Wesley%20Thompson%20Professor%20in%20Section%20of%20Neurobiology%20Info%20about%20me,%20office%20hours,%20how%20to%20reach%20me%20can%20be%20found%20on%20the%20206%20website:%20www.bio.utexas.edu/courses/bio206/

1
Wesley ThompsonProfessor in Section of
NeurobiologyInfo about me, office hours, how to
reach me can be found on the 206 website
www.bio.utexas.edu/courses/bio206/
2
Wesley ThompsonI will conduct 5 of your labs

1. Microscopy I
2. Microscopy 2
3. Introduction to Electrophysiology
4. Frog Sciatic Nerve
5. Frog Nerve/Muscle

3
Objectives of the Microscopy Labs (this week and
next week)

1. Learn the parts of the microscope and how to use
   them
2. Learn about 3 types of microscopy brightfield,
   phase contrast, fluorescence
3. Understand 3 issues in microscopy and how to
   manipulate your microscope to get what you need
   magnification, resolution, contrast
4. Review the stages of mitosis in preparations you
   make yourself
5. Learn how to measure the size of objects under
   the microscope
6. Learn about computer image capture and processing.

4
Test of CPS System

• A question will be posed
• Are you here today?
• Yes
• No

5
Microscopy

• Why do we use a microscope?

6
Microscopy

• Why do we use a microscope?
• To enable use to see (i.e.image) objects
  that are small.

7
Microscopy

• Why do we use a microscope?
• To enable use to see (i.e.image) objects
  that are small.
• What is the problem here?

8
Microscopy

• Why do we use a microscope?
• To enable use to see (i.e.image) objects
  that are small.
• What is the problem here?
• Our eyes contain light a sheet of light
  detectors. In order to discern the parts of an
  object, light from different parts of that object
  must fall on different light detectors.

9
Microscopy

• Why do we use a microscope?
• To enable use to see (i.e.image) objects
  that are small.
• What is the problem here?
• Our eyes contain light a sheet of light
  detectors. In order to discern the parts of an
  object, light from different parts of that object
  must fall on different light detectors.
• This leads us immediately to concepts of
  magnification and resolution.

10
With chalk on blackboard

• Discuss the wonder of our sense of vision
• Draw an object, its interaction with light, and
  how an organism, like us can detect this object
  and learn something useful about it.
• The wonder of lenses, the refraction of light,
  and a brief discussion of how a convex lens
  brings light from a point to a focus

11
Resolutiondef. Ability to
see individual parts of an object as unique and
separate usually measured as the smallest
distance that can separate two objects and we
still see them as two rather than blurred
together. For naked human eye this is about 0.1
mmWhat sets the limit here?
12
MagnificationBy varying the
curvature of the lens, we can produce different
magnifications. In microscope we do this with an
objective lens, attached to a nosepiece.
13
(No Transcript)
14
(No Transcript)
15
Total magnification
magnification of objective
lens X
magnification of eyepiece lens
16
Eyepieces are commonly 10XObjectives commonly
are 4X-100X Your student microscopes have a
nosepiece and 4X, 10X, 40X, and 100X
objectives
17
CPS So what is the maximum magnification
available on your microscope? A. 10X
B. 40X C. 100X D. 1000X
18
MagnificationWhats the
limit? Why do we not have objective lens ground
to so that they magnify even more than 100X?
19
MagnificationWhats the
limit? Why do we not have objective lens ground
to so that they magnify even more than 100X?
Because there are limits set by the laws of
physics. Due to a phenomenon known as
diffraction.
20
(No Transcript)
21
(No Transcript)
22
Abbes equation
23
Abbes equation What are the limits Wavelength
of light Angle of acceptance of the lens n
24
Dry lenses Use air between
lens and specimen denominator can
approach 1 Oil immersion lenses
Use oil between lens and specimen
denominator can be as high as 1.45
25
(No Transcript)
26
(No Transcript)
27

• CPS If we want better resolution, what could we
  do?
• Use a longer wavelength of light and a higher NA
  objective
• Use a shorter wavelength of light and a lower NA
  objective
• Use a longer wavelength of light and a lower NA
  objective
• Use a shorter wavelength of light and a higher NA
  objective

28
(No Transcript)
29
(No Transcript)
30
Problem of contrast
31
ContrastWe require the objects
we are viewing to differ in the color or
intensity of light that they send to our eyes.
Otherwise, NO VisionHowever, many objects are
transparent.
32
Problem of contrast
Problem Even if we magnify an object so its
different parts fall on different detectors on
the back of our eye, if those parts are of the
same intensity, we will see nothing. There have
to be differences in intensity between the parts
of the image (i.e. contrast) to see an image.
33
(No Transcript)
34
The Condenser
35
The Condenser
What do you use it for? Control illumination
Desire is to get uniform illumination
Condenser moves up and down racking the
condenser You need to know where to put it.
This process is called setting
Kohler Condenser has two diaphragms Field
diaphragm Aperture diaphragm

36
(No Transcript)
37
The condenser has 2 iris diaphragms

• Field diaphragm
• Aperture diaphragm

38
(No Transcript)
39
(No Transcript)
40
The Field DiaphragmControls the positions within
the specimen plane that are illuminated. It
controls what in the specimen plane is
illuminated. The Aperture DiaphragmControls
the angles of light passing through the specimen
plane. It controls the contrast of the specimen
but it also influences the resolution one obtains
in the image.
41
(No Transcript)
42
Another method of inducing contrast
43
Still another method of inducing contrastPhase
contrastMakes use of small phase differences
induced in the light by even transparent objects.

44
Homework for you1. How a lens maps positions
of the specimen to angles and how an lens can
take angles and map them into positions.2.
Understand scattering contrast and how the
aperture diaphragm influences this contrast.3.
Understand concept of conjugate planes in the
microscope.4. How a phase contrast microscope
uses conjugate planes in the condenser and in a
plane behind the objective to amplify the phase
differences induced by the specimen.5. Know the
stages of mitosis.