Microscopy Flashcards
What is the Compound light microscope (bright-field microscope) used for?
Examine cellular structures that are too small to be seen with the naked eye
What are the two magnifying lenses?
- Objective Lens
- Ocular lens (eye piece)
What is the Critical factor resolution achieved by the microscope?
Ability to see structures as separate and distinct
What are the 5 basic terminology of microscopy?
- Resolution
- Magnification
- Numerical Aperture (NA)
- Resolving Power
5.Definition
What is Resolution:
- Indicates how small individual objects can
be and still be recognized as distinct. - Separation of two distinct points/objects
- The more detail seen, the higher the resolution
What is the equation for calculating Magnification:
Magnification Equation: Total Magnification = Objective x Ocular
What is Numerical Aperture (NA):
light gathering ability of the system
It’s the ability of the microscope to render the finest detail distinctly visible
Empty Magnification:
Further magnification of two dots that are no longer resolvable (cannot be distinguished) is considered empty magnification
What is Resolving Power:
The ability of a lens to separate two distinct points
This is the limit of usable magnification
What is Definition:
- The ability of the objective lens to make the outline of an object distinct.
- An object, if poorly defined, is unlikely to be well-resolved even if the lens system has a high resolving power
- Definition is a function of the object and its illumination
What are the functional categories of the microscope?
- Foundational structures
- Slide Holder
- Light Controls
- Magnification System
- Focus Adjusters
What components make up the foundation Structure?
- Base
- Arm
What components make up the Slide Holder?
- Mechanical Stage
- Slide Holder
- Stage Adjustment Knobs (Co-axial knobs)
What components make up the Light Controls?
- Light Source
- Rheostat
- Field Diaphragm
- Condenser
- Condenser Adjustment Knob
- Condenser Centering Screws
- Iris Diaphragm
What components make up the Magnification System?
- Objective Lenses
- Rotating/Revolving Nosepiece
- Ocular Lens
- Diopter Rings
What components make up the Focus Adjusters?
- Coarse Adjustment Knob
- Fine Adjustment Knob
What parts of the microscope are found in the base?
1.Transformer
2.Rheostat
3.Illuminator (lamp)
What is the Transformer:
- Usually located in the base
- Some have an external transformer
- Steps down the voltage or amount of energy entering the illuminator or lamp
What is the Rheostat:
- The dimmer switch located on the base or on the external transformer
- Light intensity regulator
- Controls amount of current entering the illuminator
What is the purpose of the Illuminator (lamp):
1.Provides the major illumination to light the specimen
- Usually located at the back of the base
- If there is a separate on/off switch from the rheostat, then the rheostat must be turned down prior to turning off the microscope in order to increase the life of the bulb
- Light source is then directed up through the condenser
- The bulb must be correctly positioned for proper alignment
6.Tungsten or Tungsten-halogen bulbs are most frequently used
What parts of the microscope are found in the Condenser?
- Centring Screws
- Filter Holder
- Aperture Iris Diaphragm
Centring Screws
- Found on either side of the condenser
- Used to center the condenser over the light
- Condenser and centering screws are used in conjunction with the field diaphragm and light source to control light path from the lamp to the objective lens
- These adjustments are referred to as Koehler illumination
Filter Holder
A swing out attachment at the bottom of the condenser
Easily swings into the light path
It is used to hold light-absorbing filters (blue, green, daylight filters)
Purpose of the coloured or selective filters is to control the contrast in the image of a coloured microscopic preparation (i.e. blood film)
Should be used in precise microscopy where fine detail is important
Aperture Iris Diaphragm
Can be used to adjust the diameter of the light beam passing through the objective lens so that it will just fill the front lens of the objective
This can help to reduce spherical aberration
Spherical aberration is when many focal points produce a blurry image
Field diaphragm
Controls the circle of light in the field of view when specimen and condenser are properly focused
Light sources passes through the field iris diaphragm
The size of the field diaphragm is controlled by rotating a knurled ring, which is concentric with it
Condenser Adjusting Knob
Located in the middle of the body of the microscope (usually on the left-hand side)
Knob raises and lowers the condenser so that the lens focuses the light source directly on the specimen
The top of the condenser lens should lie just under the slide
Focusing Knobs
Located on both sides of the microscope
Knobs used to adjust the position of the stage to bring the specimen being examined into focus and to obtain a clear image
The coarse adjustment knob is the largest knob
Permits the movement over large distances and should only be used when the low power objective (10x) is in place
Then you can get a sharper focus with the fine adjustment knob
Mechanical stage
The platform just above the condenser
Supports the object to be examined (your specimen slide)
Has a central hole through which the light from the substage condenser passes through the specimen
Metal spring clips hold your slide
What is the Vernier Scale:
There are rulers on the right-hand side and the “top” of the stage
Used to quickly re-locate a particular cell or object at a later time
Slide holder function and location:
Holds your slides secure and steady during observation this is located on the stage.
Co-axial adjustment knobs:
Moves in two directions front to back and left to right.
Revolving Nosepiece:
Holds the objective lenses
Used to move objectives from one magnification to another by rotating the ring
Diopter rings
The eyepiece usually has a diopter adjustment for sharper focus.
Allow you to make minor corrections to the image, compensating for the difference in vision between the two eyes.
Microscope Head
Lies just above the revolving nosepiece
It contains the prisms that split the light into two beams (binocular)
Body/Optical Tube
Holds the objective and the eyepieces in proper alignment and at the correct optical distance
The distance from the insertion position of the objective to the top of the eyepiece is called the mechanical tube length
Objective Lens System
- Located on the revolving nosepiece, above the slide
- Responsible for primary magnification
- Objectives are usually engraved with pertinent information relating to their performance
What is Parfocal:
When you change from one objective lens to another, the focus will not be lost
If you change from your 40X high-dry power to your 100X oil immersion power, you should only have to finely tune the focus (fine adjustment knobs)
What is Low power objetcive:
Usually 10X lens with 16mm working distance
What is working distance:
The distance between the top of the slide on the stage and the objective lens
High power objective:
Also known as high-dry (does not require oil)
40X lens with a 4mm working distance
What is the Ocular lens:
Eyepieces
Where is the ocular lens located
- At the top of the microscope
- Each ocular contains a lens that magnifies the image on the slide
What is interpupillary distance:
Distance between the two eyepieces
Ocular Adjusting Wheel
Located between the eyepieces
Used to set the interpupillary distance to that of the individual using the microscope
Focusing Collar:
Used to focus the eyepiece for the left eye
The specimen is brought into focus with the focusing knobs for the right eye (left eye closed) and then with the right eye closed, the focusing collar is adjusted for the left eye
What are coverslips and what are there purposes:
Coverslips are little thin pieces of glass that adhere on top of a slide
Serves two purposes:
- Protects the mirocscopes objective lenses from coming in contact with specimen
- Creates an even thickness for viewing (i.e. for wet mounts)
Correction collar:
Helps w the variety of thickness of cover glass
Allow adjustment of the central lens group position to coincide with fluctuations in cover glass thickness.
What happens when the slide of the coverslip is too thick:
If the slide is too thick then light rays will be focused within the slide and not on the specimen
Plan-neofluar microscope lens & Plan-fluotar microscope lens
Excellent for polarization microscopy
Also transmit UV very well, making them excellent lenses for all types of fluorescence microscopy
Any lens with the term fluar in it has fluorite elements in it and that’s what makes it good for fluorescence work
Apochromatic lenses (Planapochromat)
The most highly colour-corrected objectives
They are corrected for four wavelength and are top of the line objective lenses
These most often have the highest NA (give best resolution/definition)
Microscope Ergonomics:
Seat should be adjusted for proper height
Technologist should be able to look into microscope without bending their head down or raising their head to look into the eyepieces
What is Koehler Illumination:
- A process that provides optimum contrast and resolution by focusing and centring the light path and spreading it evenly over the field of view
- Should be the first thing you do when you sit down at a microscope
- Specific to you and your eyes
Why do you need to clean a microscope:
Cleaning of the microscope maintains the life of the microscope and the lenses as well as providing good quality images
What sequence should you follow when cleaning the microscope monthly:
- Blowing
- Use compressed air source to blow dust from the lenses
- This removes larger particles of dust that could scratch the lens - Brushing
- Using camel hair brush or sable brush to remove particles that will not displace with the compressed air - Wiping
- Use ONLY lens paper (specific)
- Moisten with a lens cleaner and wipe carefully in a circular motion
- Clean low power and high power first
- Clean oil immersion last so as to not spread oil the other lenses
Cleaning the exterior of the Microscope:
- Wipe down with neutral soap and water
- Moisten gauze or soft cloth, rubbing in a circular motion
- Immediately dry exterior using a dry gauze or cloth
- Never use gauze to clean the optical components of the microscope (eyepieces, objective lenses) – Only ever use specific lens paper
What do Brightfield microscopes require of the objects being looked at:
Brightfield microscopes require the object to be stained in order to have enough contrast to view detail of the object being examined
What are the 6 different types of microscopes:
- Phase contrast
- Polarizing
- Darkfield
- Fluorescence
- Electron Microscopy
- Inverted
What is a phase contrast microscope:
Unstained objects can be viewed
Can view live preparations as in “wet preparations”
Superimposed light and dark rings
Annular diaphragm:
An annular diaphragm is put in or below the condenser
This allows a hollow cone of light through the condenser to the specimen
Condenser annular:
Each phase contrast objective has a corresponding condenser annular
What is Phase Contrast Perfect alignment:
- First, the microscope must be set for brightfield viewing
- Next, the phase contrast objective must be matched to the condenser annulus
- A phase telescope is then inserted into the ocular tube
- Bring the annular ring in to focus – White ring of light
Polarizing
Brightfield microscope is modified using a polarizer
Polarizing filter allows light waves of one orientation (i.e. north-south or east-west) to pass through the filter
Polarizing filter is placed between the light source and the specimen
A second polarizing filter is placed above the specimen between he objective and eyepiece and Objects split light into two beams
You would then see a black field when looking through the eyepieces
What does Birefringent mean:
Objects are able to polarize light
Birefringent objects will appear light against a black background
What does Non-birefringent mean:
Non-birefringent objects will not be seen as they cant polarize light.
Darkfield
- In other words, indirect light is reflected off the object
- Substage condenser causes light waves to cross on specimen.
- The field of view will be dark as no light is actually pass from the condenser to the objective
- Used to examine spirochetes
Fluorescence
Detects specific wavelengths of light emitted from objects
Some objects have the capability to fluoresce – Ability to absorb light of a short wavelength (ultraviolet) and emit a light of a longer wavelength (visible)
Electron Microscopy
Magnification of about 50,000X
Utilize a beam of electrons rather than a beam of light for illumination
Scanning electron microscope (SEM) produces a 3D image
Transmission electron microscope produces a 2D image
Inverted
Light source and condenser located above the stage pointing down
Objectives are below the stage pointing upwards
Used in blood bank to observe RBC reactions (agglutination) in test tubes
What to do when there is not enough light:
- The field diaphragm is closed
- Poor light source – Increase intensity by the rheostat
- Condenser is not properly positioned – Adjust up or down to that light is focused on the specimen
- The NA of the condenser is not adjusted properly for the objective – Remove an eyepiece and adjust the condenser diaphragm
What to do when there is incomplete illumination under low power:
The objective is not clicked in
The flip-top condenser lens is in – It should only be used with objectives of higher magnification
What to do when there is Too much glare:
The diaphragm is open too far – Set it until the light just illuminates the field
The light source is too intense – Adjust the rheostat
The condenser diaphragm is open too far
What to do when there is Poor definition with one of the objectives:
Objective may be loose
Objective is dirty – Clean with moistened lens paper, pat dry with clean lens paper
What to do when there is Failure to focus with oil immersion lens:
Coverslip is too thick
Slide may be upside down
What to do when there is Dirt obscuring image:
Dirty objective, eyepiece, or slide – clean accordingly
What to do when the Image shifts:
- Slide not secure on stage – Ensure it’s in the metal clips
- Stage screws may be loose
What to do when there is Discomfort in one eye:
The eyepiece is not properly adjusted for that eye – Focus for the right with the focusing knob and focus the left with the ocular focusing collar
What to do when you have a double image:
Interpupillary distance not correct
What to do when you have Eye fatigue:
- maybe be due to any of the other trouble shooting problems.
- Inter-pupillary distance
- Dirty Objective, slide or eyepiece
- Rheostat
