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Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials
Biosystems & Biomaterials Division, National Institute of Standards & Technology, Gaithersburg, MD, 20899, USA
Unit on Ocular and Stem Cell Translational Research, National Eye Institute, National Institutes of Health, Bethesda, MD, 20892, USA
c
The Stellenbosch Nanober Company (Pty) Ltd, Cape Town, South Africa
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 February 2015
Received in revised form
4 May 2015
Accepted 14 May 2015
Available online 15 May 2015
Despite the growing use of nanober scaffolds for tissue engineering applications, there is not a validated, readily available, free solution for rapid, automated analysis of nanober diameter from scanning
electron microscope (SEM) micrographs. Thus, the goal of this study was to create a user friendly ImageJ/
FIJI plugin that would analyze SEM micrographs of nanobers to determine nanober diameter on a
desktop computer within 60 s. Additional design goals included 1) compatibility with a variety of
existing segmentation algorithms, and 2) an open source code to enable further improvement of the
plugin. Using existing algorithms for centerline determination, Euclidean distance transforms and a novel
pixel transformation technique, a plugin called DiameterJ was created for ImageJ/FIJI. The plugin was
validated using 1) digital synthetic images of white lines on a black background and 2) SEM images of
nominally monodispersed steel wires of known diameters. DiameterJ analyzed SEM micrographs in 20 s,
produced diameters not statistically different from known values, was over 10-times closer to known
diameter values than other open source software, provided hundreds of times the sampling of manual
measurement, and was hundreds of times faster than manual assessment of nanober diameter.
DiameterJ enables users to rapidly and thoroughly determine the structural features of nanober scaffolds and could potentially allow new insights to be formed into ber diameter distribution and cell
response.
Published by Elsevier Ltd.
Keywords:
ImageJ
Image analysis
FIJI
Scaffold
Morphology
Structure
1. Introduction
Electrospun polymeric nanober scaffolds have wide applicability across a variety of elds including tissue engineering [1,2],
ltration [3e6], catalysis [7e9] and in biosensors [10e12]. In all of
these elds a strong correlation has been found between nanober
mat morphology and performance. Nanober morphology features
such as ber diameter, orientation, and mesh hole size (pore size)
have been shown to correlate strongly with mechanical properties
[13e15], surface area to volume ratios (inuencing catalytic efciency [16,17], signal to noise ratios in biosensors [18,19], and
ltration efciency [20]), and in tissue engineering to determine
cell morphology [21e23], phenotype [24,25], and differentiation
[26,27]. Thus, there is a need for characterization tools of the
* Corresponding author.
E-mail addresses: Nathan.Hotaling@gmail.com,
(N.A. Hotaling).
http://dx.doi.org/10.1016/j.biomaterials.2015.05.015
0142-9612/Published by Elsevier Ltd.
Nathan.Hotaling@nist.gov
scaffolds that can quickly and accurately assess these ber properties [28]. Currently, a variety of manual assessment techniques
are commonly used to obtain distributions of ber diameters
[29e31]. Due to the size of the bers being spun (<1000 nm) the
most prevalent tool for the assessment of nanober scaffolds is the
scanning electron microscope (SEM) [32,33]. Therefore, image
analysis tools that can analyze SEM images, would have the largest
user-base if developed.
The gold standard for assessment of nanober morphology has
historically been manual measurement in SEM images using line
tools in image analysis programs such as ImageJ/FIJI (National
Institute of Health, MD) [34e36]. In addition to being lowthroughput, manual measurements may be biased. Operators
may avoid non-representative bers, such as blebs or very thin or
thick bers as well as introduce small systemic biases in measurement. Also, because a distribution of ber diameters is present
in electrospun nanober preparations, obtaining enough manual
measurements to reduce the standard deviation to a level where
meaningful statistical comparisons can be performed is time-
328
PLGA nanobers. The PLGA nanober mat was removed from the
foil and cut into 5 mm 5 mm squares for SEM.
Nanober samples with two layers of different diameter
(bimodal dia.) were commercially available (Stellenbosch Nanober
Company; Cape Town, South Africa). The bimodal samples were
composed of PLGA 50:50 (PLGA 50:50 molar ratio of L to G, inherent
viscosity 0.4e1.0 dl/g). The manufacturer reported values for ber
diameter in the two layers were 580 nm 226 nm and
1800 nm 775 nm. All micrographs of either the monodispersed
PLGA nanobers or the bimodal PLGA bers that were taken and
analyzed can be found in Fig. 4 of reference [53].
2.5. Scanning electron microscopy
Steel wire samples (each ga. individually and mixed-ga. samples), PLGA monodisperse nanobers (removed from the aluminum
foil target) and PLGA bimodal nanobers were mounted onto SEM
stubs using carbon tape (Ted Pella, Inc.), placed under house vacuum overnight with desiccant and gold sputter-coated (120 s at
75 mA, Desk V HP, Denton Vacuum, approximately 10 nm of gold
deposition). Six scanning electron micrographs were captured of
each sample (Hitachi S4700 SEM, 5 kV, 10 mA, z13 mm working
distance). Wire with a gauge of 48 was imaged at 150X, and 50 ga.,
53 ga., and mixed-gauge samples were imaged at 200X. Monodisperse nanobers were imaged at 5000X and bimodal nanobers
were imaged at 2500X. In total 36 SEM images were analyzed (24
steel wire and 12 PLGA nanober).
2.6. Manual measurement
In order to validate DiameterJ, ber diameter for the wire
samples and nanobers were determined manually from SEM images using the Line tool in ImageJ. Two people experienced in this
practice each measured ber diameter at 25 random locations in
each micrograph. Each person analyzed six micrographs for 6
samples: wire samples (48 ga., 50 ga., 53, ga., mixed ga.), PLGA
monodisperse nanobers and PLGA bimodal nanobers, for a total
36 images and 150 data points per sample per person. The 300 data
points for each sample (150 data points from 2 people) were
plotted on a histogram and peak-tting (described below) was used
to assess the results. This manual measurement was not seen as a
ground truth for the images and the protocol was chosen by using
a method that is indicative of that which is used in the literature
[54]. All manual human measures and their histograms can be seen
in Figs. 3 and 4 of reference [53].
2.7. DiameterJ algorithm
A design requirement for the DiameterJ algorithm was to be able
to analyze an 8-bit SEM image of any resolution using a desktop
computer in less than 60 s. DiameterJ was tested on a Dell Optiplex
9010 (Windows 8, 8 GB of RAM, Intel 3rd generation i7-3770) and
functionality was also veried on an older system (Windows 8.1,
4 GB RAM, AMD Phenom II 2.5 Ghz processor, built in 2009). The
DiameterJ algorithm has been published to the ImageJ repository
and is submitted to be incorporated into the Fiji base release. The
DiameterJ plugin can be downloaded from the ImageJ Wiki, http://
imagej.net/DiameterJ, and installed into ImageJ 1.48 or newer or
the latest release of Fiji. See Fig. 1 for an overview of how the
DiameterJ algorithm analyzes ber diameter and other scaffold
properties.
2.7.1. Segmentation
The rst step to image analysis is segmenting the image into
foreground and background. SEM micrographs were segmented
329
330
Fig. 1. Diagram of ber analysis algorithm. The starting input point is the black box labeled Segmented Picture. Gray Boxes are outputs of DiameterJ. Algorithms were not able to
effectively determine ber diameter at ber intersections requiring subroutines to remove ber intersections from the analysis leading to interconnected outputs within the code as
indicated.
331
An advantage of DiameterJ Histogram is that many ber diameter measurements are made on each image enabling histograms of
ber diameter distributions to be generated and analyzed via peaktting (Fig. 3). Gaussian curves were t to histograms of Multi-Dia.
digital synthetic images to nd the mean diameter of each peak
(Fig. 3C). The data and ts for the histograms as well as the original
digital synthetic images can be seen in Fig. 2 of reference [53]. For
images with multiple line diameters that were close together
multimodal peak-tting became more difcult due to overlap between the peaks (see six line diameter image in Fig. S1). To measure
the deviation of the peak-ts, the absolute (Fig. 3D) and percent
(Fig. 3E) error between the known diameters in digital synthetic
images and the peak locations were calculated. The mean absolute
error of the peak-ts was less than 0.6 px from the known value in
digital synthetic images with a maximum error of 1.5 px. This absolute error translated to a mean percent error of less than 3% for all
Mult-Dia digital synthetic images (Fig. 3E). In Fig. 3E, the larger
percent error for the 4-diameter images was caused by the fact that
these images had a high percentage of lines less than 10 px in
diameter, leading to a high percent error even though the absolute
error was 0.5 px to 1.0 px. Measurements on 10-diameter Multi-Dia.
digital synthetic images were not successful since the peaks were
too close to one another to resolve via peak-tting. Thus, peaks
should be separated by at least 3 px in order to enable accurate
peak-tting. These results validate that Super Pixel and Histogram
were able to effectively determine line diameters in digital synthetic images. FibreQuant was not designed to analyze segmented
images (black and white images) and, thus, could not be validated
on digital synthetic images.
3.2. DiameterJ validation on wire micrographs
After validating DiameterJ with digital synthetic images, the
program was tested on SEM images of wires with known diameters. Obtaining polymer nanobers with a known diameter is
not possible because there is a distribution of ber diameters in
electrospun nanober mats. Thus, small gauge (48, 50, or 53 ga.)
316 stainless steel wire with nominally monodispersed diameter
was used to provide a measurement base for DiameterJ (Fig. 4,
Fig. S2, Fig. S3). Manual measurement of wires produced an error of
less than 1% for both the single wire and three wire micrographs
(Fig. 4, Fig. S3). The two analysts' measurements were shown to
produce statistically not different results for the wire diameter
(Fig. S3). The modes of the DiameterJ Histogram results shown in
Fig. 4D (before peak-tting) differed by less than 1.2% from known
diameter of the wires. The histograms showed a tight dispersion of
measured wire diameters with none of the Gaussian peak-ts
having a standard deviation greater than 0.64 0.1 mm. The data
and ts for the histograms as well as the original SEM images and
their segments can be seen in Fig. 3 of reference [53].
Analysis of wire micrographs with different measurement tools
(Super Pixel, Histogram, BoneJ, Manual, PrC) revealed that each tool
produced a similar amount of error when comparing measurements between the three sizes of single ga. micrographs
(Fig. 4EeF). When comparing percent error between different tools,
DiameterJ's Histogram was not statistically different from Manual
or PrC in either single ga. or mixed-ga. micrographs (Fig. 4EeF). The
Super Pixel algorithm had a statistically higher error than the
Manual measurement when measuring the 48 ga. and 50 ga. wire.
BoneJ had a statistically higher error when compared to any other
tool across all three gauges. BoneJ included all ber intersections in
its analysis which most likely led to inated average diameters as
shown in Fig. S3. For mixed-ga. image analysis in Fig. 4F, all
methods tested produced similar levels of mean percent error (less
than 1.5%) and were not statistically different from one another.
332
Fig. 2. Analysis of calibration images to determine the mean line dia. Digital synthetic images were analyzed by DiameterJ (Super Pixel, Histogram) or BoneJ. Lines in (E) and (G), are
colored by size (Blue > Red > Green) for ease of visual inspection but actual analysis was performed on images with white lines. The plot of the percent error between the diameter
produced by the programs and the ground truth from the calibration images can be seen in Figures (B), (D), (F), and (H). The red dashed line represents 10% error. N 3 images for
each data point in all graphs. Error bars were omitted for clarity. (For interpretation of the references to colour in this gure legend, the reader is referred to the web version of this
article.)
Table 1
Representative digital synthetic image analyses using DiameterJ super pixel: comparison of results from the IC and SC centerline determinations.
Image type
Actual
diameter (px)
Insensitive e
IC (px)
Sensitive e
SC (px)
Insensitive e
IC (% error)
Sensitive e
SC (% error)
Average of IC &
SC (% error)
Ordered-1D
Disordered-1D
Ordered-3D
Disordered-3D
50
15
70
35
50.7
12.7
70.4
31.7
53.5
16.0
77.9
36.9
52.1
14.4
74.1
34.3
1.4%
15.1%
0.5%
9.4%
7.1%
6.6%
11.3%
5.34%
4.3%
4.3%
5.9%
2.0%
Table 2
Percent Error of the Super Pixel, Histogram, and BoneJ Algorithms for Digital Synthetic Images with Line Diameters between 10 px and 125 px.
Super Pixel
Histogram
BoneJ
Ordered-1D (% error)
Disordered-1D (% error)
Ordered-3D (% error)
Disordered-3D (% error)
4.9 2.2
2.5 1.9
13.6 3.4
7.2 4.0
4.7 1.4
57.6 12.7
7.2 4.4
19.1 13.5
122.6 87.6
7.8 4.3
16 10.7
158.9 23.7
(BoneJ and Super Pixel only provide a global average ber dia).
Taken together, these results indicate that Super Pixel and Histogram produced ber diameter measurements from SEMs of steel
333
Fig. 3. DiameterJ Histogram algorithm peak tting analysis to determine multi-modal line diameter distributions in Multi-Dia. calibration images. Lines in the digital synthetic
images (A) are colored by size (Blue > Red > Green > Orange > Yellow > Purple) for visualization but analysis was performed on images with white bers. (B) DiameterJ's Histogram
algorithm's analysis of images in (A). Peak modes are indicated on histograms. (C) Gaussian peak ts for the 2 dia. histogram in (B). The mean absolute error and percent error of
the DiameterJ Histogram algorithm when Gaussian peaks are t to each histogram in (D) and (E), respectively. Error bars represent standard error for N 3 images. (For interpretation of the references to colour in this gure legend, the reader is referred to the web version of this article.)
334
Fig. 4. Analysis of SEM micrographs of 316 stainless steel reference wire. (A, B) SEM micrographs of steel wire. (C) Optical image of 53 ga. steel wire. (D) DiameterJ's Histogram
algorithm output. Error in mean wire diameter of the single diameter (E), or mixed three diameter (F) steel wire micrographs for 5 different methodologies/algorithms. N 6
images for all analyses. Error bars represent the standard deviation. Asterisks (*) indicate statistically different from DiameterJ's Histogram algorithm; number signs (#) indicate
statistical difference from human measure; plus signs () indicate statistical difference from DiameterJ's Super Pixel algorithm; and an alpha sign (a) indicates statistical difference
from PrC (P < 0.05).
335
Fig. 5. Analysis of SEM micrographs of PLGA nanobers. (A, B) SEM micrographs of PLGA nanobers. (C, D) Cumulative ber diameter histograms generated by DiameterJ Histogram
(left) and by human measure (right). (E, F) Mean ber diameter for different methodologies/algorithms. The mean and standard deviation in nm are given on the bars in (E). N 6
images for all histograms and graphs. Error bars are standard deviation. Money sign ($) represents statistically different (P < 0.1) from DiameterJ's Histogram algorithm.
336
Fig. 6. Segmentation algorithms and analysis of ber dia., ber orientation, mesh holes, porosity, intersection density and ber length. (A) An SEM micrograph of 53 gauge wire (dia.
16.7 mm) was segmented with the indicated algorithms. Diameter (B) and Orientation (C) histograms produced by DiameterJ's Histogram algorithm and OrientationJ given below
their corresponding segmented images in (A). (D) Summary table of DiameterJ analysis of the segmented images. (E) Original SEM image of 53 ga. steel wire.
~100 s). The large data sets afforded by DiameterJ enabled ber
diameter distributions to be plotted and analyzed via peak-tting,
providing better condence in ber diameter measurements.
DiameterJ was validated on a wide range of digital synthetic images
that mimic segmented ber images (white lines on a black background) and with SEM images of steel wires of known diameters.
Further, when analyzing ber diameter in wire SEM images,
DiameterJ results were comparable to manual operator measurements, demonstrating that throughput was not purchased at the
expense of accuracy. Most importantly, DiameterJ provides an
accessible, rapid, validated, exible, free and easy-to-use tool that
engineers, biologists, and other scientists can use to efciently
analyze ber diameter in nanober scaffolds.
Acknowledgments
We thank Daniel Krogstad for help with Igor Pro peak-tting
software, Desu Chen for help with image processing and Antonio
D'Amore for help with SEM image analysis. The standard deviation (S.D.) is the same as the combined standard uncertainty of
the mean for the purposes of this work. This article, a contribution
of NIST, is not subject to US copyright. Certain equipment and instruments or materials are identied in the paper to adequately
specify the experimental details. Such identication does not imply
recommendation by NIST, nor does it imply the materials are
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