Transcript Objectives - University of Texas at Austin

Wesley Thompson
Professor in Section of Neurobiology
Info about me, office hours, how to reach
me can be found on the 206 website:
www.bio.utexas.edu/courses/bio206/
Wesley Thompson
I will conduct 5 of your labs
1.
2.
3.
4.
5.
Microscopy I
Microscopy 2
Introduction to Electrophysiology
Frog Sciatic Nerve
Frog Nerve/Muscle
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.
Test of CPS System
A question will be posed
Are you here today?
A. Yes
B. No
Microscopy
Why do we use a microscope?
Microscopy
Why do we use a microscope?
To enable use to see (i.e.”image”) objects that are small.
Microscopy
Why do we use a microscope?
To enable use to see (i.e.”image) objects that are small.
What is the problem here?
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.
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.
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
Resolution:
def. 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 mm
What sets the limit here?
Magnification:
By varying the curvature of the lens, we can
produce different magnifications. In
microscope we do this with an objective lens,
attached to a nosepiece.
Total magnification:
=
magnification of objective lens
X
magnification of eyepiece lens
Eyepieces are commonly 10X
Objectives commonly are 4X-100X
Your student microscopes have a nosepiece
and 4X, 10X, 40X, and 100X objectives
CPS: So what is the maximum
magnification available on your
microscope?
A. 10X
B. 40X
C. 100X
D. 1000X
Magnification:
What’s the limit? Why do we not have
objective lens ground to so that they magnify
even more than 100X?
Magnification:
What’s 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.
Abbe’s equation:
Abbe’s equation:
What are the limits:
Wavelength of light
Angle of acceptance of the lens
n
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
CPS: If we want better resolution, what could we do?
A.Use a longer wavelength of light and a higher NA
objective
B.Use a shorter wavelength of light and a lower NA
objective
C.Use a longer wavelength of light and a lower NA
objective
D.Use a shorter wavelength of light and a higher NA
objective
Problem of contrast
Contrast
We require the objects we are viewing to differ
in the color or intensity of light that they send
to our eyes. Otherwise, NO Vision
However, many objects are transparent.
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”.
The Condenser
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
The condenser has 2 iris
diaphragms
Field diaphragm
Aperture diaphragm
The Field Diaphragm
Controls the positions within the specimen plane that are
illuminated. It controls what in the specimen plane is
illuminated.
The Aperture Diaphragm
Controls 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.
Another method of inducing
contrast
Still another method of
inducing contrast:
Phase contrast
Makes use of small phase
differences induced in the light
by even transparent objects.
Homework for you:
1. 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.