the suitability of using fluorescence microscopy for studying lignification … · 2010. 2. 22. ·...

THE SUITABILITY OF USING

FLUORESCENCE MICROSCOPY FOR

STUDYING LIGNIFICATION

IN BALSAM FIR

Norman P. Kutscha1 and Raymond R. McOrmond

Life Sciences and Agriculture Experiment Station

University of Maine at Orono

Technical Bulletin 62 November 1972

1 Assistant Professor, Wood Technology, School of Forest Resources, University of Maine, Orono 04473.

2 Former Graduate Research Assistant, University of Maine. Present address: Western Electric Company, Springfield. New Jersev 07081.

CONTENTS

Page

Introduction 3

Primary Fluorescence . . . 4

Disadvantages 5

Advantages 7

Secondary Fluorescence . . 7

Disadvantages 8

Advantages 8

Fluorescence in Balsam Fir . . 9

Results . . . . 10

Conclusions and Summary 13

Literature Cited 14

ACKNOWLEDGEMENT

This work was supported by McIntire-Stennis Project 5009. Review of this paper by Dr. Wilfred A. Cote, Jr., Director, Laboratory for Ultrastructure Studies, College of Environmental Science and Forestry, State University of New York, Syracuse, and Dr. Charles Camiglia, Department of Physics, University of Maine, Orono, is gratefully acknowledged.

THE SUITABILITY OF USING

FLUORESCENCE MICROSCOPY FOR

STUDYING LIGNIFICATION

IN BALSAM FIR

INTRODUCTION

Certain substances have the ability to absorb ultra-violet light which in turn stimulates the emission of visible light. This phenomenon is known as fluorescence. Many materials fluoresce naturally when exposed to ultra-violet light. Fluorescence of this type is known as primary or autofluorescence. Fluorescence can also be induced artificially by treatment with a fluorochrome, and this fluorescence is referred to as secondary fluorescence.

Fluorochromes cause a minimum of physiological change within the cell, can produce high image contrast and can exhibit chemical selectivity. These fluorochromes are derived from chemicals or plant extracts and are used in aqueous or alcoholic solution. Marts (1950, 1955a, 1955b) and Kutscha (1961) have described the application of fluorescence microscopy and certain fluorochromes to the study of wood and fiber structure.

For most biological fluorescence which utilizes ultra-violet light, wavelengths ranging from 300 nm1 to 400 nm (long or near ultra-violet) are used. Much of this range of ultra-violet light can be transmitted by glass optics although special ultra-violet transmitting glass or quartz is preferred. If shorter wavelength ultra-violet radiation is required, quartz optics must be used (Needham, 1958).

The absorption curve for lignin in the ultra-violet has maxima at 212 nm and 280 nm. Therefore, quartz optics should be used for best transmittance of these shorter wavelengths. With the addition of fluorochromes which might attach themselves to the lignin molecule, lignin could be studied using longer wave ultra-violet light (365 nm); the need for quartz optics would be reduced.

When ultra-violet microscopy is used to study lignin, the specimen is illuminated by a very narrow band of ultra-violet light. The lignin rich tissue absorbs the ultra-violet light and appears dark, instead of bright

1 nm is the abbreviation for nanometer, formerly known as millimicron (mu), as adopted by the International Committee on Weights and Measures.

4 LSA EXPERIMENT STATION TECHNICAL BULLETIN 62

as in fluorescence microscopy. For lignin, usually the 212 nm or the 280 nm band is transmitted to the wood section to study its absorption characteristics.

Different lignins can be classified by their absorption spectra by varying the wavelength of the light used to illuminate the specimen and measuring the wavelengths emitted by the specimen. Venverloo (1969) found the absorption maxima of Popuhis nigra lignin to be at 247 nm and 295 nm. Siegel (1969) studied the absorption spectra of lignin in moss gametophytes and found maxima at 280 nm and 282 nm. Bailey (1951) studied the ultra-violet absorption spectra of chromatographic fractions of lignin and concluded that the spectrographic evidence obtained supports the validity of using flourescence observation as indications of chemical differences in the lignin. Fergus and Goring (1968a) separated guaiacyl and syringyl lignins on the basis of their absorption maxima.

Lignin distribution in the cell wall has been studied using ultraviolet light (Wergin, 1965). Quantitative determinations have been made of lignin distribution within the cell wall using the ultra-violet microscope (Fergus and Goring, 1968£>; Fergus et al., 1969). A critical evaluation of the technique has been given by Scott et al. (1969). Scott and Goring (1969) observed photolysis of lignin in wood sections within two hours. They pointed out that this effect could lead to serious errors when using ultra-violet microscopy to detect lignin.

Ultra-violet microscopy in general is an excellent tool for studying lignin. In addition to the uses already mentioned, it can be effectively used to study delignification by various pulping and bleaching processes (Fergus and Goring, 1969). Ultra-violet microscopy achieves better resolution than flourescence. It also compares favorably with certain lignin stains such as potassium permanganate (Mann, 1972). One must keep in mind, however, that the instrumentation is expensive and that specimen preparation and image interpretation techniques are involved, such that flourescence microscopy should not be discounted for studying lignification.

PRIMARY FLUORESCENCE

The fluorescence microscope utilizes that principle of fluorescence which causes a substance to emit visible light upon exposure to ultraviolet light. The emitted light maximum of organic compounds is displaced by 120-150 nm toward the red end of the spectrum. Since the first absorption maxima of lignin is in the 270-290 nm band, the fluorescence of lignin should be in the near ultra-violet or short wave visible regions of the spectrum, 400-430 nm (Barskii and Bardinskaya, 1959).

FLUORESCENCE MICROSCOPY AND LIGNIFICATION 5

It is felt that the fluorescence is due to the presence of conjugated double bonds in the lignin (Frey-Wyssling, 1964).

Many workers have reported the fluorescence of lignin as a blue light (Eichler, 1935; Frey-Wyssling, 1964). The exact position (wavelength) of the maxima and intensity of the visible light are determined by several factors. The most important factors are chemical composition, molecular weight, temperature, pH and density of the absorbing material. Lignin can be broken down into separate fractions and their individual fluorescence spectra studied (Kunze, 1968; Polcin and Rapson, 1969). By studying the primary fluorescence, one can determine the presence of lignin, resin, oil. etc., in wood and can distinguish certain species of wood (Pecina, 1965). It would be difficult, however, to identify all types of wood based on their primary fluorescence alone, since too many factors affect the visible light emitted.

The primary fluorescence of lignin has been used by many workers as a tool to study the process of lignin deposition. Eichler (1935) reported a slight increase in fluorescence intensity at the beginning of lignification which then remained unchanged. This maturing fluorescence image was also observed by Wimmer (1948).

Kisser and Wittmann (1951) studied the fluorescence image in lignified cell walls, and Frey-Wyssling (1964) studied the lignification of the primary and secondary cell walls. By using densitometer curves it was found that the concentration of lignin in the primary wall was more than twice as high as in the secondary cell wall.

Hoedemaker (1967) made quantitative determinations of the concentration of lignin by its primary fluorescence.

Pfoser (1959) worked with the primary fluorescence of lignin and compared its merits with those of standard lignin tests such as the phloroglucinol-HCl and the Maule reaction. He found that the primary fluorescence may be present in very early stages of development and remain constant during lignin development. As the lignin develops, the phloroglucinol test will become positive (where at first it was negative); the Maule test will eventually become negative. Eichler (1935) and Wimmer (1948) also mentioned that the primary fluorescence of lignin is very sensitive and surpasses the use of stains.

DISADVANTAGES

The primary fluorescence of lignin as a tool for studying lignin has several basic drawbacks. Lignin absorbs ultra-violet light in the 212 nm and 280 nm region, and expensive quartz or special glass optics must be used for optimum transmittance of these wavelengths. Since the image is of low brightness, a darkened room and special light sources are required. It has been reported by several workers that lignin is subject to

° LSA EXPERIMENT STATION TECHNICAL BULLETIN 62

photodegradation. The fluorescence of lignin has been attributed to water soluble lignins that can easily be lost or altered in sample preparations. The fluorescence image can be affected by resins, cell wall thickness and variations due to very small physical and chemical changes.

The fluorescence image may decay during the time of ultra-violet illumination due to degradation of the lignin. Yellowing of lignified material normally begins by the oxidation of the phenolic hydroxyl groups of the lignin (Lcary, 1968). Lin and Kringstad (1970) have found the degradation of natural lignin to occur in three hours. Hoedemaker (1967) found that the fluorescence intensity dropped to one-half of its original value in 20 minutes if a mercury vapor lamp was used. Twenty-four hours later a recovery to 75 percent of the initial value was noted. With a quartz-iodide lamp, the drop in fluorescence intensity was only 10 percent in the first 10 minutes and was fully recovered after cooling off for 5 minutes. This evidence refutes the earlier work of Ruch and Hengartner (1960) who felt that the fluorescence remained constant over a prolonged period of time. With regard to storage of specimens, Wimmer (1948) noted that specimens could be stored for days and still produce the same fluorescence intensity while certain lignin color reactions more or less change quickly.

Kisser and Wittmann (1951) found that the primary fluorescence of the lignified cell wall cannot serve as a specific test reaction for lignification. The blue fluorescence can be made to disappear by exhaustive extraction with hot water, while the phloroglucinol-HCL reaction is still positive. They concluded that it is very probable that the blue fluorescence is conditioned by the water soluble lignins present. Pfoser (1959) indicates that other workers have found similar results, with the blue fluorescence of lignin weakening to a white-like blue with hot water extraction. He further suggests that exact determination of lignin can only be made by certain other physical and chemical methods.

Many other factors, in addition to those already mentioned, affect the blue fluorescence image of lignin. Eichler (1935) indicates that resin has an intense bluish-white fluoresence that masks the fluorescence of lignin and must be dissolved with alcohol. Hoedemaker (1967) reports a direct relationship between section thickness and fluorescence intensity. Kisser and Wittmann (1951) have shown that it could possibly be changes in cell wall structure connected with lignification that are causing the fluorescence. This can be seen by drying non-fluorescing sections which then fluoresce blue, or by drying fluorescing sections which then fluoresce a still stronger blue. In addition, Pecina (1965) points out that fluorescence intensity and wavelength may be affected by the presence of defects (decay), moisture content or direction of cut as well as various growth factors which might have affected wood


formation such as climate or winds which could cause compression wood formation.

ADVANTAGES

Primary fluorescence is suited for determining lignified elements in a section without causing any changes through the use of chemical reactions. It is sensitive, yet quick and easy, as the steps necessary to fix, embed and stain sections are eliminated if one uses fresh tissue. Even sectioning can be omitted in the case of surfaced wood blocks. Fluorescence methods can be used for the detection of lignified elements, to study the lignification process, to determine changes during delignification, answering certain questions about the properties of lignified elements and understanding the chemistry of other lignin indicators (Wimmcr, 1948). It can be used very effectively in the study of wood decay (Aufsess, et ai, 1968).

Both Frey-W yssling (1964) and Hoedemaker (1967) have used the primary fluorescence of lignin to make quantitative evaluations of the concentration and distribution of lignin in the cell wall. Eichler (1935) states that the superiority of the fluorescence test over stain reactions is plainly recognized in the first stages of lignification. This has been confirmed by other workers (Wimmer, 1948; Pfoser, 1959). Wimmer (1948) further indicated that the fluorescence image keeps its same intensity all day long while the image of certain stains changes more rapidly. This would undoubtedly only hold true for short exposures by ultra-violet light. Apparently, equipment which allows one to make extremely short exposures is currently being developed (Reinhardt, 1972). With regard to longevity. Eichler (1935) felt that glycerine preparations two years old showed only slight changes in fluorescence.

SECONDARY FLUORESCENCE

Secondary fluorescence is the introduction of a highly fluorescent substance (fluorochrome) into a material so that when it is irradiated with ultra-violet light it will fluoresce brightly in the visible spectrum. This fluoresence is considerably brighter than most natural primary fluorescence. This phenomenon has been utilized as a technique for studying the lignification process.

Jayme and Bauer (1957) were able to separate earlywood and late-wood fibers by secondary fluorescence. Using fluorochromes in aqueous solutions, they reported a distinct color difference which possibly reflects cell wall density. Yos (1957) examined the three fluorochromes, rhoda-mine red, acridine orange and titan yellow, and concluded that acridine orange can be used as a differential stain for lignin. Pfoser (1959) also


used acridine orange and found that the fluorescence begins in very early stages of lignification and has a greater ability to show lignification than either the phloroglucinol-HCl or Miiule test. The acridine orange fluorescence is superior to the phoroglucinol-HCl reaction for determining the existence of more highly lignified membranes. In addition, it has a greater ability to show less dense phloroglucinol negative membranes than the Maule test. Aufsess, et al. (1968) found that acridine orange can produce very exact indications of the degree of lignification.

DISADVANTAGES

Jayme and Bauer (1957) noted that a color change of fluoro-chromed material may take place within a few seconds. The fluorescence color and intensity of fluorochromes are pH sensitive (Drawert, 1968). Casperson and Hoyme (1964) obtained a difference in fluorescence of various layers in tension wood with a particular set of fluorochromes and connected these results with a difference in packing density of the cellulose. Pfoser (1959) felt that while acridine orange fluorescence is well suited for indicating lignified membranes, it cannot be used to prove the presence of lignin in the strictest sense.

ADVANTAGES

Since lignin retains phenolic hydroxyl groups, one would expect to have pH-independent, basic color reactions that give orthochromatic or even metachromatic2 color tones with metachromatic dyes (Drawert, 1968). Pfoser (1959) has shown that the yellow-green secondary fluorescence by acridine orange corresponds mostly to the blue primary fluorescence in lignified tissue, and that in early stages of lignification one may also obtain mixed tones of red and green with acridine orange. The beginning of tension wood formation is recognized by the hue, and its intensity and can be shown very well even in black and white photographs (Aufsess, 1968). Acridine orange has a maximum absorbance at 270 nm using 365 nm mercury burner and emits visible light at 542 nm (Porro et al, 1963). Yet, when the lignin-poor gelatinous layer takes up acridine orange (as can be seen with visible light), it does not fluoresce in ultra-violet light (Siebers, 1960). While this phenomenon may relate to lack of lignin, Casperson and Hoyme (1964) who saw a difference in fluorescence of various layers in tension wood with particular fluorochromes, connected these results with a difference in packing density of the cellulose.

2 Metachromatic dyes may impart two colors in the specimen. The color of the dye solution is termed the orthochromatic shade; the second color is called the metachromatic shade (Jensen, 1962).


Perhaps the greatest advantage in using a fluorochrome to detect lignin is the increased image intensity relative to that obtained with primary fluorescence. In general, fluorochroming is a more rapid and sensitive technique than conventional staining. A fluorochrome of very low concentration gives high contrast with very little physiological interference.

FLUORESCENCE IN BALSAM FIR

The primary and secondary fluorescence image of balsam fir, A bies balsamea (L.) Mill was observed both in freshly-cut sections and celloi-din-embedded sections. The wood selected for this study was cut on July 6, 1966, for the embedded material (killed in FAA) and on July 6, 1970, for the fresh material. On both collection dates the cambial region was active. All sections were cut at 10 microns with a sliding microtome. Observations were made on a Zeiss Photomicroscope set up for fluorescence microscopy using a mercury vapor lamp.

To obtain the fluorescent image a combination of Zeiss exciter filters and barrier filters was used. Either a UG 5. BG 3 or BG 12 exciter filter was used to permit light of certain wavelengths to be transmitted. The peak transmittance of the BG 3 filter corresponds to die very intense 365 nm line of the mercury vapor lamp. The UG 5 filter transmits light at somewhat shorter wavelengths than the BG 3, specifically with peaks at 280 and 312 nm, in addition to 365 nm. The BG 12 transmits intensity peaks at 365 and 404 nm.

Either one of two barrier filters was used. These two filters were the 47 and the 50 long wave pass filters. In addition, the -65 filter was used to cut out any possible infra-red light. A non-fluorescing immersion oil (nD 1.515) was used between the condenser lens and the slide.

Celloidin-embedded sections were soaked in a 50:50 solution of ether and absolute ethanol overnight to dissolve out the celloidin. Sections were then run through an hydration series from 80 percent ethanol to 100 percent distilled water prior to observation or staining, to swell the cell walls into their more natural state.

The sections for primary fluorescence observations, both fresh and embedded material, were mounted in glycerin on a quartz slide with a # 0 0 glass cover slip. The #00 cover slip allowed for experimentation with incident light. Three filter combinations, UG 5 and 47, BG 3 and 47 and BG 3 and 50, were used.

The sections for secondary fluorescence observation were stained using the fluorochrome acridine orange. The sections were stained in a 1:100 aqueous solution of acridine orange for 5 minutes, rinsed and


mounted in glycerin on a glass slide with a # 0 0 glass cover slip. A quartz slide would have been preferred but was unavailable. Three filter combinations, BG 12 and 47, BG 12 and 50 and BG 3 and 47, were used.

RESULTS

Figure 1 illustrates the primary fluorescence image of fresh balsam fir tissue close to the cambial region. The blue fluorescing lignin was easily detectable in the developing xylem cells. It was also possible to differentiate between highly lignified and less lignified regions of the cell wall in mature xylem cells.

The primary fluorescence of embedded balsam fir showed much the same differentiation, although there appeared to be some washing out of the fluorescent image (Fig. 2). This fading also appeared to be true in the mature xylem cells.

Fresh balsam fir tissue stained with acridine orange showed increased fluorescence which seemed to give a clearer image than that obtained with primary fluorescence (Fig. 3). The presence of lignin in the cell corners, however, was not as clear as in the unstained fresh material and seemed to show up in a later stage of cell development. There was very good differentiation between what is probably the compound middle lamella and the secondary wall in mature xylem cells.

The embedded material stained in acridine orange showed, as did the fresh stained material, that the yellow and orange colors of the fluoro-chrome helped to add clarity and brightness to the image (Fig. 4). The image of the embedded material, however, appeared to be a little less distinct when compared to the fresh material.

A stained section of fresh balsam fir compression wood was irradiated with the full spectra of ultra-violet light from the mercury vapor lamp for three hours. There was a discernible drop in the microscope exposure meter reading after three hours. The slide was moved slightly and it was easy to observe the interface between the irradiated and non-irradiated areas as well as the loss of image intensity in the irradiated area (Fig. 5).

FLUORESCENCE MICROSCOPY AND LIGNIFICATION I I

Figure 1. Primary fluorescence of fresh balsam fir indicating easily detectable lignin. Transmitted dark field illumination. UG 5 exciter filter and #47 barrier filter. (215x)

Figure 2. Primary fluorescence of celloidin embedded balsam fir illustrating washed-out image. Transmitted dark field illumination. BG 3 exciter filter and # 5 0 barrier (215x)


Figure 3. Secondary fluorescence of fresh balsam fir illustrating clear image. Transmitted dark field illumination. Stained five minutes in 1:100 aqueous acridine orange. BG 3 exciter filter and #47 barrier filter. (215x)

Figure 4. Secondary fluorescence of celloidin embedded balsam fir illustrating clear, bright image. Transmitted dark field illumination. Stained five minutes in 1:100 aqueous acridine orange. BG 3 exciter filter and #47 barrier filter. (215x)


Figure 5. Secondary fluorescence of fresh balsam fir compression wood. Indicates darkened area (center of photo) as a result of irradiation with ultra-violet light for three hours. Transmitted bright field illumination. Stained five minutes in 1:100 aqueous acridine orange. BG 12 exciter filter and #47 barrier filter. (215x)

CONCLUSIONS AND SUMMARY

The primary blue fluorescence and the secondary acridine orange fluorescence are well suited for indicating the general nature of cell wall lignification but cannot be used alone as absolute tests for lignin. The numerous factors, as outlined in this paper, which affect the fluorescence image must be isolated and understood before interpretation of any particular fluorescence image is possible.

While quantitative determinations of lignin distribution can be made using primary fluorescence, probably ultra-violet microscopy, which is somewhat more specific and less confusing, would be a better choice.

The distribution of lignin in balsam fir tissue has been shown by primary fluorescence and by the secondary fluorescence of acridine orange. In comparison, the primary fluorescence of lignin appeared to show up in the cell corners before the acridine orange fluorescence. The acridine orange secondary fluorescence, however, did demonstrate a greater ability to show the distribution of lignin in the mature tracheids. This is probably due to increased fluorescence intensity and the chemical selectivity of acridine orange.


The fluorescence of the sections of balsam fir that were embedded in celloidin appeared a little less intense than the freshly-cut sections. This might be attributed to the dissolving out of some of the water soluble lignins in the embedding processes.

Prolonged irradiation of ultra-violet light has been shown to affect fluorescence intensity. This is not a serious problem as long as one is not concerned with quantitative measurements.

LITERATURE CITED

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Bailey, A. 1951. Ultra-violet absorption spectra of chromatographic fractions of lignin. J. Am. Chem. Soc. 73(5): 2325-2326.

Barskii, I. and Baidinskaya, M. 1959. Application of ultra-violet fluorescence microscopy to the study of lignification of cell walls. Doklady Akad. Nauk SSSR. 129: 267-270.

Casperson, G. and Hoynie, E. 1964. Dber die bildung von Zellwanden bei Laiibholzern. 3 Mitt. Cber die Lumenbegrenzung beim Reaktionsholz. Fraserforsch. Textiltech. 15: 205-210.

Drawert, H. 1968. Vitalfiirbung und Vitalfluorochromierung pflanzlicher Zellen und Gewebe. Springer-Verlag. New York.

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Fergus. B. and Goring, D. 1968a. The location of guaicyl and syringyl lignins in birch xylem tissue. Post-Graduate Res. Rep. No. 12. Pulp and Paper Res. Institute of Canada.

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layme, G. and Bauer, G. 1957. Separation of early and latewood fibers by secondary fluorescence. Holzforschung. 11: 16-18.

Jensen, W. 1962. Botanical Histochemistry: Principles and Practice. W. H. Freeman. San Francisco.

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Leary, G. 1968. The yellowing of wood by light. Part II. TAPPI. 51(6): 257-260.

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Pecina, H. 1965. Anwendungsmoglickeiten der Fluorochromierung und Eigen-lumineszenz in der Holzforschung. Holztechnol. (Dresden). 6(4) : 273-277.

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Reinhardt. W. 1972. Personal communication. Zeiss Representative, Brinkmann Instruments, Westbury, N. Y.

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Scott, J. and Goring, D. 1969. Photolysis of wood microsections in the ultraviolet microscope, Post-Graduate Res. Rep. No. 16, Pulp and Paper Res. Institute of Canada.

Scott, J., Proctor, A.. Fergus, B. and Goring D. 1969. The application of ultraviolet microscopy to the distribution of lignin in wood: description and validity of the technique. Wood Sci. Tech. 3(1) : 73-92.

Siebers, A. 1960. The detection of tension wood with fluorescent dyes. Stain Tech. 35(5): 247-251.

Siegel, S. 1969. Evidence for the presence of lignin in moss gametophytes. Am. J. Bot. 56(2): 175-179.

Venverloo, C. 1969. The lignin of Populus nigra L. en. "Italica". A comparative study of the lignified structures in tissue cultures and the tissues of the tree. Acta. Bot. Neerl. 18(2): 241-314.

Wergin, W. 1965. t'ber Enstehung und Aufbau von Reaktionsholzzellen. 4. Mitt.: Nachweis der Ligninverteilung in den Zellwanden des Druckholzes durch Untersuchungen im UV-Licht. Flora Oder Allg. Bot. Zeit. 156(3): 322-331.

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Yos, D. 1957. A standardized method for evaluating the image in fluorescence microscopy. Proc. Iowa Acad. Sci. 64: 132-138.

the suitability of using fluorescence microscopy for studying lignification … · 2010. 2. 22. ·...

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