Chemical imaging of secondary cell wall development in cotton fibers using a mid-infrared focal-plane array detector

Abstract

Market demands for cotton varieties with improved fiber properties also call for the development of fast, reliable analytical methods for monitoring fiber development and measuring their properties. Currently, cotton breeders rely on instrumentation that can require significant amounts of sample, which complicates fiber development studies. Herein, we explored the use of high-resolution, Fourier-transform infrared (FT-IR) microscopy to examine cotton fiber secondary cell wall development in single fibers. Notably, there was a marked intensity increase for the C-O bending region near 1015 cm^–1 and the C-H stretch at 2900 cm^–1. These changes agree with those observed with macroscopic FT-IR tests. Chemical distribution maps and principal component analysis plots visually depict these spectral changes. Our results suggest the FT-IR microscopy can potentially be utilized as a tool to monitor and assess important fiber properties, such as cotton maturity, during fiber development.

Keywords

cotton fibers infrared microscopy fiber development imaging

Cotton remains the most used natural textile fiber in the world.¹ In 2012 alone the USA produced over 15 million bales of the commodity.² Given its commercial importance, ongoing breeding efforts seek to produce new cotton varieties with improved performance.³ However, the development of each new variety requires a multitude of tests to examine fiber growth and the commercial viability of the fibers. Developmental and fiber property tests can require large amounts of fiber samples and time; a significant complication for smaller-scale cotton breeding operations that may require evaluations of numerous fiber samples representing individual plants from large populations. As a result, there is a need to develop rapid testing methods that require small amounts of sample and allow for the examination of cotton development and fiber properties.

Vibrational spectroscopy tools (i.e. mid-infrared (mid-IR), near-infrared, and Raman spectroscopy) have been previously used to estimate fiber properties such as maturity ratio,⁴ micronaire,^4,5 fineness,^4,6 and crystallinity index.⁷ These tools offer fast and reproducible measurements that require small amounts of sample. Studies of cotton fibers with mid-IR techniques have gained recent attention, in part due to the wide-spread availability of attenuated total reflection (ATR) accessories. Notable studies include those of Abidi and coworkers,^8–10 who have utilized ATR Fourier-transform infrared (FT-IR) to examine changes in the spectra of developing cotton fibers and to estimate cotton fiber properties. In another recent study, Liu and his coworkers¹¹ relied on the intensity of a C-O bending peak to estimate the maturity of small bundles of cotton fibers. Mid-IR microscopes with high spatial resolution represent another IR research area with increasing commercial adoption. Mid-IR microspectroscopy can be a particularly useful analytical tool, since it combines the informational capability of IR with the spatial ability of microscopic techniques. This combination allows for hyperspectral analysis of samples, which can be used to examine their chemical distribution and composition. To date, published reports of cotton fiber studies with mid-IR microspectroscopy have been limited in scope. Instead, microscopy techniques have been more routinely used in the examination of other plant cell walls^12–17 or in the forensic identification of natural and synthetic fibers.^18,19 Cotton studies include the work of Himmelsbach and coworkers,²⁰ who used FT-IR microspectroscopy to describe the chemical distribution of the cotton seed–cotton fiber interface. In a limited study, Wang and coworkers²¹ used FT-IR microspectroscopy techniques to characterize bioscoured cotton samples. Still, further exploration of the applicability of FT-IR microspectroscopy techniques in the examination of cotton fibers is needed.

Herein we present the chemical imaging of developing cotton fibers with a FT-IR microscope equipped with a focal-plane array (FPA) detector. FPA detectors present significant advantages over the more traditional mercury cadmium tellurium (MCT) detectors. Notably, FPA detectors allow for simultaneous testing of hundreds of sampling points.²² This capability allows analyzing large sample areas in a reduced amount of time and with high spatial resolution (∼2 µm). For this initial study, FT-IR multi-point analysis of developing cotton fibers was performed. In addition, principal component analysis (PCA) scatter plots and high-resolution chemical distribution maps were calculated to visually differentiate between stages of cotton fiber development. Cotton fibers used were harvested beginning at 18 days post-anthesis (DPA) until the fibers were mature (fully opened cotton bolls), a time period with high secondary cell wall development (SCWD). Proper SCWD during this stage is important for obtaining cotton fibers with commercially valuable properties, such as high strength. Cotton fiber development has been recently explored with ATR sampling techniques;^8,10 hence, this study provides for a good comparison of the capabilities of FT-IR microspectroscopy to routine FT-IR studies. Our studies seek to establish the potential use of FT-IR microspectroscopy to examine cotton fiber development.

Experimental details

Plant materials

Cotton samples of the germplasm line MD 90ne were grown in 2009 under standard field conditions in New Orleans, LA. The development of the line was previously described.^23–25 Samples were harvested at different developmental stages between 18 and 40 DPA. Mature samples from open cotton bolls (60 + DPA) were also collected. Fibers for all developmental points were removed from the cotton seed by hand and allowed to dry at room temperature. Fibers from each developmental point (∼2 g) were manually blended. To ensure sampling of different areas of the fiber samples, each developmental time point was separated into three sub-samples (∼0.65 g). Examined fibers were randomly selected from each of the three sub-samples.

FT-IR examination

Cotton fibers were examined with a Hyperion 3000 FT-IR microscope (Bruker Optics, Billerica, MA) equipped with a mid-IR FPA detector and a Sony ExwaveHD color video camera. The microscope was used as installed by the manufacturer. Fiber samples from each developmental stage were individually mounted on a metal plate with a small opening. Double-sided tape was placed on each side of the opening so as to secure the fiber in place. All FT-IR microscope data was collected in the transmission mode. Scans were collected until a clear IR spectrum of each sample was obtained; typical sample scans were between 64 and 96 scans. A higher number of scans did not significantly alter the signal-to-noise ratio of the spectra. When necessary, samples were manually flattened with a knife roller before mounting. Scans were measured with a resolution of 8 cm^–1 (3800–900 cm^–1), and the resulting spectra were corrected against an air background. At these conditions, examination of a 340 µm × 340 µm sampling window was completed in 7 minutes (at 64 scans). Except when otherwise noted, multi-point spectra are the average of 21 distinct fibers. For each sample, three sampling areas were examined, with each area providing a minimum of 15 spectral sampling points. To retain spectral reliability, sampling points were selected in areas that showed high intensity for cotton spectral bands. As a result, fiber twists and edges were avoided from selection as sampling points. Spectra were corrected for atmospheric CO₂ and baseline using the OPUS spectroscopy software (version 6.5). Additional spectral manipulation was kept to a minimum. Single spectra were exported to Microsoft Excel and plotted with IGOR Pro (version 6.2, Wavemetrics, Portland, OR). Unless otherwise noted, band assignments were taken from the Maréchal and Chanzy²⁶ FT-IR study on cellulose I_β crystalline samples (Table 1).

Table 1.

Fourier-transform infrared band assignments for resolved bands observed for flattened cotton fibers.

Vibration	Observed
	(cm^–1)
Symmetric C–H stretch¹⁰	2914, 2850
C–H stretch²⁶	2899
Absorbed water¹⁰	1633
O–H bending bands²⁶	1425
C–H bending²⁹	1366
O–H bending bands²⁶	1335
O–H bending bands²⁶	1316
C–H bending²⁹	1281
C–H bending²⁹	1239
C–O vibrations, C₂–O₂–H²⁶	1107
C–O vibrations, C₃–O₃–H²⁶	1054
C–O vibrations, C₆–O₆–H²⁶	1029
C–O vibrations, C₆–O₆–H (minor)²⁶	1000
C–O vibration (unspecified)¹¹	985

Macro sampling of fibers was performed with a Vertex 70 (Bruker Optics, Billerica, MA) equipped with an ATR sampling accessory (Pike Technologies, Madison, WI) with a diamond-ZnSe reflective crystal. Cotton samples were placed on top of the ATR crystal and secured with a metal clamp. A total of 256 scans were measured for each sample point with a resolution of 4 cm^–1 (3800–900 cm^–1). Spectra are presented without ATR correction or atmospheric compensation.

Principal component analysis

FT-IR and first-derivative spectra were calculated with OPUS and were then uploaded to the CAMO Unscrambler software (CAMO Software Inc., Woodbridge, NJ). Two-dimensional PCA models and scatter plots were generated for cotton fiber sets at different developmental time points. The PCA databases consisted of either FT-IR absorbance or first-derivative pre-processed data. For both databases, 777 wavenumber variables were included. PCA sample points represent the average spectra of examined single fibers, with each sample being examined in three distinct areas and providing a minimum of 45 sampling spectral points (total).

Chemical imaging

Three-dimensional data analysis was performed with the 3D OPUS software package. Spectral bands were integrated using the measurement applications in OPUS. The default integration method was used, which draws a line between the selected frequency limits. Only the area above the line was integrated. Unless otherwise noted, chemical images are presented normalized to the highest observed integration. These points correspond to the red and pink tones observed in chemical distribution maps; a dark blue color corresponds to integrations at or below zero.

Scanning electron microscopy

The fibers were mounted on standard Cambridge scanning electron microscope (SEM) stubs using double-stick Avery photo tabs, #06001. The SEM mounts were coated with 60/40 gold/palladium using a HummerTM II Sputter Coater (Ladd Research, Williston, VT, USA) for 4 minutes. The specimens were examined in a XL30 Environmental SEM (FEI Company, Hillsboro, OR) at an accelerating voltage from 10–15 kV under high vacuum conditions.

Results and discussion

Multi-point FT-IR microspectroscopy

Figure 1 shows the video image (20× objective) of two mature cotton fibers (Figure 1(a)) and the FT-IR spectra of multiple sampling points along these fibers (Figure 1(b)). All spectra were collected simultaneously utilizing a FPA mid-IR detector. The spectra show absorption bands with positions that closely match major bands of a small cotton bundle examined with a conventional FT-IR spectrometer and an ATR accessory (Figure 2, bottom). There are, however, noticeable differences in band shapes and intensity. For example, the O-H stretching band (3600–3000 cm^–1) of the single fiber spectra show moderate noise and shape distortion when compared to the O-H band in the FT-IR ATR spectrum (Figure 2). In addition, C-O bending peaks in the fingerprint region (1185–985 cm^–1) are significantly distorted and reduced in their intensity. Previous FT-IR microspectroscopy studies have reported spectra with significant noise and distorted spectral bands for fibers with irregular pathlengths or thickness.²⁷ Mature cotton fibers do not show uniform thickness (Figure 3(a)) and cross-sections of mature fibers often show a kidney bean shape.¹ Light transmission through these uneven samples can result in lensing effects, such as light scattering, that alter detector readings.²⁷ As a result, we investigated if fiber processing could provide better-resolved cotton spectra.

Figure 1.

Multiple-point examination of mature cotton fibers with a Fourier-transform infrared (FT-IR) microscope equipped with a focal-plane array mid-infrared detector. Video image (20 × objective) of two mature MD90 cotton fibers (40 days post-anthesis; (a)) and FT-IR spectra of the indicated fiber sampling points (b). A FT-IR spectrum of an air sampling point is also show in (b) (black trace, bottom). Spectra are shown shifted along the y-axis (normalized absorbance).

Figure 2.

Fourier-transform infrared (FT-IR) microscope spectra of an uncompressed 40 days post-anthesis (DPA) cotton fiber (top, black trace) and a manually flattened 40 DPA cotton fiber (center, gray trace). A FT-IR spectrum of a small fiber bundle acquired with a conventional FT-IR spectrometer and an attenuated total reflection (ATR) accessory is also show (bottom, dashed gray trace). Single fiber spectra are the average of 10 sampling points along the fibers. Spectra are shown shifted along the y-axis (normalized absorbance).

Figure 3.

Detail of cotton fiber surface and fiber thickness as observed with a scanning electron microscope. The developmental time points shown are (a) 40 days post-anthesis (DPA), (b) 24 DPA, and (c) 18 DPA.

Manual flattening of the cotton fibers significantly alters the relative intensity of cotton spectral bands in the fingerprint region (Figure 2, center). Notably, the relative intensity and shape of the C-O region (1185–985 cm^–1) more closely compares to those observed in the FT-IR ATR spectrum of a small, uncompressed cotton bundle. The series of fingerprint region peaks observed between 1500 and 1150 cm^–1 and the water bending band^8,10 observed near 1640 cm^–1 now appear reduced in intensity. However, the shape of these peaks is not significantly altered by sample compression. Since previous research reports have identified important spectral changes that occur to C-O bending bands during SCWD,^8,10,11 manual flattening of the fibers was incorporated into our sample preparation. We note that while the O-H stretching bands of the flattened fibers appeared more symmetrical after fiber flattening, they still exhibited some noise. Better resolution of the O-H bands might require further fiber flattening with commercial tools (e.g. a diamond press) or deuteration. However, close examination of the changes in the O-H–hydrogen bonding network of developing cotton fibers is beyond the scope of this study.

Figure 4 shows FT-IR microscope spectra of MD 90ne samples harvested at 18, 24, 32, 40 DPA, and mature fibers from open cotton bolls. Each spectrum is the average of at least 15 sampling points taken from three distinct fibers. All samples were manually flattened before spectrum acquisition. Notably, the first two developmental points (18 and 24 DPA) show strong O-H bands (∼3600–3000 cm^–1) that lack the noise observed in the more developed fibers (32, 40 DPA and the fully mature fiber). This phenomenon also appears to be related to fiber thickness. During SCWD, layers of cellulose are added along the inner limits of the primary cell wall of the cotton fiber, which results in a gradual expansion of the fiber thickness. The relatively low amount of SCW in the 18 and 24 DPA samples reduces the likelihood that the samples will scatter light and alter detector readings. SEM images of uncompressed 18 and 24 DPA fibers (Figures 3(b) and (c)) illustrate our observations; the micrographs show thin, flat structures for the less developed fibers. We note that spectra of uncompressed 18 and 24 DPA show the same general features as those of the manually compressed fibers (data not shown). These findings suggest that light scattering by the fibers could be used to follow stages of SCWD in cotton fibers (see below).

Figure 4.

Fourier-transform infrared microscope spectra of cotton fibers harvested at different days post-anthesis (DPA): (a) 18 DPA; (b) 24 DPA; (c) 32 DPA; (d) 40 DPA; (e) mature fibers from open cotton bolls. Spectra are shown shifted along the y-axis (normalized absorbance).

Two additional spectral regions exhibit significant changes during fiber development: the C–H stretching region, 3000–2700 cm^–1, and a portion of the fingerprint region with multiple C–O bending vibrations, 1185–985 cm^–1 (Figure 4). In contrast, peaks in the O–H and C–H bending region, between 1500 and 1150 cm^–1, do not undergo large changes in peak shape or position during fiber development; instead, an even increase in peak intensity is observed.

Vibrational peaks associated with cotton surface waxes drop in prominence as cotton fibers mature (Figure 4). The less developed fibers show three peaks in the C-H region: 2914, 2899, and 2850 cm^–1 (18 and 24 DPA). The 2914 and 2850 cm^–1 peaks are associated with the alkyl groups of waxes in the cuticle, while the 2899 cm^–1 peak is associated with the C-H bonds in the cotton fiber.^8,10 As the SCW grows, the prominence of the 2899 cm^–1 peak increases and masks peak contributions from the waxes. While this trend mirrors what has been previously observed in FT-IR ATR examinations,^8,10 the intensity of the fiber C-H peak is much more pronounced in the microspectroscopy spectra. As ATR is a reflection technique that examines the near surface of samples, acquired spectra will highlight surface components, such as waxes, in the fiber cuticle. In contrast, the current study was performed in transmission mode, a sampling method that should better represent the totality of the cotton fiber.

Spectral changes in the C-O bending region during SCWD are given emphasis in Figure 5. Three resolved peaks are observed in the C-O bending region at 1107, 1054, and 1029 cm^–1. In addition there are shoulder bands near 1000 and 985 cm^–1, covering a region between 1015 and 970 cm^–1. The intensity of this range significantly increases with SCWD relative to the prominent C-O band at 1029 cm^–1. Changes in this spectral region as observed with ATR FT-IR – specifically changes to a resolved peak at 985 cm^–1 – have been previously used to monitor fiber development and to estimate cotton fiber maturity,¹¹ and our preliminary results suggest that FT-IR microscope spectra could be used in a similar fashion. Still, a more robust sampling method that examines a high number of fiber samples will have to be employed to develop a quantitative fiber quality measurement.

Figure 5.

Fourier-transform infrared microscope spectra of the C–O stretching region for cotton fibers harvested at different developmental time points: (a) 18 days post-anthesis (DPA); (b) 24 DPA; (c) 32 DPA; (d) 40 DPA; (e) mature fibers from open cotton bolls DPA. Spectra are shown shifted along the y-axis (normalized absorbance). A dashed line indicates the region (1015 and 970 cm^–1) that undergoes significant changes during cell wall development.

Principal component analysis of FT-IR microspectroscopy data

PCA is a mathematical method that analyses variables in a data set and, through a process of transformative reduction, produces independent variables known as principal components.²⁸ In accomplishing this variable reduction, PCA allows for the simple comparison of complex data groups. We investigated if PCA could simplify the analysis of our multi-point microspectroscopy data. A two-component depiction of cotton fiber FT-IR spectra following PCA is plotted in Figure 6(a). Separation of cotton fiber spectra into four spatial clusters is observed, with each grouping corresponding to one of the fiber developmental time points examined (18, 24, 32, and 40 DPA). Five points in the 32 DPA cluster appear somewhat separated from the high concentration of points. Most of this separation is observed along the principal component 1 (PC1) axis (left to right), which suggests that this principal component is greatly influenced by the developmental progress of the samples, and possibly by C-O peak intensity. The small overlap for the 32 and 40 DPA in the PC1 axis is differentiated along the principal component 2 (PC2) axis. The first two principal components in the model account for 95.7% of the variance observed in Figure 6(a): 89.1 % for PC1 and 6.6% for PC2. PCA of first-derivative FT-IR spectra results in a similar separation of data points by their developmental time point (Figure 6(b)). The 18, 24, and 40 DPA sample points appear closer together; however, no overlap is observed. A benefit of the first-derivative PCA is the smaller separation of 32 DPA sample points along the PC2 axis. The first two principal components in the first-derivative model account for 81.4% of the variation observed in Figure 6(b), with 61.3% coming from PC1. Taken together, these PCA plots suggest FT-IR microspectroscopy data can be used to differentiate between developmental time points of a cotton fiber.

Figure 6.

Principal component analysis scatter plots of (a) Fourier-transform infrared microscope spectra and (b) first-derivative spectra for cotton fibers and harvested at different developmental time points: 18 days post-anthesis (DPA), 24 DPA, 32 DPA, and 40 DPA.

FT-IR chemical distribution maps

FT-IR chemical distribution maps reveal changes in the composition of developing cotton fibers (Figure 7). To better understand the spectroscopic changes observed in developing cotton fibers (Figure 4), we calculated intensity maps that quantified the integration of selected spectral regions (Figure 7). Three developmental time points were examined: 18, 24, and 40 DPA. While limited in number, these sampling points were selected to showcase large spectral changes. Integration of the dominant C–O bending peak near 1054 cm^–1 resulted in maps with high intensity regions (red tones) that evenly covered space associated with cotton fiber samples (Section ii of Figures 7(a)–(c)). A notable exception includes areas near fiber twists (Figure 7(b)). Section ii maps were normalized to their own maximum integrations, which accounts for the intense integrations observed in those maps, but not for their even distribution. Section iii maps show the integration of the C–O bending region near 1016 cm^–1. To better compare changes in the C–O bending region, the second set of integration maps (iii) were normalized to the maximum intensity of the corresponding 1054 cm^–1 peak (ii). As the fiber develops SCW, the integration of the 1016 cm^–1 region (iii) transitions from showing medium intensities (green and yellow tones for the 18 DPA sample and yellow/orange tones for the 24 DPA sample; Figures 7(a) and (b)) to strong integrations (mostly red and pink areas for the 40 DPA sample; Figure 7(c)). This observation mimics the increase in relative intensity of the C–O bending region near 1016 cm^–1 described above (Figure 5).

Figure 7.

Chemical distribution in cotton fibers as determined with a Fourier-transform infrared microscope equipped with a focal-plane array mid-infrared detector; developmental points shown are for (a) 18 days post-anthesis (DPA), (b) 24 DPA, and (c) 40 DPA fiber samples. Fibers were manually flattened prior to examination. For each developmental point an optical image (i) and various integration intensity maps are shown (ii–vi). Intensity maps correspond to the integration of a primary (ii) and a less prominent (iii) C–O bending peak (∼1053 and ∼1016 cm^–1, respectively), a portion of the O–H stretching band (iv, ∼3384 cm^–1), the central portion of the C–H stretching band (v, ∼2897 cm^–1), and combination peaks in the fingerprint region (vi, ∼1315 cm^–1). The C–O maps (i and ii) are normalized to the maximum intensity of the primary C–O peak (i). The remaining maps (iv–vi) are normalized to the maximum intensity of the O–H stretching band (iv). Red and pink tones correspond to high-intensity integrations, while the dark blue color corresponds to integrations near zero. (Color online only.)

Three additional spectral regions were integrated: the O–H stretching band (iv; 3384 cm^–1), the central C–H stretching band (v; 2899 cm^–1), and a peak in the fingerprint region (vi; 1315 cm^–1). Maps were normalized to the maximum integration of the O–H stretching band. While the integrations of the O–H bands were high for most areas associated with the cotton fibers, only the integration of the mature fiber appeared uniform (Figure 7(c)). Variability might be a result of uneven participation in hydrogen bonding by these O–H groups (hydrogen bonding increases the intensity of the O–H band). The color transition in the C–H peak maps (v in Figure 7) is striking. Colors covering the fiber areas in the maps transition from a pale blue (low value integrations) for the 18 DPA fiber, to mostly green (moderate integrations) for the 24 DPA fiber, to an overwhelmingly red map (high integrations) for the 40 DPA fiber. Curiously, the areas of highest C–H stretch intensity for the 18 and 24 DPA fibers appear to be areas where the O–H stretching bands have relatively low intensity. In these early time points the C–H peaks from the surface waxes are likely contributing to the intensity of the central C–H band considered in these maps. Hence, these higher concentration C–H areas have surface waxes that might be limiting moisture content and hydrogen bonding. The intensity of the fingerprint peak ∼1315 cm^–1 (vi) remains relatively low or moderate throughout fiber development. The only exception corresponds to two red areas of the 40 DPA fiber; however, inspection of the video image and the C–O integration maps suggests that the area corresponds to the thicker sections of the sample. Taken together, our observations suggest that chemical distribution maps can be used to visually represent general changes observed during cotton cell wall development.

Large-area cotton fiber imaging

The mapping capabilities of the IR microscope and FPA detector allow for imaging large areas (>100,000 µm²) of the cotton samples. Figure 8(a) shows the chemical distribution of two flattened cotton fibers (32 DPA) over a 560,000 µm² area. Maps show the integration intensity of the 1053 cm^–1 C–O bending peak. Mapping large fiber areas could lead to better sampling of a single or multiple cotton fibers. For example, Figure 8(b) shows the average spectra of sampling points inside the red box shown in Figure 8(a) (10 points along the center of the fiber). To the best of our knowledge, Figure 8 marks the first report of a large-area chemical distribution map showing multiple cotton fibers.

Figure 8.

(a) Chemical distribution map of two compressed cotton fibers (32 days post-anthesis) over a large sampling area (560,000 µm²). Intensity corresponds to integration of the C–O bending peak at 1053 cm^–1. (b) Average spectra of 10 fiber sampling points inside the red box shown in (a). (Color online only.)

Simple examination of cotton fiber development

Absorption changes in the fingerprint region of uncompressed cotton fibers can be used to quickly examine fiber development (Figure 9). We previously discussed how uncompressed mature cotton fibers exhibited deformed and reduced absorption in the C–O stretching region. Conversely, the absorption of the C–H and O–H bending peaks showed increased intensity. This phenomenon is indirectly related to sample thickness, with the less developed fibers with an even pathlength not exhibiting this scattering-based alteration. We investigated if this phenomenon could be used to rapidly examine fiber thickness and SCWD in cotton fiber samples. Figure 9 shows video images of cotton fibers harvested at different developmental time points and infrared maps corresponding to the integration of various cotton spectral bands: the O–H region, the C–O bending region, and the C–C and O–H bending region. Integrations were normalized to the dominant O–H stretching band. As a result, the O–H FT-IR maps for all developmental points are fairly even in their high intensity (red and pink colors). The intensity of the C–O region is highest and most continuous for the immature 18 DPA fiber. As expected, the contribution of this band is visibly reduced as the fiber develops its SCW; at 40 DPA most of the fiber shows a moderate (green) contribution from the C–O bending region. A pronounced change was also observed in the C–H and O–H bending region maps. At 18 and 24 DPA these bands are barely noticeable in the FT-IR maps, but they quickly gain intensity at the 32 (green) and 40 DPA (orange and red) time points. While these observations are preliminary, future studies will try to use these observations to establish a fiber quality measurement method.

Figure 9.

Spectral band distribution in uncompressed cotton fibers as determined with a Fourier-transform infrared microscope equipped with a focal-plane array mid-infrared detector; developmental points shown are (a) 18 days post-anthesis (DPA), (b) 24 DPA, (c) 28 DPA, and (d) 40 DPA. For each developmental point an optical image and various intensity maps are shown. Intensity maps correspond to the spectral regions indicated. Integration maps are normalized to the maximum absorbance of the O–H band. (Color online only.)

Conclusions

FT-IR microspectroscopy can be used to assess general mid-IR spectral changes of cotton fibers undergoing SCWD. Measurements are fast and can be performed on a single fiber with little sample preparation (fiber flattening with a knife roller). Notably, the spectral changes observed in this study generally coincide with those observed with macroscopic sampling techniques, such as FT-IR ATR. PCA scatter plots of the microspectroscopy data also allow for the facile distinction of four of the examined developmental time points. High-resolution microspectroscopy also allows for the calculation of chemical distribution maps that can visually depict general spectral changes. These maps can cover multiple cotton fibers at high resolution, and over a larger area (560,000 µm²) than previously reported microscopy studies. Our preliminary results suggest that FT-IR microspectroscopy with a FPA detector could be used to examine cotton fiber properties and cell wall development. Future studies will explore if these general observations could lead to the development of quantitative methods for measuring cotton fiber properties.

Footnotes

Acknowledgements

The authors thank HN Cheng, Meg Yoshioka-Tarver, Aracelis Concepción James, and Anel Méndez Velázquez for their suggestions on the manuscript.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported in part by Cotton, Inc.

Disclaimer

The use of a company or product name is solely for the purpose of providing specific information and does not imply approval or recommendation by the US Department of Agriculture to the exclusion of others.

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