Week 2
Microscopic Anatomy
Expanded Studies
OBJECTIVES
• Knowing and using the
compound light microscope
• Slide preparation
• Parts of cell
• Embryonic germ layers
Objective 1:
The Compound Light Microscope
• Parts of the Microscope
• Focusing Procedures
• Useful Terms
• Microscope Care & Handling
Parts of the Microscope
Revolving
Substage light
Can you name the following
parts and their function?
How about part number:
•
•
•
•
9
11
3
1
Mechanical stage
Iris diaphragm
Arm
Ocular lens
Microscope Care & Handling
• Always use both hands when transporting the microscope
• Clean lenses before and after use
• Never use anything but lens paper to clean lenses
• Before plugging in the microscope, check to make sure all
components are in proper “starting position”
“Starting Position” of Microscope
1. On/Off switch Æ OFF
2. Objective lenses Æ Rotated such that 4X
lens (shortest) is facing down
3. Stage and Objective lenses as close
together as possible (lenses lowered or
stage raised)
4. Substage condenser lens in raised position
5. Iris diaphragm turned to dimmest setting
Focusing Procedures
1. Slide placement
2. Ocular lens adjustment & focusing
3. Light adjustment
1. Dimmer
2. Diaphragm
3. Substage condenser
4. Objective lens rules
1. 4X or 10X first
2. Objective and stage close together
5. Focusing
1. Course adjustment - 4X or 10X only
2. Fine adjustment
6. Readjusting light with increasing magnification
7. Oil immersion lens rules
Microscope Care & Handling
When finished
• Clean lenses before and after use
• Never use anything but lens paper to clean lenses
• Make sure you have removed the slide
• Turn light OFF
• Rotate the nosepiece to the lowest power objective
• Position the stage & the objective lenses as close together
as possible
• Raise condenser lens
• Switch diaphragm lever to dimmest setting
• Return the microscope to it’s proper place
Useful Terms
Field of Vision:
40X
100X
Contrast:
Use iris
diaphragm to
adjust the
contrast
Increased contrast
Decreased contrast
vs Brightness:
Use dimmer
to adjust the
brightness
Increased brightness
Decreased brightness
Working Distance:
Reminder: Do not use Course Adjustment Knob when either High Power
lens or Oil Immersion lens are down
Total magnification is calculated by multiplying the
magnification of the objective lens by the
magnification of the ocular lens
TM =
(Objective lens)
x
(Ocular lens)
X
4X
10X
40X
100X
10X
TM = (Objective lens) x (Ocular lens)
Try these:
The total magnification of this Ə is 40X.
The magnification of the ocular lens is 10X.
Which objective lens was used?
The total magnification of this Ə is 400X.
Which objective lens was used?
Objectives 2:
Specimen Preparation
Always remember:
Structures, organs,
and even individual
cells can be
sectioned in
different planes.
Does this sound
familiar?
LS
WM
CS
There are several ways a specimen can be
prepared for light microscopy:
• Wholemount - the entire structure is mounted on the slide
• Section - a thin slice of a tissue or organ is placed
on a glass slide
• Smear - most suitable when viewing cell suspensions
such as blood, urine, mucus, cyst fluid, etc...
• Squash - cells are broken by applying pressure to the
slide, intracellular contents are liberated,
e.g., chromosomes
Whole Mount Preparation
Different planes of section through the
seminiferous tubules of the testes
Examples of Sections
Nerve Fibers, CS
Nerve Fibers, LS
Longitudinal Section
Cross Section
lum
en
en
m
u
l
Cross Section
Lumen
Lumen
Cross Section
e
Lum
n
Lumen
Examples of a Smear Preparation
Sperm
Blood
High magnification vs Oil immersion
Example of a Squash Preparation
Chromosomes
Staining
The majority of specimens are stained with a
combination of dyes that highlight
differences between cellular structure
This is an H&E stained section
of the kidney. The darker blue
structures are the nuclei of the
cells
See Lab Manual Appendix
Some Common Stains Used in Histology
or:
You can’t judge a slide by it’s color
Liver
Hematoxylin and Eosin
(H & E)
Liver
Iron stain
Several steps are used to make a section
Fixation: a specimen is treated with a chemical solution to:
(1) prevent bacterial action and self destruction by cellular
enzymes
(2) ensure preservation and hardening
Dehydration and Embedding
In order for the tissue to be sectioned, it must be “supported”
to ensure accurate cutting. Paraffin wax is often used for
this purpose, using the following procedure:
– dehydration of the tissue by passing it through a series
of alcohol/water mixtures, since wax won’t mix with the
water naturally found in the tissue
– clearing, which removes the alcohol
– transfer into molten wax inside an embedding oven
Dehydration
Embedding
Sectioning
•
•
•
a microtome is used to slice the embedded tissue into thin slices
(1-150 micrometers thick)
the cut sections are then floated onto a water covered slide to
remove any creasing
the slides are then drained and placed over a gentle heat to dry off
the water and ensure that the tissue adheres to the slide
Objective 3:
Generalized Mammalian Cell
Some cells of the body
(Simple columnar)
(Osteocyte)
(Adipocyte)
You will be responsible for knowing the various cells of the
body by name and their parts
Parts of a generalized cell
Plasma Membrane
The other organelles are not visible under the light microscope
Identify these parts of a cell as seen under
the microscope:
Nucleus
Cytoplasm
Plasma
membrane
Renal Tubule, cs
Objectives 4:
Embryonic Germ Layers
Pluripotent Stem Cells
Stem Cell Tutorials
Click on the links below to learn more about stem cells &
stem cell research:
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/S/Stem_Cells.html
http://www.umich.edu/stemcell/tutorial/
- requires Flash Player
- check the links above for more information, or if you do not have Flash Player
Totipotent vs. Pluripotent Stem Cells
The embryonic germ layers are:
Ectoderm
Mesoderm
Endoderm
They will give rise to all the different tissues and
organs of the body
Embryonic Germ Layers
Section taken from a blastocyst, around the
2nd week of development
For the Practical:
• Know the microscope parts
• Be able to calculate TM
• Identify the specimen under the microscope
(vessel, tubule, chromosomes, sperm)
• Identify the mount preparation
(wm, cs, ls, sq, sm)
• Identify the embryonic germ layer &
• Name one adult tissue (organ) that is
formed from this layer