Lecture 2 Flashcards
Compound Microscope
Works by passing visible light through a specimen
Uses 2 separate lens systems:
Objective -forms a magnified image of the specimen in the optical tube
Eyepiece -further magnifies the image
Compound Microscope
Key characteristics:
Magnification – ability to make a sample appear larger than it is in real life
Resolving power (or Resolution) – a measure of how close two objects can be to each other before they appear as one object
Contrast – ability to enhance parts within a cell
route at looking at something in a compound microscope
eyes -EYEPIECE- OBJECTIVES-SPECIMEN
CONDENSER (IRIS DIAPHRAGM)-FIELD DIAPHRAGM-LAMP
OCULAR LENS
AKA Eyepieces (binocular at Michener)
10x lenses (degree of magnification is 10x)
magnify the intermediate image
limit the area of visibility
INTERPUPILLARY CONTROL
Adjusts the lateral separation of the eyepieces (different for each user)
Adjust Interpupillary Distance (IPD) so that user should be able to focus both eyes comfortably on the specimen and visualize ONE clear image
REVOLVING/ROTATING NOSEPIECE:
3 or 4 different Objective lenses
each has specific power of magnification
engraved on it are their numerical aperture
Numerical Aperture (NA):
how well the lens is able to gather light
Larger NA – greater resolution
Microscope Resolution
needs detail
ability of a lens to separate or distinguish small objects
Wavelength of light used is major factor in resolution shorter wavelength -> greater resolution
the object needs to be distinct, clear sharp
the resolution can be limited by the wavelength and numerical apeture
Resolving Power
Ability of the lens to achieve resolution
Dependent on the objective used and the medium
Air / Oil
Immersion oil
Has the same refractive index as glass
Used with the 50x or 100x objective lenses
Allows the objective lens to collect light from a wide NA
Allows for high resolution of detail
Coverslips provide the same effect as immersion oil
when is Light is refracted (bent)
When passing from one medium to another
Direction and magnitude of bending depends on the two mediums it passes through
Glass and air
Glass and immersion oil
Refractive Index (RA)
measure of how greatly a substance slows the velocity of light
The speed at which light travels in air, divided by the speed at which light travels through a substance, such as immersion oil
OBJECTIVE LENSES
powers of magnification and NA
Low power 10x / 0.25
High dry power 40x / 0.65
Oil immersion 50x / 0.90
Oil immersion 100x / 1.25
The smaller the magnification the larger the viewing field (x10 objective) ZOOM OUT
The larger the magnification the smaller the viewing field (x100 objective) ZOOM IN
Total magnification = magnification of eyepiece x magnification of objective lens
Total Magnification = 10x (eyepiece) x 100x (oil immersion lens) = 1000x
OBJECTIVE LENS SEE THE REAL IMAGE WE SEE THE VIRTUAL
Working Distance
WHY THE OBJECTIVE LENSES ARE DIFFERENT IN LENGTH
Distance between the front of the microscope objective lens and the surface of the specimen or slide coverslip at the point where the specimen is completely in focus
working distance decreases and total magnification increases
Image Aberrations
light rays are deviated through the lens causing the image to be blurred and distorted
Chromatic Aberrations
Result in colour distortion
gives an outline of specimen is blurred and has colored rings
caused by lights of different wavelengths at different focal point
Spheric Aberrations
Produce an image in which the center of the field of view is in focus when the periphery may not be
consequence of using lenses with spherical surfaces
blurred because the light travels through different thicknesses of the lens
Achromatic lenses
Objective lens
Corrects for chromic & spheric aberrations
Brings light of two colours into one common focal point
Plan achromatic lenses
Corrects for chromic & spheric aberrations
Parfocal Objective lens
set of lenses with corresponding focal points all in the same plane
Parcentric Objective lens
A set of lenses where an object in the center of the field of view remains centered when another objective is rotated into use
HEAD (BODY/OPTICAL TUBE)
Houses optical parts of microscope
Connects eyepieces to the objective lenses
NECK/ARM
Attachment site for revolving nosepiece
Used to carry the microscope
STAND
Supports the stage assembly, condenser and base
BASE
Supports the microscope and houses the lamp and field diaphragm
LIGHT SOURCE – Bulb in base – turn on using main switch – adjust amount of light using the Brightness adjustment or Light Intensity Knob
When burns out, lamp must be replaced
STAGE
Horizontal surface that supports the microscope slide for observation
Move slide by hand or translational controls
Specimen holder/Stage clip(s) holds the slide in place
VERNIER SCALE
Graduated locator markings found on the stage
Ability to record the exact location of important specimen details or cells on a slide
X&Y axis coordinates are noted
you can go back to the exact spot with the measurements
STAGE CONTROLS
Located under stage
Can be on right or left (x axis)
front back (y axis)
FOCUS CONTROLS
Allow the specimen to be focused moving the stage up and down
Coarse adjustment knob – Focuses the image under lower power (generally, larger in size and located on arm of microscope)- DO FIRST
Fine adjustment knob – Sharpens the image under all objective powers (generally, smaller in size and located inside coarse adjustment knob)
FIELD DIAPHRAGM
An opaque structure with a central aperture
Located between the light source and the condenser
OPEN ALL LIGHT THROUGH
CLOSED ALL LIGHT CLOSED
CONDENSER
Works in conjunction with the Aperture or Iris Diaphragm
Lens system located directly under stage (made up of one or more lenses)
Can be raised or lowered, to help focus light passing through and can be centered in the field of view
IRIS DIAPHRAGM
Located under stage, incorporated with condenser system
Controls angle and amount of light
VERTICAL ADJUSTMENT CONTROL
Adjusts condenser lighting focus
CONDENSER CENTERING SCREWS
Centers image of field diaphragm
Kohler Illumination
method of specimen illumination
helps to generate even illumination , reduce artifact and high sample contrast
uses diaphragm and condenser
CONSIDERATIONS
for Kohler Illumination
After setting Kohler, when examining a new slide, always bring the specimen into focus using 10x objective first, and then move to higher magnifications
especially important when oil immersion
rotate the nosepiece so the the objectives dont come in contact with the oil
start with the lowest objective with low light intensity and then move on up with higher light intensity
Care of the Microscope
Transport microscope using one hand on the base as a support and one hand firmly around the arm
Clean objective lenses and eyepieces with lens paper and lens cleaner or 70% isopropyl (use sparingly)
Dry lenses with lese paper
NEVER USE:
KIMWIPES or PAPER TOWEL - scratches the lenses
KLEENEX - leaves lint behind
Clean off all residual oil/debris
Avoid placing fingers on lens surfaces
Set Kohler and document in log
Ensure to remove slides from stage when complete
turn off microscope when not in use and set back to 10x
Issue: Microscope light is not ‘on’ even when microscope is ‘switched on.’
check the light source
bulb
plug
plugged in
Issue: Unable to see a clear image, even while focusing on slide
Oculars - Objective lens (both clean or loose) - Condenser (clean, adjusted, kohler set) - Slide (upside down?) - Coverslip
If slides are too thick, rays of light will be focused within the slide and not on the specimen
List common strains caused by improper microscope workstation ergonomics.
strains in the neck, back, forearms, wrists, hands and legs/feet may result, which can lead to Musculoskeletal Disorders (MSDs)
eyestrain
List three steps to ensure the eyepieces (oculars) of your microscope are properly positioned.
Interpupillary distance of binocular eyepieces should be adjusted to ensure that both eyes are focusing comfortably
Microscope eyepieces should be at an angle of no more than 30°above the horizontal
Describe proper positioning of your neck and back while sitting at the microscope workstation.
Neck and Head should bend as little as possible, preferably no more than 10-15º below the horizontal
Back should be:
Completely upright and flat against the back rest and feet supported equally on the floor or footrest
Describe the proper positioning of your legs
feet should rest firmly on the floor or a footrest,
wrists should be straight or neutral
List five strategies used to reduce fatigue and prevent strain.
adequate leg room
height-adjustable chair
breaks
stretch
take breaks - 15 mins
