CHAPTER 10
Microscopy Techniques
Contents
10.1 I ntroduction 183
10.2 O ptical Microscopy 184
10.2.1 Particle Size Testing 184
10.2.2 Morphology 185
10.2.3 Thermal or Hot-Stage Microscopy 188
10.2.4 Evaluation of Particulate Matter 188
10.2.5 Evaluation of Foreign Particulate Matter 188
10.2.6 Evaluation of Material Compatibility 189
10.2.7 Testing of Tablets 189
10.2.8 Ingredient-Specific Particle Sizing 189
10.2.9 Fourier-Transform Infrared Microscopy 189
10.3 Scanning Electron Microscopy 189
10.4 Transmission Electron Microscopy 190
10.5 Atomic Force Microscopy 191
10.6 Conclusion 192
References192
10.1 INTRODUCTION
Microscopy is a very useful tool in pharmaceutical research, formulation
development, process development, and quality control of manufactured
products. Microscopy is routinely used for the examination of the drug
material (the API—Active Pharmaceutical Ingredient), excipients (inactive
ingredients present in the finished dosages), or the drug product [1,2]. Cur-
rently, there are two main types of microscopes classified based on the illu-
mination sources—optical and electron microscopes. Optical microscopes
use light sources in the visible range of the electromagnetic spectrum,
whereas electron microscopes use an electron beam. Microscopes that use
illumination sources in the ultraviolet (UV) and the infrared (IR) ranges are
also available and their application in pharmaceutical product development
is gaining popularity. Other, relatively newer, high-resolution microscopy
techniques such as scanning tunneling microscopy and atomic force micros-
copy (AFM) are available, and have been used in the research phase of
nanoparticle-related drug delivery systems. From the practical standpoint,
Essential Chemistry for Formulators of Semisolid and Liquid Dosages © 2016 Elsevier Inc.
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184 Essential Chemistry for Formulators of Semisolid and Liquid Dosages
optical microscopy is still the most widely used imaging technique in
pharmaceutical product development and quality control.
10.2 OPTICAL MICROSCOPY
Optical microscopy is the most widely adopted imaging technique used in
pharmaceutical product development and quality control. The reasons for
this are the versatility of application for raw materials and finished products
in their native state, ease of use, time taken to generate data, and cost. One
of the primary applications of optical microscopy is to characterize particles
of the raw material either by itself or in the finished product. This charac-
terization includes generating information relating to the size of particles,
particle morphology, and crystallinity.
10.2.1 Particle Size Testing
Raw materials, as well as suspended particles in the formulation (e.g., gels
and ointments), can be characterized by optical microscopy. The resolution,
d, of an optical microscope is given by the equation d = 0.61*λ/NA, where
λ is the wavelength of light in μm and NA is the numerical aperture (μm).
In practical terms, the smallest particles that can be detected by optical
microscopy are approximately 1 μm.
There are several software packages available that give an automatic
count and determine the size distribution and morphology of particles.
However, all of them have limitations when it comes to identifying particle
boundaries, and distinguishing between the particle of interest and other
particles. As a result, one critical aspect of measuring particle size by micros-
copy is the preparation of the sample slide. If the sample to be tested consists
of well-separated particles, the software can count thousands of particles
within a minute and provide meaningful size distribution data. However,
the preparation of slides in which the particles are well separated becomes
challenging when testing gels, ointments, or products with a viscous matrix.
Consequently, fully automated particle counting and characterization may
not be suitable for all types of samples because the data generated may not
be accurate.To address this situation, software packages generally provide an
option to manually correct the data.This process, however, can become very
time-consuming due to the large number of particles that need to be mea-
sured to generate accurate/representative values for size. As for determining
the number of particles to count, if all the particles are of the same size, just
one particle is enough. In reality, however, this is not the case. Statistical
Microscopy Techniques 185
software packages can be used to calculate the number of particles (sample
size) required based on the accuracy to which the particle size is needed
(e.g., the required standard error of the mean, standard deviation, and desired
confidence interval). International Organization for Standardization (ISO)
13322-1 provides guidance on the number of particles required to be
counted based on a geometric standard deviation and a 95% confidence
level. However, products are generally mixtures of both coarse and fine par-
ticles; therefore, as part of method development, one may establish an opti-
mum number of particles to be counted by comparing the results obtained
from counting different numbers of particles—for example, in the range of
100–5000 particles. When the size distribution is independent of the num-
ber of particles counted, the data are likely to indicate a representative dis-
tribution of the material in question.
Although U.S. Pharmacopeial Convention (USP) 〈776〉 provides some
guidance on particle size limit tests, the description of particle shapes, and
the measurement of irregular particles, it does not provide guidelines regard-
ing method validation for particle size measurement, or the acceptable vari-
ance. For quality control testing under Current Good Manufacturing
Practice (CGMP) conditions, the method for measuring particle size must
be validated. Accuracy, sensitivity, specificity, robustness, and reproducibility
need to be established as part of method development and validation. Under
USP 〈1225〉 analytical method classification, particle size is a Category-III
method, for which establishing precision is critical for validation. National
Institute of Standards and Technology traceable standard particles can be
used to verify the calibration and normal performance of the microscope
and software. A placebo sample can be spiked with standard particles to
establish accuracy and sensitivity. In the absence of USP guidelines for
acceptable differences between measured and certified values for these
standard particles, self-established (and justified) acceptable variance can be
used.
10.2.2 Morphology
The quantitative evaluation of particle shape is critical when powder flow is
key to the performance of the product, such as in the case of dry powder
inhalation dosage forms. Some common particle descriptors from USP
〈776〉 are:
• Lamellar: Stacked particles.
• Aggregates: Mass of adhered particles.
• Agglomerate: Fused or cemented particles.
186 Essential Chemistry for Formulators of Semisolid and Liquid Dosages
• Conglomerate: Mixture of two or more types of particles.
• Spherulite: Radial clusters.
• Drusy: Particles covered with tiny particles.
Particle morphology is quantitatively evaluated using various geometrical
factors. ISO 9276-6 provides descriptive and quantitative representations
of particle shape and morphology.
• Feret diameter: The distance between two parallel tangents on opposite
sides of the image of a randomly oriented particle.The maximum Feret’s
diameter, Fmax, also called the maximum distance in some references, is
defined as the longest distance between any two parallel tangents on the
particle. Likewise, the minimum Feret’s diameter, Fmin, also called the
minimum distance in some references, is defined as the shortest distance
between any two parallel tangents on the particle.
• Martin diameter: The diameter of the particle at the point that divides
a randomly oriented particle into two equal projected areas.
• Length: The longest dimension from edge to edge of a particle oriented
parallel to the ocular scale.
• Width: The longest dimension of the particle measured at right angles
to the length.
• Convex hull perimeter: Length of an imaginary rubber band stretched
around the particle.
• Convex hull area: Area covered within the convex hull perimeter.
Commonly used nomenclature for shape descriptions are:
• Acicular: Slender, needle-like particle of similar width and thickness.
• Columnar: A long thin particle with width and thickness greater than
those of an acicular particle.
• Flake: Thin flat particle.
• Plate: Flat particle of similar length and width, however, thicker than a
flake.
• Lath: Long thin blade-like particle.
• Equant: Cubical or spherical particle.
Commonly measured shape factors are [3,4] circularity (roundness),
convexity, elongation, aspect ratio, diameter of circle of equivalent area,
sphericity, and ratio of diameters of inscribed and circumscribed circles.
Different shape factors for characterizing particles by image analysis are
shown below.
• Aspect ratio = FMin length/FMax length
• Elongation = 1-(Aspect ratio)
• Sphericity = 4πA/P2 (in which A = area and P = perimeter of the particle)
Microscopy Techniques 187
• Circularity is defined as the degree to which the particle is similar to a circle.
Circularity = Pc/P (in which Pc = perimeter of a circle of equal area as the
particle, P = perimeter of the particle). Also Circularity = √(sphericity)
• Roundness = 4A/π(Lmax)2 (in which A = particle area, Lmax = Fmax length)
• Convexity, Cx, is a measurement of the particle edge roughness.
Cx = Ph/P in which Ph is the convex hull perimeter and P is the actual
perimeter
• Solidity (compactness), S, is a measure of overall concavity of the particle.
S = A/Ac, in which A = area of the particle and Ac = Convex hull area.
Figure 10.1 shows the convex hull. Table 10.1 shows shape factors for
some of the common shapes.
Figure 10.1 An imaginary particle is shown by the bold boundaries. The convex hull
perimeter is shown as the line joining corners to corners, as if an elastic rubber band is
stretched around the particle projection.
Table 10.1 Shape factors for some common geometrical shapes and objects
Shapes Aspect ratio Solidity Circularity Convexity
Circle 1 1 1 1
Square 1 1 0.89 1
Hexagon 0.9 1 0.95 1
Ribbon 0.11 0.85 0.49 0.99
Fiber 0.2 0.1 0.14 0.89
Equilateral triangle 1 1 0.78 1
A star-like object 0.81 0.69 0.49 0.63
188 Essential Chemistry for Formulators of Semisolid and Liquid Dosages
10.2.3 Thermal or Hot-Stage Microscopy
Controlled heating and cooling stages are available for optical microscopes.
These accessories can be used to heat specimens up to 350 °C in a con-
trolled manner.Temperatures can be held fixed for a length of time to allow
any transitions occurring within the specimen (active drug or excipient) to
be observed. Hot-stage microscopy is a very useful tool during product
development as well as for characterizing the finished product for thermal
behavior. For example, with semisolid dosage forms, the drug substance
might dissolve in the excipients at high temperature. When the product is
subsequently cooled to room temperature, the drug may crystallize out.The
crystalline form of the recrystallized drug, however, may be different from
the initial form. In this situation, preformulation studies by hot-stage
microscopy can provide critical information relating to the recrystallized
drug substance or excipient. For ointments containing a suspended drug
substance, particle imaging by hot-stage microscopy can be performed at
elevated temperatures (40–50 °C). At this temperature the ointment matrix
becomes fluid and the drug particles become clearly visible through the
microscope. A calibrated hot-stage accessory is also very useful for deter-
mining the melting point of a drug or an excipient, and can be used to
distinguish between two different types of suspended particles provided
they have distinct melting points.
10.2.4 Evaluation of Particulate Matter
USP 〈788〉 (Particulate Matter in Injections) Method 2 is a microscopic par-
ticle count test. A known amount of test solution is filtered through a 1 μm
(or finer) filter membrane, and is then examined using an optical microscope.
Particles of 10 μm or larger, and 25 μm or larger, are counted. The product
complies with the test if the average number of particles present in the units
tested do not exceed 3000 per container that are equal to or greater than
10 μm, and 300 per container that are equal to or greater than 25 μm.
10.2.5 Evaluation of Foreign Particulate Matter
The optical microscope will probably be the first tool used in any investiga-
tion of the presence of “foreign” particulate matter found in development
or commercial products. Optical microscopy under reflectance mode can
provide information relating to the surface features of the particles, and may
help in assessing the nature of the subject particle (e.g., metallic or nonme-
tallic, crystalline/birefringent or amorphous).
Microscopy Techniques 189
10.2.6 Evaluation of Material Compatibility
The physical compatibility of manufacturing components (e.g., mixing tank
scraper blades, gaskets, seals, transfer pipes) or the container closure system
with the formulation may be evaluated by microscopic examination. A sam-
ple of the component in question is placed in contact with the formulation
for a given period of time and then examined for any discoloration, depos-
its, or defects compared to the “control” material.
10.2.7 Testing of Tablets
The US FDA has released guidance [5] relating to the size and shape of generic
tablets and capsules. In this testing, optical microscopy will be a critical tool
for the quantitative assessment of the size and shape parameters.
10.2.8 Ingredient-Specific Particle Sizing
Raman chemical imaging coupled to optical microscopy can be used to
determine the chemical identity of different particles found during micro-
scopic evaluation (e.g., in cases in which drug material is mixed with excip-
ient particles). Use of this coupled technique also enables the particles of
interest to be selected for size analysis [6]. Although this technique appears
to be very appealing, it is not necessarily applicable to all sample types (e.g.,
where particles are embedded in a complex matrix such as a gel or
ointment).
10.2.9 Fourier-Transform Infrared Microscopy
Fourier-Transform Infrared (FTIR) microscopy can be used to help detect
any defects in a specimen—for example, in pharmaceutical package materi-
als, or contamination in a formulation [7]. With this technique, a small area
of the specimen is selected and an FTIR spectrum is recorded. The FTIR
microscopy technique is also suitable for characterizing drug materials for
polymorphism and hydration content.
10.3 SCANNING ELECTRON MICROSCOPY
In scanning electron microscopy (SEM), a focused beam of high-energy
electrons (e.g., 20 keV) is scanned across the surface of the sample [7].When
these high-energy electrons strike the sample, three types of signal are
generated—secondary electrons, backscattered electrons, and X-rays. These
signals reveal details regarding the morphology and elemental composition
of the sample.
190 Essential Chemistry for Formulators of Semisolid and Liquid Dosages
Secondary electrons are emitted from the atoms occupying the top sur-
face of the sample. Backscattered electrons are primary beam electrons
which are “reflected” by atoms in the solid. Both secondary and backscat-
tered electrons are used to produce a readily interpretable image of the
surface. The image produced is of very high magnification (100,000×) with
a resolution of approximately 5 nm.
Interaction of the primary beam with atoms in the sample causes excita-
tion of the orbital electrons. When these excited electrons return to a lower
energy state they emit X-rays of fixed wavelengths. The emitted X-rays have
an energy that is characteristic of the parent element. Detection and measure-
ment of this energy permit elemental analysis (Energy Dispersive X-ray Spec-
troscopy [EDX or EDS]). SEM analysis is “non-destructive”––that is, there is
no sample loss and the sample may be analyzed multiple times, if needed.
SEM is commonly used in product development to analyze the mor-
phology and size of nanoparticles. SEM/EDX is a very useful tool for inves-
tigating any “foreign” particulates. The limitation of SEM is that the sample
needs to be placed under high vacuum; therefore, this technique is not suit-
able for wet samples.
10.4 TRANSMISSION ELECTRON MICROSCOPY
Transmission electron microscopy (TEM) is a very powerful research tool in
which a highly accelerated electron beam (100–1000 keV) is passed through
an ultra-thin sample to reveal the details of nanostructures (such as lipo-
somes, solid lipid nanoparticles). TEM requires extensive skills with respect
to both sample preparation and operation of the instrument. Sample prepa-
ration involves depositing a thin film of carbon on the TEM grid, placing
the liquid sample (e.g., liposome solution) on the carbon film, and then
blotting away the excess liquid.The background of the sample is then stained
with a staining agent such as uranyl acetate solution, forming a “shadow”
around the sample (this is known as negative staining). The sample appears
as a negative picture when the grid is examined under a microscope.
To visualize the nanostructures in their native state (i.e., without stain-
ing), a technique known as Cryo-TEM is used. In Cryo-TEM, the sample
is mounted on a perforated grid and examined at a very low temperature
(∼100 K). The analyst looks for the nanostructures of interest overlying one
of the holes in the supporting carbon film.
Structural details of the internal and/or external parts of the nanoparti-
cles can be obtained using Freeze–Fracture TEM (FF-TEM). The solution
Microscopy Techniques 191
Figure 10.2 Freeze-fracture micrographs of liposomes showing surface texture at room
temperature. (a) Liposome of palmitoyl oleoyl phosphatidylcholine (POPC) showing a
smooth surface texture. (b) Liposome of a mixture of POPC and Galactosylceramide
showing surface undulations in which the undulations are registered through several lay-
ers of the liposome. (c) Liposome of 24:1 Sphingomyelin showing a rough surface texture.
of nanoparticles is frozen in liquid ethane, and then held and fractured at
liquid nitrogen temperature.The fractured sample is coated at an angle with
carbon, and the carbon film (which is a replica of the sample) is then trans-
ferred to an electron microscope grid and examined by TEM at room tem-
perature. In this method, it is the replica of the sample that is examined and
not the sample itself. FF-TEM is very useful for viewing the internal core of
liposomes or nanoparticles. Figure 10.2 compares the undulated surface tex-
ture of the vesicles formed by (1) a mixed lipid system of palmitoyl oleoyl
phosphatidylcholine (POPC) and galactosylceramide (GalCer), and (2) pure
24:1 sphingomyelin to the smooth surface exhibited by POPC alone.
The limitations of the various TEM techniques are that they require a
very high level of operator training and skill, and are very time-consuming
and expensive.
10.5 ATOMIC FORCE MICROSCOPY
Scanning probe microscopy covers a group of technologies that are used for
imaging and measuring surfaces at a molecular/group of atoms level. In
AFM, the surface of the specimen is scanned with an extremely fine tip
(often less than 100 Å in diameter and approximately 2 μm in length) that is
mounted on a flexible cantilever, thus allowing the tip to follow the surface
profile of the sample. Van der Waals forces between the tip and the surface
of the specimen cause the cantilever to bend or deflect. A sensor measures
the deflection of the cantilever as either the tip is scanned over the sample,
or the sample is scanned under the tip. Based on the movement of the
192 Essential Chemistry for Formulators of Semisolid and Liquid Dosages
cantilever, a topographic map (image) is generated. AFM can be operated in
a “contact” or “non-contact” mode.
The use of AFM in the pharmaceutical field is not widespread in either
product development or manufacturing. Nonetheless, it is a very useful tool
during the research phase for characterizing nanodrug delivery systems [8,9].
10.6 CONCLUSION
Microscopy techniques are very useful for determining the morphology of
particles. The advantages of using microscopy for particle size analysis are
that it can be ingredient specific, and is complementary to other particle
sizing techniques. As such, microscopy may be the most applicable tech-
nique for determining the size of particles within some semisolid products,
such as gels and ointments. The challenges of using microscopy for particle
sizing are (1) deciding the sample size (number of particles) for accurate size
analysis of the product, and (2) the level of skill required by the operator.
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