Microscopy

Notes for cbse net jrfForensic science

By – priya tamang

It is the most efficient image-processing system available to date. All the appliances provided by technology are no match for the eye

as regards speed and resolution. The structure of the eye is related to that of a camera. Together with the muscle-adjusted lens (1a), the curved surface of

the cornea (1) projects an optical image onto the retina (2). The amount of incident brightness is controlled via the variable diameter of an iris (3).

A sharp image is provided by the flexible lens, the focal length of which is changed by muscles in such a way that focusing is possible on any subject at a distance between approx. 20 cm and infinity.

The image itself is picked up on the retina by approx. 130 million receptor rods (recognition of grey levels) and 7 million cones (color recognition) and transferred to the brain on the shortest possible path via the optic nerve.

Eye as the visual organ

The light rays shown in the following illustration form a viewing angle of 30°. The following objects, for example, are seen under this angle:

The spire of the Ulm Minster with a height of 161 m, seen from a distance of 300 m

a photo with a height of 13 cm seen from a distance of 25 cm. The details we want to see have a diameter of only 1/100 or

even 1/1000 of a millimeter. However, we are unable to go any closer to the object than

approx. 20 cm. As a consequence, the viewing angle becomes extremely small, which is why we are unable to recognize any details.

A similar situation is experienced if we view the spire already mentioned from a distance of 300 m. The many intricate details created by the stonemasons cannot be recognized from such great a distance because the viewing angles are too small.

For example

A remedy has been known for such cases for centuries: the use of a “magnifying glass” which – when put between the eye and the object – makes everything appear larger.

However, there is a limit to this method: a magnification of more than 8-fold or 10-fold is not possible. Anyone who wants to see more must use the “compound” microscope.

First Aid: The magnifier

If one lens is not sufficient, several lenses can be arranged one behind the other. The magnifying effect is thus multiplied, allowing magnifications of up to 2000x.

The classic microscope magnifies in two steps: The objective produces a magnified image of the object in the so-called intermediate image plane, and the eyepiece or ocular (Latin: oculus = eye) magnifies the intermediate image in the same way as a magnifier.

The beam path on the right shows how light is emitted from an object ( ← ) and processed in the three lenses. We shall only look at the rays originating in the two ends of the object.

This will be sufficient to explain the process of magnification. The illustration shows the ICS principle (ICS=Infinity Color corrected System) also used with the Axio lab microscope.

A further step is included in this modern microscope featuring “infinity optics”: a tube lens is added to support the objective.

Microscopes magnify in steps.

The objective projects an image at an “infinite” distance, the tube lens with its focal length f = 164.5 mm then forms the intermediate image from these parallel beams.

First, let us assume that nothing decisive for the image formation happens in the space between objective and tube lens.

The light rays coming from the focused specimen plane are parallel in this space anyway.

The eyepiece, in turn, serves as a magnifying glass to make this small intermediate image appear even more magnified to the eye.

Polarizing microscopes

The optical microscope is used extensively in pharmaceutical development with the primary application being solid-state analysis.

The applications range from simple images of drug substance to illustrate particle size and shape to full optical crystallography.

The range of utility of the microscope is considerably extended by the use of polarized light which allows us to obtain crystallographic data on small individual crystals.

I will use the term Polarized Light Microscopy (PLM) for all light microscopy discussions in this chapter.

Polarized light microscopy provides us a unique window into the internal structure of crystals and at the same time is aesthetically pleasing due to the colors and shapes of the crystals.

The use of PLM as a tool for crystallography extends back at least 200 years. For many of those years, it was the prime tool for examining the crystal properties of minerals and inorganic chemicals, as well as organics.

Pasteur’s seminal studies in the handedness of organic chemicals were initially conducted on an optical microscope.

Needham (1958) provides some information on the development of PLM but frankly I am not aware of any good articles or books that detail the history of both the development of the polarizing light microscope and its applications.

Introduction

The use of polarized light on the optical microscope allows us to determine the optical crystallographic properties of the crystal. Optical crystallography is related to but different from X-ray crystallography.

Each technique provides unique information about the crystal structure and the combination of the two is powerful indeed.

For example, optical crystallography uniquely yields the sign and magnitude of the angle between optic axes in a biaxial crystal.

This value, among others, can be used for the definitive identification of the solid-state form.

This determination can be made on crystals as small as ~5 mm and can be done in a mixture of particles of different forms. PLM was originally developed for studies in mineralogy and petrography.

It has since expanded so that virtually every field that deals with crystalline materials uses the techniques of optical crystallography.

Optical crystallographic measurements can yield as many as 20 different characteristics, many of which are numerical.

This set of values is unique for each solid-state form. A hydrate will have different optical crystallographic

values than its anhydrate and different polymorphs of the anhydrate (or hydrate for that matter) will have different optical properties.

Optical crystallography, then, is a superb tool for the in situ identification of different solid-state forms.

There are two caveats: First, we must have good reference data for each of

the forms; second, the microscopist must be skilled in the art

and science of optical crystallography. It seems to me that the decline in the use of this

science is related to both of these requirements.

There are a number of excellent texts on light and optics in relation to optical microscopy.

Much of the discussion below follows Slayter and Slayter (1992), McCrone et al. (1984), and Needham (1958).

Gage (1943) and Martin (1966) provide a more detailed and extensive discussion of light and optics as they relate to microscopy.

Born and Wolf (1980) is the standard for optics. Visible light is a narrow part of the electromagnetic (EM) spectrum

usually considered to extend from 400 to 700 nm (10−9 m) in wavelength. Wavelengths of the entire electromagnetic spectrum extend from 10−16 m for gamma rays to 1010 m for long radio waves.

The spectrum is divided into the following categories based on short to long wavelength (high to low frequency): gamma rays, X-rays, ultraviolet radiation, visible radiation, infrared radiation, microwave radiation, short radio waves, and long radio waves.

These categories are somewhat arbitrary in nature and all electromagnetic radiation share common features.

Properties of Light

The key properties of electromagnetic radiation are intensity, frequency (wavelength), polarization, phase and angular orbital momentum.

The first four properties are directly used in PLM. Intensity is defined as power over area, for example,

watts/m2. We have an intuitive sense of intensity in normal life as we may turn on more lights in an area if we feel it is dimly lit.

While intensity is of great interest in microscopy, we do have some control over the property since most microscopes use artificial illumination with variable power bulbs.

Historically, intensity was a major issue in microscopy since most microscopes used the sun or candles as the light source.

Even today, we are interested in increased intensity for low light level applications like fluorescence microscopy.

For that reason, some specialized techniques also have specialized high-intensity light sources.

Optical crystallographic references are scattered and incomplete and there are relatively few scientists that are skilled in the necessary techniques.

I believe that there are a number of good reasons to attempt a revival of optical crystallography in pharmaceutics.

First, it does provide a set of unique values for the identification of solid-state forms.

Second, optical crystallography can be quite useful in the study of thermodynamic form relationships.

Third, if the optical and X-ray crystallographic characteristics have been related, then it is possible to determine which faces and thereby which functional groups of the chemical are exposed by comminution processes (Nichols, 1998).

Fourth, a thorough understanding of optical crystallography is an excellent tool for the understanding of crystallo-graphy overall.

Electromagnetic radiation can be considered as a wave phenomenon. As such, the waves have a frequency (number of waves in a unit measure) and the inverse – a wavelength. Wavelength is frequently used in optical microscopy and is defined as the wave distance from peak-to-peak or from trough-to-trough. These relationships can be mathematically represented as follows:

where f is the frequency, u is the velocity of the wave, and l is the wavelength. For the electromagnetic spectrum,

Since the velocity of the wave is the speed of light. We often use the term and concept of wavelength in optical microscopy and commonly use nanometers as the unit of measure (spectroscopy more commonly uses wavenumber,

Various filters of specific wavelengths of light are used with the microscope and the most common of these are 589 nm (D line), 486 nm (F line), and 656 nm (C line). The D line (589 nm) is yellow light and is situated in the middle of the visible spectrum.

Some microscopists also use monochromators with their microscope to produce narrow wavelengths of light, although the practice is not as common today as it once was.

Polarization refers to limitations on the direction of wave oscillation.

A polarizer acts like a filter to allow only light oscillating in one orientation to pass.

We use polarized light for many applications in optical microscopy.

For example, we can determine crystallinity of a sample using a few crystals and crossed polars. The test is sensitive, fast, and prone to only a few errors.

Polarization is one of the more difficult concepts to describe since there really are not many large scale analogous phenomena to act as an example.

As Needham (1958, pg 164) states, “It is much easier to demonstrate polarized light than to clearly describe what it is.” I think I could title this entire chapter with that statement.

Bloss (1961) presents a cogent and wellillustrated introduction to polarized light.

Phase refers the time progression of the wave. In other words, at t0 the wave may be at a peak, t1 halfway

between peak and trough, t2 at the bottom of the trough, etc. In general, we do not use phase information directly in microscopy

but only as the phase relates to other waves through interference. Light waves can either constructively or destructively interfere. The waves may add in amplitude, subtract in amplitude, or cancel

each other. If the phase of the combining light waves is the same, the waves

will constructively interfere and add amplitudes. If the waves are out of phase, then amplitudes will be diminished

or even canceled. Phase differences are used in a variety of microscopy techniques

primarily to improve contrast. Phase contrast, differential interference contrast, and Hoffman

modulation contrast are just a few of the methods utilizing phase differences to enhance contrast.

These latter techniques are more commonly applied to biological than to physical pharmacy.

Some crystals have the property of double refraction and incoming light is split into different optical axes (directions where speed of light is different).

When this light is recombined, such as in a polarizing light microscope, we get interference and with PLM a visual interferogram is formed at the back focal plane of the objective.

The properties of this interference figure are characteristic of the material and can be used in understanding the crystallography and molecular properties of that material.

The underlying physical principle is alteration of light speed by interactions of light with different functional groups in the molecule.

The properties of the interference figure can also be used to identify the crystal and even to distinguish among polymorphs, hydrates, and solvates.

Double refraction is the phenomenon underlying all of optical crystallography and it behooves us to understand it well. Simple refraction occurs when a light beam traverses a refractive index interface.

Another important property of light waves is diffraction as the light interacts with a slit or small opening. In a sense, light bends around the opening so that it appears that the small slit or hole is the source of new waves.

It is not necessary to be an expert at optics to intelligently use the microscope, but it is necessary to know and understand a few basic concepts.

There are many excellent texts on optics in the microscope and I recommend Slayter and Slayter (1992), McCrone et al. (1984), and Chamot and Mason (1958) along with Needham (1958) for further study.

At the heart of optics is the interaction of light with materials and, of course, for microscopy the material of most interest is glass in different shapes.

When visible light irradiates an object, the light can be reflected, transmitted, or both.

Reflection and transmission can occur such that the light retains all of its energy or some energy can be absorbed in the process.

The rich complexity of the interaction of light with objects, in particular glass, allows for the versatility and power of modern microscopes.

Basic Optics

The law of reflection states that the angle of reflection is equal to the angle of incidence for any particular light ray irradiating a smooth, reflective surface.

If the surface is irregular and a broad beam of light is used, then we get diffuse reflection.

If the surface is polished and we use a narrow beam of light, then we get specular reflection and this reflected light can be polarized.

Fishermen are familiar with this phenomenon and use polarizing sunglasses to reduce light reflection so as to better see the fish below the surface of the water.

It is easy to see that if we make mirrors of different shapes, particularly concave mirrors, we can use the curved surface to focus light rays to a point and produce an image.

According to Webster’s Ninth New Collegiate Dictionary (Merriam Webster 1983), crystallography is defined as the “Science dealing with the system of forms among crystals, their structure, and their forms of aggregation.”

The atoms or molecules of a crystalline solid usually are oriented in space with a repeating pattern in three dimensions.

Think of bricks in a wall or even better, of patterned ceramic tiles. This is the crystalline state.

In some solids, there is no long-range repeating pattern, though there may be short range order and this condition is referred to as the amorphous or glassy state.

There are also imperfections in most real crystals and this condition is referred to as disorder.

Crystallinity and disorder are important topics in physical pharmacy since the degree of Crystallinity can affect some important bulk properties of a solid such as the amount of water it can absorb as a function of temperature and relative humidity.

Crystallography

Surprisingly, there are only a few repeating patterns. The patterns can be categorized as follows into six crystal systems

with decreasing degrees of symmetry: cubic, tetragonal, hexagonal, orthorhombic, monoclinic, and triclinic.

The trigonal system, which is referenced in older literature, is now considered part of the hexagonal system.

Optical crystallography is most commonly applied in mineralogy but is also directly applicable to organic chemicals.

Optical crystallography is based on the fact that light travels with different speeds in different directions in the crystal.

The methods of optical crystallography take advantage of this property.

Given the range and specificity of optical crystallographic properties of pharmaceutical compounds and a good reference database, it is possible to identify single small crystals even to the point of distinguishing between similar polymorphs.

Add in thermal microscopy, IR and Raman microscopy, and SEM/EDS and we have a powerful identification scheme, indeed.

Optical Crystallography

Table Description of optical properties of crystals

Optical property Description Color Color of thick sections in brightfield Habit, shape Habit for well-shaped crystals, morphology of particles

Aggregation, Crystals or particles mechanically or chemically fused together

agglomeration Appearance and resistance to breakage Twining Individual crystals sharing face(s) Cleavage Description of manner in which crystals break Surface texture Appearance of crystal, particle surface Transparency How easily does light pass through particle Edge angles Angles between crystal faces Dispersion staining colors Central stop colors in high dispersion R.I. liquids near refractive index of particles Pleochroism Color change on stage rotation Refractive indices Formally, speed of light in vacuum divided by speed of light in particle. Measured by comparing particle relief in known R.I. liquids. Number of principal indices dependant on crystal system R.I. Dispersion Variation of refractive index with wavelength Interference colors Color of crystal between crossed polars, depends on birefringence, crystal thickness, and crystal orientation. Saturation of color dependant on order

Anomalous interference colors Different sequence of colors related to crystal thickness

Birefringence Difference between high and low refractive indices Extinction position All crystals and crystal fragments will go dark four times on rotation of the stage (unless the crystal is oriented so that the optic axis in parallel to the light path). Refractive index measurements are generally made at extinction positions for anisotropic crystals

Extinction angle The angle of a crystal edges with the positions of the crossed polars (generally aligned with crosshairs of the eyepiece) when

the crystal is dark. Only pertinent to crystals in the monoclinic and triclinic crystal systems

Dispersion of extinction angles Variation of extinction angle with wavelength Interference figure. An image of light interference at the back focal plane of an objective for anisotropic crystals. Uniaxial interference figures have Maltese cross appearance or part of the cross depending on

crystal orientation. Biaxial interference figures have appearance of hyperbola or parts of the hyperbola depending on crystal orientation and optic axial angle

Optic sign Sign of birefringence, for uniaxial crystals if e > w optic sign if positive, negative if opposite. For biaxial crystals, if value of b closer to that of g, optic sign if negative, if closer to a then optic sign is positive

Optic axial angle Angle between optic axes, evidenced as angle between dark regions in interference figure. Is 90º for uniaxial and orthorhombic crystals, and between 0 and 90º for biaxial crystals (monoclinic and triclinic)

Dispersion of optic axes Variation of angle of optic axes with wavelength

Acute bisectrix Vibration direction isecting acute angle between optic axes

Obtuse bisectrix Vibration direction bisecting obtuse angle between optic axes

Optical orientation Relationship between crystallographic directions and optical directions. Fixed in uniaxial crystals, but varied in biaxial

Sign of elongation For elongated uniaxial crystals (and fibers that act like uniaxial crystals), if the high index is associated with the length then the sign is positive, negative otherwise. Not related to crystal symmetry

This is the simplest type of microscope in terms of both construction and use. The stereomicroscope consists of two compound microscopes which are aligned

side by-side at the correct visual angle to provide a true stereoscopic image. The long working distance (space between the specimen and objective lens),

upright non reversed image and large field of view make these the instruments of choice for performing preliminary examinations of evidence as well as manipulating small particles and fibers to prepare them for more detailed microscopical or instrumental analyses or comparisons.

An additional advantage which results from the long working distance and illumination by reflected light is that specimens rarely require any sample preparation.

The specimen is simply placed under the microscope and observed. The useful magnification range of stereomicroscopes is typically between 2.5 x

and about 100 x . Modern stereomicroscopes incorporate a number of features which increase

their utility and ease of use. A choice of illuminators which can provide brightfield and darkfield reflected,

fluorescence and transmitted light permit the microscopist to visualize microscopic objects and features which might otherwise appear invisible, and thus escape detection.

Stereomicroscopy

Attaching the microscope to a boom stand permits it to be swung out over large objects such as clothing, piles of debris or even entire vehicles.

Both photographic and video cameras can be attached to record images for inclusion in a report, as a courtroom exhibit or to display to colleagues.

Even the least experienced members of the laboratory staff can use these instruments with very little training

The comparison microscope is used to compare microscopic items side by side.

Although the human eye can be very good at discerning minute differences in color and morphology, the brain has a more difficult time remembering and processing these subtle differences.

This problem is overcome by a comparison microscope in which the images from two microscopes are observed side by side in a single field of view.

Reflected light instruments are used by firearms examiners to compare rifling marks on bullets as well as ejector marks and firing pin impressions on cartridge cases.

Tool marks and cut and polished layered paint chips can also be compared with the same equipment.

Transmitted light microscopes are used to compare hairs, fibers and layered paint chips which have been thin-sectioned. Polarizing and fluorescence equipment may added to a comparison microscope which is to be used for fiber comparisons to enhance its capabilities.

Human hair comparisons, particularly the final stages of an examination, are conducted almost exclusively under a comparison microscope.

Comparison Microscope

The phase contrast microscope is used primarily in serological and glass examinations.

Its principal use in serology is to observe cells in biological fluids or after reconstitution in aqueous mountants.

Under these conditions cells exhibit a very small optical path difference with respect to the medium in which they are immersed.

Such specimens are referred to as phase objects. The human eye cannot observe phase differences, but it can discern amplitude (dark and light) differences.

A phase contrast microscope uses half-silvered rings and disks placed in the optical system to change these phase differences into amplitude differences which can then be observed and photographed.

Spermatozoa, epithelial cells, and other cellular matter can be studied in detail without staining using this technique.

One of the principal methods of glass comparison is based on a very accurate measurement of the refractive indexes of the known and questioned samples.

The measurement is conducted in a hot stage mounted on a phase contrast microscope.

The crushed glass fragment is mounted between a slide and cover-slip in a specially prepared and characterized silicone oil which is placed in the hot stage.

As the temperature is raised, the refractive index of the silicone oil decreases while that of the glass remains essentially constant.

Other Optical Microscopes

Since even small differences between the refractive indexes of the glass and oil are easily seen with phase contrast, the true match point (i.e. temperature at which the silicone oil has the same refractive index as the glass) can be observed with great precision.

The refractive index of a glass particle can be measured to 0.00002 using this technique.

A commercial instrument in which the phase contrast microscope, hot stage and camera are all connected to a computer makes these measurements automatically and objectively.

Fluorescence microscopy is based on the property of certain substances to emit light of a longer wavelength after they have been irradiated with light of a shorter wavelength.

This emitted light is called fluorescence and differs from luminescence in that the emission of light stops after the exciting radiation is switched off.

The fluorescence may originate from fluorescent ‘tags’ attached to proteins or other compounds which cause the substance they react with to fluoresce

The comparison microscope is used to compare microscopic items side by side. Although the human eye can be very good at discerning minute differences in color and morphology, the brain has a more difficult time remembering and processing these subtle differences.

This problem is overcome by a comparison microscope in which the images from two microscopes are observed side by side in a single field of view.

Reflected light instruments are used by firearms examiners to compare rifling marks on bullets as well as ejector marks and firing pin impressions on cartridge cases.

Tool marks and cut and polished layered paint chips can also be compared with the same equipment.

Transmitted light microscopes are used to compare hairs, fibers and layered paint chips which have been thin-sectioned.

Polarizing and fluorescence equipment may added to a comparison microscope which is to be used for fiber comparisons to enhance its capabilities.

Human hair comparisons, particularly the final stages of an examination, are conducted almost exclusively under a comparison microscope. after the nonreacting remainder of the reagent is washed away, or it may originate from autofluorescence.

The first technique is the basis for detecting antigen-antibody reactions which occur on a cellular level and has been applied to a limited extent in forensic serology.

Autofluorescence may originate from either organic or inorganic compounds or elements.

When it occurs, autofluorescence is a useful comparison characteristic. It may originate from organic dyes or optical brighteners on fibers; it may be

observed in layers of paint in cross-section where it originates from organic pigments or inorganic extenders and may be observed on certain varieties of mineral grains and be absent from others

A modern fluorescence microscope is equipped with a vertical illuminator which directs the light from a mercury burner through a series of lenses and filters designed to focus the light on the specimen and select a narrow or wide range of wavelengths to excite fluorescence in the specimen.

Since the intensity of the fluorescence from a specimen does not depend on absorption and the image is formed with the emitted fluorescent light rays, fluorescence images are bright and well resolved.

These images can be recorded and make excellent exhibits for use in reports and courtroom presentations

The hot stage microscope permits the microscopist to observe the behavior of specimens as they are exposed to temperatures from ambient up to approximately 350°C.

Melting temperatures can be used to help in the identification of unknown substances and as an aid in certain types of comparisons; particularly those involving thermoplastic polymers.

For example, infrared microspectroscopy is of only limited use in distinguishing nylon fibers.

It can be used to determine if fibers have been spun from nylon 6 or nylon 6,6 polymer.

Much finer distinctions can be made by comparing melting points of nylon fibers since these are a function not only of the type of polymer from which the fiber was spun, but also the average molecular weight, crystallinity, presence of additives, etc.

Although the contributions from each of these factors cannot be individually assessed from a melting point determination alone, the actual melting points of two fibers result from all of these factors and thus form a useful point of comparison or discrimination.

Although other instrumental methods of analysis have largely superseded hot stage microscopy as a tool for the identification of unknown compounds, it is still a useful technique which can add information and make distinctions which are difficult or impossible by other methods.

The identification of poly-morphs of drugs of abuse, for example, is better studied by thermal methods than by spectroscopic ones.

Determination of the melting range can also give information on the purity of a minute sample which could be difficult to assess by other means.

Electron microscopes make use of electrons rather than photons to form their image. The transmission electron microscope (TEM) was developed first, followed some years later by the scanning electron microscope (SEM).

Transmission instruments are generally more difficult to use and require more painstaking sample preparation than scanning microscopes and thus have found very few applications in forensic science.

Specimens for TEM must be extremely thin to permit penetration by the electron beam.

The image in an SEM is formed from collected secondary or backscattered electrons emitted from (and just beneath) the surface of the sample and not by transmitted electrons as in the TEM.

Since the SEM only looks at the surface of a specimen, sample preparation is often much simpler and frequently consists simply of placing the specimen on a piece of conductive carbon tape.

It may be necessary to vacuum deposit a layer of carbon or gold over nonconductive specimens to make them conductive, although the new ‘environmental SEMs’ can image nonconductive samples in a low vacuum. SEMs are now in use in many forensic laboratories around the world.

Most of these microscopes are equipped with energy dispersive X-ray spectrometers for elemental analysis.

Electron microscope

X-ray spectrometers collect the X-rays which are produced along with the secondary and backscattered electrons when a specimen is bombarded in a vacuum with electrons.

These X-rays are collected and then sorted in a multichannel analyzer according to their energy which is directly related to atomic number.

Both qualitative and quantitative analyses can be performed on microscopic specimens from boron all the way up in the periodic table.

The detection limit for each element varies, but typical limits of detection for most elements, excluding some of the light elements, is about 0.1%. One of the principal uses of analytical SEMs in forensic science laboratories is the detection and analysis of gun shot residue (GSR) particles.

Conductive sticky tape, attached to the back of a sample stub, is pressed over a suspect’s hands to collect any residue which might be present.

The stub is placed in the microscope and searched, either manually or automatically, for particles with a spherical morphology which contain lead, antimony and barium.

The combination of the spherical morphology with this elemental composition provides better proof of the presence of GSR than an elemental analysis alone.

Other types of microscopic evidence which can be examined in the SEM include items as diverse as pollen grains, diatoms, paint, glass, inorganic explosives and general unknowns.

The combined abilities of the SEM to resolve fine structures and provide the elemental composition of these small particles is a tremendous aid in the examination of many small items of trace evidence