Porometer(2019) – Delta-T Devices

AP4 Leaf Porometer
AP4 Leaf Porometer in the field
AP4 Leaf Porometer
AP4 5


“We use the AP4 porometers to study stomatal responses to nutrient availability in legume crops. One of the most useful aspects of using the AP4 is the rapid data collection allowing for large and accurate data sets both in the field and glasshouse environment. We can highly recommend them to anyone interested in studying stomatal physiology.”Shane Rothwell, PHD Student
Lancaster Environment Centre, Lancaster University

  • Direct readout of stomatal conductance or stomatal resistance 
  • Simple absolute calibration in the field
  • Minimises leaf stress during measurement
  • Ideal for phenotyping based research  
  • Award-winning user interface

Stomatal aperture is the dominant factor in the diffusion conductance of leaf surfaces, which controls both the water loss from plant leaves and the uptake of CO2 for photosynthesis. Measurements of diffusion conductance are therefore important indicators of plant water status and provide a valuable insight into plant growth and plant adaptation to environmental variables.

AP4 Leaf Porometer features

The AP4 Leaf Porometer measures diffusion conductance by comparing the precise rate of humidification within a small cuvette (chamber) to readings obtained with a calibration plate. The plate has 6 diffusion conductance settings whose values have been accurately determined by finite element analysis.

Quoted accuracy figures for other porometers and gas analysis systems are based on time-consuming laboratory set-up and calibrations which bear little comparison to field conditions. In contrast, the AP4 Leaf Porometer features simple direct calibration in the field against a tested physical standard.

The AP4 Leaf Porometer has many other features designed to ensure that accurate, reproducible readings can be taken as easily as possible:

  • Sophisticated temperature compensation
  • Unstirred leaf chamber minimises unwanted stomatal closure
  • Lightweight ergonomic sensor head
  • Large clear LCD display (8 lines by 40 characters)
  • Full QWERTY keypad for annotating up to 1500 readings
  • A  rugged and reliable tool for phenotyping projects
  • Over 2,000 Delta-T porometers in use worldwide