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6. LIGHT MICROSCOPES

Light, or optical, microscopes are essential for histological studies because they allow us to visualize cells and morphological features of tissues. Light microscope relies on glass lenses and visible light to magnify tissue samples. It was invented in XVII century, and has been improved over the years resulting in the powerful modern light microscopes. The most remarkable improvement has been getting better glass lenses to obtain sharper and non-distorted images, as well as adding devices to explore new ways of visualizing tissular features.

The resolution power of light microscope is 0.2 µm. It is the shortest distance between two point where they can be distinguished from each other. This limit is a consequence of visible light wavelength.

Light microscopes contain two main lenses: objective and ocular (eyepieces). Objective gathers the light that goes through the tissue, whereas the ocular project the tissue image on the eye. The total magnification is the result of multiplying the objective magnification by the ocular magnification. For example, if we have a 40X objective (40 times magnification) and a 10X ocular (10 times magnification), the total magnification is 400 times. More advanced microscopes can get 1000 to 1500 magnifications (100x objective and 10X or 15X ocular). Some light microscopes may contain additional internal lenses between the objective and the ocular that can change the total magnification. Magnification and resolution power must not be confused because not matter how we can magnify an image, including digital methods, the resolution cannot be increased.

Light microscopes are made up of several components (Figures 1 and 2):

Light microscope
Figure 1. Basic components of light microscopes.
Light and lenses
Figure 2. Optical pathway of light going through the different lenses of a typical compound optical microscope.

Ocular lenses, or eyepieces. They form the final image projected into our eyes. There are two ocular lenses, one per eye, and that is why current microscopes are called binoculars. The first light microscopes had only one ocular lenses, so they were monocular microscopes. Nowadays, each ocular lens (as well as objective lens) are made up of several lens. In the microscope, at least one of the ocular lenses can be adjusted, that is, change the focus of the sample to be adapted to the particular diopters of the observer.

Objective lenses. They are one on the most important part of the microscopy because they are the first lenses that gather the light passing through the tissue. Current microscopes have a nose-piece with several objectives, each of one with a different magnification power. The more frequent magnifications are: 4X, 10X, 20X, 40X and 100X (Figure 3). By rotating the nose-piece, the objective can be selected and therefore a particular magnification for studying the tissue. Besides magnification, objectives have other features to improve the quality of the image. They can be achromatic, apochromatic, contain fluorite, flat field feature to prevent peripheral curvatures, interference contrast to increase border contrast, etcetera.

Ampliaciones
Figure 3. Epidermis visualized by using objectives with different magnificaton power.

When 100X objective is used, a small drop of a specific type of oil, called immersion oil, is placed between the objective and the coverslip (or the sample). Visible light refraction is high in the air and results in image alterations when observing at high magnifications. Immersion oil minimizes the light refraction rendering sharp images.

The stage is the platform where the slide with the sample is placed. It has a device to hold the slide and another to move the slide manually in the X and Y axis.

The condenser is a light concentrating lens that focuses the light coming from the lamp into the sample.

The diaphragm is placed between the lamp and the condenser. It increases the contrast of the image and the field depth, that is, the distance in the Z axis of the sample that looks sharp or is in focus.

The lamp is the light source. The light goes through the tissue section and is gathered by objective lenses. At the beginning of the microscopy, Sun light was used, which was focused on the tissue with concave mirror lenses. Nowadays, electric lamps are used with a light beam that is adjusted with a diaphragm and a condenser before hitting the tissue. The light intensity can be selected by a brightness control dial.

Fine and coarse adjustment (or fine and gross adjustment) are intended to get the sample in focus by changing the distance between the sample and the objective lens. It is done by either rising or lowering the stage where the slide is lay on. The distance depends on the objective: shorter as magnification increase. It also depends on the specs of the objective. Fine and gross means the amplitude of the distance change that can be got it with each of them.

Other types

Phase contrast. It is a modification of the clear-field microscopy by using special objective lenses that take advantage of the very small variations in phase of the light when going through the different tissular structures. These small differences are translated into changes in amplitude, which can be visualized as image contrast. Then, different tissular structures are visualized more or less brilliant. Phase contrast microscopy can be used to observe non stained samples, aqueous solutions, and living cell cultures.

Dark field. Dark field microscopy can replace phase contrast microscopy to visualize unstained samples or aqueous samples. Dark field microscopy includes a dark disc under the condenser, between the lamp and the condenser. The dark disk lets pass the more lateral light only, which hits the sample in an oblique manner. Only the light scattered by the sample will be gathered by the objectives. Those areas without tissue are black and different tissue densities reflect a variety of light intensities.

Differential interference contrast (DIC) or Nomarski microscopy. This type of microscopy needs special objectives and filters, and enhances the contrast of the sample. It is based on a polarizing filter that separates the light into two orthogonal rays, which go through the condenser and the sample. Each ray has different path in the sample because of tissue densities, and the ray changes in phase compared to the other. The objective focuses the sample and the rays pass through a prism filter that produces interference between the two rays, resulting in bright and dark areas. At the end, the images look like 3D images with different levels of luminosity.

OTHERS

Stereo microscope. It is also known as dissecting microscope. It is commonly used for manipulate small samples and to visualize features that not need large magnifications, about 7X to 40X, although 100X can be reached. Samples can be visualized at different magnifications by using a zoom system or changing objectives, which is less common. Samples can be studied tridimensionally since stereo microscopes are binoculars. In addition, they have a wide focal distance, that is, there is a long distance between the sample and the objective that makes much comfortable to manipulate samples. Illumination of the sample may come from different sources and angles. Light sources can be external lamps.

FLUORESCENCE

Fluorescence microscopy is used to visualize fluorochromes, fluorescent chemical compounds. Fluorescent molecules are able to be excited by electromagnetic radiation with a particular wavelength and emit electromagnetic radiation with other wavelength, usually in the visible light spectrum. Fluorochromes are used to visualize tissue structures, and are commonly used conjugated with antibodies during immunofluorescence protocols (Figure 3). The more popular fluorochromes are excited by ultraviolet light, commonly produced by mercury lamps, and emit in visible light range. Each fluorochrome is excited by a narrow wavelength range, which is selected by special filters placed between the lamp and the sample. It is possible to excite several fluorochromes present in the sample by using a number of filters with different specs.

Fluorescence
Figure 3. Fluorescence microscopy images. On the left, immunohistochemistry for neuropeptide Y in rat brain. Antibodies were conjugated with Texas-red fluorochrome. On the right, motoneurons of the lamprey brain labeled with fluorescein tracer, which is a fluorochrome. Each fluorochrome has been excited with a specific wavelength, and they emit visible light: red and green, respectively.

More than one fluorochrome, in the same sample at the same time, can be visualized with fluorescence microscopy. It can be done if the absortion wavelength of each fluorochrome are not overlapped. Otherwise, they cannot be distinguished between each other.

Confocal microscopy is performed by an advance fluorescence microscope that is able to reduce light diffusion from out of focus fluorochromes. Thus, confocal microscopes get sharper images from different depths in the sample. Furthermore, they are usually aconnected to a computer so that the digital images obtained from different depths of the sample can be rendered in 3D images.

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