medical school histology basics introduction to microscopy larry johnson texas a&m university...
TRANSCRIPT
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Medical School Histology Basics Introduction to Microscopy
Larry Johnson Texas A&M University
VIBS 289 lab
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Objectives
• Learn the difference in magnification and resolution
• Learn about different types of staining for LM and observe details of cells by EM
• Learn how cell/organelles look different at different magnifications
• Learn about different types of EM
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Use your atlas!
pancreas
blood
testis
stomach
Use your atlas!
Use your atlas!
Use your atlas!
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MAGNIFICATION VS. RESOLUTION
1. MAGNIFICATION - INCREASE IN IMAGE SIZE
2. RESOLUTION - SMALLEST DISTANCE BETWEEN TWO POINTS THAT CAN BE SEEN (DISTINGUISHED)
RESOLUTION is CALCULATED BY
0.61 (WAVELENGTH)/NUMERICAL APERTURE
0.25 um FOR LIGHT MICROSCOPE
0.1 nm FOR ELECTRON MICROSCOPE
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Blood
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Blood
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Peripheral blood smear (May-Grunwald-Giemsa)113
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Peripheral blood smear (Leishman-Giemsa)110
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Peripheral blood smear (Leishman-Giemsa)basophil, and neutrophils110
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Slide 113 human blood
Neutrophils
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Slide Histo021 human blood
Neutrophils
Neutrophils
Platelets
Red blood cells
Neutrophil cytoplasm will merely have a granular appearance
Nucleus
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EM 8f: Peripheral blood cells; 9,000x
1. Monocyte
2. Lymphocyte
3. Neutrophil
Granules
Neutrophils
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Pancreas158
Islets of Langerhans = light-staining endocrine portion produces insulin
Acinar cells = exocrine produces pancreatic enzymes
In H&E staining, the acid dye is eosin (stains proteins red) and the basic dye is a completed form of hematoxylin (stains ribosomes and nuclei blue). Hence, color provides distinguishing characteristics.
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Pancreas158
Secretory granules are red as they are protein rich with enzymes
Islets of Langerhans
Base cytoplasm is blue with ribosomes as in RER
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Pancreas, monkey (toluidine blue)156
Secretory granules
The entire pancreatic acinar cell is blue with varying intensities depending on the density of structures. Shape, size, and darkness are used to identify structures.
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Secretory granules
Smooth cytoplasm region =high density of ribosomes in this case
Secretory granules are red as they are protein rich
Base cytoplasm is blue with ribosomes as in RER
156
158
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Mucosa of stomach
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Fundic stomach145
Mucosa
Connective tissue of submucosa
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Fundic stomach: mucosa145Chief cells Parietal cells
Chief cells
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Fundic stomach, rabbit (toluidine blue)244
Chief cells Parietal cells
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244
145
Dark spots visible with toluidine blue staining are mitochondria in parietal cells .
Mitochondria are not distinguishable with H&E staining
Nuclei
Secretory granules in chief cells
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Human testis - blood and lymph vessels 19680 Toluidine blue staining
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19709 Transparency of unstained tissue
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UT165 human testis165 H & E staining and right insert toluidine blue staining – note differences in details of cytoplasm
19680Leydig cells
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EM 8f
EM 12a
EM 4c
EM 2b
EM 7
EM 6aCompare sizes of membranes ribosomes mitochondria as transmission electron microscopy (TEM) provides more cellular detail than light microscopy
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EM 8f: Peripheral blood cells; 9,000x
1. Monocyte
2. Lymphocyte
3. Neutrophil
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EM 12a: Bone marrow; 13,200x. Note the reticular cell and
developing red blood cells.
1. Reticular cell
2. Developing red blood cell
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EM 4c: Intestinal absorption cell; 60,000x
1. Budding RER
2. Coated vesicle
3. Golgi
4. Mitochondria
5. Nucleus
6. Plasma membrane
7. Primary lysosome
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EM 2b: Liver; 60,000x; cytoskeletal elements. Microtubes,
microfilaments, and intermediate filaments can be compared in this
cell, which has a high concentration of cortical microfilaments.
1. Microtube
2. Microfilaments
3. Intermediate filaments
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EM 7: Ascites fluid; 80,000x. Clear examples of Golgi apparatuses with their cisternae and vesicles
are present in this cell
1. Golgi apparatus
2. Ribosomes
3. Lipofuscin
4. Mitochondrion
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EM 6a: Centriole-microtubules; 200,000x. Centriolar region of a cell showing both the stable, triplet microtubule arrays within the
centriole, and the labile, individual microtubules originating from pericentriolar
material.
1. Centriole
2. Stable microtubule
3. Labile microtubule
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EM 8f 9,000x
EM 12a 13,200x
EM 4c 60,000x
Compare sizes of membranes ribosomes mitochondria
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EM 2b 60,000x
EM 7 80,000x
EM 6a 200,000x
Compare sizes of membranes ribosomes mitochondria
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EM 18b
EM 7
EM 8
EM 6
EM 4a
EM 2b
Conventional TEM, SEM carbon replica TEM
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EM 18b
EM 7
EM 8
EM 6
EM 4a
EM 2b
Conventional TEM, SEM carbon replica TEM
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EM 18b
EM 7
EM 8
EM 6
EM 4a
EM 2b
Conventional TEM, SEM carbon replica TEM
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EM 18b
EM 7
EM 8
EM 6
EM 4a
EM 2b
Conventional TEM, SEM carbon replica TEM
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Use your atlas!
pancreas
blood
testis
stomach
Use your atlas!
In summary
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QuestionsWhich microscope type/staining is/are better for observing
cellular details:
a. Light microscopy/ H&E
b. Light microscopy/ toluidine blue
c. Transmission electron microscopy (TEM)/ typical EM staining
d. a and b
e. a, b, and c
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Santa Elena Canyon Big Bend National Park, TX
Mexico USA
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