Gilbert Patrick L.

Manrique 2C-Ph #22

Types of Microscopes

Dark field Microscopy

A dark field microscope is arranged so that the light source is blocked off, causing light to scatter as it hits the specimen. A dark field microscope can offer brilliant, light images against a dark background of otherwise difficult to view specimens. The entire field appears dark when there is no sample on the microscope stage; thus the name dark-field microscopy. When a sample is on the stage, the light at the apex of the cone strikes it. The rays scattered by the sample and captured in the objective lens thus make the image. In darkfield microscopy the condenser is designed to form a hollow cone of light. The entire field of view appears dark when there is no sample on the microscope stage. However, when a sample is placed on the stage it appears bright against a dark background.

Uses
Dark-field microscopy has many applications in microbiology. It allows the visualization of live bacteria, and distinguishes some structure (rods, curved rods, spirals, or cocci) and movement. It is also used in viewing blood cells , Bacteria, different types of algae , hairline metal fracture, diamonds and other precious stones, shrimp or other invertebrates . Dark field microscopy has recently been used in computer mouse pointing devices, in order to allow an optical mouse to work on transparent glass by imaging microscopic flaws and dust on its surface.

Advantages
Dark-field microscopes are ideal for viewing objects that are unstained, transparent and absorb little or no light. It is also used to study marine organisms such as algae and plankton, diatoms, insects, fibers, hairs, yeast and protozoa as well as some minerals and crystals, thin polymers and some ceramics. It provide more accurate, higher contrasted images and can be used to observe a greater number of specimens.It is more useful in examining external details, such as outlines, edges, grain boundaries and surface defects than internal structure.

Disadvantage
Dark field images are prone to degradation, distortion and inaccuracies. The preparation and quality of the slides can grossly affect the contrast and accuracy of a dark field image. Similarly, if you need to use oil or water on the condenser and/or slide, it is almost impossible to avoid all air bubbles. Dark field needs an intense amount of light to work.

Phase-contrast microscopy
Phase-contrast microscopy visualizes differences in the refractive indexes of different parts of a specimen relative to unaltered light. It is an optical microscopy technique that converts phase shifts in light passing through a transparent specimen to brightness changes in the image. Phase shifts themselves are invisible, but become visible when shown as brightness variations. Phase contrast is a method used in microscopy and developed in the early 20th century by Frits Zernike. Phase is only useful on specimens that are colorless and transparent and usually difficult to distinguish from their surroundings. We call these specimens "phase objects". Examples of phase objects include cell parts in protozoans, bacteria, sperm tails and other types of unstained cells. Principle Highly refractive structures bend light to a much greater angle than do structures of low refractive index. The same properties that cause the light to bend also delay the passage of light by a quarter of a wavelength or so. A phase ring in condenser allows a cylinder of light through in phase. Light that is unaltered hits the phase ring in the lens and is excluded. Light that is slightly altered by passing through a different refractive index is allowed through. Light passing through cellular structures is retarded because they have a higher refractive index than the surrounding medium.

Use
Phase contrast is preferable to bright field microscopy when high magnifications (400x, 1000x) are needed and the specimen is colorless or the details so fine that color does not show up well. Cilia and flagella, for example, are nearly invisible in bright field but show up in sharp contrast in phase contrast. Today, two main types of phase contrast are positive and negative.Positive phase contrast reveals medium to dark gray images on a lighter grey background; these images often have a bright halo along the edge of the sample.Negative phase contrast is the opposite. The specimen appears lighter with a dark background; they also have a dark halo outlining the image. Advantages The advantages of the phase contrast microscope are its capacity to observe living cells in a natural state, can provide far more information than specimens that need to be killed, fixed or stain to view under a microscope, High-contrast, high-resolution images, Ideal for studying and interpreting thin specimens, Ability to combine with other means of observation, such as fluorescence, Modern phase contrast microscopes, with CCD or CMOS computer devices, can capture photo and/or video images Disadvantages Disadvantages and limitations of phase contrast are Annuli or rings limit the aperture to some extent, which decreases resolution.This method of observation is not ideal for thick organisms or particles because thick specimens can appear distorted. Images may appear grey or green, if white or green lights are used, respectively, resulting in poor photomicrography. Shade-off and Halo effect will also occur.Shade-off occurs with larger particles, results in a steady reduction of contrast moving from the center of the object toward its edges. Halo effect, where images are often surrounded by bright areas, which obscure details along the perimeter of the specimen

Differential Interference Contrast

Differential Interference Contrast is also known as Nomarski microscopy or imaging. It takes advantage of differences in the light refraction by different parts of living cells and transparent specimens and allows them to become visible during microscopic evaluation. Interference microscopy is a variation of phase-contrast microscopy that uses a prism to split a light beam in two. In differential interference contrast microscopy (DIC), the optical path difference is determined by the product of the refractive index difference (between the specimen and its surrounding medium) and the thickness traversed by a light beam between two points on the optical path. Interference microscopy is superior to phase-contrast microscopy in its ability to eliminate halos and extra light. Images produced by DIC have a distinctive shadow-cast appearance.

Uses
Differential interference contrast microscopy allows transparent objects to be seen by using the difference in lights refraction when transmitted through the varying thicknesses of a specimen.This technique gives a greater depth of focus allowing thicker specimens to be observed under higher magnifications. Producing a monochromatic image, areas of the object appear brighter as the optical path of the transmitted light increases and darker in areas of decreased length of optical path. How DIC Works Differential interference contrast produces contrast by visually displaying the refractive index gradients of different areas of a specimen.This process begins with light from an incandescent illumination source passing through a polarizing filter placed between the light source and the condenser. This orients the light waves in one direction so the electromagnetic waves oscillate in only one plane. Next, this beam is directed through a two-layered modified Wollaston prism which splits the beam into two beams which are spatially separated by a distance equal to the resolution of the microscopes objective lens.The path of one beam is directed through the specimen and the other, or reference beam, merely passes through the background and the two beams are again combined by an upper Wollaston prism placed above the objective lens.

Different parts of the specimen have different refractive indices and when the beams are compiled by the second prism and a second polarizing filter, or analyzer, reconstitutes the vibrational planes of the beams, this causes amplitude variations that are visualized as differences in brightness Differential Interference Contrast Components Nearly all research grade brightfield microscopes can be retrofitted with the necessary components to employ this imaging technique. Linear Polarizer - Inserted in microscope train between the incandescent illumination source and the condenser, this component is designed to produce linearly polarized or plane-polarized light necessary for the interference detection. When the contrast is perfectly adjusted the image of the specimen will have a three dimensional effect. Nomarski Prism Also known as a Wollaston prism or condenser prism, this beam-splitting prism divides the polarized light beam emanating from the polarizer into two beams. Objective Prism Either adjustable or fixed mounted, this upper prism recombines the separated beams into elliptically polarized light. Like the lower prism, this prism is formed by affixing two optical quartz wedges together.The wedges are cut differently so that one of them has its optical axis parallel to the prisms surface and the other ones optical axis is at an angle to the prisms flat surface. Analyzer Termed an analyzer, this is really a second polarizing filter and is usually located behind the objective prism and is oriented perpendicular to the transmission path of the lower polarizer. This is where the interference occurs that generates the differential interference contrast.

Advantages
An advantage of DIC is that the specimen will appear bright in contrast to the dark background. No halo effect occurs with differential interference contrast and it can be used to produce very clear images of thick specimens. It can also be used in conjunction with digital imaging systems to add further definition to the image. Differential interference contrast imaging can be used in conjunction with fluorescence microscopy to provide a better fluorescence image and to pinpoint specific areas on a specimen before switching to the fluorescence mode to further examine the object. A major advantage of the differential interference contrast technique is in examining living specimens when normal biological processes might be impeded by normal staining procedures.

Disadvantages
The three-dimensional image of a specimen may not be accurate. The enhanced areas of light and shadow might add distortion to the appearance of the image.

Fluorescence microscope
The fluorescence microscope is the most used microscope in the medical and biological fields. Fluorescence microscopy is used to study specimens that are chemically manipulated to emit light.These types of microscopes use high-powered light waves to provide unique image viewing options that are unavailable with traditional microscopes. In fluorescence microscopy, specimens are first stained with fluorochromes and then viewed through a compound microscope by using an ultraviolet (or near-ultraviolet) light source. Microorganisms appear as bright objects against a dark background. Fluorescence microscopy is used primarily in a procedure called fluorescent-antibody (FA) technique, or immunofluorescence.

How it works
The fluorescent microscope uses a high-pressure mercury, halogen, or xenon vapor lamp that emits a shorter wavelength than that emitted by traditional brightfield microscopy. These light sources produce ultraviolet light. When ultraviolet light hits an object, it excites the electrons of the object, and they give off light in various shades of color. Since ultraviolet light is used a larger amount of information can be gathered; thus, the resolution of the object increases.

Uses
Medical Sciences- A natural use for this technology is studying organisms that naturally have fluorescence characteristics. Environmental Sciences - Usually air and water samples are passed through membrane filters, and these filters are examined using fluorescence microscopy to determine if there is bacterial contamination.Industrial - There are many possible uses of fluorescence microscopy within a variety of industries. A very common example is the use of this technology within the food industry. You can directly count microbes in many food products and thus determine the contamination of the food in question. This method is far more sensitive than standard bacterial culturing.

Advantage
Fluorescence microscopy are highly sensitive and selective, Versatile (antibodies to label proteins, probes for nucleic acids, probes for a variety of cellular components, fluorescent proteins for live studies), potentially provide superb contrast because objects are self-luminous against a dark background.It has also the ability to separate absorbtion spectrum from emitted spectrum by virtue of Stokesshift and an extremely small number of fluorescent molecules (as few as 50 molecules per cubic micrometer) can be detected.

Disadvantages
Fluorescence detection sensitivity is compromised by background signals, either from endogenous sample constituents (autofluorescence) or from unbound or non-specific reagents. There are well-developed specimen preparation methods to manage and minimise background signal. There can also be blurring, bleaching, bleedthrough and autofluorescence.

Confocal Microscopy
Confocal microscopy requires immunoflurescence staining of biological samples. The key to the confocal approach is the use of spatial filtering techniques to eliminate out-of-focus light from biological samples. Confocal microscopy serves to control depth of field, eliminate background, and collect optical sections.The use of confocal microscopy has expanded to study both fixed and live cells with the ability to quantify targets.

Advantage
There are many advantages to using this type of microscope including the detailed study of minute objects within a sample and the ability to filter fluorescence colors providing views of a sample not visible using white light.These microscopes provide better resolution on both the vertical and horizontal planes, in some cases up to 0.2 microns on the horizontal and 0.5 on the vertical plane.Utilizing

state of the art technology and lasers that separate light waves, you can view images without blurred edges and in higher resolutions.

Mechanism
Confocal microscopes use dichromatic mirrors to bounce light from the objective lens onto a second mirror and past a laser that separates the different colors of light waves.

Application and Uses

Confocal Microscopy allows a small section of the sample to be seen at a time, but allows many images to be taken quickly. By collecting these images and combining them using a computer, you can construct two and three-dimensional pictures of the sample. Confocal microscopy has multiple applications in microbiology such as the study of biofilms and antibiotic-resistant strains of bacteria. Development of modern confocal microscopes has been accelerated by new advances in computer and storage technology, laser systems, detectors, interference filters, and fluorophores for highly specific targets.

Scanned-probe microscopy
Scanned-probe microscopy has enabled researchers to create images of surfaces at the nanometer scale with a probe. Scanned-probe microscopy uses a

fine probe rather than a light-beam or electrons to scan the surface of a specimen and produce a 3D image. The probe has an extremely sharp tip that interacts with the surface of the specimen. A scanning tunneling microscope, or STM, is a microscope commonly used in fundamental and industrial research.It is invented in 1981 by Gerd Binnig and Heinrich Rohrer from IBM's Zurich Research Center in Switzerland, it helped them win the 1986 Nobel Prize in Physics. High-End Resolution For an STM, good resolution is 0.1 nm lateral resolution and 0.01 nm depth resolution. The high resolution of STMs enable researchers to examine surfaces at an atomic level. The microscopes help scientists get a picture of how the atoms are arranged on a surface, by looking at the electron density of the surface atoms. Quantum Tunneling STMs are based on the idea of quantum tunneling, when a conducting tip is brought very close to the surface and a voltage difference between the tip and the surface is applied. When the voltage difference is applied, electrons can tunnel through the vacuum between the tip and the surface, causing a tunneling current. Using this principle, STMs work by passing a sharp wire made of metal over the surface that is to be examined. The tip passes very close to the surface at the same time that the microscope applies an electrical voltage to the tip. This creates an image that shows miniscule details on an atomic level.

Advantages
STMs are helpful because they can give researchers a three dimensional profile of a surface, which allows researchers to examine a multitude of characteristics, including roughness, surface defects and determining things about the molecules such as size and conformation. Other advantages of the scanning tunneling microscope include its capability of capturing much more detail than lesser microscopes. This helps researchers better understand the subject of their research on a molecular level. STMs are also versatile. They can be used in ultra high vacuum, air, water and other liquids and gasses. They will operate in temperatures as low as zero Kelvin up to a few hundred degrees Celsius.

Disadvantages
STMs can be difficult to use effectively. There is a very specific technique that requires a lot of skill and precision. STMs require very stable and clean surfaces, excellent vibration control and sharp tips. STMs use highly specialized equipment that is fragile and expensive.

Atomic force microscope

An atomic force microscope is a type of high resolution scanning probe microscope that has a resolution that you can measure in fractions of a nanometer. It was pioneered in 1986 by Nobel Prize Winner Gerd Binnig along with Calvin Quate and Christoph Gerber.

Atomic Force Microscopy One of the most important tools for imaging on the nanometer scale, Atomic Force Microscopy uses a cantilever with a sharp probe that scans the surface of the specimen. When the tip of the probe travels near to a surface, the forces between the tip and sample deflect the cantilever according to Hookes law. Atomic force microscopy will measure a number of different forces depending on the situation and performs more specialized measurements, such as temperature. Contact and Non-Contact Modes There are two primary modes of operation for an atomic force microscope, namely contact mode and non-contact mode depending on whether the cantilever vibrates during the operation. In contact mode, the cantilever drags across the sample surface and it uses the deflection of the cantilever to measure the contours of the surface. In non-contact mode, the tip vibrates slightly above its resonance frequency and does not contact the surface of the sample. Any long range forces, like van der Waals forces, decreases the resonant frequency of the cantilever. Tapping Mode In tapping mode, the cantilever uses a piezoelectric element mounted on the top to oscillate it at near to its resonance frequency with an amplitude of up to 200nm.The forces cause the amplitude to decrease as the tip gets close to the surface, and the height of the cantilever adjusts to keep the amplitude constant. Advantages The atomic force microscope is a powerful tool that is invaluable if you want to measure incredibly small samples with a great degree of accuracy. Unlike rival technologies it does not require either a vacuum or the sample to undergo treatment that might damage it.At the limits of operation however, researchers have demonstrated atomic resolution in high vacuum and even liquid environments. Disadvantages One of the major downsides is the single scan image size, which is of the order of 150x150 micrometers, compared with millimeters for a scanning electron microscope. Another disadvantage is the relatively slow scan time, which can lead to thermal drift on the sample.