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Questions and Answers
The wavelength of an electron in an electron microscope is inversely proportional to the velocity of the electron.
The wavelength of an electron in an electron microscope is inversely proportional to the velocity of the electron.
True (A)
The electromagnetic lenses in electron microscopes are fixed in position, similar to light microscopes.
The electromagnetic lenses in electron microscopes are fixed in position, similar to light microscopes.
False (B)
The maximum resolution achievable with a light microscope is approximately 0.2µm or 200nm.
The maximum resolution achievable with a light microscope is approximately 0.2µm or 200nm.
True (A)
In electron microscopes, the convergence of a beam of electrons is achieved via a circular magnetic field, similar to light microscopes.
In electron microscopes, the convergence of a beam of electrons is achieved via a circular magnetic field, similar to light microscopes.
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The strength of the magnetic lens in an electron microscope can be regulated by adjusting the amount of current flowing through the electrical coils, thereby altering the focal length and magnification.
The strength of the magnetic lens in an electron microscope can be regulated by adjusting the amount of current flowing through the electrical coils, thereby altering the focal length and magnification.
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A 100x objective lens has a higher numerical aperture than a 40x objective lens.
A 100x objective lens has a higher numerical aperture than a 40x objective lens.
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Numerical aperture is a measure of the objective lens's ability to gather light and resolve fine details.
Numerical aperture is a measure of the objective lens's ability to gather light and resolve fine details.
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The resolving power (RP) of a microscope is inversely proportional to the numerical aperture (NA).
The resolving power (RP) of a microscope is inversely proportional to the numerical aperture (NA).
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Spring loaded objectives are used to increase the working distance of high-power objectives.
Spring loaded objectives are used to increase the working distance of high-power objectives.
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The working distance is the distance between the specimen and the objective lens when the specimen is in focus.
The working distance is the distance between the specimen and the objective lens when the specimen is in focus.
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Microscopy filters are used to increase contrast, resolution, and to remove harmful ultraviolet or infrared light.
Microscopy filters are used to increase contrast, resolution, and to remove harmful ultraviolet or infrared light.
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Direct sunlight can be used for microscopy as it is a good source of illumination.
Direct sunlight can be used for microscopy as it is a good source of illumination.
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Par-focal objectives are designed so that once an object is in focus with one objective, it will remain in focus when switching to another objective.
Par-focal objectives are designed so that once an object is in focus with one objective, it will remain in focus when switching to another objective.
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Modern microscopes use a fixed working distance for all objective lenses to prevent damage.
Modern microscopes use a fixed working distance for all objective lenses to prevent damage.
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A 40x objective lens typically has a numerical aperture (NA) of 0.65.
A 40x objective lens typically has a numerical aperture (NA) of 0.65.
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A concave lens is thicker at the middle than at the edges.
A concave lens is thicker at the middle than at the edges.
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A convex lens can form both real and virtual images depending on the object's distance from the lens.
A convex lens can form both real and virtual images depending on the object's distance from the lens.
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The image formed by a concave lens is always larger than the object.
The image formed by a concave lens is always larger than the object.
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Inverted images are formed by convex lenses when the object is closer than the focus.
Inverted images are formed by convex lenses when the object is closer than the focus.
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The light rays passing through a concave lens spread apart as they diverge.
The light rays passing through a concave lens spread apart as they diverge.
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A virtual image formed by a concave lens appears on the opposite side of the lens from the object.
A virtual image formed by a concave lens appears on the opposite side of the lens from the object.
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A convex lens always forms an upright image regardless of the object's position.
A convex lens always forms an upright image regardless of the object's position.
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The dark-field microscope illuminates objects brightly against a light background.
The dark-field microscope illuminates objects brightly against a light background.
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Cedar wood oil is used in microscopy because it has a similar optical density to glass.
Cedar wood oil is used in microscopy because it has a similar optical density to glass.
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Treponema Pallidum can be demonstrated using a dark-field microscope.
Treponema Pallidum can be demonstrated using a dark-field microscope.
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The resolving power of a microscope is solely determined by the magnifying power of the objective lens.
The resolving power of a microscope is solely determined by the magnifying power of the objective lens.
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Fluorescence microscopy relies on a substance emitting light of shorter wavelength.
Fluorescence microscopy relies on a substance emitting light of shorter wavelength.
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The dark-field microscope requires the specimen to scatter light to be visible.
The dark-field microscope requires the specimen to scatter light to be visible.
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Resolving power can be quantitatively defined as the ability to distinguish two adjacent points as separate entities.
Resolving power can be quantitatively defined as the ability to distinguish two adjacent points as separate entities.
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Empty magnification occurs when further magnification reveals two points as distinct.
Empty magnification occurs when further magnification reveals two points as distinct.
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Using a dirty slide can enhance the viewing experience in dark-field microscopy.
Using a dirty slide can enhance the viewing experience in dark-field microscopy.
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The numerical aperture is a measurement of the angle of light collected by the objective lens.
The numerical aperture is a measurement of the angle of light collected by the objective lens.
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The presence of air bubbles in immersion oil can improve visualization in a dark-field microscope.
The presence of air bubbles in immersion oil can improve visualization in a dark-field microscope.
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The limit of useful magnification increases with decreasing resolving power.
The limit of useful magnification increases with decreasing resolving power.
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Dark-field microscopy can be effectively used for examining very dense preparations.
Dark-field microscopy can be effectively used for examining very dense preparations.
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The dark-field condenser has a special blacked-out area that allows light to enter the microscope objective.
The dark-field condenser has a special blacked-out area that allows light to enter the microscope objective.
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Oil immersion helps conserve light that would be lost through refraction.
Oil immersion helps conserve light that would be lost through refraction.
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The resolving power of an objective lens does not depend on the wavelength of light used.
The resolving power of an objective lens does not depend on the wavelength of light used.
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The light emitted by fluorescent substances has more energy than the light absorbed.
The light emitted by fluorescent substances has more energy than the light absorbed.
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In dark-field microscopy, the light comes up through the specimen.
In dark-field microscopy, the light comes up through the specimen.
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Increasing the magnifying power of a microscope will always result in increased resolving power.
Increasing the magnifying power of a microscope will always result in increased resolving power.
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Flashcards
Microscope
Microscope
An instrument used to view tiny objects not visible to the naked eye.
Compound Microscope
Compound Microscope
A type of laboratory microscope with multiple lenses for enhanced magnification.
Lens
Lens
A transparent object with one or two curved surfaces that refracts light.
Concave Lens
Concave Lens
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Convex Lens
Convex Lens
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Virtual Image
Virtual Image
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Real Image
Real Image
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Cedar Wood Oil
Cedar Wood Oil
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Resolving Power (RP)
Resolving Power (RP)
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Numerical Aperture (NA)
Numerical Aperture (NA)
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Empty Magnification
Empty Magnification
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Oil Immersion Technique
Oil Immersion Technique
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Limit of Useful Magnification
Limit of Useful Magnification
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Light Conservation
Light Conservation
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Wavelength (λ) in RP Formula
Wavelength (λ) in RP Formula
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Focal Length Impact
Focal Length Impact
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Focal Length
Focal Length
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Working Distance
Working Distance
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Spring Loaded Objectives
Spring Loaded Objectives
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Microscopy Filters
Microscopy Filters
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Ambient Light Management
Ambient Light Management
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Immersion Oil Objective
Immersion Oil Objective
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Par-focal Objectives
Par-focal Objectives
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Light Sources for Microscopy
Light Sources for Microscopy
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Dark-Field Microscope
Dark-Field Microscope
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Principle of DFM
Principle of DFM
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Object Appearance
Object Appearance
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Application of DFM
Application of DFM
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Limitation of DFM
Limitation of DFM
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Fluorescence Microscope
Fluorescence Microscope
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Fluorescence Principle
Fluorescence Principle
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Blue Light Function
Blue Light Function
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Emission Types
Emission Types
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Applications of Fluorescence
Applications of Fluorescence
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Resolution limit of light microscope
Resolution limit of light microscope
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Electron wavelength
Electron wavelength
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Whenalt cap function
Whenalt cap function
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Condenser lens in EM
Condenser lens in EM
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Variable lens currents
Variable lens currents
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Study Notes
Microscopy
- Microscopy is used to view tiny objects that cannot be seen with the naked eye.
- An ordinary magnifying glass is a simple microscope, while a laboratory microscope is a compound microscope.
- Compound microscopes have a complex arrangement of lenses.
- A lens is a transparent object with one or two curved surfaces, typically made of glass (or clear plastic for contact lenses).
- A lens refracts (bends) light to form an image. The image is a copy of the object formed by the refraction (or reflection) of visible light.
- The more curved the lens surface, the more it refracts the light passing through it.
- There are two basic types of lenses: concave and convex.
- Concave lenses are thicker at the edges and cause light rays to diverge (spread apart). The image formed by a concave lens is on the same side of the lens as the object, smaller than the object, and right-side up. It is a virtual image.
- Convex lenses are thicker in the middle than at the edges and cause light rays to converge (come together) at a point called the focus. A convex lens forms either a real or virtual image, depending on the object's distance from the focus.
Properties of Lenses: Concave Lens
- Concave lenses are thicker at the edges than in the middle.
- They cause light rays to diverge (spread apart) as they pass through.
- The image formed by a concave lens is on the same side as the object.
- The image is smaller than the object.
- The image is right-side up.
- It is a virtual image.
- Light rays actually pass through the lens and spread out in all directions (divergent rays).
Properties of Lenses: Convex Lens
- Convex lenses are thicker in the middle than at the edges.
- They cause light rays to converge at a point called the focus (F).
- A convex lens forms either a real or virtual image.
- Formation of a real image depends on the object's distance to the focus.
- Image is real and inverted if the object is further than the focus.
- Image is virtual and upright if the object is between the lens and the focus.
Parts of a Microscope
- Eye Piece Lens (Ocular Lens): Used to magnify the image.
- Diopter Adjustment: A knob used to adjust the focus for different eyesight needs.
- Nose Piece: Holds multiple objective lenses, allowing for different magnifications.
- Objective Lens: A system of lenses that magnifies the image of the sample.
- Stage Clip: Holds microscope slides in place.
- Aperture: An opening in the stage that controls the amount of light passing through.
- Diaphragm: Adjusts the amount of light.
- Condenser: Concentrates light on the specimen.
- Illuminator (Light Source): Provides light to view the sample.
- Eye Piece Tube: Supports the eyepiece lens.
- Head: Contains the eyepiece lenses.
- Arm (Carrying Handle): Holds the microscope.
- Glass Slide: Holds the specimen.
- Mechanical Stage: Allows for precise movement of the slide.
- Coarse Adjustment Knob: A knob that moves the stage to quickly focus the image.
- Fine Adjustment Knob: A knob for precise focusing.
- Stage Control Knobs: Moves the stage left, right, up, or down.
- Base: The bottom of the microscope.
- Brightness Adjustment: Adjusts the intensity of the light source.
- Light Switch: Turns the light source on or off.
Microscope Use and Care
- Always grip the microscope by the arm and put your hand beneath its base.
- Hold the scope upright at all times.
- Only the 100X (oil immersion) objective uses oil.
- Do not get oil on any of the other objectives; the oil will ruin them, and you will not be able to focus at those powers.
- Large specimens should be examined at low power only.
- When putting the scope away, remove the slide, clean any oil off the 100X objective, clean the other objectives, and turn off the light.
- Loop the cord up on itself, secure it, and don't let it dangle freely.
- Replace the cover.
- Do not tamper with any of the components of the microscope.
- Always be certain that the low-power objective is in place before putting the microscope away.
- Always unplug the electrical cord by pulling on the plug, not the cord.
- Never look through the microscope while rapidly reducing the distance between the objective lens and the slide.
- Always carry the microscope with two hands.
- Keep the microscope at least 6 inches from the edge of the lab table, and keep the excess electrical cord on the table top.
- Do not touch the glass lens with your fingers.
- Clean the lenses with lens paper only, and wipe the lenses before and after each use.
- Always wipe the oil from the oil-immersion objective with lens paper before putting the microscope away.
Refraction
The bending of light rays from the 'normal' when it passes from one optical medium to another. It is caused by changes in the passage of light when it passes from one medium to another of different optical density. When light enters a denser medium it bends toward the normal line. The normal line is the line perpendicular to the surface. When light enters a less dense medium, it bends away from the normal line.
Snell's Law
Snell's law states that the ratio of the sines of the angles of incidence and refraction is equivalent to the ratio of phase velocities in the two media, or equivalent to the reciprocal of the ratio of the indices of refraction.
Limitation of Lenses
Limitations caused by light and the shape of the lens. These limitations create defects such as:
- Spherical aberration.
- Chromatic aberration.
Spherical Aberration
- The indistinct or fuzzy appearance of images due to non-convergence of light rays to a common focus.
- This occurs when the edge of the lens gives a slightly higher magnification than the center of the lens.
- Spherical aberration results in loss of contrast, resolution, clarity, and overall focus. Spherical aberration is the property of lenses that have less than perfect spherical shape. It increases with an increase in the thickness of the biconvex lens.
- It can be corrected by compounding with a biconcave lens that brings the image into sharp focus.
Chromatic Aberration
- Fuzzy appearance of the image due to non-convergence of rays of white light.
- The image is surrounded by a multi-colored fringe with the blue light being slightly more magnified than the red.
- It is caused by splitting white light into its constituent colors while passing through the lens, which acts as a prism.
- As white light passes through the lens, the light of shorter wavelength is refracted more strongly than the light of longer wavelength.
- Chromatic aberration is controlled by proper combination of lenses in the modern microscope. Achromatic lenses correct for two colors, while apochromatic lenses correct for three colors.
Compound Microscope
- Magnification is produced by two sets of lenses.
- The objective lens produces a real, magnified, inverted image.
- The eye piece lens brings the inverted image into sharp focus and produces a magnified virtual image.
- Total magnification is the magnification of the objective multiplied by the magnification of the eye piece lens.
- Useful magnification reveals more details, whereas empty magnification fails to show more details and loses its sharpness.
Bright-field Microscopy
- Bright-field microscopy is the simplest technique for sample illumination in light microscopes and is widely used.
- In bright-field microscopy, the sample appears dark against a bright background.
- A direct light source illuminates the object to make it visible.
- Amplitude specimens possess color and are able to decrease the brightness of the passing light all on their own. These specimens are often stained.
Dark-field Microscopy
- Dark-field microscopy brightly illuminates the object.
- Use a special condenser with a blacked-out light source and angled light path to make the background appear dark.
- The light from the specimen that scatters reaches the eye, creating a bright object against a dark background.
- A special condenser and objective are critical for visualization.
- Limitations include difficulties in setting, centering, and focussing, using dirty slides and cover slips, and problems with dense specimens or air bubbles.
Fluorescence Microscopy
- A substance is fluorescent when it can absorb light of shorter wavelengths and energy (e.g., blue light) and emit light of longer wavelengths and lesser energy (e.g., green light).
- In practice, microbes are stained with a fluorescent dye and then illuminated with blue light. The dye absorbs the blue light and emits green light.
- Components like an exciter filter, dichroic mirror, emission filter are necessary for proper viewing.
- Photobleaching, a phenomenon where fluorophores lose their ability as they are illuminated, is a limiting factor. Techniques to minimize photobleaching include using robust fluorophores, minimizing illumination, or using photoprotective drugs.
Phase-contrast Microscopy
- An advantage of this method is the ability to study a structure without dyes or harming the cell.
- Phase contrast is a type of light microscopy that enhances contrasts between differing structures.
- Bright-field microscopy shows unstained objects as they pass through light based on their optical density.
- This technique translates minute differences in optical density or refractive indices into amplitude changes, and these are easier to distinguish with the human eye.
Electron Microscopy
- Electron microscopy provides much higher resolution than light microscopy and is vital for viewing minute structures.
- A beam of electrons having a very short wavelength is used rather than light to increase resolution.
- The electron beam travels through a vacuum-sealed column, and the image is formed by the scattering of electrons.
- The image is produced on a fluorescent screen, and lenses (condenser, objective, and intermediate) are crucial for focusing and magnification.
- Biological stains are not fully applicable as these depend on light absorption which is minimal with electron microscopy.
- Electron microscopy is often used on dead or dried specimens since gas molecules would affect the electron beam and disrupt their path in the vacuum.
Micrometry
- Micrometry measures the dimensions of microorganisms using calibrated eyepiece and stage micrometers.
- The stage micrometer has a precise, known scale.
- The calibrated eyepiece scale is viewed in the same field of vision.
- The calibration allows you to precisely measure specimen dimensions.
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Description
Test your knowledge on the principles and functions of electron microscopes. This quiz covers topics such as wavelength, resolution, numerical aperture, and the workings of electromagnetic lenses. Ideal for students studying microscopy techniques.
