The Principles of Microscopy.pptx

The Principles of Microscopy

1. The Principles of Microscopy The Light Microscope

2. Microscope "Microscope" was first coined by members of the first " Academia dei Lincei " a scientific society which included Galileo “An instrument that produces an enlarged image of an object” Light microscope: uses a “light source” to illuminate the specimen Simple or Compound Upright or Inverted Phase contrast or Dark field or Interferance Köhler Illumination Fluorescence Illumination

3. Earliest Microscopes 1590 - Hans & Zacharias Janssen of Middleburg, Holland manufactured the first compound microscopes 1660 - Marcello Malpighi , considered the father embryology and early histology, was one of the first great microscopists - observed capillaries in 1660 1665 - Robert Hooke (1635-1703)- book Micrographia , published in 1665, devised the compound microscope most famous microscopical observation was his study of thin slices of cork. He wrote: “. . . I could exceedingly plainly perceive it to be all perforated and porous. . . these pores, or cells, . . . were indeed the first microscopical pores I ever saw, and perhaps, that were ever seen, for I had not met with any Writer or Person, that had made any mention of them before this.”

4. 1673 - Antioni van Leeuwenhoek (1632-1723) Delft, Holland, worked as a draper (a fabric merchant); he is also known to have worked as a surveyor, a wine assayer, and as a minor city official Created a “simple” microscope that could magnify to about 275x, and published drawings of microorganisms in 1683 His simple microscope could reach magnifications of over 200x with simple ground lenses However compound microscopes were mostly of poor quality and could only magnify up to 20-30 times Discovered bacteria, free-living and parasitic microscopic protists , sperm cells, blood cells, microscopic nematodes In 1673, Leeuwenhoek began writing letters to the Royal Society of London - published in Philosophical Transactions of the Royal Society In 1680 he was elected a full member of the Royal Society, joining Robert Hooke, Henry Oldenburg, Robert Boyle, Christopher Wren

5. Secondary Microscope 1740-1772 : George Adams Sr . made many microscopes but he was predominantly just a good manufacturer not inventor ~ 1730 : a barrister names Chester More Hall observed that flint glass (newly made glass) dispersed colors much more than “crown glass” (older glass) He designed a system that used a concave lens next to a convex lens which could realign all the colors This was the first achromatic lens . George Bass was the lens-maker who actually made the lenses, but he did not divulge the secret until over 20 years later to John Dolland who copied the idea in 1759 and patented the achromatic lens.

6. 1827 : Giovanni Battista Amici , built high quality microscopes and introduced the first matched achromatic microscope in 1827 He had previously (1813) designed “reflecting microscopes” using curved mirrors rather than lenses He recognized the importance of cover-slip thickness and developed the concept of “ water immersion ” © J.Paul Robinson

7. Limitations faced by cell biologists Exceedingly small dimensions of the cells and cell organelles Transparent nature of cell and its components Can be overcome by Increasing the resolving power of microscope Increasing the contrast of cellular structures to counteract the transparency of the cell

8. Abbe’s Law Ernst Abbe together with Carl Zeiss published a paper in 1877 defining the physical laws that determined resolving distance of an objective which is now known as Abbe’s Law “minimum resolving distance (d) is related to the wavelength of light ( λ ) divided by the Numeric Aperture, which is proportional to the angle of the light cone ( θ ) formed by a point on the object, to the objective ” Abbe and Zeiss developed oil immersion systems by making oils that matched the refractive index of glass They were able to make the a Numeric Aperture (N.A.) to the maximum of 1.4 allowing light microscopes to resolve two points distanced only 0.2 microns apart (the theoretical maximum resolution of visible light microscopes ).

9. Resolution Limit of resolution: The minimum distance between two points which allows their discrimination as two separate points: where: 0.61 is a geometrical term, based on the average 20-20 eye, λ = wavelength of illumination, N.A. = Numerical Aperture: a measure of the light gathering capabilities of an objective lens. N.A. = n sin α Where: n = index of refraction of medium, α = semi-angle of aperture subtended by the lens Sin α cannot exceed 1 RI for most optical material does not exceed 1.6 Maximal NA of lenses (using oil immersion) is about 1.4

10. Factors Affecting Resolution Resolution improves if the wavelength (λ) of illumination is shorter R.I (n) is larger α is larger The eye is more sensitive to blue than violet

11. Magnification Ability of a microscope to produce an enlarged image An object can be focussed generally no closer than 250 mm from the eye (depending upon how old you are!) This is considered to be the normal viewing distance for 1 x magnification Young people may be able to focus as close as 125 mm so they can magnify as much as 2 x because the image covers a larger part of the retina - that is it is “magnified” at the place where the image is formed

12. Magnification The overall magnification is given as the product of the magnification of lenses and the distance over which the image is projected: where: D = projection (tube) length (usually = 250 mm); M1, M2 = magnification of objective and ocular. 250 mm = minimum distance of distinct vision for 20/20 eyes

13. Depth of Focus DOF or the vertical resolution : This is the ability to produce a sharp image from a non-flat surface Depth of Focus is increased by inserting the objective aperture (an iris that cuts down on light entering the objective lens) However, this decreases resolution

14. Noteworthy… All microscopes are similar in the way lenses work and they all suffer from the same limitations and problems. Magnification is a function of the number of lenses and their individual magnifications. Resolution is a function of the ability of a lens to gather light. Apertures can be used to affect resolution and depth of field if you know how they affect the light that enters the lens. Simple microscopes could attain around 2 micron resolution, while the best compound microscopes were limited to around 5 microns because of chromatic aberration.

15. Inverted Microscope

16. Specialized LM Techniques Bright & Dark field Microscopy Phase Contrast Microscopy & Differential Interference Contrast Microscopy: Convert phase differences to amplitude differences; Enhancement of Contrast Fluorescence Microscopy : mainly organic materials Confocal Scanning Optical Microscopy (new) Three-Dimensional Optical Microscopy : Inspects and measures sub-micrometer features in semiconductors and other materials Hot- and Cold-stage Microscopy : Examines melting, freezing points and eutectics, polymorphs, twin and domain dynamics, phase diagram In situ microscopy E-field, stress, etc. Special environmental stages-vacuum or gases

17. Bright Field Microscopy The condenser is used to focus light on the specimen through an opening in the stage After passing through the specimen, the light is displayed to the eye with an apparent field that is much larger than the area illuminated Typically used on thinly sectioned materials Drawback: Bright field illumination does not reveal differences in brightness between structural details - i.e. no contrast Structural details emerge via phase differences and by staining of components The edge effects (diffraction, refraction, reflection) produce contrast and detail

18. Contrast Contrast is defined as the difference in light intensity between the specimen and the adjacent background relative to the overall background intensity Image contrast, C is defined by:

19. Dark Field Microscopy or Ultramicroscopy Light is scattered at the boundaries between regions of different refractive indices An opaque disc is placed underneath the condenser lens, so that only light that is scattered by objects on the slide can reach the eye Ordinary condenser is replaced by one that illuminates object obliquely (Dark-field condenser) No direct light enters the objective Instead of coming up through the specimen, the light is reflected by particles on the slide Object appears bright because of the scattered light Everything is visible regardless of color, usually bright white against a dark background Smaller objects can be detected but resolution is poor

20. OM images of the green alga Micrasterias

21. Phase Contrast Microscopy Although biological structures are highly transparent to visible light, they cause phase changes or retardations in the transmitted radiations As the light wave impinges on a NON-ABSORBENT, TRANSPARENT, REFRACTIVE material that has RI different from that of the medium, amplitude is not affected but velocity changes If the RI of the material is higher than the medium, velocity is RETARDED As the wave emerges from the medium, velocity is restored but retardation is maintained If material is ABSORBENT, amplitude is reduced

22. Principle In PC Microscope, small differences are intensified Lateral light passing through the objective is advanced or retarded by 1/4 th wavelength w.r.t . the central light Annular phase plate introduces a variation of ¼ wavelength in the back focal plane of the objective The phase effect results from the interference between the direct geometric image produced by the central part of the objective and the lateral image that has been advanced or retarded by ½ wavelength Bright image: Negative contrast : the 2 sets of rays are added; object appears brighter than the surrounding Dark image: Positive contrast : the 2 sets of rays are subtracted making the image darker than the surrounding

23. Uses… Minute phase changes in the object are amplified and translated into amplitude changes But reveals only sharp differences Transparent object appears in various shades of grey PCM is used routinely to observe living cells and tissues Particularly useful for in vitro studies of cell cycle progression, etc.

24. Interference Microscopy (DIC) Similar principles as the PCM but gives quantitative data Used to detect small continuous changes in RI The variations of phase can be can be transformed into vivid color changes Nomarski Interference Microscope: a special variation in which the image obtained gives a “relief effect” Particularly useful for study of mitosis in live cells in vitro

25. Contrast and Illumination Brightness-contrast arises from different degrees of absorption at different points in the specimen Color-contrast can also arise from absorption when the degree of absorption depends on the wavelength and varies from point to point in the specimen Phase contrast arises from a shift in the phase of the light as a result of interaction with the specimen Polarization-dependent phase contrast arises when the phase shift depends on the plane of polarization of the incident light. Fluorescence contrast arises when the incident light is absorbed and partially reemitted at a different wavelength.

26. Modern Microscopes Early 20th Century Professor Köhler developed the method of illumination still called “ Köhler Illumination” Köhler recognized that using shorter wavelength light (UV) could improve resolution Köhler illumination creates an evenly illuminated field of view while illuminating the specimen with a very wide cone of light Two conjugate image planes are formed – one contains an image of the specimen and the other the filament from the light

27. Electron microscope An electron microscope is a type of microscope that uses a particle beam of electrons to illuminate the specimen and produce a magnified image. Electron microscopes (EM) have a greater resolving power than a light-powered optical microscope Like a beam of light, a stream of electrons also have corpuscular and vibratory character Wavelength of electrons is about 100,000 times shorter than visible light (photons), λ e = . 005 nm; λ l = 550 nm EM can achieve better than 50 pm resolution and magnifications of up to about 10,000,000x Ordinary non- confocal light microscopes are limited by diffraction to about 200  nm resolution and useful magnifications below 2000 x

28. The electron microscope uses electrostatic  and electromagnetic "lenses" to control/deflect the electron beam and focus it to form an image These lenses are analogous to, but different from the glass lenses of an optical microscope that form a magnified image by focusing light on or through the specimen

29. Cathode or Thermionic Gun: Electron source – A metal filament placed in vacuum and heated emits electrons Condenser: a magnetic coil that focuses the electrons on the plane of the object Objective: 2 nd magnetic coil that deflects electron beam from the object giving a magnified image Projection lens or Occular : 3 rd magnetic lens that receives and magnifies the image from the objective Final magnified image formed on a fluorescent screen

30. Image in EM is principally formed due to electron scattering Electrons colliding with the atomic nuclei in the object are dispersed and may fall outside the aperture Elastic dispersion : image on the fluorescent screen results from absence of electrons blocked by the aperture Inelastic dispersion : results from multiple collision Electron dispersion is a function of Thickness of the objective Molecular packing of the objective Depends on the atomic number of the atoms in the object

31. Electron microscopes are used to observe a wide range of biological and inorganic specimens including microorganisms, cells, large molecules, biopsy samples, metals and crystals Industrially, the electron microscope is primarily used for quality control and failure analysis in semiconductor device fabrication

32. Transmission electron microscope (TEM) The original form of electron microscope, the  Transmission electron microscope (TEM) uses a high voltage electron beam to create an image The electrons are emitted by an electron gun commonly fitted with a Tungsten filament cathode as the electron source Electron beam is accelerated by an anode typically at +100 keV  ( 40 to 400 keV ) with respect to the cathode Focused by electrostatic and electro-magnetic lenses Transmitted partially through the specimen (that is in part transparent to electrons) and partially scattered out of the beam by the opaque part of the specimen

33. Emerging electron beam carries information about the structure of the specimen that is magnified by the objective lens system of the microscope Magnified “electron image” (caused by spatial variation) is viewed by projection onto a fluorescent viewing screen coated with a phosphor or  scintillator  material such as zinc sulfide The image can be photographically recorded by exposing a photographic film or plate directly to the electron beam High-resolution phosphor may be coupled by means of a lens optical system or a  fibre optic light-guide to the sensor of a CCD (charge-coupled device) camera The image detected by the CCD may be displayed on a monitor or computer.

34. Resolution of the TEM is limited primarily by  spherical aberration Advanced aberration correctors have been able to partially overcome spherical aberration to increase resolution Hardware correction of spherical aberration for the high-resolution transmission electron microscopy (HRTEM) has allowed the production of images with resolution below 0.5 Angstrom ( 50  pm) at magnifications above 50 million times The ability to determine the positions of atoms within materials has made the HRTEM an important tool for nano -technologies research and development

35. Scanning electron microscope Electron beam of the SEM does not at any time carry a complete image of the specimen The SEM produces images by probing the specimen with a focused electron beam that is scanned across a rectangular area of the specimen ( Raster Scanning) At each point on the specimen the incident electron beam loses some energy, and that lost energy is converted into other forms Heat Emission of low energy secondary electrons Light emission ( cathodoluminescence ) or X-ray emission.

36. The display of the SEM maps the varying intensity of any of these signals into the image in a position corresponding to the position of the beam on the specimen when the signal was generated Generally, the image resolution of an SEM is ~ 1 order of magnitude poorer than that of a TEM SEM image relies on surface processes rather than transmission It is able to image bulk samples up to many cm in size Depending on instrument design and settings has a great depth of field Can produce images that are good representations of the three-dimensional shape of the sample. Environmental scanning electron microscope (ESEM) can produce images of sufficient quality and resolution with the samples being wet or contained in low vacuum or gas This greatly facilitates imaging biological samples which are unstable in the high vacuum of conventional electron microscopes.