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Light Microscopy   Richard Cole Research Scientist IV Director: Advanced Light Microscopy & Image Analysis Core P.O. Box 509 Albany N.Y. 12201-0509 518-474-7048.

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Presentation on theme: "Light Microscopy   Richard Cole Research Scientist IV Director: Advanced Light Microscopy & Image Analysis Core P.O. Box 509 Albany N.Y. 12201-0509 518-474-7048."— Presentation transcript:

1 Light Microscopy Richard Cole Research Scientist IV Director: Advanced Light Microscopy & Image Analysis Core P.O. Box 509 Albany N.Y Website

2 Light Our vision is limited to a very small range of total light waves. Generally, we see between the violet (380nm) upwards to red (750nm). However, there are wavelengths on either side of this narrow range that can also be used for imaging. For example, ultraviolet (UV) is used to excite specific fluorescent dyes and X-rays are used not only in the microscopic world of crystallography, but also in the more common place diagnostic tool of the same name. The shortest distance is not a straight line. At least when talking about light, it does not move in a straight line. Instead it follows a wave motion.

3 Simple Lenses Apertures Diffraction:
All lenses refract light (photons). For optical microscopes there are two basic lens types, convex and concave lenses. Each shape has the ability to either reduce or enlarge an image. Inherent in all lenses are aberrations. Spherical aberrations are most common in poorly made lenses. If a lens is not ground with great care, images will not be brought to an even focus across the entire image plane. Chromatic aberrations caused by different light wavelengths being refracted at different angles. In essence, the color spectrum becomes defocused (separated) where the image is projected. This would leave color halos at the edges of specimens. Apertures One way to reduce the problems of aberrations is to limit the area of the lens being used. By using an aperture, you can select only the image being refracted in the middle of the lens. However, there are two problems with apertures. Reduces the amount of light Light diffraction limits the how small an aperture can be. Diffraction: light wave theory and airy discs Light has wave-like properties. This is evident when you place an aperture in the path of a light source. Instead of a single pinpoint of light, there is a bright central disk surrounded by a diffuse ring of light. This image is called an airy disc. Light diffraction is the reason that an exact image of a specimen is never achieved.

4 Limit of resolution. d = 0.612 x l / n sin a
d = minimal separation of two objects being resolved l = (wavelength of light) n = refractive index of medium between the objective lens and the specimen. a = the aperture angle, which is half the angle of the cone of light from the specimen accepted by the front lens objective. This is also called the half-aperture angle. Today objectives already have n sin a calculated and is know as numerical aperture (N.A.). d = x l /N.A. Under this equation, the smaller the wavelength of light the greater the resolution. In addition the larger the numerical aperture the greater the resolution. What does this mean for microscopy? In simple terms, resolution is the minimum distance between objects that can be visually detected.

5 Microscopes Hooke microscope
Introduction Microscopes are instruments designed to produce magnified visual or photographic images of small objects. The microscope must accomplish three tasks: produce a magnified image of the specimen, separate the details in the image, and render the details visible to the human eye or camera. Hooke microscope The microscope illustrated is a simple compound microscope invented by British microscopist Robert Hooke sometime in the 1660s. This early microscope suffered from chromatic (and spherical) aberration, and all images viewed in white light contained "halos" that were either blue or red in color.

6 Human eye For an image to be seen clearly, it must spread on the retina at a sufficient visual angle. Unless the light falls on non-adjacent rows of retinal cells we are unable to distinguish closely-lying details as being separate (resolved). Further, there must be sufficient contrast between adjacent details and/or the background to render the magnified, resolved image visible. This same criteria apply to CCD camera!

7 Köhler Illumination Most important variable in achieving high-quality images in microscopy. Köhler illumination was first introduced in 1893 by August Köhler of the Carl Zeiss corporation as a method of providing the optimum specimen illumination.

8 Conjugate planes Conjugate focal planes are often useful in troubleshooting a microscope for contaminating dust, fibers, and imperfections in the optical elements. If these artifacts are in sharp focus, it follows that they must reside on or near a surface that is part of the imaging-forming set of conjugate planes. Members of this set include the glass element at the microscope light port, the specimen, the reticle in the eyepiece, and the bottom lens element of the eyepiece. Alternatively, if these contaminants are fuzzy and out of focus, look for them near the illuminating set of elements that share conjugate planes. Suspects in this category are the condenser top lens (where dust and dirt often accumulate), the exposed eyepiece lens element (contaminants from eyelashes), and the objective front lens (usually fingerprint smudges).

9 Microscope Objectives
There is a wealth of information inscribed on the objective barrel, objective lens has inscribed on it: magnification (e.g. 10x, 20x or 40x etc.) was designed to give its finest images (usually 160 millimeters or the Greek infinity symbol) thickness of cover glass, correcting for spherical aberration (usually 0.17 millimeters). If the objective is designed to operate with a drop of oil between it and the specimen, the objective will be engraved OIL or OEL or HI (homogeneous immersion). In cases where these latter designations are not engraved on the objective, the objective is meant to be used dry numerical aperture (NA) value. This may vary from 0.04 for low power objectives to 1.3 or 1.4 for high power oil-immersion apochromatic objectives.


11 Many high-performance apochromat dry objectives are fitted with correction collars, which allow adjustment to correct for spherical aberration by correcting for variations in cover glass thickness Optical correction for spherical aberration is produced by rotating the collar, which causes two of the lens element groups in the objective to move either closer together or farther apart. A majority of the correction collar objectives have an adjustment range for cover glass thickness variations between 0.10 and 0.23 millimeters. Phase contrast objectives designed for observing tissue culture specimens have an even broader compensation range of 0 to 2 millimeters.

12 The standard thickness for cover glasses is 0
The standard thickness for cover glasses is 0.17 millimeters, which is designated as a number 1½ cover glass. Unfortunately, not all 1½ cover glasses are manufactured to this close tolerance (they range from 0.16 to 0.19 millimeters) and many specimens have media between them and the cover glass. Compensation for cover glass thickness can be accomplished by adjusting the mechanical tube length of the microscope, or (as previously discussed) by the utilization of specialized correction collars that change the spacing between critical elements inside the objective barrel. The correction collar is utilized to adjust for these subtle differences to ensure the optimum objective performance.

13 Oil immersion objectives
Objective numerical aperture can be dramatically increased by designing the objective to be used with an immersion medium, such as oil, glycerin, or water. By using an immersion medium with a refractive index similar to that of the glass coverslip, image degradation due to thickness variations of the cover glass are practically eliminated whereby rays of wide obliquity no longer undergo refraction and are more readily grasped by the objective. Typical immersion oils have a refractive index of 1.51 and dispersion similar to that of glass coverslips. Oil immersion objectives Objectives that use water and/or glycerin as an imaging medium are also available for applications with living cells in culture or sections of tissue immersed in physiological saline solution. A primary problem with common immersion oils is their inherently high absorption characteristics in the ultraviolet region of the spectrum (below about 375 nanometers). This does not affect a majority of the visible light optical microscopy done with immersion media, but it can lead to trouble when attempting to image specimens at wavelengths below 400 nanometers. The immersion media listed in Table have a wide spectrum of refractive indices Most of the media with refractive indices equal to or lower than that of glass are used in common biological brightfield, phase contrast, DIC, and fluorescence microscopy. High refractive indices, such as bromonaphthalene and methylene iodide, are used with specialized objectives to achieve the highest numerical aperture and resolution possible and are also very useful in reflected light microscopy.

14 Common Immersion Media
Material Refractive Index Air 1.0003 Water 1.333 Glycerin 1.4695 Paraffin oil 1.480 Cedarwood oil 1.515 Synthetic oil Anisole 1.5178 Bromonaphthalene 1.6585 Methylene iodide 1.740 Modern advances in tissue and cell culture techniques have sparked an interest in being able to image living cells and tissues that are bathed in physiological saline solution. The average refractive index of living cells is 1.35, very close to that of the surrounding buffered aqueous-saline solution (n = 1.33) long working distance dry can create reflections from the surface of the liquid that will obscure details of the specimen. Likewise, the use of oil immersion objectives to observe living cells is also problematic due to refractive index differences between the aqueous imaging medium and the glass of the objective's front lens. Dipping lens, where the lens in submerged into the medium solves some of these issue for live cell imaging, at least on a upright scope!.

15 Depth of field Magnification Numerical Aperture Depth of Field (mm) 4x 0.10 15.5 10x 0.25 8.5 20x 0.40 5.8 40x 0.65 1.0 60x 0.85 100x 0.95 0.19 The field diameter in an optical microscope is expressed by the field-of-view number or simply field number, which is the diameter of the viewfield expressed in millimeters and measured at the intermediate image plane. The clearance distance between the closest surface of the cover glass and the objective front lens is termed the working distance.

16 Image Brightness brightness of the microscope source illumination is determined by the square of the condenser working numerical aperture, brightness of the specimen image is proportional to the square of the objective numerical aperture. Unlike the situation for the microscope illuminating system, however, objective magnification also plays an important role in determining image brightness. In fact, the image brightness is inversely proportional to the square of the lateral magnification: Image Brightness µ (NA/M)2 where NA is the objective numerical aperture and M is the magnification. The ratio given in the equation above expresses the light-gathering power of the objective in transillumination cases where an objective is used for transillumination, image brightness decreases rapidly as the magnification increases in a series of objectives having identical correction. The result is that brightness of the specimen image is directly proportional to the square of the objective numerical aperture as it reaches the eyepiece (or camera system), and also inversely proportional to the objective magnification.

17 Contrast in Optical Microscopy
When imaging specimens in the optical microscope, differences in intensity and/or color create image contrast, which allows individual features and details of the specimen to become visible. Contrast is defined as the difference in light intensity between the image and the adjacent background relative to the overall background intensity. Transmitted light contrast modes

18 Differential Interference Contrast (DIC)
In the mid-1950s, a French optics theoretician named Georges Nomarski modified the Wollaston prism used for detecting optical gradients in specimens and converting them into intensity differences. Living or stained specimens, which often yield poor images when viewed in brightfield illumination, are made clearly visible by optical rather than chemical means. Light passed through a polarizer located beneath the substage condenser, next in the light path (but still beneath the condenser) is a modified Wollaston prism that splits the entering beam of polarized light into two beams traveling in slightly different directions, emerging light rays vibrate at 90 degrees relative to each other with a slight path difference These two rays travel close together but in slightly different directions (see Figure 2). The rays intersect at the front focal plane of the condenser, where they pass traveling parallel and extremely close together with a slight path difference, but they are vibrating perpendicular to each other and are therefore unable to cause interference. The distance between the rays, called the shear, is so minute that it is less than the resolving ability of the objective.

19 The split beams enter and pass through the specimen where their wave paths are altered in accordance with the specimen's varying thickness, slopes, and refractive indices. These variations cause alterations in the wave path of both beams passing through areas of any specimen details lying close together. When the parallel beams enter the objective, they are focused above the rear focal plane where they enter a second modified Wollaston prism that combines the two beams at a defined distance outside of the prism itself. This removes the shear and the original path difference between the beam pairs. As a result of having traversed the specimen, the paths of the parallel beams are not of the same length (optical path difference) for differing areas of the specimen. In order for the beams to interfere, the vibrations of the beams of different path length must be brought into the same plane and axis. This is accomplished by placing a second polarizer (analyzer) above the upper Wollaston beam-combining prism. The light then proceeds toward the eyepiece where it can be observed as differences in intensity and color. The design results in one side of a detail appearing bright (or possibly in color) while the other side appears darker (or another color). This shadow effect bestows a pseudo three-dimensional appearance to the specimen. The color and/or light intensity effects shown in the image are related especially to the rate of change in refractive index, specimen thickness, or both. Orientation of the specimen can have pronounced effect on the relief-like appearance and often, rotation of the specimen by 180 degrees changes a hill into a valley or visa versa. The three-dimensional appearance is not a representation of the true geometric nature of the specimen, but is an exaggeration based on optical thickness. It is not suitable for accurate measurement of actual heights and depths.

20 Advantages in DIC microscopy as compared to phase microscopy:
Fuller use of the numerical aperture, unlike phase contrast microscopy, there is no substage annulus Images can be seen in striking color (optical staining) and in 3-dimensional shadowed-like appearance. The visibility of outlines and details is greatly improved, and the photomicrography of these images is striking in color and detail. Regular planachromats or achromats (also suitable for ordinary brightfield work) can be used if the manufacturer states that such objectives are designed for their apparatus. Disadvantages or limitations in DIC: Expensive, because of the many prisms that are required. Birefringent specimens, many kinds of crystals not suitable Specimen carriers, culture vessels, Petri dishes, made of plastic not suitable. Apochromatic objectives may not be suitable, such objectives themselves may significantly affect polarized light.

21 Phase contrast Unstained specimens that do not absorb light are called phase objects because they slightly alter the phase of the light diffracted by the specimen, usually by retarding such light approximately 1/4 wavelength as compared to the undeviated direct light passing through or around the specimen unaffected. Phase Contrast microscopy-- making unstained, phase objects yield contrast images as if they were amplitude objects. Zernike's method was to speed up the direct light by 1/4 wavelength so that the difference in wavelength between the direct and deviated light for a phase specimen would now be 1/2 wavelength. As a result, the direct and diffracted light arriving at the image level of the eyepiece would be able to produce destructive interference. Such a procedure results in the details of the image appearing darker against a lighter background. Limitations of phase contrast microscopy: Phase images are surrounded by halos. Such halos are optical artifacts, which sometimes obscure the boundaries of details. The phase annuli do limit the working numerical aperture of the optical system to a certain degree, thus reducing resolution. Phase contrast does not work well with thick specimens because of shifts in phase occur from areas slightly below or slightly above the plane that is in focus. Such phase shifts confuse the image and distort image detail. Phase images appear gray if white light is used and green if a green filter is used.

22 Polarized Light Microscopy
Although much neglected and undervalued as an investigative tool, polarized light microscopy (Figure 1) provides all the benefits of brightfield microscopy and yet offers a wealth of information, which is simply not available with any other optical microscopy technique. The technique exploits optical properties of anisotropy to reveal detailed information about the structure and composition of materials, which are invaluable for identification and diagnostic purposes. Isotropic materials, which include gases, liquids, unstressed glasses and cubic crystals, demonstrate the same optical properties in all directions. Anisotropic materials, in contrast, which include 90 percent of all solid substances, have optical properties that vary with the orientation of incident light with the crystallographic axes. They demonstrate a range of refractive indices depending both on the propagation direction of light through the substance and on the vibrational plane coordinates. Polarized light microscopy is perhaps best known for its geological applications--primarily for the study of minerals in rock thin sections, but it can also be used to study many other materials. Polarizing microscopy can be used both with reflected and transmitted light. Reflected light is useful for the study of opaque materials such as mineral oxides and sulphides, metals and silicon wafers (Figure 3). Reflected light techniques require a dedicated set of objectives that have not been corrected for viewing through the coverslip, and those for polarizing work should, again, be stress free.

23 (a) surface features of a microprocessor integrated circuit
(a) surface features of a microprocessor integrated circuit. Birefringent elements employed in the fabrication of the circuit are clearly (b), which shows birefringent crystalline areas with interference colors interspersed in a matrix of isotropic binder. Metallic thin films are also visible with reflected polarized light. (c) illustrates blisters that form imperfections in an otherwise confluent thin film of copper (about 0.1 micron thick) sandwiched over a nickel/sodium chloride substrate to form a metallic superlattice assembly. Polarized light image Garnet NaHPO4


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