Why do specimens need to be thin

These specimens are often termed reflected light specimens. Phase changes are primarily due to differences in thickness and refractive index between the specimen and its surrounding medium. A typical example is living tissue culture cells, which appear almost transparent under brightfield illumination. When passing through a transparent specimen, the illuminating light wavefront is not modified in a brightfield microscope, nor is the image-forming wave absorbed by the specimen.

If the specimen is somewhat out of focus, a thin grayscale shadow produced by refracted light is observed through the eyepieces. However, when the microscope is properly focused, the image of a thin, transparent specimen disappears specimens having a significant thickness continue to be observed due to refracted light from numerous focal planes along the optical or z -axis. The effect on incident illuminating light rays of amplitude and phase specimens is presented in Figure 3.

The uppermost sine wave in the figure illustrates a typical undisturbed surround light wave that does not pass through the specimen. When light waves enter a stained specimen represented by the green box in Figure 3 b having the same refractive index as the surrounding medium, the waves experience a reduction in amplitude as a result of absorption by the stain, but their relative phase remains unchanged. However, when light waves enter a specimen having a refractive index that is different from the surrounding medium Figure 3 c , the amplitude is not affected, but the phase is retarded by approximately 90 degrees.

In reality, a majority of the specimens encountered exhibit a combination of amplitude and phase effects Figure 3 d , producing changes to both the amplitude and phase relationships between the incident and emerging light waves.

Optical path differences and phase gradients experienced by incident wavefronts passing through a transparent specimen are utilized to full advantage by several popular contrast enhancing techniques, including phase contrast and differential interference contrast.

In most cases, the portion of an incident wavefront that traverses the specimen, but not through the surrounding medium, is either slightly advanced or retarded, depending upon the refractive index differential between the specimen and the medium. When considering transparent phase specimens that are poorly imaged in brightfield illumination, the role of the specimen in altering the optical path length in effect, the relative phase shift of waves passing through is of paramount importance. A majority of phase specimens that are observed in culture vessels or sandwiched between a microscope slide and coverslip are relatively flat or plate-like as illustrated in Figure 4 for a hypothetical specimen immersed in a homogeneous medium.

In this figure, the specimen has a thickness denoted by the variable t and a refractive index, n s. The refractive index of the surrounding medium is n m. Incident light waves yellow arrows approach the specimen, bathed in its surrounding medium, from the left and pass through at a velocity dictated by the product of the refractive index and thickness.

When light passes from one medium into another for example, from an aqueous nutrient tissue culture medium into the cytoplasm of a cell , the velocity is altered according to the refractive index differences between the two media.

Thus, when a coherent light wave emitted by the focused microscope filament passes through a phase specimen having a thickness t and a refractive index n s , the wave is either increased or decreased in velocity. If the refractive index of the specimen is greater than that of the surrounding medium, the wave is reduced in velocity while passing through the specimen and is subsequently retarded in relative phase when it emerges from the specimen.

Alternatively, when the refractive index of the surrounding medium exceeds that of the specimen, the wave is advanced in phase upon exiting the specimen. Light traveling exclusively through the specimen experiences an optical path OP that is determined by the product of the specimen refractive index n s and the specimen thickness t. The optical path difference OPD between the specimen and its surrounding medium can be expressed as:. The optical path difference is the product of two terms: the thickness t and the difference in refractive index n.

In many cases, the optical path difference can be quite large even though the thickness of the object is quite thin. On the other hand, when the refractive indices of the specimen and the surrounding medium are equal, the optical path difference is zero even if the specimen thickness is very large. In this case, light traveling through the specimen is merely delayed a phase difference relative to the light passing an equal thickness of the surrounding medium.

The phase contrast microscope is designed to take advantage of phase differences between the various components in a specimen and the surrounding medium.

However, it is not simply a phase difference that is necessary, but also diffraction by the specimen must occur for the phase contrast microscope to produce a suitable image. By comparison, differential interference contrast relies on phase gradients to generate contrast in otherwise transparent specimens, resulting in the classical pseudo three-dimensional images for which the technique is widely known.

The most common shape of a phase object is one of continuously changing optical path or density, such as the hemispherical specimen illustrated in Figure 5. In this example, the sides of the phase object can be approximated mathematically by a prism shape, as discussed below.

The refractive index of the phase object in Figure 5 is designated n s and that of the surrounding medium, n m. Radical geometrical transitions in shape for the phase object occur only at edges A and B see Figure 5 a. Incident light impacts the object perpendicular to the plane AB , while the plane wavefront is parallel to AB.

The boundary at 1 the apex of the rounded phase object in Figure 5 a is essentially parallel to the incident wavefront whereas the boundaries at A and B are perpendicular. Regions 2 through 4 are miniature prisms defined by a tangent to the rounded surface of the phase object Figure 5 b.

The "prism" angle is lesser at region 2 than at region 4 , which is opposite to region 4'. At edges A and B diffraction is strongest. Thus, curved or hemispherical specimens are composed of many prisms, and opposite sides of the specimen have prisms oriented in opposite directions. The steepest prisms are at the equator of a spherical specimen, while prisms with the least slope are located at the top and bottom.

These miniature prisms form an optical gradient :. The change in direction can be large, even though the slope or boundary gradient may be small, if the difference in refractive index is large. If the refractive indices are identical, the light wave passes through the phase object unrefracted. The direction of light exiting the prism is dependent on the relative difference in refractive indices between the phase object n s and its surrounding medium n m ; see Figure 5 c and 5 d.

As discussed above, rounded phase objects have continuously varying optical gradients, and each individual optical gradient "prism" creates a different angle of light deflection. To summarize the "prism" effect of optical gradient boundaries, when the refractive index of the phase object exceeds that of the surrounding medium, then gradients of equal size on each slope of the object deflect at the same angle. When the refractive index of the medium n m is greater than the refractive index of the object n s , the deflection is opposite from when the situation is reversed.

When there is no gradient, there is no deflection of light passing through. The edges, gradients, and thickness of a specimen, regardless of whether the specimen is roughly classified as amplitude or phase, affect the refraction and diffraction angles of light wavefronts passing through.

Only a portion of this scattered light is captured by the objective, a factor that is dependent upon the numerical aperture. The remaining scattered light, which is not collected, represents specimen information that is lost to the resulting image. The aperture of the objective rear focal plane mimics a low pass filter affecting large specimen details for diffracted light, which must be focused at the intermediate image plane to undergo interference and form an image.

Because smooth rounded surfaces of phase specimens have relatively few or no diffraction sites, they suffer a significant lack of contrast when imaged without the benefit of auxiliary contrast enhancing optical components.

When considering optical methods to enhance specimen contrast, it is useful to consider various characteristics of a specimen that can be manipulated to create intensity variations that will result in rendering the specimen visible.

A primary question is which characteristic of the object will be transformed into a difference of intensity under a unique set of circumstances. Minute specimen details and edges that have a size approximating the wavelength of imaging light will diffract or scatter light, provided there is a difference in refractive index between the specimen and its surrounding medium.

Refractive index is classically defined as the ratio of the speed of light through air or a vacuum divided by the speed of light through the object. Because the speed of light through any material is less than the speed of light in a vacuum, the refractive index always exceeds a value of 1. In order to resolve small distances between objects and to reproduce their shape with reasonable fidelity, a large angle of diffracted light must be captured by the microscope objective.

Diffracted or deviated light gathered by the objective must be brought into a sharp focus at the image plane in order to generate specimen detail. At the image plane, light waves comprising the diffracted light undergo interference with undiffracted light that passes through and around the specimen.

The quality of the image generated by interference is highly dependent upon the coherency of light illuminating the specimen, and image quality generally is dramatically improved by increasing the coherence.

In the optical microscope, the condenser aperture diaphragm opening size partially controls the spatial coherence of light incident on the specimen.

Decreasing the diaphragm opening size yields a greater spatial coherence. Illumination of the specimen with light waves that are at least partially coherent is critical to the role of diffraction and interference in image formation, and is required in all forms of interference optical microscopy, including phase contrast, differential interference contrast DIC , and polarization. In effect, the light waves passing through the specimen surround or undeviated and those diffracted by the specimen have a mutual degree of coherent character, which must be preserved throughout the microscope optical train to enable constructive and destructive interference to occur at the image plane.

For example, cheek cells are often stained with methylene blue, to make individual cells more distinguishable from each other, and also to make the nucleus inside a cell distinguishable from the rest of the cell since this particular dye stains nucleic acids. Additionally, iodine is often used to stain plant cells to reveal the presence of starch.

Home » Blog. Prev: what is the function of the objectives on a microscope. Instead, the samples must undergo complex preparation steps to help them withstand the environment inside the microscope. For scientists who wish to view biological samples, this poses a challenge — how can the sample be preserved so that it looks as much as possible like it would in the living organism, while still being able to withstand being visualised in the electron microscope.

The electron beam inside a transmission electron microscope TEM causes problems for biological samples because of its high energy. It needs to have enough energy to pass right through the sample and out the other side.

This temperature is far too high for living cells to survive. Scanning electron microscopes SEMs use a lower-energy electron beam, but it can still be damaging to the sample. The vacuum inside an electron microscope is important for its function. Without a vacuum, electrons being aimed at the sample would be deflected knocked off course when they hit air particles. But liquid water, which is abundant in biological samples, evaporates immediately in a vacuum. If this happened, a biological sample would vaporise in front of your eyes!

To be visualised by an electron microscope, biological samples need to be:. Right from the word go, from the moment you collect your sample, you have to be thinking about preserving it in as close to the living state as possible. The first — and perhaps most important — step in the preparation process is fixation. In this step, living tissue is chemically treated to stabilise it.