25 Introduction to Microscopy

Vinay Gupta

  Introduction

Optical microscopy is widely used for the study of materials with the samples frequently examined in the reflection and transmission modes; in addition phase contrast and interference microscopy offer certain special advantages. In these techniques light from a source is condensed on a specially prepared sample; the reflected or transmitted beam goes through the objective lens where the first image is formed; the latter is magnified with the help of the projector lens. The magnification range available in an optical microscope is typically 50X to 500 X. While it is possible to enhance the magnification the information obtained is restricted by the resolution limit of the microscope. In scanning electron microscopy (SEM), a fine probe of electrons scans the surface of the sample and the signals emanating from the incident side are processed and quantified. High energy electrons passing through a specially prepared thin sample (thickness 10 to 50 nm) are characterized in the transmission electron microscope (TEM) to provide information about the sample. In this chapter we shall briefly review the salient features of optical microscopy and indicate the advantages of electron optical techniques over the former.

Components of the Microscope:

The following are the basic components of the microscope:

• Light or source
• Condenser lens
• Objective lens
• Projector lens
• Detectors for the signals.

It is necessary to have a suitably prepared specimen to maximize the information obtained.

The principles of image formation in the reflected and transmitted modes are illustrated in Fig. 1.1 and 1.2 respectively. Light from the source, condensed with the help of the condenser lens and the condenser aperture, falls on a half silvered mirror and then on to the specimen (position A in figure 1.1). The reflected light is used to form the first image by the objective lens; the projector lens magnifies the first image. The use of specially prepared thin samples allows light to go through the sample in the transmitted mode; the other steps in imaging are the same as those in the reflected mode.

The lens system:

The condenser lens collects light to direct it at the small area of the object which is to be examined. It makes the object brighter (better contrast) and enables to control the angle at which the illumination reaches the object. The condenser lens can converge the light beam on object or can illuminate it with parallel rays. The Condenser aperture: controls the area of specimen to be illuminated.

Objective lens is closest to the object and is the most important single part of the microscope. The quality of objective lenses varies widely from manufacturer to manufacturer and the only way to evaluate objective lenses is to physically compare one with another. They can be of several type, Achromat, Semi-Plan, Super High Contrast, and Plan. Achromatic Lenses are corrected for the lens defect chromatic aberration (will be explained later in this chapter) by using two different types of glass (sandwiched into a “lens doublet”) with different refractive indices. They are the most common type sold with microscopes. The Super High Contrast (ASC) lenses are standard achromats with an internal field stop which offers a higher contrast image. For image quality, these lenses are excellent for the price. Like the laundry detergent commercials on TV, they produce whiter whites and darker darks to offer a very nice sharp high contrast image. The standard achromatic objectives guarantee flat focus quality for 60% of the field of view. In contrast semi-plan lenses guarantee a flat field for 80% of the field of view and plan lenses (the best) are a full 100%. These lenses are also achromats, correcting for the color dispersion effects. Phase contrast objectives are only useful on specimens that do not absorb light (they are called “phase objects”) and are very useful in showing details in certain specimens such as cell parts in protozoans, bacteria, sperm tails and other types of unstained cells.

A comparison of the components of the optical, scanning and transmission electron microscopes is shown in Fig. 1.3. The transmission electron microscope is similar to an optical instrument in that lenses are used to form images. The scanning electron microscope is not similar to an optical instrument (no image forming lens) but uses electron optics to form a fine probe on the specimen which is scanned back and forth over the specimen and the signals emitted at every point are detected and characterized.

Fig. 1.3: Comparison of optical, scanning and transmission electron microscopes

The total magnification of the microscope is given by the product of the magnification of the objective and projection lenses. In contrast with the optical microscope, where the magnification of the objective and projector lenses need to be considered, the TEM would have an extra lens to contribute to magnification. Also both SEM and TEM have two condenser lenses.

Abbe (Diffraction) limit

The Abbe limit is the minimum resolvable distance between the images of two point objects using a perfect lens; it represents the ability to discern fine details. In any magnifying system, a point object (i.e. zero dia) can not be imaged as a point but as a bright disc with several subsidiary intensity maxima. There are several intensity maxima with the peak value reducing as we move away from the centre of the feature (Fig. 1.4). As two point objects approach each other, the main peaks get more closely spaced and finally start overlapping. The resolution limit is reached when the shoulder observed in the intensity curve in Fig. 1.4 (b) has a value of 0.81 the peak intensity.

Fig. 1.4(a): Diffraction effects around an aperture and overlapping of peaks as the features approach each other, (b) Defining the criterion for resolution limit; minimum distance between two diffraction maxima still projected separately

The resolving power or resolution limit (ρ) is defined by the expression

where λ is the radiation used, η is the refractive index of the medium between the lens and the specimen and α is the semi-acceptance angle (Fig. 1.5). η sin α is also referred to as the numerical aperture (NA) of the lens.

Fig. 1.5: Illustrating the semi-acceptance angle of the lens

Taking a value of 400 nm for the wave length of the radiation (green light) and is ηsinα of 1.6 (sinα taken as 1 and refractive index as 1.6), ρ = λ 0.61/N.A. = 0.61x 400/1.6 = 152 nm; the resolution is about 150 nm (0.15 µm). The realistic value of resolution is about 200 nm (0.2 µm). In electron microscopy, λ is small (small fraction of nm depending the accelerating voltage) and µ sin α is very small, because µ is unity and α can have a maximum value of a few degrees (reason for low α will be given later). For wavelength of 0.0037 nm (100 keV electrons) and α = 0.1 radians, the resolution is about 0.02 nm

Important features for imaging

Wave length λ of the electrons accelerated by a potential of V volts is given by the expression,

λ   = h / √2meV

where h is Planck’s constant (6.626×10-34 Js), m is electronic mass (9.11×10-31kg) and e is electronic charge (1.602×10-19C). For large values of V, electrons can attain velocities comparable with the speed of light and relativistic increase in mass should be taken into account. This is done by replacing V by Vc (relativistic accelerating voltage)

Vc = V[1 + eV/2m0c²]

where c is the velocity of light (2.998×108 m/s). For 100 kV electrons, wavelength is 0.04Å (without relativistic correction) and 0.037Å with correction.

Lens Aberrations:

The lenses suffer from several aberration effects which affect the resolution; the most important of these defects are

• Spherical aberration
• Chromatic aberration
• Astigmatism

    Spherical aberration arise from the fact that the focussing action of outer regions of lens is more than that of the regions close to the optic axis. Point in the optic axis is imaged as a disc in the image plane (Fig. 1.6). This disc has minimum size on a particular imaging plane; the radius of this disc of least confusion, ∆rs, is given by

∆r_s = C_sα³

where Cs is the spherical aberration coefficient (typically 1 – 5 mm) and α is the semi-aperture angle. Note that even for a small value of α (~10-²), ∆rs ~ 1nm.

Fig. 1.6: Illustration of spherical aberration

Chromatic aberration arises from variations in accelerating voltage or lens current and the consequent energy spread of the electrons. The lower energy electrons are bent more (Fig. 1.7). Note that in the case of white light this effect is due to different wavelength of the components of white light. ∆rc, radius of the disc of least confusion, is given by

∆r_c = C_cα(∆E/E).

Typical value of Cc is 1- 4 mm. Electrons from W filament have a spread of energy of 0 – 3 eV and HT and lens current stability are ~ 10-6/min. Electron energy loss from inelastic scattering (20-50 eV) would be the resolution limiting factor.

Fig. 1.7: Illustration of chromatic aberration

Astigmatism refers to the imaging of a point in the object as a disc due to differing focal lengths depending on the plane of the ray paths (Fig. 1.8). Radius of the disc of least confusion, ∆rA, is given by

∆rA = ∆fAα

where ∆fA is the maximum difference arising from astigmatism. Aberration is corrected by the use of electromagnetic stigmators in the electron microscope (recall the use of cylindrical lenses for correcting astigmatism in the eye).

Fig. 1.8: Illustration of astigmatism

Scanning electron micrograph of a magnetic tape is shown in Fig. 1.9. The appearance of the image is considerably changed when astigmatism is not corrected and the image is improperly focused.

Fig. 1.9: Scanning electron micrograph of magnetic tape (a) no astigmatism, (b) & (c) images with astigmatism, (b) is overfocused & (c ) is underfocused

This point is again emphasized in Fig. 1.10 where the effects of astigmatism and improper focus are illustrated.

Fig. 1.10 : Dependence of image appearance on focus and astigmatism

Resolution of the Electron Microscope

A compromise value for the resolution of the electron microscope can be obtained by considering the two opposing factors, diffraction effects favoring a high value of α and lens defects requiring small values for α.

Resolution is given by the expression,

ρdiff = (0.61λ/µsinα) = (0.61λ/α),

since µ is 1 (vacuum in the electron microscope column) and α is small.

Compromise angular aperture size with diffraction and spherical aberration into account is

β   ~ 0.88 (λ/Cs)^1/4.

Corresponding value for resolution is given by

ρ                                                                            = 0.69λ3/4Cs^1/4.

Depth of Field represents the distance parallel to the optical axis of the microscope that a feature on the specimen can be displaced without loss of resolution. For the optical microscope it is given by the expression

where d is the depth of field and M is the total magnification of the microscope. Some values for the depth of field of optical and scanning electron microscope are given in Table 1.2. The large depth of field of the SEM is clearly seen in the micrographs of blood corpuscles (Fig. 1.11).

Fig. 1.11: Micrographs of blood corpuscles (a) optical, (b) scanning modes

We will conclude this chapter by comparison of the characteristics of optical and scanning electron microscopes (Table 1.3); the advantages of using the SEM are in terms of better resolution, larger depth of field and possibilities of obtaining images in several modes.

Table 1.3: Comparison of optical and scanning electron microscopes

References

1. Fundamentals of Light Microscopy and Electronic Imaging by Douglas B. Murphy.
2. Introduction to Light Microscopy by Savile Bradbury.
3. The microscope and its revelations by William Benjamin Carpenter.
4. Electron Microscopy: Principles and Fundamentals by S. Amelinckx (Editor), Dirk van Dyck (Editor), J. van Landuyt (Editor), Gustaaf van Tendeloo (Editor).
5. Scanning Electron Microscopy and X-ray Microanalysis: Third Edition by Dale E. Newbury, David C. Joy, and Joseph I. Goldstein.
6. Electron microscopy by John J. Bozzola.
7. Transmission Electron Microscopy: A Textbook for Materials Science, Authors: Williams, David B., Carter, C. Barry