Algae to angiosperms: Autofluorescence for rapid visualization of plant anatomy among diverse taxa.

PREMISE: Fluorescence microscopy is an effective tool for viewing plant internal anatomy. However, using fluorescent antibodies or labels hinders throughput. We present a minimal protocol that takes advantage of inherent autofluorescence and aldehyde-induced fluorescence in plant cellular and subcellular structures to markedly increase throughput in cellular and ultrastructural visualization. METHODS AND
RESULTS: Twelve species distributed across the plant phylogeny were each subjected to five fixative treatments: 1% paraformaldehyde and 2% glutaraldehyde, 2% paraformaldehyde, 2% glutaraldehyde, formalin-acid-alcohol (FAA), and 70% ethanol. Samples were prepared by embedding and mechanically sectioning or via whole mount. A confocal laser scanning system was used to collect micrographs. We evaluated and compared fixative influence on sample structural preservation and tissue autofluorescence.
CONCLUSIONS: Formaldehyde fixation of Viridiplantae taxa samples generates useful structural data while requiring no additional histological staining or clearing. In addition, a fluorescence-capable microscope is the only specialized equipment required for image acquisition. The minimal protocol developed in this experiment enables high-throughput sample processing by eliminating the need for multi-day preparations.

Technological advances in the past two decades have changed the amount and types of data collected in the biological sciences. Most notably, the increased availability of bioinformatics pipelines combined with exponential decreases in DNA and RNA sequencing costs have revolutionized biological research, changing not just the scale of data collection but also the types of questions that can be addressed (Buermans and den Dunnen, 2014). Considerable advances in non‐molecular phenotypic data collection have also been made. Recent efforts incorporate robotics, remote sensing, and automated image analysis pipelines to enable high‐throughput morphological and ecophysiological data collection (Perez‐Sanz et al., 2017; Baker et al., 2018). Combining high‐throughput phenotyping with second‐ and third‐generation sequencing technologies has led to detailed analyses of genotype–phenotype connections and environmental interactions with previously unimaginable statistical power, enabling candidate gene identification even in non‐model organisms (Yang et al., 2013; Großkinsky et al., 2018).

Examining the genetic architecture of relationships between internal plant anatomic structures and ecophysiological function or organismal morphology promises to open up new avenues for plant breeding and new arenas for modeling complex ecological and evolutionary dynamics in natural systems (Baker et al., 2017; He et al., 2017). However, high‐throughput methods for assessing internal anatomy remain elusive, precluding the widespread inclusion of internal anatomy in many modern ‐omics‐level studies (Yadav et al., 2021). Internal anatomy remains particularly difficult to assess for several reasons. First, most techniques involve destructively sampling from the plant. Live‐imaging techniques exist (Fang and Spector, 2010; Mathers et al., 2018); however, these can be cost prohibitive, are often relatively low throughput, and typically require that plants are grown in artificial conditions. Second, although it is possible to reconstruct internal anatomy at fairly high resolution after destructive sampling (Miki et al., 2020), destructive sampling followed by lengthy fixation, embedding, sectioning, and staining protocols for light microscopy are time consuming even if equipment such as automated embedding stations or powered microtomes are available.

We present a set of minimal sample preparation and microscopy protocols that take advantage of inherent autofluorescence in plant internal anatomic structures to achieve markedly increased throughput in cellular and ultrastructural visualization. Fluorescence is observed when visible light is emitted from a sample that has previously absorbed electromagnetic radiation (i.e., “excited”) with higher energy and shorter wavelengths (Lichtman and Conchello, 2005). Many proteins, lipids, and polysaccharides important to structural biology studies may be labeled with fluorescent proteins to identify specific tissues or organs (Neef and Schultz, 2009; Modesti, 2011; Sun et al., 2018). Many living organisms possess cells, tissues, and pigments that fluoresce naturally in a process termed “autofluorescence” (Roshchina, 2012; García‐Plazaola et al., 2015).

Autofluorescence in plants derives from multiple sources. Pigments such as chlorophyll possess chemical ring structures that become excited by ultraviolet, blue, or green light and strongly emit red light wavelengths in response (Billinton and Knight, 2001; Lamb et al., 2018; Donaldson, 2020). Lignin, a chemical found in secondary cell walls of xylem and other sclerenchymatous tissues, fluoresces when exposed to ultraviolet or blue wavelengths (De Micco and Aronne, 2007; Donaldson, 2020). Additional cell wall components such as ferulate, cutin, suberin, sporopollenin, and flavonoids also display varying degrees of autofluorescence.

Autofluorescence may be induced, or enhanced, in selective morphological structures by preserving plant samples in aldehyde‐based fixatives. Fixatives such as paraformaldehyde, glutaraldehyde, and formalin increase protein autofluorescence (i.e., aldehyde‐induced fluorescence) and help differentiate lignified cells from non‐fluorescent hemicellulose and pectin cell wall components in immunohistochemical localization experiments (Donaldson, 2020; Pegg et al., 2020). Aldehyde‐induced fluorescence is also often utilized to image organelles and proteins in cell protoplasts (Donaldson, 2020).

Here, we expand upon these results by examining the applicability of autofluorescence among diverse plant taxa spanning two major groups of green algae and four major groups of embryophytes. We present a protocol utilizing several types of aldehyde fixation in conjunction with whole‐mount confocal fluorescence microscopy for rapid observation of 2D and 3D anatomic features in plants. We report on alterations to cell ultrastructure and tissue fluorescence in species from a diverse array of plant taxa and suggest optimal fixations to preserve specific morphology. We demonstrate the use of this protocol as a general, standalone tool for plant anatomic studies.

METHODS AND RESULTS

Study design and tissue collection

We selected 12 species to demonstrate the applicability of our protocol across a phylogenetically broad range of plant taxa (Appendix S1): Arabidopsis thaliana (L.) Heynh. (thale cress, Brassicaceae), Brassica rapa L. (field mustard, Brassicaceae), Chara L. sp. (Characeae), Coleochaete Bréb. sp. (Coleochaetaceae), Glycine max (L.) Merr. (soybean, Fabaceae), Marchantia L. sp. (Marchantiaceae), Pinus strobus L. (white pine, Pinaceae), Zea mays L. (maize, Poaceae), Acetabularia J. V. Lamour. sp. (Polyphysaceae), Selaginella P. Beauv. sp. (spikemoss, Selaginellaceae), Ulva L. sp. (sea lettuce, Ulvaceae), and Volvox L. sp. (Volvocaceae). A detailed protocol, including a materials list with sample source information, is provided as Appendix 1.

Leaf tissue (A. thaliana, B. rapa, G. max, P. strobus, Z. mays, and Selaginella sp.), thalloid gametophyte (Ulva sp. and Marchantia sp.), cap segments (Acetabularia sp.), stipe tissue (Chara sp.), and individual colony (Coleochaete sp., Volvox sp.) samples were collected and placed separately into the fixative solutions listed in Table 1 for at least 24 h. Following fixation, samples were washed three times with deionized water. Samples from A. thaliana and G. max were embedded in 3.5% agarose (Millipore‐Sigma, St. Louis, Missouri, USA; Chemical Abstracts Service [CAS] no. 9012‐35‐6) and sectioned to a thickness of 150 µm on a Ted Pella Vibratome 1000 (Ted Pella Inc., Redding, California, USA). Samples from Acetabularia sp., B. rapa, Chara sp., Marchantia sp., P. strobus, Ulva sp., Selaginella sp., and Z. mays were harvested as 1 mm × 1 mm hand sections without further embedding or sectioning. Samples from Volvox sp. and Coleochaete were harvested as individual colonies via pipettor. All samples were then mounted in glycerol on a glass slide and covered with a coverslip.

TABLE 1

Fixative solutions used in this study.

Fixative no.	Abbreviation	Solution composition
1	PFA	1% paraformaldehyde in 0.1 M sodium cacodylate buffer (pH 7.4)
2	Glut	2% glutaraldehyde in sodium cacodylate buffer (pH 7.4)
3	PFA/Glut	1% paraformaldehyde and 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4)
4	FAA	Formalin‐acid‐alcohol (Ruzin, 1999)
5	EtOH	70% ethanol

Confocal microscopy

All images were observed using an Olympus FV500 Confocal Laser Scanning system (Olympus, Tokyo, Japan) using 20×/0.70 numerical aperture (NA) or 40×/1.30 NA dry objectives. Excitation of aldehyde‐induced fluorescence was achieved with 405‐nm and 433‐nm laser lines, while chlorophyll autofluorescence was obtained with 633‐nm laser lines. Each sample was recorded as a confocal Z‐stack comprising approximately 150 optical “slices” of 0.6‐micron thickness taken over a 15‐min period. Exposure time was 5.5 s per individual Z‐stack optical section. Three channels were recorded for each sample image, representing aldehyde‐induced fluorescence (ultraviolet and fluorescein isothiocyanate [FITC] bandpass filter sets: 400–499 nm and 467–498 nm, respectively) and autofluorescence belonging to chlorophyll (Texas Red bandpass filter set: 542–582 nm range). Images were recorded using a Photometric (Teledyne Photometrics, Tucson, Arizona, USA) HQ cooled CCD camera and stored in TIFF format. Each TIFF file consists of a confocal Z‐stack comprising approximately 150 individual images.

Image processing

Analysis and preparation of fluorescence micrographs were performed using the ImageJ distribution Fiji imaging software (Schindelin et al., 2012). Enhancement of fluorescence micrographs was limited to contrast/brightness adjustments and assignment of green, blue, and red lookup table values (LUTs) for image channels corresponding to aldehyde‐induced fluorescence emission from the 405‐nm, 433‐nm, and 633‐nm excitation wavelengths, respectively. Each channel Z‐stack was processed into a single extended depth of focus image with Fiji software. Channels were then overlaid to form a composite image for each sample. Final images were stored in both TIFF and PNG formats.

Effect of fixation on fluorescence among taxa

Trends in fluorescence were examined among treatments and species with the goal of clearly identifying cellular and subcellular structures. Samples preserved with PFA, Glut, and PFA/Glut (see Table 1 for fixative abbreviations) demonstrated notable fluorescence of cell walls across all taxa (Figs. 1, 2A–C, F–H; Appendices [Link], [Link], [Link], [Link], [Link], [Link], [Link], [Link], [Link], [Link]). Plants belonging to the tracheophytes (Figs. 1D–F, 2F–J; Appendices [Link], [Link], [Link], [Link], [Link]) showed bright, consistent fluorescence with leaf tissue from Zea mays (Fig. 2F–H) and Pinus strobus (Fig. 1E, Appendix S8A–C) representing excellent examples of clear cell wall, xylem, phloem, nuclei, and chloroplast definition.

FIGURE 1

Comparison of fixative treatments among representative taxa. Images show results of fixation with 1% paraformaldehyde and 2% glutaraldehyde for (A) Ulva sp. thallus and protonemal section with insert highlighting a nucleus, (B) Chara sp. stipe longitudinal section, (C) Marchantia sp. thallus and rhizoid section with insert highlighting a nucleus, (D) Selaginella sp. microphyll cross section with insert highlighting a nucleus, (E) Pinus strobus needle cross sections with insert highlighting a nucleus, and (F) Glycine max (soybean) leaf cross section. False‐color represents cell walls (green), nuclei (blue), and chlorophyll (red). Cell walls, nuclei, and chloroplasts are indicated by yellow wedges, white wedges, and white brackets, respectively. Scale bars = 50 µm.

FIGURE 2

Comparison of the five fixation treatments in Volvox colonies and Zea mays leaf tissue. Images display (A–E) Volvox colonies and (F–J) Zea mays leaf cross sections. Fixatives represented are (A, F) 1% paraformaldehyde and 2% glutaraldehyde, (B, G) 2% paraformaldehyde, (C, H) 2% glutaraldehyde, (D, I) formalin–acetic acid (FAA), and (E, J) 70% ethanol treatment. False‐color indicates cell walls (green), nuclei (blue), and chlorophyll (red). Cell walls and nuclei are indicated by yellow wedges and white wedges, respectively. Scale bars = 50 µm.

Samples preserved with FAA demonstrated less cell wall fluorescence and poor or mottled chlorophyll fluorescence—shown as a red false‐color image—in taxa belonging to the chlorophytes (Ulva, Acetabularia, and Volvox) (Fig. 2D; Appendices S2D, S3D) and in streptophycean algae (Coleochaete and Chara) (Appendices S4D, S5D). By contrast, taxa belonging to the tracheophytes displayed adequate definition of cell walls, but similar loss of chlorophyll fluorescence (Fig. 2I, Appendices [Link], [Link], [Link], [Link], [Link]D). Zea mays, Glycine max, Marchantia, and several green algae samples (e.g., Ulva, Chara, Coleochaete) fixed with 70% ethanol demonstrated similar patterns of fluorescence, suggesting non‐aldehyde‐induced cell wall fluorescence emission wavelengths partially overlap with those of aldehyde‐induced fluorescence (Fig. 2J; Appendices S2E, S4E, S5E, S6E, S9E). By comparison, Volvox (green algae) and most of the tracheophytes showed little cell wall fluorescence in samples preserved with alcohol (Fig. 1D–F, J; Appendices [Link], [Link], [Link], [Link], [Link]E). All samples demonstrated diminished chlorophyll fluorescence when preserved with 70% ethanol, similar to observations made with FAA‐preserved samples.

Effect of fixation on structural preservation

There were differing effects of fixative treatments among taxa (Figs. 1, 2; Table 2). Tracheophyte samples demonstrated little change in structural quality across fixative treatments, with clearly delineated regions of mesophyll, epidermal, and vascular tissues in many samples (Fig. 2F–J; Appendices [Link], [Link], [Link], [Link], [Link]). However, fixatives containing alcohol caused substantial damage in tissues belonging to aquatic plants, or those with thinner cell walls. For example, fixation with FAA resulted in structural preservation for tracheophytes with minimal cellular damage but caused prominent shrinkage and lysis of cells in chlorophycean and streptophycean algae and Marchantia (Appendices S2D, S3D, S4D, and S6D). The poor structural preservation of FAA‐treated samples may be the result of rapid infiltration with ethanol, as similar cell damage was also observed when these samples were preserved with 70% ethanol (Appendices S2D, S3D, S4D, and S6D). In addition, ethanol content appeared to leach chlorophyll from numerous samples, resulting in limited damage to chloroplasts (Fig. 2E, J; Appendices S9–S11E).

TABLE 2

Comparison of fixative treatment influence on cell structure visibility across taxa.

	Fixatives
Species	1% PFA and 2% Glut	2% PFA	2% Glut	FAA	70% EtOH
Ulva	Nuc, CW, Chl	Nuc, CW, Chl	Nuc, CW, Chl	Nuc, CW	Nuc, CW
Acetabularia	Nuc, CW, Chl	Nuc, CW, Chl	Nuc, CW, Chl	‐‐‐	‐‐‐
Volvox	Nuc, CW, Chl	Nuc, CW, Chl	Nuc, CW, Chl	CW	CW
Chara	CW, Chl	CW, Chl	CW, Chl	CW	CW
Coleochaete	Nuc, CW, Chl	Nuc, CW, Chl	Nuc, CW, Chl	Nuc, Chl	Nuc, Chl
Marchantia	Nuc, CW, Chl	Nuc, CW, Chl	Nuc, CW, Chl	CW	CW
Selaginella	Nuc, CW, Chl	Nuc, CW, Chl	Nuc, CW, Chl	Nuc, CW, Chl	CW
Pinus strobus	Nuc, CW, Chl	Nuc, CW, Chl	Nuc, CW, Chl	Nuc, CW, Chl	Nuc, CW, Chl
Glycine max	CW, Chl	CW, Chl	CW, Chl	CW, Chl	CW, Chl
Zea mays	Nuc, CW, Chl	Nuc, CW, Chl	Nuc, CW, Chl	Nuc, CW	Nuc, CW
Brassica rapa	Nuc, CW, Chl	Nuc, CW, Chl	Nuc, CW, Chl	Nuc, CW, Chl	Chl
Arabidopsis thaliana	Nuc, CW, Chl	Nuc, CW, Chl	Nuc, CW, Chl	Nuc, CW	Nuc, CW

Chl = chlorophyll; CW = cell wall; Nuc = nucleus.

Fixative solution abbreviations are provided in Table 1.

CONCLUSIONS

An improved understanding of internal anatomical structures may lead to new insights for plant breeding and evolutionary ecology (He et al., 2017). Such studies often require large sample sizes, but versatile, high‐throughput methods for assessing internal anatomy remain elusive. Here, we compare several conventional fixation solutions and find that preservation of plant tissues in aldehyde‐based fixatives is an effective protocol for rapid fluorescence imaging of cellular and subcellular structures. Samples from taxa spanning the Viridiplantae phylogeny fixed in formaldehyde‐based fixatives—commonly used in light and electron microscopy—require no additional histological staining or clearing, and sample preparation and image acquisition require minimal specialized equipment. The lack of additional tissue clearing, histochemical staining, and antibody labeling enables increased throughput by eliminating the need for multi‐day tissue preparation protocols.

Sample throughput partially depends on the passive diffusion rate of fixatives. Fixative diffusion typically occurs at an average rate of 2.5 mm/h for formaldehyde‐based fixatives (Baker, 1958), although paraformaldehyde and the formaldehyde in FAA penetrate at a faster rate than glutaraldehyde (Huebinger et al., 2018). However, paraformaldehyde fixatives cross‐link proteins at a slower rate than glutaraldehyde does (Fox et al., 1985; Howat and Wilson, 2014), resulting in a potential for poor structural preservation and fluorescence despite faster tissue penetration. As a result, the fixation protocols demonstrated here are best suited to single‐cell algae and small plant tissue sections to minimize differences in fixation rate.

There is also a trade‐off between sample preparation and image acquisition throughput. Image acquisition time can be minimized using a simple, rapid technique to embed samples and section them on a vibratome (Appendices S7, S9). Alternatively, sample preparation throughput can be increased by omitting embedding and mechanical sectioning and instead preparing either whole‐mount or hand‐sectioned specimens for optical sectioning via confocal microscopy (e.g., Fig. 2). However, decreasing sample preparation time comes at the cost of increased time to acquire images using confocal microscopy. Hand‐ or whole‐mount sections require focusing the confocal beam on tissue layers below the uneven surfaces generated by these techniques. Confocal microscopes are normally utilized to image thicker biological samples than standard optical microscopes (often 100–200 µm) (Reihani and Oddershede, 2009; Wang and Larina, 2017), but resolution is still limited by sample thickness and multiple refractive index interfaces such as those encountered by light penetrating multiple plant cell wall layers (Timmers, 2016). As a result, optical slices acquired from deep within thick tissue may require extending scan times to collect sufficient data. We found that thicker samples most benefited mechanical sectioning to limit the exposure time required to collect high‐resolution images. Furthermore, if surfaces are very uneven, it may not be possible to consistently collect large Z‐stacks. One advantage of Z‐stacks is that they can be used to generate a 3D wire frame and rotate the image to the optimal plane of section. For many research questions, a single optical slice may be sufficient, and the plane of section may not be critical. Institution‐specific fees for microscopes at core facilities, microscope‐specific scan times, and the specific research goals (2D or 3D data) are all worth considering when adopting a protocol for maximizing throughput and minimizing cost.

We found that plant taxa influence the quality of cellular preservation as revealed by aldehyde‐induced fluorescence, with notable differences between green algae (Ulva sp., Acetabularia sp., Volvox sp., Chara sp., and Choleochaete sp.) and embryophytes. For example, for chlorophycean algae (i.e., Ulva lactuca, Acetabularia sp., and Volvox sp.) and algae in the Streptophyta (e.g., Chara and Choleochaete), paraformaldehyde and glutaraldehyde fixation produced well‐preserved cell walls and chlorophyll content that resulted in bright fluorescence across multiple excitation wavelengths. However, these same taxa had poor structural preservation when exposed to alcohol‐containing fixatives such as FAA and 70% ethanol, resulting in cell wall and cell membrane shrinkage, loss of chlorophyll pigments, and low cell wall fluorescence when compared to embryophytes. These differences may be due to the osmotic sensitivity that green algae possess due to their aquatic habitat (Shetty et al., 2019), leaching of chlorophyll by alcohols, and lack of significant phenolic chemicals within their cell walls (Delwiche et al., 1989; Martone et al., 2009), which natively autofluoresce when exposed to ultraviolet wavelengths (Harris and Hartley, 1976; Donaldson, 2013).

Compared to green algae, embryophytes demonstrated better preservation of cellular structures (i.e., nuclei and chloroplasts) and cell walls (Table 2). The presence of thicker primary cell walls and multiple phenolic compounds, such as lignin, may have contributed to excellent structural preservation and fluorescence with all paraformaldehyde, glutaraldehyde, and mixed paraformaldehyde/glutaraldehyde fixatives. FAA also preserved cell structure but led to poor chlorophyll fluorescence, and some signs of cytoplasmic shrinkage, caused by the high alcohol content of the fixative solution and the caustic nature of the glacial acetic acid component (Nikara et al., 2020). Mediocre structural preservation and cell fluorescence observed with 70% ethanol fixation likely resulted from the absence of protein cross‐linking normally induced by the presence of formaldehydes (Thavarajah et al., 2012; Hoffman et al., 2015; Nikara et al., 2020).

Historically, plant cell morphology research has relied on time‐consuming techniques such as histological staining and immunolabeling protocols, electron microscopy preparations, or limitations in what may be revealed in standard bright‐field microscopy. Fully integrating internal anatomy with modern molecular and morphological data sets requires generating anatomical information easily and quickly (Li and Chen, 2014). Here, we demonstrate how simple fixation protocols may be used to rapidly observe aldehyde‐induced fluorescence with confocal light microscopy to gather useful cellular structure data from multiple plant phyla. Our protocols serve as a guide for proper plant fixative selection and an illustration of possible preservation artifacts. Using these protocols to enhance structural preservation and increase throughput for anatomical studies may improve our understanding of plant evolutionary biology, such as the development of cell walls.

AUTHOR CONTRIBUTIONS

T.J.P. and R.L.B. conceived of the research questions and performed data interpretation. T.J.P. grew plants, harvested tissue samples, performed the fixation protocol, and acquired image data. T.J.P, D.K.G., and R.L.B. drafted and revised the manuscript. T.J.P, D.K.G., and R.L.B. gave their final approval for publication and are accountable for all aspects of the work.

APPENDIX S1. Phylogenetic tree representing evolutionary relationships of the 12 species used in this protocol (adapted from Leebens‐Mack et al., 2019).

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APPENDIX S2. Images of an Ulva sp. thallus and protonemal sections. Each image demonstrates fluorescence of tissues subjected to different fixation treatments. Fixatives represented are (A) 1% paraformaldehyde and 2% glutaraldehyde, (B) 2% paraformaldehyde, (C) 2% glutaraldehyde, (D) formalin–acetic acid (FAA), and (E) 70% ethanol treatment. False‐color indicates cell walls and nuclei (green and blue), and chlorophyll (red). Cell walls, nuclei, and chlorophyll are indicated by yellow wedges, white wedges, and magenta wedges, respectively. Scale bars = 50 µm.

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APPENDIX S3. Images of Acetabularia sp. radial cap sections. Each image demonstrates fluorescence of tissues subjected to different fixation treatments. Fixatives represented are (A) 1% paraformaldehyde and 2% glutaraldehyde, (B) 2% paraformaldehyde, (C) 2% glutaraldehyde, (D) formalin–acetic acid (FAA), and (E) 70% ethanol treatment. False‐color indicates cell walls and nuclei (green and blue), and chlorophyll (red). Cell walls and nuclei are indicated by yellow wedges and white wedges, respectively. Scale bars = 50 µm.

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APPENDIX S4. Images of Chara sp. stipe longitudinal sections. Each image demonstrates fluorescence of tissues subjected to different fixation treatments. Fixatives represented are (A) 1% paraformaldehyde and 2% glutaraldehyde, (B) 2% paraformaldehyde, (C) 2% glutaraldehyde, (D) formalin–acetic acid (FAA), and (E) 70% ethanol treatment. False‐color indicates cell walls and nuclei (green and blue), and chlorophyll (red). Cell walls and chlorophyll are indicated by yellow wedges and white brackets, respectively. Scale bars = 50 µm.

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APPENDIX S5. Images of immature Coleochaete sp. colonies. Each image demonstrates fluorescence of tissues subjected to different fixation treatments. Fixatives represented are (A) 1% paraformaldehyde and 2% glutaraldehyde, (B) 2% paraformaldehyde, (C) 2% glutaraldehyde, (D) formalin–acetic acid (FAA), and (E) 70% ethanol treatment. False‐color indicates cell walls and nuclei (green and blue), and chlorophyll (red). Cell walls, nuclei, and chlorophyll are indicated by yellow wedges, white wedges, and orange wedges, respectively. Scale bars = 50 µm.

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APPENDIX S6. Images of Marchantia sp. thallus and rhizoid cross sections. Each image demonstrates fluorescence of tissues subjected to different fixation treatments. Fixatives represented are (A) 1% paraformaldehyde and 2% glutaraldehyde, (B) 2% paraformaldehyde, (C) 2% glutaraldehyde, (D) formalin–acetic acid (FAA), and (E) 70% ethanol treatment. False‐color indicates cell walls and nuclei (green and blue), and chlorophyll (red). Cell walls, nuclei, and chlorophyll are indicated by yellow wedges, white wedges, and white brackets, respectively. Scale bars = 50 μm.

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APPENDIX S7. Images of Selaginella sp. microphyll cross sections. Each image demonstrates fluorescence of tissues subjected to different fixation treatments. Fixatives represented are (A) 1% paraformaldehyde and 2% glutar­aldehyde, (B) 2% paraformaldehyde, (C) 2% glutaraldehyde, (D) formalin–acetic acid (FAA), and (E) 70% ethanol treatment. False‐color indicates cell walls and nuclei (green and blue), and chlorophyll (red). Cell walls, nuclei, and chlorophyll are indicated by yellow wedges, white wedges, and white brackets, respectively. Scale bars = 50 μm.

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APPENDIX S8. Images of Pinus strobus (white pine) needle cross sections. Each image demonstrates fluorescence of tissues subjected to different fixation treatments. Fixatives represented are (A) 1% paraformaldehyde and 2% glutaraldehyde, (B) 2% paraformaldehyde, (C) 2% glutaraldehyde, (D) formalin–acetic acid (FAA), and (E) 70% ethanol treatment. False‐color indicates cell walls and nuclei (green and blue), and chlorophyll (red). Cell walls, nuclei, and mesophyll (i.e., containing chlorophyll) are indicated by yellow wedges, white wedges, and white brackets, respectively. Scale bars = 50 μm.

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APPENDIX S9. Images of Glycine max (soybean) leaf cross sections. Each image demonstrates fluorescence of tissues subjected to different fixation treatments. Fixatives represented are (A) 1% paraformaldehyde and 2% glutar­aldehyde, (B) 2% paraformaldehyde, (C) 2% glutaraldehyde, (D) formalin–acetic acid (FAA), and (E) 70% ethanol treatment. False‐color indicates cell walls and nuclei (green and blue), and chlorophyll (red). Cell walls and palisade mesophyll (i.e., containing chlorophyll) are indicated by yellow wedges and white brackets, respectively. Scale bars = 50 μm.

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APPENDIX S10. Images of Brassica rapa leaf cross sections. Each image demonstrates fluorescence of tissues subjected to different fixation treatments. Fixatives represented are (A) 1% paraformaldehyde and 2% glutaralde­hyde, (B) 2% paraformaldehyde, (C) 2% glutaraldehyde, (D) formalin–acetic acid (FAA), and (E) 70% ethanol treatment. False‐color indicates cell walls and nuclei (green and blue), and chlorophyll (red). Cell walls, nuclei, and chlorophyll are indicated by yellow wedges, white wedges, and white brackets, respectively. Scale bars = 100 μm.

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APPENDIX S11. Images of Arabidopsis thaliana leaf cross sections. Each image demonstrates fluorescence of tissues subjected to different fixation treatments. Fixatives represented are (A) 1% paraformaldehyde and 2% glutar­aldehyde, (B) 2% paraformaldehyde, (C) 2% glutaraldehyde, (D) formalin–acetic acid (FAA), and (E) 70% ethanol treatment. Cell walls, nuclei, chlorophyll, and xylem vessel elements are indicated by yellow wedges, white wedges, white brackets, and yellow brackets, respectively. Scale bars = 100 μm.

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