Biology
234 (Spring, 2004)
Cellular
Ultrastructure
and
Transmission Electron Microscopy
The Bio 234 graduate laboratory is
designed to give you a working knowledge of and competence in the standard
techniques of transmission electron microscopy.
Those techniques include specimen preparation, sectioning, poststaining
of thin and thick sections and negative staining, operation of a TEM,
photographic processing and printing, evaluation of negative and print quality,
and identification of subcellular structures.
Scheduled laboratory meetings for the first half of the semester will be
devoted to demonstrations of the essential procedures. Limited time in the first six weeks will be
allotted to individual work. However,
you will have access to the EM lab (DH 442) and prep room (DH 449) for the
entire semester to fulfill the requirements of the course. Individual attention will be provided during
lab periods of the second half of the semester.
Lecture (course
code 20692) TTh 0730-0820, DH 245
Lab (code
20693) TTh 0830-1120, DH 442 (EM lab; 924-4907) or
449 (prep room; 924-4888)
Instructors OFFICE PHONE OFFICE HOURS
Dr. David K. Bruck DH 349 924-4837 W
230-430, others TBA
Reza Ehsanian DH 242 924-4887 TBA
Required Text Bozzola, J.J. and
Russell, L.D. 1999. Electron Microscopy. Principles and Techniques for
Biologists. 2nd edition. Jones and
Bartlett, Boston. 670 pp.
Recommended Text
Cross, P.C. and Mercer, K.L. 1993. Cell and Tissue Ultrastructure. A Functional Perspective. W.H. Freeman, N.Y.
420 pp.
Materials
Provided
|
- 100 grids |
- 6 rolls film (Kodak Technical Pan
6415; 120 mm |
|
- grid box |
- vials, envelopes, or boxes for
block storage |
|
- glass knife holder |
-
glassine envelopes |
|
- slide box - jeweler’s forceps |
-
25 sheets photographic paper (Kodak Polycontrast F: multigrade, RC,
8x10" |
Note that if these supplies are depleted,
you will need to obtain your own. Please
return the jeweler’s forceps to me at the end of the semester.
Units
4
Prerequisites Biol 3 (Cell biology)
or equivalent; Phys 2A, B or equivalent; Chem 135 or equivalent; Biol 100W or
equivalent; permission of instructor
Attendance
Attendance is required during scheduled
lecture sections throughout the semester.
As this class is primarily concerned with lab activities, formal
lectures will occasionally be substituted by the start of a lab procedure. Attendance in lab is required during all
scheduled sessions. During sessions
designated on the schedule for completion of projects, you may choose your own
hours unless notified of required demonstrations, typically each day at the
beginning of the laboratory period. The
instructors, however, will be available during each regular lab period for
questions and individual help. The EM
lab and prep room will be locked at all times, but I will authorize keys for
them.
Reserve lists for the ultramicrotomes, EM,
and darkroom(s) will be posted.
Reservations can be made for 2-hour blocks two weeks in advance. Please try not to miss a reserved session, as
others will not be able to plan on the vacancy.
If a reserver is late by 10 minutes, that person forfeits the block to
whomever claims it.
Laboratory
Policies
Lab cleanliness and order are
necessities in any electron microscopy lab.
The slightest amount of debris will ruin a section. Please keep the lab and prep room clean, and
put all materials in their proper place when you have finished with them. Walk softly when sectioning, staining, and
Formvar coating are being done. Replenish all photographic chemicals
whenever a container is emptied. Cover
all instruments with plastic covers, when applicable. Return all instruments to their original
settings. Above all, make an effort to
get along and be considerate of your classmates. Equipment (knife maker, ultramicrotomes, EM)
may not be used in the absence of an instructor until a checkout has been run.
Grading and Assignments
The laboratory grade will constitute the
majority of the course grade with possible lecture exams used to adjust for
borderline cases. The lab grade will be
based on individual performance and the final write-up. The write-up will consist of a minimum of 12
micrographs, legends, and a description of the individual project. The micrographs must be of publishable
quality and fully labeled, including a magnification bar. The legends must fully explain the
micrographs; they should include the organisms, organ, and cell type shown, a
description of the content of the micrograph, and the designation for each label
in the micrograph. The project
description should be about three pages and should include a description of the
structure and function of the organ studied that uses the information in your
own micrographs to at least partially illustrate that description. Under
no circumstances will any copying, even of partial sentences, from any source be
permitted; lifting phrases, sentences, and paragraphs from the literature is
plagiarism and it will be dealt with severely. The write-up will be judged by the quality of
the micrographs, quality of the writing, and the completeness of the
descriptions. Each of the required 12
micrographs must be no smaller than 3 x 4 inches. The micrographs required are as follows:
1) Mouse (2 organs) 5
2) Negative-Stained Bacterium or Virus 1
3) Individual Project (1 mouse organ) 6 (different from any of those
in no. 1 above)
These
are minimum numbers; you may produce as many additional micrographs as you
wish. One micrograph of my choice (preferably
one from the individual project) with full legend will be taken for display of
course activities at the end of the semester.
University and departmental guidelines
require serious and compelling reasons to drop a course. Grades alone do not constitute reason for
dropping a course (see university catalogue).
A lab fee has been charged among your registration fees for this
course. Note that Incompletes are
strongly discouraged and will be awarded only with exceedingly good
reasons. They will not be given when any
equipment in the EM Facility must be used to complete the project.
Individual Project
Each student must conduct an individual
study concentrating on one rodent organ.
Examples of past projects will be available. The project will be presented orally to the
class during Finals’ Week.
Bio 234
Tentative Laboratory Schedule
Date Topic Reading in B&R
Th,
Jan 29 Introduction; Mouse perfusion
and primary fixation 18-21,
24, 25-27
T, Feb 3 Specimen
prep: secondary fixation, dehydration, infiltration 21-23, 24, 34-35
Th,
Jan 5 Specimen prep: infiltration,
embedment, resin curing 35-37
T, Feb 10 Block
trimming; knife making 75-78
Th,
Feb 12 Knife making; block trimming 82-86,
89
T, Feb 17 Formvar
coating; thick sectioning 90-96
Th,
Feb 19 Thick sectioning; Formvar
coating 74,
79-82
T, Feb 24 Ultramicrotomy:
thin sectioning 74,
97-109
Th,
Feb 26 Ultramicrotomy: thin sectioning
T, Mar 2 Ultramicrotomy:
thin sectioning
Th,
Mar 4 TEM 163-201
T, Mar 9 TEM
Th,
Mar 11 TEM
T, Mar 16 TEM
Th,
Mar 18 TEM
T, Mar 23 TEM:
camera use; film processing 243-249
Th,
Mar 25 Section staining 122-130
M-F, Mar 29-Apr
2 Spring Break
T, Apr 6 Printing;
print mounting and labeling; mag bars 250-257
Th,
Apr 8 Carbon coating; negative
staining 130-133,
135-145
T, Apr 13 Projects
Th,
Apr 15 Projects
T, Apr 20 Projects
Th,
Apr 22 Projects
T, Apr 27 Projects
Th,
Apr 29 Projects
T, May 4 Projects
Th,
May 6 Projects
T, May 11 Projects
Th,
May 13 Projects
T, May 18 Projects;
slide making 257-260
W, May 26 Oral
presentations (0945-1200, DH 249)
Bio 234 Lecture
Topics and Reading List
Rodent
Handling, Sedation, Dissection, and Perfusion; Specimen Preparation
Resolution and Magnification
-
Introduction 1:4-6
-
Historical Perspective 1:6-9
-
Development of the Electron Microscope 1:9-10
-
Contributions to Biology and the Future of Electron Microscopy 1:12
-
Introduction 6:150-151
-
Equation 6-2: Calculation of Optimal Resolving Power of Light Microscope 6:155
-
Electrons, Waves, and Resolution 6:155-156
-
Magnification 6:162
Fixation
-
Introduction 2:18-19
-
Fixation 2:19-20
-
The Mechanism of Chemical Fixation for Electron Microscopy; Glutaraldehyde
2:20-21
-
Osmium Tetroxide 2:21-22
-
Selection of a Fixative and A Buffer 2:22-23
-
Obtaining and Preparing Buffered Glutaraldehyde Fixative 2:24
-
Obtaining and Preparing Osmium Fixative
-
Fixation Conditions 2:26-27
-
Microwave-Assisted Specimen Preparation 2:27-30
-
Freezing Method for Specimen Preparation 2:30-31
- Popular Fixation Protocols Other than
Glutaraldehyde-Osmium Tetroxide; Karnovsky’s Fixative 2:31
- Osmium-Reduced Ferrocyanide 2:31-33
-
Potassium Permanganate 2:33
- Fixative Additives 2:33
-
Embedding Cell Fractions 2:41-42
-
Embedding Tissue Culture Cells 2:43
Fixation
Quality
- Tissue Volume Changes During Specimen
Preparation 2:45
- Judging Adequate Specimen Preparation
2:45-46
- Fixation Artifacts 19:453-455
-
Interpreting Dynamic Processes from Static Images 19:474
Washing,
Prestaining, and Dehydration
- Washing 2:34
- Preemebedding, Positive Staining with
Uranyl Salts 5:111-112
- Dehydration 2:34-35
- Use of Transitional Solvents 2:35
Infiltration
and Embedment
- Infiltration
of Resin 2:35-36
- Embedding 2:36
- Epon Embedding 2:36
- Measuring Embedding Media 2:36
- Mixing Embedding Media 2:36
- Other Embedments and Their Use 2:36-39
-
Curing of the Embedment 2:39-41
Sectioning
and Staining Quality
- Introduction 5:122
- Positive Staining 5:122-123
- Preembedding, Positive Staining Uranyl
Salts 5:123-124
- Postembedding Staining with Uranyl Salts
5:124-126
- Postembedding Lead Staining 5:127-129
- Microwave Staining 5:129
- Staining Many Grids 5:129-130
- Dehydration, Infiltration, and Embedding
Artifacts 19:455-456
- Sectioning Artifacts 19:456-464
- Staining Artifacts 19:464-466
Optics
and Diffraction
- Visible Light, Electrons, and Lenses,
Electromagnetic Radiation and the Diffraction Phenomenon 6:151-153
- Effect of Diffraction on Resolution
6:153-155
Lens
Aberrations
- Defects in Lenses 6:158-161
- Apertures in Objective Lens 6:176-177
- Equation 6.8: Depth of Field 6:177
- Equation 6.9: Depth of Focus 6:179
Electromagnetic
Lenses
- General Design of Lenses 6:156-157
- Design of Electromagnetic Lenses
6:157-158
TEM
Column
- Comparison of Light Microscope to
Transmission Electron Microscope 6:163
- Basic Systems Making Up a Transmission
Electron Microscope 6:163-164
- Illuminating System 6:164
- Electron Gun 6:164-171
- Condenser Lenses 6:171-173
- Specimen Manipulation System 6:173-175
- Imaging System 6:175
- Objective Lens 6:175-178
- Intermediate (Diffraction) Lens
6:178-179
- Projector Lens 6:179
- Viewing System and Camera 6:179-180
- Alignment Theory 6:170-173
- Major Operational Modes of the
Transmission Electron Microscope 6:192
- High Contrast 6:192-193
- High Resolution 6:193-194
- Electrical Stability 6:196
- Image Drift 6:196
- Contamination 6:197
- Magnification 6:197
- Magnification Calibration 6:197-198
Microscope
and Photographic Mishaps
- Introduction to Viewing Biological
Electron Micrographs 19:444-446
- Interpretation of Normal Tissue
Structure 19:446
- Magnification and Resolution 19:446-447
- Membranes 19:447-448
- Shape, Kinds, and Number of Structures
19:448-453
- Microscope Artifacts 19:466-473
- Photographic Artifacts 19:473
Vacuum
Systems
- Vacuum System 6:180
- Vacuum Terminology 6:180-181
- Rotary and Diffusion Pumps 6:181-184
- Reading Vacuum Levels 6:184
- Total Vacuum Systems 6:185
- Vacuum Problems and Safety Features
6:185-187
- Other Types of Vacuum Pumps (Ion pumps)
6:187-188
Ultrastructure
of the Cell Surface
- The Cell Surface 20:480
- The Lipid Bilayer of the Plasmalemma
20:480-482
- The Glycocalyx 20:482
- Cell Junctions 20:483488
- Cell Surface Specializations 20:488-505
- Collagen 20:571-574
- Basal Lamina 20:575
- The Cell Wall 20:583-584
Endoplasmic
Reticulum and Golgi Apparatus
- Free Ribosomes 20:535
- Membrane-Bound Ribosomes 20:535
- Rough Endoplasmic Reticulum 20:535-537
- Smooth Endoplasmic Reticulum 20:537-538
- The Golgi Apparatus 20:538-542
Lysosomes
and Peroxisomes
- The Lysosomal System 20:556-558
- Multivesicular Bodies 20:558-562
- Peroxisomes or Microbodies 20:563-
Mitochondria
- Mitochondria 20:528-534
Cytoplasmic
Inclusions
- Secretory Products 20:542-548
- Glycogen 20:564-567
- Lipid 20:568
- Crystalloids 20:568-571
Cytoskeletal
Elements
- The Cytoskeleton 20:506
- Microtubules 20:506-509
- Microfilaments 20:509-510
- Intermediate Filaments 20:510-512
- Centrioles 20:549-551
- Cilia and Flagella 20:551-555
Nuclei.
- The Nucleus 20:513
- The Nuclear Envelope and Nuclear Lamina
20:514-515
- Chromatin 20:515-518
- The Nucleolus 20:518
- Dividing Animal Cells 20:518-521
- Cells without Nuclei 20:521
- The Synaptonemal Complex 20:521-527
Immunocytochemistry
- Cryoultramicrotomy 4:109-117
- Chapter 9 (Immunocytochemistry), pp.
262-280
Autoradiography
- Chapter 11 (Autoradiography;
Radioautography), pp. 292-308
Localization
Techniques
- Chapter 10 (Enzyme Cytochemistry), pp.
282-291
- Chapter 12 (Miscellaneous Localization
and Enhancement Techniques), pp. 310-318
- Chapter 17 (Tracers), pp. 407-413
Additional
Methods
- Negative Staining 5:130-134
- Metal Shadowing Techniques 5:135
- Metal Evaporation Procedures 5:136-140
- Some Applications of Metal Shadowing and
Negative Staining 5:140-145
- Chapter 15 (The Analytical Electron
Microscope), pp. 368-395
- Chapter 16 (Intermediate and High
Voltage Microscopy), pp. 396-405
- Chapter 13 (Quantitative Electron
Microcroscopy), pp. 320-340