Microscopy-Light microscope (Part-2)

ü  Phase- Contrast Microscope

The phase-contrast microscope was invented by Frits Zernike in the early 1930s. He won a Nobel Prize in 1953.

A phase-contrast microscope converts slight differences in refractive index and cell density into easily detected variations in light intensity. The phase-contrast microscope is used to visualize unstained living cells. It enables the visualization of living cells and life events.

Working

·         The condenser of a phase-contrast microscope has an annular stop, an opaque disk with a thin transparent ring, that produces a hollow cone of light.

·         As this cone of light passes through a cell, some light rays are bent due to variations in density and refractive index within the specimen, and are retarded by about ¼ wavelength.

·         The deviated light is focused to form an image of the object.

·          Undeviated light rays strike a phase ring in the phase plate, an optical disk located in the objective, while the deviated rays miss the ring and pass through the rest of the plate.

·         If the phase ring is constructed in such a way that the undeviated light passing through it is advanced by ¼ wavelength, the deviated and undeviated waves will be about ½ wavelength out of phase and will cancel each other. When they come together to form an image.

The production of contrast in Phase-contrast microscopy- The behavior of deviated and undeviated (i.e., undifferacted) light rays in the dark-phase-contrast microscope. Because the light rays tend to cancel each other out, the image of the specimen will be dark against a brighter background.

 

The background, formed by undeviated light, is bright, while the unstained object appears dark and well defined. This type of microscopy is called dark-phase-contrast microscopy.

 

Color filters often are used to improve the image.

 

The optics of a dark-phase-contrast microscope

Applications

·         Phase-contrast microscopy is especially useful for studying microbial motility, determining the shape of living cells, and detecting bacterial structures such as endospores and inclusions and various cell organelles (mitochondria, nucleus and vacuoles).

·         These are clearly visible because they have refractive indices markedly different from that of water.

·         Helps to study cellular events such as cell division, phagocytosis, cyclosis, etc.

·         Phase-contrast microscopes also are widely used to study eukaryotic cells.

 

Advantages

·         The capacity to observe living cells and as such the ability to examine cells in a natural state.

·         Observing living organisms in its natural state and/or environment can provide far more information than specimens that need to be killed, fixed or stain to view under a microscope.

·         High contrast, high-resolution image.

·         Ideal for studying and interpreting thin specimens.

·         Ability to combine with other means of observation, such as fluorescence.

·         Modern phase-contrast microscope, with CCD (Charge-Coupled Device) or CMOS (Complementary Metal Oxide Semiconductor). Computer devices, can capture photos and/or video images.

·         In addition advances to the phase contrast microscope especially those that incorporate technology, enable a scientist to hone in a minute internal structures of a particle and can even detect a mere small number of protein molecules.

Disadvantages

·         Annuli or rings limit the aperture to some extent which decreases the resolution.

·         This method of observation is not ideal for thick organisms or particles.

·         Thick specimens can appear distorted.

·         Images may appear gray or green, if white or green lights are used, respectively, resulting in poor photomicrography.

·         Shade-off and halo effect referred to a phase artifacts.

·         Shade-off occurs with larger particles, results in a steady reduction of contrast moving from the center of the object toward its edges.

·         Halo the effect, where images are often surrounded by bright areas, which obscure details along the perimeter of the specimen. The formation of the halo is due to the particle or incomplete separation of direct and deviated rays.

 

 

ü  Fluorescence microscope- using emitted light to create an image

 

The light microscopes thus for considered produce an image from light that passes through a specimen. An object also can be seen because it emits light: this is the basis of fluorescence microscopy. When some molecules absorb radiant energy, they become excited and release much of their trapped energy as light. Any light emitted by an excited molecule has a longer wavelength (i.e., has lower energy) than the radiation originally absorbed. Fluorescent light is emitted very quickly by the excited molecule as it gives up its trapped energy and returns to a more stable state.

 A fluorescence microscope excites a specimen with a specific wavelength of light and forms an image with the fluorescent light emitted by the object. The most commonly used fluorescence microscopy is epifluorescence microscopy, also called incident light or reflected light fluorescence microscopy. Epifluorescence microscopes employ an objective lens that also acts as a condenser so the specimen is illuminated from above rather than below.

 

Epifluorescence microscopy- The principles of operation of an epifluorescence microscope

 

Working

·         A mercury vapor arc lamp or other source produces an intense beam of light that passes through an exciter filter.

·         The exciter filter transmits only the desired wavelength of light.

·         The excitation light is directed down the microscope by the dichromatic mirror.

·         This  mirror reflects light of shorter wavelength (i.e., the excitation light) but allows the light of longer wavelengths to pass through.

·         The excitation light continues down, passing through the objective lens to the specimen, which is usually stained with molecules called fluorochromes.

·         The fluorochrome absorbs light energy from the excitation light and fluoresces brightly.

·         The emitted fluorescent light travels up through the objective lens into the microscope. Because the emitted fluorescent light has a longer wavelength, it passes through the dichromatic mirror to a barrier filter, which blocks out any residual excitation light.

·         Finally, the emitted light passes through the barrier filter to the eyepieces.

 

Commonly used fluorochromes

Fluorochrome	Uses
Acridine orange	Stains DNA
Diamidino-2-phenyl	Stains DNA
Fluorescein isothiocyanate (FITC)	After attached to DNA probes or to antibodies that bind specifically cellular components
Tetramethyl rhodamine isothiocyanate (TRITC or rhodamine)	Often attached to antibodies that bind specific cellular components

 

Applications

·         The fluorescence microscope has become an essential tool in microbiology. Bacterial pathogens can be identified after staining with fluorochromes or specifically tagging them with fluorescently labeled antibodies using immunofluorescence procedures.

·         In ecological studies, fluorescence microscopy is used to observe microorganisms stained with fluorochrome-labeled probes or fluorochromes that bind specifically cell constituents.

·         Microbial ecologists use epifluorescence microscopy to visualize photosynthetic microbes, as their pigments naturally fluoresce when excited by light of specific wavelengths.

·         It is even possible to distinguish live bacteria from dead bacteria by the color they fluoresce after treatment with a specific mixture of stains. Thus, the microorganisms can be viewed and directly counted in a relatively undistributed ecological niche.

·         Another important use of fluorescence microscopy is the localization of specific proteins within cells.

·         One the approach is to use genetic engineering techniques that fuse the gene for the protein of interest to a gene isolated from jellyfish belonging to the genus Aequorea. This jellyfish gene encodes a protein that naturally fluoresces green when exposed to the light of a particular wavelength and is called Green Fluorescent Protein (GFP). Thus when the protein is made by the cell, it is fluorescent. GFP has been used extensively in studies on bacterial cell division and related phenomena.

 
ü  Confocal microscope

When three-dimensional objects are viewed with traditional light microscopes, light from all areas of the object, not just the plans of focus, enters the microscope, and is used to create an image. The resulting image is murky and fuzzy. This problem has been solved by the Confocal Scanning Laser Microscope (CSLM), or simply, confocal microscope. An optical imaging technique for increasing optical resolution and contrast of a micrograph.

The confocal microscope uses a laser beam to illuminate a specimen,  usually one that has been fluorescently stained cause sample to fluoresce. Uses pinhole screen to  block out-of-focus (eliminates out of focus) light in the image formation and produce high-resolution images. So images have better contrast and are less hazy. It generates a 3–dimensional image. Is an updated version of fluorescence microscopy.

Principle

In confocal microscopy, two pinholes are typically used. A pinhole is placed in front of the illumination source to allow transmission only through a small area. This illumination pinhole is imaged on to the focal plane of the specimen, i.e. only a point of the specimen is illuminated at one time. Fluorescence excited in this manner at the focal plane is imaged onto a confocal pinhole placed right in front of the detector.

Only fluorescence excited within the focal plane of the specimen will go through the detector pinhole and eliminates stray light from the parts of the specimen above and below the plane of focus. Thus the only light is used to create an image is from the plane of focus, and a much sharper image is formed. Scanning of small sections is done and joined them together for a better view.

Working mechanism

·         Confocal microscope incorporates two ideas:

1.      Point-by-point illumination of the specimen

2.      Rejection of out of focus of light

·         Light source of very high intensity is used- laser light source

1.      Laser provides intense blue excitation light.

2.      The light reflects off a dichoric mirror, which directs it to an assembly of vertically and horizontally scanning mirrors.

3.      These motors driven mirrors scan the laser beam across the specimen.

4.      The specimen is scanned by moving the stage back and forth in the vertical and horizontal directions and optics are kept stationary.

·         Dye in the specimen is excited by the laser light and fluoresces.

·         The fluorescent (green) light is descanned by the same mirrors that are used to scan the excitation (blue) light from the laser beam.

·         Then it passes through the dichoric mirror.

·         Then it is focused on pinhole.

·         The light passing through the pinhole is measured by the detector such as photomultiplier tube.

·         For visualization, the detector is attached to the computer, which builds up the image at the rate of 0.1-1 second for a single image.

·         Computers are integral to the process of creating confocal images

·         A computer interfaced with the confocal microscope receives digitized information from each plane in the specimen that is examined.

·         This information can be used to create a composite image that is very clear and detailed or to create a three-dimensional reconstruction of the specimen.

 

 

Applications

·         One is the study of biofilms, which can form on many different types of surfaces, including indwelling medical devices such as hip joint replacements.

·   It allows analysis of fluorescent-labeled thick specimens without physical sectioning (example- zebrafish embryo).

·         Three-dimensional reconstruction of the specimen (example-biofilm)

·         It is possible to detect more color differences (more color possibilities) example- actin filaments ina cancer cell.

·         The improved resolution allows detecting easily each area of the specimen.

 

  Advantages

·         The specimen is everywhere illuminated axially, rather than at different angles, thereby avoiding optical aberrations.

·         Entire field of view is illuminated uniformly.

·         The field of view can be made larger than that the static objective by controlling the amplitude of the stage movements.

·         Image formed are of better resolution.

·         Cells can be live or fixed.

·         Serial optical sections can be collected.

·         Taking a series of optical slices from different focus levels in the specimen generates a 3D data set.

 Disadvantages

·         Resolution – It has an inherent resolution limitation due to diffraction. Maximum best resolution of confocal microscopy is typically about 200nm.

·         Pin hole size- The strength of optical sectioning depends on the size of the pinhole.

·         Intensity of the incident light.

·         Fluorophores

1.      The fluorophore should tag the correct part of the specimen.

2.      Fluorophore should be sensitive enough for the given excitation wavelength.

3.      It should not significantly alter the dynamics of the organism in the living specimen.

·         Photobleaching – Photochemical alteration of a dye or a fluorophore molecule such that it permanently is unable to fluoresce.