FLUORESCENCE MICROSCOPY

Fluorescence microscopy is an essential tool in molecular and cellular biology. It is a technique that allows one to study and visualize the cellular structures and dynamics of tissues and organelles, and macromolecular assemblies inside the cell. It was devised in the early twentieth century by various scientists like Köhler, Lehmann, Reichert and others.

The wide utilization of fluorescent proteins since their discovery have revolutionized the applications and use of the microscope in biological studies.

A fluorescence microscope uses the property of fluorescence to generate an image. It uses a high-intensity light source that excites the fluorescent molecule that may be inherently present in the sample to be studied or may be artificially labelled with a fluorescent molecule. The fluorescent molecule is called the fluorophore which is usually present in the fluorescent dye. 

Therefore, one could say that any microscope that works on the same basis to study the properties of organic or inorganic substances is a fluorescent microscope.

A fluorescence microscope is a type of optical microscope that uses fluorescence (ability of a substance to emit light on excitation) and phosphorescence (ability of a substance to continue emitting light even after the removal or withdrawal of the excitation factor). It may use these properties instead of or in addition to the properties of scattering, absorption, reflection and attenuation. 

The setup for the microscope may be simple as in an epifluorescence microscope or it may have a complicated design like that of a confocal microscope. A confocal microscope uses optical sectioning to provide a better resolution of the fluorescence image.

Principle:

Fluorescent substances are the substances that absorb light of a particular energy and wavelength and then emit light of a longer wavelength and lesser energy.  

This phenomenon of fluorescent substances can be applied to the working of the fluorescent microscope. Fluorescent dyes (also called fluorochromes or fluorophores) are molecules that have the ability to absorb excitation light at a given wavelength, and then emit light of a comparatively longer wavelength after a delayed time interval.

In practical use, the sample is stained with a fluorescent dye and then illuminated with a blue light. The blue light (short wavelength) is absorbed by the fluorophores of the fluorescent dye, and the green light (which has longer wavelength) is emitted. This change is called the Stokes shift.

The light source that is used in fluorescent microscopy is a high intensity mercury arc lamp. The lamp emits white light when then passes through a device called an ‘exciter filter’. (as shown in the figure) This device filters the emission light to reveal the location of the fluorophores. It allows only the blue component of white light (white light comprises of coloured light of all wavelengths) to pass through and prevents the passage of light of other colors.

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The dichroic mirror is used to reflect the blue light and allows the green light to pass. The angle of the mirror is fixed in such a way that the blue light is reflected towards the specimen placed below. It allows the passage of green light.

Finally, when the light reaches the ‘barrier filter’, it blocks out or removes all the remnants of the residual blue light from the specimen which may not have been ideally reflected by the dichroic mirror.

Thus, enabling the observer to perceive the glowing green portions of the specimen against the jet-black background of the dark field condenser that is used. The portions of the specimen that have not been stained remain invisible to the eye and this is how fluorescence microscopy provides a sharp image for the observation of the fine and intricate components of the sample to be studied.

Components:

The essential components of the fluorescence microscope are:

• Fluorescent dyes (fluorophore): Chemical compounds that have the ability to re-emit light upon excitation. Examples include; nucleic acid stain like DAPI and Hoechst, phalloidin etc.
• Light source: This is provided by a bright mercury vapor arc lamp, xenon lamp or LEDs with a dichroic excitation filter, lasers etc.
• Heat filter: The lamp produces infrared rays which generate considerable heat. No other major uses of the heat filter exist.)
• Exciter filter: The light undergoes cooling and passes through the exciter filter which allows the passage of the shorter waves which play a role in excitation of the fluorochrome dye coated sample on the slide and does not allow the other wavelengths to pass through.
• Dichroic mirror: An accurate colour filter/mirror which selectively allows the passage of light of a particular wavelength and reflects the others. 
• Condenser: A dark field condenser is usually used because it provides a dark background and it is easy to detect even mild fluorescence exhibited by the sample
• Barrier filter: It removes all the remnants of the exiting light and is situated in the body tube of the microscope between the objectives and the eye piece. 

Applications: 

• Identify structures in fixed and live biological samples in microbiological studies.
• Used in food chemistry for the assessment of the structural organization and spatial distribution of the components of food.
• Used for the study of mineral like coal and graphene oxide in minerology.
• Used in the textile industry for analysis of fibre dimensions. 

Article By- Shaily Sharma (MSIWM041)

References:

Fluorescence Microscopy (nih.gov)

Immunofluorescence staining – PubMed (nih.gov)

Fluorescence Microscopy – Explanation & Labelled Images (microscopeinternational.com)

The Simple Microscope

BY- K. Sai Manogna (MSIWM014)

Principle : 

A single lens, historically called a loupe, consists of a simple microscope. A reading or magnifying glass is the most familiar example nowadays. Higher-magnification lenses are often made of two glass elements that create a color-corrected image. They can be worn in a cylindrical shape around the neck, which can be kept immediately in front of the eye. These are commonly referred to as eye loupes or lenses for jewelers. A single magnifying lens was used to create the standard simple microscope, which was often of good optical quality to enable the study of microscopic species, including Hydra and protists. 

Magnifications 

1. When one wants to study an object’s information, it is instinctive to put it as close to the eye as possible. 

2. The closest the object is to the eye, the greater the angle it subtends to the eye, and thus the more extensive the object appears. 

3. However, the human eye can no longer form a clear picture of an object brought too close. 

4. The magnifying lens between the viewer and the object makes it possible to construct a “virtual image” that can be viewed comfortably.

5. The magnifier should be positioned in front of the eye to obtain the best possible image. To make out the object of interest, one should position the object at the microscope’s lens’s focus.

6. The highest magnification possible without lenses is when the object is carried to the closest location where a clear virtual image is observed. 

7. This distance from the picture is around 25 cm for many people. The closest point of separate vision recedes to greater distances as individuals age, making a magnifier a valuable adjunct to a vision for older people. 

8. The optical system’s geometry is related to magnifying force, or the degree to which the object is seen appears to be expanded, and the field of view, or the scale of the object that can be seen. 

9. The working value of the magnifying power of the lens can be measured by dividing the minimum distance of separate vision by the focal length of the lens, which is the distance from the lens to the plane where the incoming light is centered

10. A lens with a minimum different vision distance of 25 cm and a focal length of 5 cms would also have a magnifying power of around 5 percent, for example. 

11. If the magnifying lens diameter is adequate to fill or exceed the eye’s pupil diameter, the viewed virtual image will appear to be of significantly the same brightness as the original object. 

12. As the focal length of the magnifier is increased. the field of view will be determined by the degree to which the working diameter is exceeded by the lens and the distance between the lens and the eye. The clearer the virtual picture, the more dependent it will be on the irregularities of the lens, its contours, and the conditions of its use.

Aberrations:

Various aberrations affect the picture’s sharpness or consistency. 

1. Chromatic aberrations create colored fringes around the image’s high-contrast regions since longer light wavelengths (such as red) are brought to focus slightly further from the lens than shorter wavelengths in a plane (such as blue). 

2. Spherical aberration creates a picture in which, while the periphery may not be, the center of the field of view is in focus and uses lenses with spherical (rather than nonspherical or aspherical) surfaces. 

3. The distortion produces curved images from straight lines in the object. The apparent shape and degree of distortion are closely related to the magnifier’s possible spherical aberration and is usually the most severe in high-powered lenses.

4. As relative aperture, i.e., the working diameter divided by the lens’s focal length, increases, the aberrations of a lens increase. 

5. The aberration of the lens with a smaller diameter than the focal length would be more important than that of the other lens with a greater diameter.

6. Thus, there is a conflict between a short focal length that allows for high magnifying power but a narrow field of view and a longer focal length that offers a lower magnifying power but a wider linear field of view. 

7. The 1670s high-powered lenses of Leeuwenhoek had a focal length and a few millimeters’ working size. This made it hard to use them, but they produced remarkable pictures that have not been changed for a century. 

Magnification: 

Several kinds of magnifiers are available. The option of an optical design for a magnifier depends on the power needed and the magnifier’s intended use. 

For low powers:

a. A simple double convex lens is applicable for low forces, around 2-10x. Early simple microscopes, such as the microscope of Leeuwenhoek, magnified up to 300x. 

b. If the lens has unique aspherical surfaces, as can be easily obtained in a plastic molded lens, the image can be enhanced. 

c. When an aspheric lens is used, a reduction in distortion is noted, and the manufacture of such low-power aspheric plastic magnifiers is an important industry. 

For high powers:

There are various types of magnifiers in which the basic magnifier is replaced by a compound lens consisting of multiple lenses mounted together for higher powers of 10-50x. 

Using two simple lenses, usually plano-convex, which are flat on one side, angled outward on the other side, with curved surfaces facing each other, would automatically increase the distortion expected from a loupe. This form of magnifier is based on the Huygenian telescope eyepiece, in which the chromatic lateral aberration is corrected by removing the elements from the focal length. Since two components provide and share the image properties, the magnifier’s spherical aberration and distortion are substantially reduced compared to those of a single lens of the same strength. 

Coddington lens:

To choose the imaging light portion with the lowest aberrations, a Coddington lens incorporates two lens components into a single thick element, with a groove cut in the center of the element. This is a simple and inexpensive system that suffers from the requirement that the distance between the optic beam and the target of imaging is small.

More complex magnifiers:

Three or more components are used by more advanced magnifiers, such as the Steinheil or Hastings types, to achieve better correction for chromatic aberrations and distortion. In general, the use of aspherical surfaces and fewer components is a safer solution. 

– Often, mirrors are used. The British physicist C.R. brought reflecting microscopes, in which the image is magnified by concave mirrors rather than convex lenses, to their height of excellence in 1947. 

– Burch, who made a set of giant tools that used ultraviolet rays. 

– Using a magnifying mirror, chromatic aberration will not be minimized, although, with the use of an aspheric mirror carefully contoured, distortion and spherical aberration are reduced.

– The reflecting microscopes of today are limited to analytical instruments that use infrared rays.

Microscope

BY- K. Sai Manogna (MSIWM014)

 

The microscope is an instrument that creates enlarged photographs of small objects, enabling the viewer to display minute structures in a too near manner at a scale that is convenient for observation and study. Although optical microscopes are the focus of this article, many other types of wave, like acoustic, X-ray, or electron beam, can also enlarge the image and receive it by direct or digital imaging or a combination of these methods. The microscope can give a dynamic picture (as with traditional optical instruments) or a static one (as with conventional scanning electron microscopes).

The Resolution (or) Magnification: 

1. The magnification of a microscope tests the amount of times the object tends to increase in size and the magnification ratio.

2. It is typically expressed in the 10-fold form (for a 10-fold magnified image), often misrepresented as “ten eks,” as though the algebraic symbol were an algebraic symbol, rather than the correct “ten times.” form.

3. A microscope’s resolution is a measure of the smallest object detail that can be observed. Resolution is expressed in linear units, which usually are micrometers.

Different types of microscopes: 

Optical Microscope or Light Microscope:

The optical, or light microscope, consists of glass lenses used to shape the image, which is the most familiar microscope type. Optical microscopes, consisting of a single lens or compound consisting of many in-line optical components, may be straightforward.

a. The Resolution: 

The hand magnifying glass will magnify approximately 3 to 20 points. Single-lensed simple microscopes, though compound microscopes can magnify up to 2,000, can magnify up to 300, and can expose bacteria. Under 1 micrometer (μm; one-millionth of a meter), a simple microscope can resolve; a compound microscope can resolve down to around 0.2 μm.

b. Image of Interest: 

1. By photography through a microscope, a process is known as photomicrography, photographs of interest may be captured.

2. This has been done with film since the 19th century, but digital photography is now used widely instead. Some optical microscopes have dispensed with an eyepiece and view images on the computer screen directly.

3. A modern series of low-cost digital microscopes with a wide variety of imaging possibilities, including time-lapse microscopy, has made previously challenging and costly tasks much easier for the beginner or amateur microscopist.

Other types of microscopes use the wave nature of different physical processes. The electron microscope uses an electron beam in its image creation, which is the most important. There are magnifying powers of more than 1,000,000 in the transmission electron microscope (TEM). In a near-vacuum, TEMs shape images of thin specimens, usually parts. A scanning electron microscope typically has a lower resolution than a TEM, which produces a mirrored image of relief in a contoured specimen, but can display concrete surfaces in a way that the traditional electron microscope cannot. Microscopes that use lasers, sound, or X-rays are also available. The Scanning Tunnelling Microscope (STM) can generate atoms, and the Environmental Scanning Electron Microscope (ESEM), which produces images of specimens using electrons in a gaseous atmosphere, use other physical effects to broaden further the types of objects that can be examined.

History of Microscopes:

1. The theory of magnification has been established for a long time. “About 1267 In Perspectiva, English philosopher Roger Bacon wrote, “Because of the greatness of the angle under which we can see them, we can number the smallest particles of dust and sand,” and in 1538 in Homocentrica, Italian physician Girolamo Fracastoro wrote, “If someone can look through two spectacle lenses, one superimposed on the other, the image seems something much larger”. 

2. Hans Jansen and his sons, Zacharias Jansen and Hans Lippershey, three Dutch spectacle makers, received credit for inventing the compound microscope around 1590.

3. The first illustration of a microscope was drawn in the Netherlands around 1631. It was a compound microscope with an oculus and an objective lens. In the mid-17th century, this kind of instrument, which came to be made of wood and cardboard, often decorated with polished fish skin, became increasingly popular and was used by the English natural philosopher Robert Hooke to provide the new Royal Society with frequent demonstrations.

4. These demonstrations began in 1663, and Hooke published a folio volume titled Micrographia two years later, which offered a wide variety of microscopic views of recognizable objects such as fleas, lice, and nettles. He coined the word cell in this book.

5. The description of how a single high-powered lens could be made into a serviceable microscope is concealed in the unnumbered pages of Micrographia’s preface. Using this template, the Dutch civil servant Antonie van Leeuwenhoek began his pioneering observations of freshwater microorganisms in the 1670s.

6. He made his postage-stamp-sized microscopes by hand, and details of about 0.7 μm could be resolved by the best of them. More than three centuries later, his fine specimens found at the Royal Society in excellent condition show what a great technician he was.

7. Leeuwenhoek’s simple microscopes launched microbiology in 1674, and single-lensed microscopes remained popular until the 1850s.

8. The Scottish botanist Robert Brown used them in 1827 to illustrate the ubiquity of the cell nucleus, a phrase he invented in 1831.

9. Using single lenses, simple microscopes can produce fine images; but they can also produce spurious colors in which various wavelengths of light do not come to the same focus due to chromatic aberration.

10. In the compound microscopes of the time, the aberrations were worse than magnifying the images.

11. They produced inferior images, although the compound microscopes were beautiful objects that bestowed status on their owners.

12. In 1733, by trial and error, the amateur English optician Chester Moor Hall discovered that a combination of a convex crown-glass lens and a concave flint-glass lens could help correct chromatic aberration in a telescope. 

13. In 1774, Benjamin Martin of London designed an important set of color-corrected lenses for a microscope.

14. In the 19th century, the advent of new optical glasses stimulated continued microscope growth, and substantial advances were made to understand image forming’s geometric optics.

15. In 1791, Dutch optician Francois Beeldsnijder eventually introduced the idea of an achromatic (non-color-distorting) microscope target, and in 1830 the English scientist Joseph Jackson Lister published a work outlining a theoretical approach to the complete design of microscope goals.

16. The German physicist Ernst Abbe studied the physics of lens design. He developed an apochromatic lens system in 1868, which had even better color correction than achromatic lenses, and he published a detailed lens theory study in 1873.

17. The successful limits of optical microscopy were reached by light microscopes developed in the closing quarter of the 19th century.

18. Subsequent methods, such as phase-contrast microscopes, confocal microscopes, and interference microscopes, solved particular problems while examining specimens such as living cells.