Modern Inverted Microscope Design

Modern Inverted Microscope Design

In critical illumination, the image of the light source falls in the same plane as the image of the specimen, i.e. the bulb filament is visible in the final image. In Kohler illumination, the collector lens collects light from the bulb and focus it at the plane of the condenser diaphragm. The condenser projects this light, without focusing it, through the sample. This illumination scheme creates two sets of conjugate image planes, one with the light source (aperture) and one with the specimen. Aperture planes: Lamp, condenser diaphragm, objective back focal plane, pupil Image planes: Field diaphragm, Specimen, the eyepiece diaphragm, retina or camera

Conjugate Planes of a Microscope

Infinity Optics Microscope systems with finite tube length were produced in the past. When objectives and tube lengths are mismatched, image quality often suffers due to the introduction of spherical aberrations because the optical tube length is changed When optical element is inserted in the light path between the back of the objective and the eyepiece, the mechanical tube length changes infinity-corrected objectives generate emerging parallel rays and provide flexibility to the user to modify the path.

Diversity of Cells

E. Coli (model prokaryotic cell) What is Inside Cells?

Fibroblast (model higher eukaryotic cell) What is Inside Cells?

What is Inside Organelles? Mitochondria (power plant of a cell, model organelle) Play Power Plant of A Cell Movie

Biological Scale http://learn.genetics.utah.edu/content/begin/cells/scale/

Bright Field Microscopy Some specimens are considered amplitude objects because they absorb light partially or completely, and can thus be readily observed using conventional brightfield microscopy. Limitations of standard optical microscopy: (bright field microscopy) lie in three areas; The technique can only image dark or strongly refracting objects effectively. Diffraction limits resolution to approximately 0.2 micron. Out of focus light from points outside the focal plane reduces image clarity. Live cells in particular generally lack sufficient contrast to be studied successfully, internal structures of the cell are colorless and transparent. Solution: Kill the cell, stain it with dark material to generate contrast

What do you see? Overall cell shape, membrane, nucleus and large organelles. Most of the internal structures of the cell are invisible. Most biological specimens do not absorb much light to produce contrast, but they diffract the light and cause a phase shift of the transmitted wave.

Phase Contrast Microscopy Other specimens do not absorb light and are referred to as phase objects. Because the human eye can only detect intensity and color differences, the phase changes due to objects must be converted to intensity differences. The phase contrast microscope is designed to take advantage of phase differences between objects in a specimen and in the surrounding medium. However, it is not simply a phase difference that is necessary, but also diffraction from the specimen must occur for the phase contrast microscope to work. One of the major advantages of phase contrast microscopy is that living cells can be examined in their natural state without previously being killed, fixed, and stained. As a result, the dynamics of ongoing biological processes can be observed and recorded in high contrast with sharp clarity of minute specimen detail.

Phase Specimens Phase is generated by the refractive index and thickness Optical Path Difference (OPD) = Δ = (t n s - t n m ) = t (n s - n m ) phase shift (δ) δ = 2πΔ/λ If Δ is λ/2, two waves become out of phase

Interaction of Light Through Phase Specimens An incident light is divided into two components upon passing through a phase specimen. The primary component is undiffracted surround (S) wave, which passes through and around the specimen, but does not interact with it. A diffracted D-wave is also produced, which is scattered in many directions. Typically, only a minority of incident waves are diffracted by cellular objects. Both S and D waves are collected by the objective lens and focused in the image plane at, where they undergo interference and generate a resultant particle wave (P wave). P = S + D Only when the amplitudes of the P and S waves are significantly different in the image plane can we see the object in the microscope.

Phase Differences in Biological Specimens For individual cells in tissue culture, the optical path difference is relatively small. A typical cell in monolayer culture has a thickness around 5 micrometers and a refractive index of approximately 1.36. The cell is surrounded by a nutrient medium having a refractive index of 1.335 A typical optical path difference is 0.125 micrometer, or about a quarter wavelength (of green light). D is retarded by ~λ/4 (first order diffraction), amplitude is low Amplitudes of P and S are similar P is shifted by only ~λ/20 Since the amplitudes of P and S are the same, the phase variations do not create a contrast and these objects remain invisible in the bright field microscopy

Optical Design of the Phase Contrast Microscope The key elements of the optical design are: 1. separation of the direct zeroth order light (S wave) from the first order diffracted light (D wave) at the rear focal plane of the objective. (Condenser annulus) 2. Introduce a phase shift so that the S-wave gives rise to completely destructive or constructive interference. (Phase Plate) 3. Decrease the intensity of the S wave to make it comparable to D wave (ND filter) 4. Combine the direct and diffracted light so that the resulting interference generates contrast

Under conditions of Koehler illumination, the objective s back focal plane is conjugate to the condenser s front aperture Plane. S waves (nondiffracted, 0th-order) that do not interact with the specimen are focused as a bright ring in the back focal plane of the objective (the diffraction plane). In some phase contrast objectives, the phase plate is a plate of glass with an etched ring of reduced thickness to selectively advance the phase of the S wave by λ/4. The same ring is coated with a partially absorbing metal film to reduce the amplitude of the light by 70 75%.

λ/4 phase shift to S wave is introduced positively (S and D are out of phase, λ/2) or negatively (S and D are in phase). Only S wave is observed for nondiffracted regions (water) Positive: Objects with high n d look darker from the environment Negative: Environment looks darker than the high n d object

Comparison of Phase Contrast with Brightfield Bright Field Phase Contrast

Interpreting the Phase Contrast Image What can we see? Objects with high density (Cytoplasm, nucleus and nucleolus) can be seen as progressively darker objects Objects with low density (some vesicles, vacuoles and lipid droplets) look brighter than cytoplasm Live cells! Limitations: Relies on index of refraction Sample thickness changes the phase Small object with high n d have same intensity with large object with low n d Objects with large phase retardations (phase shift of the D wave ~λ/2), interference becomes constructive, making the objects appear brighter than the background. In order to create a sharp edge in the image, all of the spatial frequencies diffracted by the specimen must be represented in the final image.

Image Artifacts in Phase Contrast Halos: Phase ring transmits some diffracted light. A problem accentuated by the fact that the width of the annulus generated by the 0th-order surround waves is smaller (~25%) than the actual width of the annulus of the phase plate. Low spatial frequency diffracted light remain 90 degree out of phase (e.g. no destructive interference). As a result, these waves cause a localized contrast reversal that is, a halo around the object. Halos are especially prominent around large, low-spatial-frequency objects such as nuclei and cells. Dark objects have a bright surrounding ring, vice versa.

Shade Off frequently observed on large, extended objects such as extended or flattened cells, flattened nuclei, and planar slabs of materials, for example, mica or glass. Center and the edges of an object diffract light differently. In central regions, the amount of diffraction and the angle of scattering are greatly reduced. Object rays, although retarded in phase, deviate only slightly from the 0th-order component, and fall within the annulus of the phase plate. As a result, the amplitude and intensity of the central region are essentially the same as the background.

Dark Field Microscopy For unstained transparent specimens, the component of nondiffracted background light is very large, resulting in bright, low-contrast images. In dark-field microscopy, the nondiffracted rays are removed altogether so that the image is composed solely of diffracted wave components. This technique is very sensitive because images based on small amounts of diffracted light from minute phase objects are seen clearly against a black or very dark background.

Image Interpretation Dark-field microscopy is most commonly used for imaging small light-diffracting specimens such as diatoms, bacteria and bacterial flagella, isolated organelles and polymers such as cilia, flagella, microtubules, and actin filaments, and silver grains and gold particles in labeled cells and tissues. Edges diffract the greatest amount of the light, dominate the image Provides high contrast Background is black Lysosomes, flagella, fibrins and even microtubules (24 nm width) are visible Requires high laser power, potential damage of the sample www.arizonahomeopathic.org

Polarization Microscopy The bulk light from most illuminators is nonpolarized, the E vectors of different rays vibrating at all possible. In a ray or beam of linearly polarized light, the E vectors of all waves vibrate in the same plane. Absorptive Polarizer Beam Splitting Polarizer Metallic wire grid Distance is less than the wavelength Parallel beam oscillates the electrons, and reflected back Perpendicular beam travels through the grid

Brewster Angle Electric dipoles in the media respond to p-polarized light. Light incident on the surface is absorbed, and then reradiated by oscillating electric dipoles at the interface between the two media. The polarization of freely propagating transmitted light is always perpendicular to the direction in which the light is travelling. These oscillating dipoles also generate the reflected light. However, dipoles do not radiate any energy in the direction of the dipole moment. Consequently, if the direction of the refracted light is perpendicular to the direction in reflected light, the dipoles cannot create any reflected light. Solving for θ B gives: For a glass medium (n 2 1.5) in air (n 1 1), Brewster's angle for visible light is approximately 56, while for an air-water interface (n 2 1.33), it is 53.

Polarizing Sunglasses Polarizing sunglasses mount sheets of polarizing material with the transmission axis of the Polaroids oriented perpendicularly in the field of view. Bright reflections off horizontal surfaces, such as the roofs of cars or water on a lake, are efficiently blocked, while the random light is partially blocked.

Polarizing Crystals The most efficient polarizers are made of transparent crystals such as calcite The term anisotropy refers to a non-uniform spatial distribution of properties, which results in different values being obtained when specimens are probed from several directions within the same material. Anisotropic crystals (i.e. quartz or calcite) exhibit double refraction (or birefringence) Light polarized parallel to the crystal orientation has a different index of refraction (that is to say it travels at a different velocity) than perpendicular polarized light.

Double Refraction: Birefringence Ordinary ray (O ray) observes the law of refraction Extraordinary ray (E ray ) follows a different path. Electric fields of O and E rays are perpendicular. Birefringence (B) = n e - n o Birefringence can be either positive or negative depending on the crystal orientation, and is not a fixed value.

Unique angles in birefringence a) When light enters the unique optical axis of anisotropic crystals, it behaves in a manner similar to the interaction with isotropic crystals, and passes through at a single velocity. b) Incident beams that are perpendicular to the optic axis are split into O and E rays, but the trajectories of these rays are coincident. The O and E rays emerge at the same location on the crystal surface, but have different optical path lengths and are therefore shifted in phase. This geometry pertains to most biological specimens that are examined in a polarizing microscope. c) Incident rays that follow trajectories parallel to this axis behave as ordinary rays and are not split into O and E rays. B = 0.

Wave Plates Optical axis is perpendicular to incident beam. O and e rays are phase shifted (n 2 -n 1 )t Thickness of the crystal generates λ/4 phase shift At 45, intensities of E and O rays become equal Resulting wave of E and O rays has a constant intensity but Electric vector rotates around its own axis. (circularly polarized light)

Half Wave Plate λ/2 retardation Useful for rotating the polarization of the incoming light http://optics.byu.edu/animation/polarwav.mov

π = λ/2 Relative Phase Shift

Birefringence in Microscopy Optical Path Difference Γ = (n e - n o ) t (Thickness) 1. Non-polarized white light enters the polarizer and is linearly polarized 2. The polarized light enters the anisotropic crystal where it is refracted and divided into two separate components vibrating parallel to the crystallographic axes and perpendicular to each other 3. The polarized light waves travel through the analyzer, which allows the components of parallel waves to pass.

Polarization Microscopy Polarized light microscopy can distinguish between isotropic and anisotropic materials 90 percent of all solid substances are anisotropic and have optical properties that vary with the orientation of incident light with the crystallographic axes. compensator Polarizer and analyzer orientations are perpendicular to each other (extinction). Compensator (birefringent crystal) is optional and can increase the contrast http://academic.brooklyn.cuny.edu/geology/powell/

Alignment Koehler illumination. Insert the fixed polarizer. Insert the rotatable analyzer, and rotate it until the two polars are crossed and maximum extinction is obtained. Insert a telescope eyepiece and focus on the back aperture of the objective lens. A dark polarization cross is observed at extinction with brighter regions of intensity between the horizontal and vertical arms of the cross. The horizontal and vertical arms are broader in the center and narrower at the periphery. This is caused by the depolarizing effects of spherical lens surfaces. Perfect alignment with respect to an azimuth at 0 is essential if quantitative measurements of azimuthal angles or birefringence are to be made with a compensator. Using a Bertrand lens, partially close the condenser aperture diaphragm to block bright outer regions of depolarized light.

Compensators Compensator makes polarization microscope an analytical instrument. Since the angle of rotation of the analyzer at extinction is equal to onehalf of the full phase shift between the O and E rays, the relative retardation is given as Γ = 2θ. de Senarmont compensator is a λ/4 plate. Linearly polarized light passes through a specimen, whose optical axis is at 45º relative to the polarizer. Elliptical or circularly polarized light from the specimen is converted back into linearly polarized light by the compensator. The azimuth is rotated 45º relative to that of excitation light. When the analyzer transmission axis is crossed, half of the light is transmitted. Rotating the analyzer clockwise by 45º produces maximum transmission, while rotating the analyzer counterclockwise by 45 degrees yields maximum extinction.

Other Uses of Compensators The image background looks very dark, approaching black. Small (λ/20) retardation can be introduced with a compensator to see the background by a small amount. The compensator can also be used to increase or decrease the amount of phase displacement between the O and E rays to improve the visibility of details in the object image.

What can be Seen? Mitotic Spindle Actin and myosin filaments Condensed DNA Lipid Bilayers Isotropic specimen (i.e. gases, liquids and cubic crystals) are not visible since they do not rotate the polarization of incoming beam, which is blocked by analyzer.

Birefringent objects look bright or dark depending on their molecular orientation or geometry of the object Spherical objects with radially symmetric structures will show four bright quadrants. Linear objects (striated muscle) will appear bright at 45 135 225 315 and dark at 0 90 180 270. http://www.olympusmicro.com/primer/virtual/polarizing/index.html

Full Wave Retardation Plates When placed in front of the analyzer, oriented 45 with respect to the crossed polars, the plate introduces a relative retardation between O and E rays of exactly one wavelength for green wavelengths of 551 nm. Green wavelengths therefore emerge from the retardation plate linearly polarized in the same orientation as the polarizer and are blocked at the analyzer. O and E waves of all other wavelengths experience relative phase retardations of less than 1 λ; they emerge from the plate as elliptically polarized waves and are only partially blocked by the analyzer. Full-wave plates introduce vivid colors to birefringent objects and are useful for making quantitative assessments of relative retardation, and the orientation of index ellipsoids.

Differential Interference Contrast Microscopy The DIC microscope employs a mode of dualbeam optics that transforms local gradients in optical path length in an object into regions of contrast in the object image. DIC microscopy is a beam-shearing interference system in which the reference beam is sheared by less than the diameter of an Airy disk. The technique produces a monochromatic shadow-cast image that effectively displays the gradient of optical paths in the specimen. Those regions of the specimen where the optical paths increase along a reference direction appear brighter, while regions where the path differences decrease appear in reverse contrast.

Wollaston Prism Wollaston Prism generates parallel coherent wave bundles from all of the incoming polarized light. The separation of the two beams depends on the shear axis. The interference fringes appear to lie inside the prism at a location termed the beam splitting plane. This makes it difficult to use a conventional Wollaston prism for certain objective lenses, where the interference plane of the prism must lie within the back focal plane (the diffraction plane) of the lens. Modified Wollaston prisms where the beam splitting plane is displaced several milimeters away from the center of the prism.

The condenser prism acts as a beam splitter The objective prism recombines the beams and regulates the amount of retardation between O and E wavefronts. Koehler illumination is required to correctly position the interference planes of the DIC prisms in the conjugate aperture planes of the condenser and objective lenses.

Optical Axes in DIC Polarizer and Analyzer are perpendicular, to eliminate the background Nomarski prisms are parallel to each other (to recombine E and O rays) Nomarski prisms are fixed at 45 to the polarizer to obtain same intensity for E and O rays.

Formation of DIC Image Physical separation of the distance (shear axis) can be as low as 180 nm. In the absence of OPD, the beam is blocked by analyzer. If E and O rays encounter a phase object, they become differentially shifted in phase, resulted in elliptically polarized light.

DIC image of human red blood cells having thick edges and thin center. E and O rays pass through a phase object and are differentially shifted in phase. The shear axis is the axis of lateral displacement of the O and E wavefronts at the specimen. Amplitude of the transmitted wave is the derivative of OPD as a function of distance. As the shear distance is reduced, resolution improves, at some expense to contrast, until the shear distance is about one half the objective s maximum resolution.

Typical DIC Image a) Phase shift between the E and O rays. Difference is only observed at the edges of the sample, in the direction of the shear axis. b) Amplitude is the derivative of the OPD. c) DIC image of oil drops.

Introducing bias retardation makes objects much easier to see, because phase gradients in the specimen are now represented by bright and dark patterns on a gray background. The resultant image exhibits a shadow-cast, three-dimensional appearance and makes objects look like elevations or sunken depressions depending on the orientation of phase gradients.

de Senarmont Retardation A phase displacement between the O- and E-ray wavefronts was introduced by a retardation plate. Microscope contains a λ/4 plate. The objective DIC prism is fixed. The bias adjustment is made by rotating the polarizer. The amount of displacement between the O and E rays caused by the objective DIC prism is small, usually λ /10.

Image Orientation shadow and highlight intensity is greatest along the shear axis of the microscope

Comparison of DIC and Phase Contrast Phase contrast yields an image as a function of specimen optical path length magnitude, with regions having large path lengths appear darker. In DIC, optical path length gradients are primarily responsible for introducing contrast into specimen images. In DIC, steep gradients in path length generate excellent contrast, and images display a pseudo three-dimensional relief shading that is characteristic of the DIC technique. Extended flat object look like background with high contrast edges. Phase contrast is not susceptible to orientation effects

DIC the nucleus, cytoplasmic inclusions, and numerous bacteria on the upper surface. Phase Contrast cheek cell Pronounced halos around the cellular periphery and nucleus, which are absent in DIC image. Optical sectioning DIC microscopy investigations (not illustrated) reveal that the bacteria are present on the membrane surface as opposed to lying on the underside of the cell. This fact cannot be unambiguously determined with phase contrast.

differential interference contrast reveals a bundle of cells enclosed within a tubule. A phase contrast image of the same area is confusing and disturbed by the presence of phase halos outside the plane of focus. However, several of the cellular nuclei appear visible in the phase contrast image, which are not distinguishable in DIC. thick section of murine kidney tissue

Phase Contrast DIC Halos are absent in DIC, but images have shadow-cast appearance oriented along the shear axis.

Phase Contrast DIC Phase contrast images become ambiguous when optical path length fluctuates over a large range, the technique should be restricted to specimens having a path difference of one-tenth wavelength or less. Thicker specimens can be imaged with DIC by using optical sectioning techniques. http://www.olympusmicro.com/primer/java/dic/dicphaseos/index.html

Phase Contrast DIC Because DIC relies on polarized light, differential absorption of ordinary and extraordinary waves leads to confusing images. Phase contrast does not require the use of polarized light, and is free of optical disturbances generated by birefringent specimens.