Microscopy Basics
We must understand clear distinctions in resolution and magnification between microscopes, telescopes, and cameras
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What is phase contrast?
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Compare 1 millimeter to micrometers and nanometers:
1 millimeter equals 1000 micrometers and 1 micrometer equals 1000 nanometers
Köhler illumination requires what 4 components?
1. Collector lens and/or field lens 2. Field diaphragm 3. Condenser diaphragm 4. Condenser lens
What are two different main types of microscopes?
1. Compound (using transmitted light) 2. Stereoscopic (using reflected light)
100 microns is how many micrometers?
100. (100 µm) Microns and micrometers are the same thing.
What is an apochromatic lens?
A lens that has better correction of chromatic and spherical aberration than the much more common achromat lenses.
What is Köhler illumination?
most common setup for modern microscopes. Creates really even illumination by defocusing the image of the light source in the sample plane.
The microscopic techniques requiring a transmitted light path
brightfield, darkfield, Zernicke phase (or just phase) and differential interference contrast (or Nomarski) optics. Other not as commonly used transmitted light techniques include Hoffman modulation, Varel optics, and polarization optics.
Define Magnification:
how much bigger an object appears when viewed with a lens in comparison to its actual size (when viewed without a lens)
Purpose of a condenser
invented to concentrate the light on the specimen in order to obtain a bright enough image to be useful.
Parts found in a compound microscope that are not present in a stereoscopic microscope:
iris diaphragm(which is the condenser diaphragm), condenser, nosepiece (holds multiple objectives, rotates)
What is a plan-apochromatic?
A modern, high-numerical aperture microscope objective lens designed with high degrees of corrections for various aberrations. It is corrected for spherical aberration in four wavelengths (dark blue, blue, green, and red), for chromatic aberration in more than these four wavelengths, and for flatness of field. A single plan Apo objective may contain as many as 11 lens elements.
What's the difference between Achromatic and Apochromatic?
Achromatic lenses are corrected to bring two wavelengths (typically red and blue) into focus in the same plane. Apochromatic lenses are designed to bring three wavelengths (typically red, green, and blue) into focus in the same plane.
Numerical Aperture: what is it?
Has to do with RESOLUTION of the OBJECTIVE The numerical aperture is the measure of an OBJECTIVE'S ability to gather light and to RESOLVE fine specimen detail while working at a fixed distance. Th higher the NA, the higher the resolution. Numerical Aperture (NA) = n(sin µ) n = refractive index of the imaging medium between the front lens of the objective and the specimen cover glass, a value that ranges from 1.00 for air to 1.51 for specialized immersion oils µ = the angle of the light to objective from specimen. (The angle µ is one-half [half the cone] of the angular aperture [the whole cone]) An increased angle increases the NA. With a condenser, you add that angle as well, and the NA increases even more The sum of the aperture angles of the objective and the condenser is referred to as the working aperture. If the condenser aperture angle matches the objective, maximum resolution is obtained.
What is birefringence? What is anisotropic?
In latin, birefringent --'refracting light in two ways'. In greek, Anisotropic -- not treating all directions the same way; aniso (unequal) tropic (turns); anisotropy refers to properties that depend on the spatial direction, while birefringence doesn't.
What software do you use to set up a monitor for your Canon 7D with the microscope?
EOS Utility. 2:41; usb connection http://digital-cameras.wonderhowto.com/how-to/use-your-laptop-as-monitor-with-canon-5d-7d-332469/
How is it we can see any object at all ever? (not just with microscopes, but looking with our eyes, period)
Interference of light by an object allows us to see an object
What is a bertrand lens?
It allows observation of the back focal plane of the objective lens and its conjugate focal planes. Any optical components at the back focal plane of the objective (e.g. the phase ring for phase contrast microscopy) and at the condenser diaphragm (e.g. the annulus for phase contrast microscopy) should also appear in focus.
What is the sine of 90 degrees equal to for numerical aperture?
It equals 1. Therefore, to get NAs higher than .90, you really need to mess with increasing the refractive index.
Transmitted light microscope
Light is transmitted from a source on the opposite side of the specimen from the objective
What's a MIC-rometer versus and mic-RAHM-eter
MIC-rometer refers to the measurement that is one-millionth of a meter. Mic-RAHM-eter is a measuring device
Does Klaus Kemp know the following people?
Mortimer Abramowitz, had many photomicrographs published and wrote the book Solliday gave you Bill Dailey Hummelink
Where's the best location for a focusable condenser?
Most often it's where the condenser is closest to the stage as possible
What resolution specifically is NA often determining?
Numerical aperture determines the resolving power of an OBJECTIVE, but the total resolution of a microscope system is also dependent upon the numerical aperture of the substage condenser. The higher the numerical aperture of the total system, the better the resolution.
Steps to test Köhler illumination:
On top of field lens: The field diaphragm is partially closed; the edges of the diaphragm should be in the same conjugate image planes as the specimen, therefore should appear in focus. The focus can be adjusted by raising or lowering the condenser lenses and diaphragm. Finally, the field diaphragm is reopened to just beyond the field of view. In order to test the alignment of components on the light source image plane, the eyepiece must be removed to allow observation of the intermediate image plane (the position of the eyepiece diaphragm) either directly or by using a phase telescope/Bertrand lens. The light source (e.g. the bulb filament) and the edges of the condenser diaphragm should appear in focus. Any optical components at the back focal plane of the objective (e.g. the phase ring for phase contrast microscopy) and at the condenser diaphragm (e.g. the annulus for phase contrast microscopy) should also appear in focus.
Concerns to address with Klaus Kemp:
Proper use of adhesive; not using too much How to keep slides clean from various contaminates/fibers How to get needle to grip diatom and drop diatom easily Advice on cover slip thickness for diatom slides (should probably be for a 10X to 20X?). The lower right number on an objective (if given) refers to the thickness of the glass cover slip (in millimeters) assumed by the lens designer for best performance of the objective. With the dividing of diatoms into pennate or centric, does this happen in the class or the order? Is this still done today? Should be order, and there's three: centric, pennate with raphe, pennate without raphe Diatoms all belong to the class Bacillariophyceae Where to buy a camera lucida. What would work with my microscope.. Or is it possible to build one?
How does the wavelength of light affect the resolution?
Shorter wavelengths are capable of resolving details to a greater degree than are the longer wavelengths. Relationship between numerical aperture, wavelength, and resolution: R = λ / 2NA R = 0.61λ / NA Ernst Abbe formula R = 1.22λ / (NA(obj) + NA(cond)) Ernst Abbe and Carl Zeiss were friends! Zeiss wanted to make the production of lenses a scientific process, and he hired Abbe to help with the endeavor. In his lifelong association with Zeiss's company, Abbe made a number of notable achievements. One of these was the Abbe sine condition, a formula to create a lens that produces sharper images. Another was the development of the apochromatic lens system, which helps to correct chromatic, or color, distortion of light.
What is the colored ring on the objective referring to?
Sometimes objectives have a color ring to aid in identifying the magnification: black (1x), brown (2x), red (4x), yellow (10x), green (20x), turquoise (25x), light blue (40x), dark blue (60x), white (100x).
What is working aperture?
The sum of the aperture angles of the objective and the condenser is referred to as the working aperture. If the condenser aperture angle matches the objective, maximum resolution is obtained. http://zeiss-campus.magnet.fsu.edu/articles/basics/resolution.html
In rheinberg illumination filters, what do the outer annular ring and inner filter provide color to?
The outer ring "becomes" the color of the specimen and the central filter "becomes" the color of the background.
What are the four numbers on an objective?
The upper left number is the magnification factor of the objective. For example, 4x, 10x, 40x, and 100x. The upper right number is the numerical aperture of the objective. For example 0.1, 0.25, 0.65, and 1.25. The lower right number (if given) refers to the thickness of the glass cover slip (in millimeters) assumed by the lens designer for best performance of the objective. Example: 0.17. The lower left number is the tube length in millimeters. This is related to the standardization of microscopes and the particular standard used for the manufacture of your microscope. Most microscopes employ the Deutsche Industrie Norm, or DIN standard configuration. The Japanese Industrial Standard (JIS) is less commonly used. DIN microscopes begin with an object-to-image distance of 195 mm, and then fix the object distance at 45 mm. The remaining 150 mm distance to the eyepiece field lens sets the internal real image position, which is defined as 10 mm from the end of the mechanical tube (which gives the 160 mm tube length). DIN standard eyepieces have an international standard 23 mm diameter. DIN standard objectives often times have "DIN" etched on the side and have a standard 0.7965 in diameter thread, 36 TPI, 55° Whitworth threading. Celestron microscopes are made to DIN standards. The tube length for the DIN standard is 160 mm, while for the JIS it is 170 mm. NOTE: JIS objectives can be used on a DIN microscope and vice versa The threads on both types are interchangable. However, since the optical distances are different, there will be a difference in magnification. A JIS objective used on a DIN microscope will have a slightly lower magnification than the rated magnification since the DIN tube is shorter. And a DIN objective used on a JIS microscope will have a slightly higher magnification than the rated magnification since the JIS tube is longer.
What are ocular lens?
They are the eyepiece lens.
Possible hints in proper Rheinberg set-up for annular ring/inner filter width with various objectives
Whatever color the whole image is will be the filter that needs to be slimmed down Rheinberg illumination is suitable for objectives ranging from 2x to 100x. However, in order to clearly separate the inner and outer colors, opaque metal or paper rings should be placed around the central filter. For example, the opaque rings might have an outside diameter of 15-18 millimeters for a 10x objective, and an outside diameter of 22 millimeters might be best for a 60x oil immersion objective. It is a good idea to experiment using central filters like the ones illustrated in Figure 4 to achieve the best results with Rheinberg illumination.
Define Resolution:
ability to see 2 objects as 2 separate objects. A microscope has better resolution than our naked eyes. Microscopes allow objects to be closer together than our naked eyes for us to see objects as being separate.