Where is a light microscope used

The advantage of light microscopes and stereomicroscopes in particular is that objects can be looked at with little or no preparation. This makes them very useful for looking at living things, such as flower parts, insects, earthworms and human skin. This is important because these samples often lack contrast, which makes it hard to distinguish between parts of the sample.

Stains interact with a specific part of the sample, turning it a different colour from its surroundings. For example, iodine is often used to stain plant cells because it colours the starch stored within the cells a blue colour and other structures a pale brown.

In fluorescence microscopy, fluorescent stains are used to highlight specific parts of the cell or tissue. Stains can be used on living or non-living biological material.

Some stains require the sample to be treated beforehand, and in this case, the tissue is no longer living. Other stains are used on living tissue, which is important for observing biological processes under the microscope. Variations of thickness and refractive index across the sample result in different colors. Credit: R. Figure 16 : Sample preparation for expansion microscopy. A cell is first stained and then linked to a polymer gel matrix.

The cell structure itself is then dissolved digested , allowing the stained parts to expand isotropically with the gel, allowing the stained structure to be imaged with more detail. Figure Image deconvolution. Left: Original fluorescence image. Right: Image after deconvolution, showing increased detail.

Credit: Author. Low contrast, fully transparent objects cannot be imaged directly and may require staining. Imaging colored or stained samples 15 and partially transparent materials Reveals small structures and surface roughness, allows imaging of unstained samples.

High illumination power required can damage the sample, only scattering image features seen. Imaging particles in cells, 17 surface inspection Complex optical setup, high illumination power required can damage the sample, generally darker images. Tracking cell motion, 19 imaging larvae Differential interference contrast microscopy.

High resolution imaging of live, unstained cells 21 and nanoparticles Strong background suppression from non-birefringent areas of a sample, allows measurement of sample thickness and birefringence.

Imaging collagen, 23 revealing grain boundaries in crystals Allows individual fluorophores and particular areas of interest in a sample to be singled out, can overcome the resolution limit. Requires a fluorescent sample and a sensitive detector, photobleaching can diminish signal. Imaging cell components, single molecules, proteins Visualize specific biomolecules using antibody targeting.

Extensive sample preparation, requires a fluorescent sample, photobleaching. Identifying and tracking cells 26 and proteins Low background signal, possible to create 3D images.

Slow imaging speed, requires a complicated optical system. Deep sample penetration, low background signal, less photobleaching. Slow imaging speed, requires a complicated optical system and high-power illumination.

Neuroscience, 29 deep tissue imaging Images only an extremely thin slice of the sample, can create 3D images by rotating the sample. Total internal reflection fluorescence microscopy. Strong background suppression, extremely fine vertical sectioning. Imaging limited to thin area of sample, requires a complicated optical system, sample needs to be in aqueous medium.

Single molecule imaging, 31 imaging molecular trafficking Increases effective resolution of standard fluorescence microscopy. Requires chemical processing of the sample, not suitable for live samples. Second, since light is selectively collected from a narrow focal plane, the information collected consists of a series of two-dimensional sections which can be analyzed individually, or assembled to construct a three-dimensional image.

The disadvantages of using confocal microscopy include the requirement of more complex and expensive optical elements to scan and focus the illumination. Also, since the technique is inherently a point-by-point analysis, there can be a trade-off between scanning speed and image quality.

In this type of microscope, two slightly offset optical paths exist between the specimen and the eyepiece, so that the user has depth resolution of the sample. This technique is useful when work is being done on the specimen, for example dissection or assembly of small pieces. Additional optics are available to improve the contrast in certain types of samples, which include phase contrast, differential interference Nomarski contrast, and dark field techniques.

These techniques require specialized condenser and objective optics. Further modifications can be made to collect spectroscopic data on specimens in parallel to optical images, infrared or Raman data for example, which can be useful in determining material composition.

As with any lab analysis, the first step to using light microscopy is to develop a plan and set of goals for the analysis. One way of doing this is to list the questions you hope to answer using this technique. The next step is sample preparation. For normal optical microscopy, this is relatively straightforward. For liquid samples that will be analyzed using transmitted light, common steps are:.

The analysis of opaque samples by reflected light microscopy is similar, but instead of preparing a slide, the specimen is mounted on a support that will prevent it from moving during analysis. The most common form of damage to a light microscope is contamination or scratches on the sensitive precision lenses and other optical components. This can be avoided by:. As an Amazon Associate Conductscience Inc earns revenue from qualifying purchases The modern pipette has had a colorful history as a standard tool in the.

Stereotaxic Accesories. Conduct Lifestyle Grants Academia. Quote Lab Basics , Science. The Light Microscope. Introduction The light or optical microscope is a common lab tool that can be used to visualize structures with sizes below that which can be seen by the human eye.

Following the path of the light, the parts are: Light source: there are a variety of different types of lamps which can be used as the light source in optical microscopy, including tungsten filament, various types of arc lamps, and LEDs.

The light source is generally chosen based on a number of factors including illumination intensity, emitted spectrum, and temperature. Condenser, which collects light from the source and projects it on to or through the sample. Sample stage, which holds the specimen and usually has multiple axes of motion. Objective, which collects light from the sample. The objective is a critical component, because, it determines the magnification and quality of the image. Magnification can therefore be varied, according to the size of the specimen to be viewed and the level of detail required.

The magnification of a lens is shown by a multiplication sign followed by the amount the lens magnifies. The total magnification of a microscope is:. Microscopes use lenses to magnify the image of a specimen so that it appears larger. The formula to calculate magnification is:. The same formula shown in a magnification triangle:. The real size of the cell shown above is 0.