Beruflich Dokumente
Kultur Dokumente
(Spring 2018)
Lectures:
Mon. & Wed. 8:40 - 9:55am Tudorita Tumbar (guest lecturer)
175 Warren Hall Dept. of MBG
Biotech Bldg 258 (tt252@cornell.edu, 255-6542)
Instructors:
Chun Han TAs:
Dept. of MBG and Weill Inst. Cell & Mol Bio Hui Ji (Ph.D. student in GGD Field)
Weill Hall 435 (CH599@cornell.edu, 255-7855) (hj377@cornell.edu)
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Course objectives
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How to achieve the learning goals?
• How do we learn?
– Constructivism
– People construct their own understanding and knowledge of the world, through experiencing
things and reflecting on those experiences.
– Knowledge cannot be transferred from one person to another; knowledge must be created by
each learner.
– The learner needs to play an active role, by asking questions, exploring, and assessing
what’s known.
• Ways to enhance learning: techniques you can use on your own
– Retrieving information: rephrasing
– Drawing: diagrams, structures
– Relate to what you have already known: building analogy
– Synthesize concepts to solve problems
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Learning cell biology
• Understanding vs. Memorizing
– Knowledge is understanding of facts, concepts, and the relations among them
– Role of facts and memorizing
– Connection is the key
• The role of lectures
– Clarify key concepts: what’s important and why it is important
– Answer representative or difficult questions
– Give you opportunities to solve problems
– Assess the effect of learning
• Required reading
– Pre-class reading
– Post-class reading: read on your own slides; slide notes
• Finding answers to your questions
– Textbook
– Internet search
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– Discussion
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Lecture format
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Textbook
• Molecular Cell Biology, by Lodish et al, 8th edition (2016).
– Most of the figures in lectures will be from this text and edition, unless marked otherwise.
Thus, for example, “Fig. 8-12” means that this is Figure 12 in Lodish et al, Chapter 8. A
few figures are from the earlier editions of the same text, which then are labeled, for
example, “Old Fig. 5-2”. A conversion table from 7th to 8th is available on Blackboard.
– LODISH et al is on 2h RESERVE IN MANN LIBRARY (both 7 th and 8th).
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Resources on Blackboard
• Lecture slides will be uploaded to Blackboard
– In both PPT and PDF notes
– In the “Content” folder.
– Upload by 6 pm the evening before lecture
• Study questions (problem sets)
– Posted weekly or bi-weekly on Blackboard; answers will be posted later.
– Not graded.
• Other materials
– Instructions for the essays
– Answer keys for the prelims and quizzes.
– Videos shown in lectures
• Be sure that Blackboard knows that you are taking this course. Otherwise
attendance scored by i>clicker won’t work. GET A CLICKER and register it,
or GET APP FOR YOUR SMART PHONE.
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Use Blackboard Piazza for questions and discussion
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Grading
The final grade will be based on three exams, a short essay, biweekly 15 minute in-class quizzes, and
attendance as scored by i>clicker. Each essay (1000 words) will focus on an assigned review article in cell biology.
NOTE: All three exams must be completed. No exam grades are thrown out. Make-up exams will be given only in
case of conflicts with other prelims, serious medical or other problems. (Studying for MCATs is not considered a
conflict.)
In previous years the median course grade in BIOMG 4320 was near the border between B and B+, in the low B+
range.
For the 10% of the final course grade that is for attendance, here is the letter grade
conversion: Missed 0 - 3 lectures = A; missed 4 - 6 lectures = B; missed 7 - 9 lectures = C;
missed 9 - 12 lectures = D; missed >12 lectures = F. (No “plus” or “minus” grades will be
given. Attendance will be taken by Clicker starting Mon Jan 30. It is YOUR RESONSIBILITY to
have a functioning Clicker.
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Instructors
Chun Han Volker M. Vogt
Research interest: dendrite morphogenesis and dendrite Research interest: structure and assembly of retroviruses
degeneration of neurons using Drosophila (fruit flies) as like HIV
the model system.
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You don’t need to remember these, but if you are curious, the EM samples are:
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BIOMG 4330
• 1-credit “companion course” to 4320: “Research Papers in Cell Biology”.
• If you are Molec & Cell Bio concentrator, have two 3-credit courses from the list and thus
need 1 more credit to get to the 7 required, consider 4330.
• Enrollment limited to 20 students. Letter grade. Recommended especially for pre-grads.
Meets once a week on Friday mornings (same time as lectures), in Comstock B106.
• Go over experimental logic and data from an assigned research paper each
meeting. Subject will be related to the subject that week in 4320 lectures.
• Enrolled students present the papers (a pair of students for each paper). Meet with Dr.
Vogt to help prepare presentation. Everyone joins discussion during presentation.
• Homework before each meeting: everyone sends answers to ~3 straightforward questions
given out the previous week, to establish that you have read and thought about the paper.
Send to Vogt by email before 8:40am Friday.
• There will be one quiz the last day of the course, May 4, which will NOT count toward your
course grade, but is intended only as feedback for the instructor.
• Come to organizational meeting THIS FRIDAY 9:05am in Comstock B106. (All future
meetings of the class will start at 8:40am.)
• As of yesterday, the 4330 had 12 students enrolled. 11
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Overview of the course
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What we will cover today
• Tree of Life
– Prokaryotes vs eukaryotes
– Model organisms useful for cell biology studies
• Methodology of cell biology: microscopy
– Definition of resolution and resolutions of various type of microscopy
– Dimensions of representative cellular structures
– Light microscopy
• Transmitted light microscopy
• Fluorescence microscopy
– Confocal microscopy
– Other types of fluorescence microscopies
• De-convolution microscopy
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Diversity of cells
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To learn:
Nothing to remember for this slide. Understand the concept.
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Mouse,
Tree of life Human
Yeast
• Biological systems follow the rules of chemistry and physics, but biological
structures and processes have evolved along different paths under the
pressures of natural selection for billions of years.
• Although highly diverged, all biological systems are composed of cells
containing the same types of chemical molecules and employing similar
principles of organization at the cellular level.
• Similarities across biological systems make investigations of model organisms
informative for understanding fundamental cellular processes.
FIGURE 1-1 All living organisms descended from a common ancestral cell.
• All organisms evolved from a single-celled ancestor along three major branches of the
family tree.
• Gene duplications and mutations gave rise to new organisms during evolution.
• Tree branch relationships were assigned by similarities in organismal morphological
features and in DNA and protein sequences.
• Mitochondria (first, in all eukaryotic cells) and chloroplast (later) organelles were formed
from bacteria incorporated as endosymbionts into precursor eukaryotic cells.
Questions:
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1. What are good bases for comparing distantly related species?
2. What about closely related species?
To learn:
Major life domains. Bases of classification.
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Prokaryotic and Eukaryotic cell structures
Prokaryotic Eukaryotic
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Figs 1.11, 1.12
This course is about eukaryotic cells. What are their distinguishing features?
*A most obvious feature: many compartments bounded by single or double membranes.
You should be familiar with pictures of cells like the one at the bottom (TEM).
To learn:
Main similarities and differences between prokaryotes and eukaryotes
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Similarities in prokaryotic and eukaryotic cells
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*Does all of life have same genetic code? No-- some variation, eg. Yeast mitochondria…
*RNA polymerase that makes RNA, and ribosomes that make proteins, clearly stem from
common ancestor-- homologous polypeptides in many cases with obvious sequence
similarity, but prokaryotic proteins are usually simpler
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Features found in eukaryotic but not prokaryotic cells
*Cells have nucleus and cytoplasm, separated by nuclear membrane.
*Complex chromosomes composed of DNA and proteins capable of compacting DNA
into mitotic structures.
*Complex membranous organelles (ER, Golgi, lysosomes).
*Specialized structures for respiration (mitochondria) and photosynthesis
(chloroplasts).
*Complex cytoskeleton (microfilaments, microtubules, intermediate filaments).
*Complex cilia and flagella.
*Capable of ingesting fluid and particulate material by endocytosis (for nutrients and
host defense).
*Cell division via complex microtubule-based spindle apparatus to separate
chromosomes.
*Sexual reproduction requiring meiosis and fertilization.
*Presence of two copies of genes (diploid), one from each parent.
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These things in this list are what much of this course is about. Get some impression first,
but many topics will be dealt with in more depth later.
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Model organisms used in cell biology
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FIGURE 1-22 Each eukaryotic organism used in cell biology has advantages for
certain types of studies.
• Model unicellular and multicellular eukaryotic organisms have distinct advantages for
investigation of fundamental cell processes.
To learn:
1. Some of these model organisms will be discussed in more details in future lectures. It’s
helpful to come back to this slides later to appreciate how each organism helped biological
research.
2. You don’t need to remember all details. But knowing these will help you to understand
biological discoveries in the context of biological systems.
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Different types of microscopy to ‘see’ cells and into cells
Fluorescence microscopy Transmission (thin section)
electron microscopy
Fig 4-14
Super-resolution microscopy
Scanning electron microscopy
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Old Fig 9-24
http://ibidi.com/
We will talk about these techniques in more details. But you can keep these questions in
mind: what are the advantages and limitations of each technique? What types of objects
are expect to see with each?
To learn:
Be able to recognize specific microscopy technology used to generate an image. More later.
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Optical microscope and definition
of ‘resolution’
Resolution:
Fig 4-9
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The most important property of any microscope is not its magnification, but its resolving
power, or resolution: the ability to distinguish between two very closely positioned objects.
*Resolution (D) is defined as the smallest distance between two points
at which the points can be recognized as two entities instead of one.
In the formula: α is the angular aperture, or half-angle, of the cone of light entering the
objective lens from the specimen; N is the refractive index of the medium between the
specimen and the objective lens (i.e., the relative velocity of light in the medium compared
with the velocity in air), and λ is the wavelength of the incident light.
Questions:
The smaller the value of D, the higher or lower is the resolution?
What would make a high resolution objective?
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Lenses for high-resolution microscopy are designed to work with oil between the lens and
the specimen since oil has a higher refractive index (1.56, compared with 1.0 for air and 1.3
for water). To maximize the angle α, and hence sin α, the lenses are also designed to focus
very close to the thin coverslip covering the specimen. The term N sin α is known as the
numerical aperture (NA) and is usually marked on the objective lens. A good high-
magnification lens has an NA of about 1.4, and the very best lenses—which cost as much as
a medium-sized car—have a value approaching 1.5. Owing to limitations in the values of α, λ,
and N based on the physical properties of light, the limit of resolution of a light microscope
using visible light is about 0.2 µm (200 nm). This is also called “diffraction limited”
resolution.
To learn:
Resolution; numerical aperture; diffraction limit of visible light
You don’t need to know the formula of the resolution, but only the approx. limit
of resolution, and on what this depends.
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Resolutions of different types of microscopy
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Lec 16
Lec 9
Lec 7
Lec 3
Lec 5
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This are examples of structures that are too small to ‘see’ by light microscopy, but about
which we know a lot from biochemical analysis and from other types of microscopy.
*You should have some knowledge of the sizes of structures in the cell. But don’t
“memorize”, just to have an overview.
*For example, is a microtubule larger or smaller in diameter than a DNA molecule?
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Common light microscopes
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FIGURE 4-9 Optical microscopes are commonly configured for bright-field, phase-
contrast, or fluorescence microscopy.
b) In bright-field light microscopy, light from a tungsten lamp is focused on the specimen
(usually stained with dyes to enhance contrast) by a condenser lens below the stage.
c) In phase-contrast (and differential-interference; DIC) microscopy , which increases
contrast of biological specimens, incident light passing through an annular diaphragm
focuses a circular annulus (ring) of light on the specimen. Light passing unobstructed
through the specimen is focused by the objective lens onto the thicker gray ring of the
phase plate, which absorbs some of the direct light and alters its phase by one-quarter
of a wavelength. If a specimen refracts (bends) or diffracts the light because of the
refractive index of the material, the phase of some light waves is altered (green lines),
and the light waves pass through the clear region of the phase plate. The refracted and
unrefracted light is recombined at the image plane to form the image.
d) In fluorescence microscopy, a beam of light from a mercury lamp (gray lines) is directed
to the excitation filter, which allows only the correct wavelength of light to pass
through (green lines). The light is then reflected off a dichroic mirror and through the
objective lens, which focuses it on the specimen. The fluorescent light emitted by the
specimen (red lines) passes up through the objective lens, then through the dichroic
mirror, and is focused and recorded on the detector at the image plane.
In phase contrast: Some of the light stream passes through the phase plate,
retarding phase by one quarter wave length. Some of the light stream passes
through the object of interest. Enhancement of contrast is due to interference
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between these two streams of light when they are brought back together. It is the
density of an object that gives it contrast, NOT light absorption.
*In phase contrast microscopy, edges (e.g. of a cell) look bright (see picture of cell
next slide).
This commonly used for looking at mammalian cells growing in culture, for example.
Principle is that cells are thin and do not absorb light (not pigmented), and to enhance
contrast various tricks are used to enhance contrast. Here, light passes either directly or
through a ring, and these two streams of light can interfere. Changing the phase of the light
that passes through a dense object, because of refraction, will therefore give an enhanced
image. Helps visualize edges and dense objects (eg. Nucleoli).
Question:
What is the role of “dichroic mirror” in fluorescence microscope?
To learn:
Read on your own. Don’t need to remember the exact light paths, but you should be able
recognize the main differences among the techniques.
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Examples of images by transmitted light microscopy
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FIGURE 4-10 Live cells can be visualized by microscopy techniques that generate
contrast by interference.
• Micrographs show the same live, cultured macrophage cells viewed by bright-
field microscopy (left), phase-contrast microscopy (middle), and DIC microscopy
(right).
• In the phase-contrast image, cells are surrounded by alternating dark and light
bands; in-focus and out-of-focus details are simultaneously imaged in a phase
contrast microscope.
• In a DIC image, cells appear in pseudorelief because only a narrow in-focus
(optical slice) region is imaged.
To learn:
You should be able to recognize the microscopy technique used to generate a typical
image.
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Fluorescence microscopy
Examples of Fluorescent Dyes
excitation emission
wavelength
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Modern microscopes used for observing fluorescent samples are configured to pass the
excitation light through the objective lens into the sample and then selectively observe the
emitted fluorescent light coming back through the objective lens from the sample. This is
achieved by reflecting the excitation light with a special type of filter, called a dichroic
mirror, into the sample and allowing the light emitted at the longer wavelength to pass
through to the observer (review Figure 4-9d)
Note that the emitted light is always of longer wavelength than exciting light (except in two
photon microscopy, a technique pioneered here at CU). This technique is so powerful in
cell biology because fluorescent molecules can be used to ‘tag’ specific proteins or other
structures.
To learn:
Principles of fluorescence.
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Examples of fluorescence microscopy
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*Two (or even three, as in this picture) fluorescent dyes of different ‘colors’ can be
visualized in the same sample, if the fluorescence wavelengths are sufficiently different and
if suitable filters are available.
*Dyes are coupled to specific antibodies, which are used to mark the structure of interest.
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CLICKER QUESTION (example, not scored today)
The numerical value for the “resolution” in a
microscope is:
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Fluorescence Microscopy: Laser scanning confocal microscope
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Nowadays much or most fluorescence microscopy is carried out with an instrument called
the confocal microscope. On laser scanning confocal, a pinhole blocks out-of-focus light,
and therefore only the plane that is in the focus is imaged. By imaging serial optical
sections, one can visualize objects in the Z dimension as well as X and Y.
To learn:
The principle of confocal microscopy
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Two types of confocal microscopy
Watch “Laser Scanning confocal light path.mov” in “Movies and animations” folder on BB to understand the role of Dichroic mirror.
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Again, you don’t need to remember the light paths or the parts, but you need to
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understand the principles and the main differences between these two types of
microscopies.
Question:
What are the advantages of the spinning disk microscope, compared to point-scanning
confocal?
To learn:
The differences between the two types of confocal microscopy
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Confocal is a big advantage when the object is thick, as in this large cell.
Since images are captured electronically in digital form, they can also be manipulated.
Thus all the sections can be combined (superimposed) for example. Or the cell can be
turned 90 degrees, and visualized from the side.
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Fluorescence Microscopy: De-convolution microscopy
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*Like laser scanning confocal microscopy, this is a way to obtain sharp optical sections, for
example through a cell. The method, however, is largely computational (very intensive).
*In this example all the optical sections were superimposed (or “projected”) to give a 3D
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look. Projection also can be done with confocal microscopy.
To learn:
The principle. How is it different from confocal microscopy.
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Types of microscopy
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Questions for the next lecture
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