Histology and Embryology - ahmu.edu.cnjcyxy.ahmu.edu.cn/_upload/article/files/7d/9e/e5bb813d...Anhui Medical University Anhui Medical University 2016/10/19 28 Tissue collecting Fresh,

Anhui Medical University


Histology and Embryology

Department of Histology and EmbryologySchool of Basic Medicine

School of International EducationAnhui Medical University

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Contact Information

Shoubing Zhang, Ph.D.E-mail: [email protected]

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Chapter 1.

Introduction of Histology



• The four fundamental tissues;• The principle of H&E staining;• Function of important cell apparatus.

Key points



Contents

I. What’s Histology? (Nature)II. Why we study it ? (Reasons and Significances)III. How to study it ? (Techniques and Methods)

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Students should know

Definition and study contents of histology.

Principle of HE staining.

Methods for preparing a paraffin section.



I. What’s Histology?

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In Greek words, Histology=“Histos”(a tissue)+ "-logos“(the study of). A branch of anatomy, which is at the

microscopic level (Microscopic Anatomy)

LM/EM microstructure

Functions！

Histology is a science which studies the microstructureof human body and the relationship between thestructure and function.



Cells---tissues---organs & systems

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Three Structural Levels



Cells

The cell is the smallest structural and functional unit. There are many kinds of cells in human body such as epithelial cells, fibroblasts, plasma cells and nervous cells . 1015 cells in the human body 200+ cell types in the body

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Purkinje cells

cerebellum



Groups of cells and extracellular matrix that are similar in morphology and function make up the body tissues. There are four fundamental tissues: epithelial tissueconnective tissuemuscular tissuenervous tissue.

Tissues

Skeletal muscle

striation

collagen (yellow).

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Organs

Several different kinds of tissues are further

organized to form organs (such as heart, stomach or

kidney) .

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Anhui Medical UniversitySystem

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Several organs with related functions

Epithelium: transitional epithelium

Cells:

Facet cell

System: urinary system Organ: urinary bladder

kidney ureter

urinary bladder

urethra



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II. Why We Study Histology?

normal structure and normal function.

basis of physiology, pathology and disease.



Normal

Abnormal

Structural lability of mitochondria

2016/10/19 14mitochondrial myopathy

C, cristae;M, matrix



• Neurofibrillary tangles ----one characteristic of Alzheimer’s disease

normal slice pathological slice



Learning methodsHistology Techniques and Methods

II. How to Study Histology?



Histology Learning Methods

• A section is a slice of a tissue• Orientation that the section is performed affects

what you see.

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2 dimensional image 3 dimensional image



Sections cut through a curved tube

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Different sections-different results



Pictures Texts



Relaxation

Contraction

Morphology

Function



skeletal muscle smooth muscle cardiac muscle

Integration Comparison

Long cylindrical；striation

Fusiform；unstriated

short cylindricalStriated；have branch



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LM EM Useful magnification: 1500X 800,000X resolution: 0.2um 0.2nm

Histology ToolsHistology studies the microstructures. So, we should have the aid of microscope to study. Several types of

microscopes are available. According to the light/beam source used, microscopes can be classified as:

Light microscope (abbreviate LM)Electron microscope (abbreviate EM)



LM

With the LM, stained specimens are usually examined by means of light that passes through the specimens.

The maximal resolving power of LM is approximately 0.2um. That means that the smallest distances between two particles at which they can be seen as separate objects is 0.2um.

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Light Microscope (LM)

• Oculars (eyepieces)• Stage• Objective Lens• Condenser• Light source• Focus knobs

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Traditional Histological Methods

Preparation of a specimen for LM

Before a piece of tissue can be observed with a light microscope, it must be made into a section.

The routine histological preparation for light microscope examination is a paraffin section

stained with hematoxylin and eosin (HE staining).

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The procedure for making a section is as follows:

Tissue collecting—Fixation—Dehydration—

Clearing—Embedding—Sectioning —Staining



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Tissue collecting Fresh, small tissue blocks are get from a organ. The size of a tissue block should be less than 1.2cm×0.5cm×0.2cm. （centimeter）

Fixation Then put the tissue blocks into a fixative. The process of fixation preserves a tissue by denaturing its proteins. Numerous fixatives are known, the most commonly used is 4% formaldehyde (4% formaldehyde is also called formalin).



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Dehydration Then use ethyl alcohol to get rid of water from the tissue blocks.

Clearing Then use xylene to get rid of alcohol.*alcohol and xylene are embedding mediums

Embedding Before a tissue can be sectioned it has to be given a firm consistency. One way of doing this is embedding a tissue in paraffin wax .



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Sectioning Usually sections of 5～10 micron (μm) thick are cut with a microtome and mounted on glass slides. When the sections are dry, they are ready for staining. Staining Numerous staining methods are available for demonstrating specific tissue elements. The most extensively used staining method is HE Staining .



HE Staining

H, Hematoxylin/Haematoxylin is a basic dye. It can bind to the acidic components of cells and tissues, which then show a blue colour. Such components, e.g. nuclei, are said to be basophilic. RER and ribosomes incytoplasm are basophilic too. E, Eosin is an acidic dye. It can bind to basic constituents and give them a pink colour. Such constituents, e.g. cytoplasm and most of other components, are termed acidophilic.

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acidophilic

basophilic


Anhui Medical UniversityLM

Intestinal villi

Renal tubule



nuclei

cytoplasm



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Some components can not be stained by hematoxylin, nor by eosin. Those components are termed neutrophilic .



Some components can be stained directly by silver. Those components are termed argentaffin.Some components can be stained by silver after adding reductants. Those components aretermed argyrophil. The components which are stained by silver show aa dark colour .

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Silver staining, cerebral cortex neurons

Silver staining



Gold chloride staining，motor end plate



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In addition to the above routine paraffin sections, frozen sections may also be cut with a freezing microtome (cryostat).

Frozen sections can preserve some chemical components and enzymes better. Preparation of frozen sections is the fastest method of examining a tissue. The technique allows the examination of pieces of tissue removed by a surgery, while the patient is still on the operating table.



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Specific light microscopes

(1). Phase-contrast microscope

(2). Differential interference microscope

(3). Fluorescence microscope

(4). Confocal microscope



Bu, bulge; Bb, bulb. Bar: 200μm.



propidium iodide, PI



Fluorescein-isothiocyanate, FITC; propidium iodide, PI



EM

The EM uses an electron beam instead of light; and electromagnetic fields in place of lenses. With the EM magnification of 100,000 (hundred thousand) times can be achieved. The maximal resolving power is 0.2 nm under the EM.

The structures of a cell or tissue as seen with the EM are referred to as ultrastructures.

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There are two kinds of EMs: Transmission electron microscope (TEM)Scanning electron microscope (SEM).

TEM can observe the ultrastructures inside a cell.SEM can observe the surface appearance of a cell and obtain three-dimensional images.





Transmission electron microscope (TEM)LM

Synapse



Synapse



Scanning electron microscope(SEM).



Scanning electron microscope(SEM).

Neuron Cell body

Dendrite



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Besides the sections we can also get a specimen as follows:Smear preparation is used to observe blood, semen or other secretions.Spread preparation is used to observe mesothelium and connective tissue. Grinding preparation is used to observe bone or tooth.



osteon

bone lacuna



Histochemical techniques or cytochemical techniques combine histological methods with chemical and biochemical methods and reveal the chemical composition of tissues and cells in situ. Many substances, such as proteins, amino acids, nucleic acid, lipids, carbohydrates and enzymes et al , can be detected in situ by histochemical and cytochemical techniques.

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Histochemical/cytochemical techniques



Immunocytochemistry/Immunohistochemistry

Antigen-antibody reaction.

Protein molecules (antigen) within cells can be identified by

treating cells/tissue sections with antibodies specific to the

molecules.

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Tissue section or cells with Antigen + labelled antibody labelled Ag-Ab complex

Fluorescence labelling

enzyme labelling

colloidal goldlabelling



Alpha-6 integrin/PI

Fluorescein-isothiocyanate, FITC; propidium iodide, PI



In Situ HybridizationIn situ hybridization (abbreviate ISH), also termed insitu hybridization histochemistry (ISHH), is a methodfor detection of specific RNA or DNA sequencesdirectly in cells or tissue sections.

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nucleotide probe



Vital Staining: observation of living cells and tissue

When the dyes are injected into living animalscertain cells or structures can be stained by the dyesor can be visualized by their selective absorption ofcolouring substance. For example, trypan blueinjected into living animals is removed from theblood by macrophages and accumulated in the theircytoplasm. This renders the cytoplasm ofmacrophage conspicuous.

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Vital staining of trypan blue. Macrophages engulf trypan blue granules in their cytoplasm.



PAS reactionThe periodic acid-

Schiffs (PAS) reaction is one of histochemical

techniques most extensively used to

localize polysaccharides, such as glycogen and

glycoprotein, in tissues and cells.

Purple or magenta color

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polysaccharidesan intestinal villus

Flow cytometer

BD FACSVantageBD-Calibur



Flow cytometer

BD FACSVantageBD-Calibur





Flow cytometer is a special microscope

Flow cytometer microscopeFlowing cells Static cells

In great number Small amount

Subpopulation analysis Cellular signaling



Blanpain C et al. Cell, 2004

Mouse HFSCs were isolated by sorting



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Cell Culture and tissue engineering

Cell and tissue culture is a technique for the study ofliving cells. Isolated cells or fragments of tissues arecultured in a sterilized culture medium at theappropriate temperature. Since the medium containsessential nutrients, such as amino acids, vitamins,etc., cells and tissues grow in vitro. This technique isquite useful in detecting the effect of variousreagents on living cells.



A, a HFSC colony at the fifth day of culture. Co-cultured with fibroblasts, single HFSC from the initial colonies formed new colonies; B, logarithmic growth of HFSCs; C, Formation of large colonies after two-weeks of growth; D, colony-forming efficiency was 49%; Bar: A, 100μm; E-F, 1cm.

HFSC passage



HF, hair follicle; HS, hair shaft; Epi, epidermis; Der, dermis; Con, control of adjacent nude mouse skin; SS, skin surface. White arrow, GFP-expressing hair follicle stem cell descendants; Asterisk, air bubble; Black arrow head, sebaceous gland. Bar: 100 μm.

A

B

C



Fibroblasts

Fibroblasts to induced pluripotent stem (iPS) cells



Shinya Yamanaka, was awarded the 2012 Nobel Prize for Physiology or Medicine, together with John Gurdon



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ES cells to cardiac muscle cells

See the video



Tissue-engineered ear



Hope: Valery Spiridonov wants to be the first person to undergo a head transplant so he can have a shot at having a healthy body having suffered from the Werdnig-Hoffman muscle wasting disease当地时间2015年6月26日，俄罗斯弗拉基米尔，即将接受世界首例头颅移植手术的俄罗斯男子Valery Spiridonov出席发布会。他天生就患有罕见的遗传病脊髓性肌肉萎缩症，找到一具健康的新身体是他生存下去的唯一机会。



'Am I afraid? Yes, of course I am. But it is not just very scary, but also very interesting,' said Mr Spiridonov from his home in Vladimir, a city 120 miles east of Moscow. 'But you have to understand that I don't really have many choices', he said. 'If I don't try this chance my fate will be very sad. With every year my state is getting worse.‘Controversial: Surgeon Dr Sergio Canavero claims all the necessary techniques already exist to transplant a head onto a donor body, despite widespread scepticism


Anhui Medical UniversityCell structureA knowledge of cytology is necessary for the study of histology.



Cytoplasmic Organelles

• Ribosomes• Mitochondria• Golgi apparatus• Endoplasmic reticulum• Lysosome• Microfilaments• Secretory vesicles• Pigment granules• Lipid droplets

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① Mitochondria

Inner membrane projects and folds—cristae, which contain enzymes and components of oxidative phosphorylation and electron transport system



Mitochondria EM x25 000



Function: Energy release and storageelectron transport system



② Ribosomes

20x30 nm in size, small electron-dense particles.

Composed of four types of rRNA and almost 80 different proteins

Protein synthesis



③ Endoplasmic reticulum (ER)

Smooth ER

Rough ER



Function of ER

RERModify newly formed polypeptides; Synthesize secretory proteins and membrane proteins.

SERSynthesize phospholipids for the membranes; Hormones synthesis.



numerous ribosomes

Rough Endoplasmic Reticulum



EM x 40,000 sER and rER



④ Golgi Complex (Golgi Apparatus)

EM x 30 000



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Protein Synthesis

transport of a glycoprotein

modification



⑤ lysosome

Rich in hydrolytic enzymes. They are responsible for degradation of macromolecules and senescent organelles.



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Questions?

1. What is histology?2. What is the principle of H&E staining?3. What are the procedures for preparing a paraffin section?4. What are the differences between SER and RER?

Documents

Histology and Embryology - ahmu.edu.cnjcyxy.ahmu.edu.cn/_upload/article/files/7d/9e/e5bb813d...Anhui Medical University Anhui Medical University 2016/10/19 28 Tissue collecting Fresh,