Introduction to Histological Tissues Elena Stark, MD, PhD Stephen Schettler, PhD Department of Pathology & Laboratory Medicine David Geffen School of Medicine at UCLA   Contributor: Paul Frank, DGSOM Class of 2014 THIS FILE HAS COPYRIGHTED MATERIAL. IT IS INTENDED FOR YOUR USE ONLY. DO NOT DISTRIBUTE OR SHARE IT. Non-graphical content and organization Copyright 2012 M.E. Stark, MD, PhD. 1

Main Menu 3 1. Introduction to Histological Tissues – Basic Cell Histology 4 2. Microscopy – Introduction 6 2.1 Microscopy – Light Microscopy Tissue Preparation 9 2.1.1 Microscopy – Light Microscopy Tissue Preparation – Slicing 10 2.1.2 Microscopy – Light Microscopy Tissue Preparation – Staining 12 2.1.3 Microscopy – Light Microscopy Tissue Preparation – Frozen Sections 13 3. Tissue Types 14 3.1 Tissue Types – Muscular 15 3.2 Tissue Types – Nervous 16 3.3 Tissue Types – Connective 17 3.4 Tissue Types – Epithelial 18 3.5 Tissue Types – Summary 19 4. Image Sources The “Back to Main Menu” buttons on pages 21 to 28 have been disabled (these are the hyperlink/highlight pages). To go to the Main Menu: first, go back to the original page by clicking or ; second, use the “Back to Main Menu” buttons on pages 3 to 20 (as they are functional). BACK 2

1. Introduction to Histological Tissues – Basic Cell Histology Please review the fundamentals of the cell on your own. Before you proceed with this module make sure you are familiar with the terms listed below. We recommend the following textbooks to learn the basics of the cell: Young B, et al. Wheater's Functional Histology. 5th ed. Churchill Livingstone, Elsevier Limited; March 14, 2006. Mescher AL. Junqueira’s Basic Histology. 12th ed. McGraw-Hill Medical; August 28, 2009. Nucleus Nucleolus Chromatin Plasma membrane Cytoplasm Cytosol Cytoskeleton Mitochondria Ribosome Endoplasmic reticulum Golgi complex Lysosomes Peroxisomes Secretory granule Inclusions 3

2. Microscopy – Introduction In the next several slides, we will provide a brief introduction to the subject of microscopy (including the concepts of slicing tissue and staining tissue). Please do not memorize this information, just read through it, it’s only FYI. There are 3 main modalities of microscopy used to study tissues: 1- Light microscopy (LM) that includes several types, such as bright-field, fluorescence, phase-contrast, and confocal and polarizing microscopy (however, we do NOT expect you to know the difference between these subtypes). 2- Electron microscopy (EM) that includes transmission electron microscopy (TEM) and scanning electron microscopy (SEM). 3- Scanning probe microscopy. Note: our modules use images taken with the light microscope (LM) and the electron microscope (EM), so we will focus on these types in general (1 and 2 above). 4

2. Microscopy – Introduction The main difference between light microscopy (LM) and electron microscopy (EM) is their capacity to reveal detail. LM can magnify up to 1000 times (x1000); while EM can magnify up to 100,000 times (x100 000). Electron microscopy (EM) includes 2 subtypes Transmission electron microscopy (TEM), also referred to simply as EM, is an electron beam that goes through the structure to form an image. This requires ultrathin slices of the structure that is under observation. The result is a two-dimensional (2-D) image. Scanning electron microscopy (SEM) produces three-dimensional (3-D) images of a structure’s surface. Ignore the i.ds in the images. SEM image of villi in the intestinal wall (x100). TEM image of the surface of a villus in the intestinal wall (x56 000). LM image of 2 villi in the intestinal wall (x150). © Elsevier. Young et al. Wheater’s Functional Histology 5e – www.studentconsult.com 5

2.1 Microscopy – Light Microscopy Tissue Preparation Since we will use LM images most often, our focus will be on sample preparation for LM. For study through LM, samples of tissues are obtained. Biopsies (small pieces of tissue) are taken from organs. The sample must be sliced and stained. The biopsy is immediately placed in a tissue cartridge (also called a cassette); which is then immersed in a fixative, such as formaldehyde or glutaraldehyde. The fixative helps: – Arrest cell metabolism – Prevent enzymatic degradation – Harden the tissue Tissue cartridges/tissue cassettes. 6

2.1 Microscopy – Light Microscopy Tissue Preparation Once the sample is fixed, it is then infiltrated with an embedding medium** (usually paraffin wax or plastic resin) in order to be cut into very thin sections, and then mounted onto glass slides. **Most tissues must be dehydrated between fixation and embedding. This example shows paraffin-embedded tumor blocks: portions of a tumor have been enclosed in paraffin wax. This can be done with any tissue, NOT just tumor tissue. 7

2.1 Microscopy – Light Microscopy Tissue Preparation So far, here are the steps of tissue preparation for LM: Fixation Dehydration (needed in most cases) Embedding (4.) Now, the sample is ready to be sectioned into very thin slices. (5.) Next, the slices are stained to visualize the tissue components. The process is as follows: Fixation > Dehydration > Embedding > Sectioning > Staining > Microscope ready These steps sometimes result in some distortions in the cell structure or the architecture of the tissue sample. We will see examples of this during the course. In the next slides, we will present more information on those last 2 very important steps: sectioning and staining. 8

2.1.1 Microscopy – Light Microscopy Tissue Preparation – Slicing These figures will help you visualize the way three-dimensional (3-D) structures (that have been sliced) appear in two-dimensional (2-D) histological slides. This is a very simple concept, but essential in understanding histology. Note: we will look at slides during lab to review this concept. 9

Reference: medscape.com 2.1.2 Microscopy – Light Microscopy Tissue Preparation – Staining A After being sliced, tissues are stained in order to visualize their components. There are several stains for observation with the light microscope (LM) that are commonly used: H&E (hematoxylin and eosin) (image A, at the top) Stains acidic structures purple/blue (nuclei, ribosomes, and RER). Stains basic structures pink (most cytoplasmic proteins). Masson’s trichrome (image B, in the middle) Stains extracellular structures blue (e.g. collagen). Giemsa (image C, at the bottom) Stains blood cells – nuclei stain blue/violet, cytoplasm stains light bluish, red cells stain pale pink. There are many other types of stains like these that we will discuss throughout the course. However, in tissue preparation for observation with an electron microscope (EM), there are NO common colored stains (like the ones listed above). Instead, the appearance is black and white with shades of gray (refer to the example shown on slide # 5, “2. Microscopy – Introduction”). Other techniques are applied to prepare tissue for EM (see next slide). B C Reference: medscape.com 10

2.1.2 Microscopy – Light Microscopy Tissue Preparation – Staining Histochemical techniques are applied to stain specific components of cells and tissues. In sample preparation for both LM and EM. Particularly helpful in understanding tissue function – often used for diagnosis of pathological tissues. Periodic acid–Schiff reaction (PAS) is an example where complex carbohydrates, mucus, and glycogen stain in deep red/magenta color (image A, at the top). Immunohistological techniques are applied for the identification of specific cell structures – by conjugating antibodies to a fluorescent stain that binds to specific antigens (image B, at the bottom). Important for diagnosis and research. A Reference: www.urmc.rochester.edu B Again, these descriptions are very simplistic. We will elaborate on staining techniques as the course progresses. Reference: www.hsc.wvu.edu 11

Thin sections are then cut in a refrigerated chamber 2.1.3 Microscopy – Light Microscopy Tissue Preparation – Frozen Sections In situations where an urgent diagnosis is needed (e.g. when the patient is undergoing surgery and a biopsy has been taken, the surgeon needs to know the results right away in order to proceed), the tissue is biopsied and then rapidly frozen to -150ºC to -170ºC by immersion in liquid nitrogen. Thin sections are then cut in a refrigerated chamber (called a cryostat) and stained without previous fixation, dehydration, or embedding. This way of tissue preservation is NOT as good as when using the regular method (previously described). Frozen sections are usually stained with H&E, but can also be treated with histochemical and immunohistological techniques. Nonfrozen section. Frozen section. 12

3. Tissue Types Cells in the human body are organized into tissues. In the next few slides, we will mention the 4 basic types of tissue that are present in the human body. We will elaborate on these tissues throughout the year. It is important that you understand the basic differences between tissues. If you find this introduction too simplistic, please refer to these 2 textbooks: Junqueira's Basic Histology by Mescher AL. 12th edition 978-0-07-163020-7 Wheater's Functional Histology by Young B, et al. 5th edition 978-0-443-06850-8 Cells in the human body are organized into tissues. There are 4 types of tissues: 1. Muscular 2. Nervous 3. Connective 4. Epithelial Tissues have CELLS and EXTRACELLULAR MATRIX or material (ECM) surrounding the cells. The proportion of these 2 components varies depending on the type of tissue: – Muscular tissue has a high cell density; – Nervous tissue is formed almost exclusively of cells and has virtually NO extracellular material; – Connective tissue is very rich in extracellular material and has a low proportion of cells; – Epithelial tissue has a very high proportion of cells and very limited amount of extracellular material. 13

3.1 Tissue Types – Muscular There are 3 types of muscle tissue: 1. Skeletal (image A, at the top) 2. Smooth (image B, in the middle) 3. Cardiac (image C, at the bottom) Muscle cells specialize in contraction. Muscle tissues have high cell density and little ECM. Muscle cell (1 in each type of muscle) ECM in muscle tissue Note: we will talk more about muscle in Block 2, where we cover the heart (cardiac muscle) and blood vessels (smooth muscle); and also in Block 4, where we cover skeletal or voluntary muscle (muscles we can contract at will). B ORIENT YOURSELF: Images A, B, and C are histological slides of muscular tissue that have all been stained for LM observation at medium magnification. Image A: thin slices of voluntary muscle Image B: thin slices of the wall of an organ Image C: thin slices of the heart (To train your eye to the differences between tissues, we will practice with many slides in lab.) For now, focus on the general concepts. C 14

An organ showing several types of cells and tissues. 3.2 Tissue Types – Nervous Nervous cells specialize in generation and conduction of electrical impulses. (We will cover nervous tissue in detail in Block 5.) Nervous tissue has abundant cells of different types and virtually NO ECM. For our purposes (before Block 5), we will identify nerves in terms of the structure highlighted below. This is a small peripheral nerve showing the long neuronal axons, as they appear in a cross-section. It looks like a bundle of cross-sected wires. In this particular section, some of the axons are running obliquely – appearing oval in shape (vs. perfect circles). An organ showing several types of cells and tissues. The purpose of this slide is to start becoming familiar with the appearance of a cross-sected peripheral nerve in an organ. 15

3.3 Tissue Types – Connective Connective tissue has much ECM and few cells . Thus, connective tissues are good “supporters” (since ECM is usually stronger than cells). They are found mainly forming the framework of organs or supporting other tissues. This week’s lab will cover connective tissue in detail. 16

3.4 Tissue Types – Epithelial Epithelia have many cells and little ECM . Epithelia are found mainly covering and lining organs. This week’s lab will cover epithelia in detail. Ignore the i.ds in the image. 17

Extracellular matrix (ECM) 3.5 Tissue Types – Summary Tissue type Cells Extracellular matrix (ECM) Function Muscular Contractile cells Very small amount Movement of body (skeletal) Involuntary movement (smooth) Beating of heart (cardiac) Nervous Neurons containing long processes, called axons and dendrites Supporting cells Virtually NONE Conduction of nerve impulses Connective Fibroblasts Other cells Large amount Support of organs Protection of organs Epithelial Tightly-packed cells with mostly flat edges Very small amount Lining and covering surfaces of body and body cavities Glands: secretion (to be discussed in the next module: “Epithelial Tissue”) 18

UCLA David Geffen School of Medicine, Integrative Anatomy. 4. Image Sources Young B, et al. Wheater's Functional Histology. 5th ed. Churchill Livingstone, Elsevier Limited; March 14, 2006. Mescher AL. Junqueira’s Basic Histology. 12th ed. McGraw-Hill Medical; August 28, 2009. www.medscape.com www.urmc.rochester.edu www.hsc.wvu.edu UCLA David Geffen School of Medicine, Integrative Anatomy. 19

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3.1 Tissue Types – Muscular BACK 3.1 Tissue Types – Muscular A There are 3 types of muscle tissue: 1. Skeletal (image A, at the top) 2. Smooth (image B, in the middle) 3. Cardiac (image C, at the bottom) Muscle cells specialize in contraction. Muscle tissues have high cell density and little ECM. Muscle cell (1 in each type of muscle) ECM in muscle tissue Note: we will talk more about muscle in Block 2, where we cover the heart (cardiac muscle) and blood vessels (smooth muscle); and also in Block 4, where we cover skeletal or voluntary muscle (muscles we can contract at will). B ORIENT YOURSELF: Images A, B, and C are histological slides of muscular tissue that have all been stained for LM observation at medium magnification. Image A: thin slices of voluntary muscle Image B: thin slices of the wall of an organ Image C: thin slices of the heart (To train your eye to the differences between tissues, we will practice with many slides in lab.) For now, focus on the general concepts. C 21

3.1 Tissue Types – Muscular BACK 3.1 Tissue Types – Muscular A There are 3 types of muscle tissue: 1. Skeletal (image A, at the top) 2. Smooth (image B, in the middle) 3. Cardiac (image C, at the bottom) Muscle cells specialize in contraction. Muscle tissues have high cell density and little ECM. Muscle cell (1 in each type of muscle) ECM in muscle tissue Note: we will talk more about muscle in Block 2, where we cover the heart (cardiac muscle) and blood vessels (smooth muscle); and also in Block 4, where we cover skeletal or voluntary muscle (muscles we can contract at will). B ORIENT YOURSELF: Images A, B, and C are histological slides of muscular tissue that have all been stained for LM observation at medium magnification. Image A: thin slices of voluntary muscle Image B: thin slices of the wall of an organ Image C: thin slices of the heart (To train your eye to the differences between tissues, we will practice with many slides in lab.) For now, focus on the general concepts. C 22

An organ showing several types of cells and tissues. BACK 3.2 Tissue Types – Nervous Nervous cells specialize in generation and conduction of electrical impulses. (We will cover nervous tissue in detail in Block 5.) Nervous tissue has abundant cells of different types and virtually NO ECM. For our purposes (before Block 5), we will identify nerves in terms of the structure highlighted below. This is a small peripheral nerve showing the long neuronal axons, as they appear in a cross-section. It looks like a bundle of cross-sected wires. In this particular section, some of the axons are running obliquely – appearing oval in shape (vs. perfect circles). An organ showing several types of cells and tissues. The purpose of this slide is to start becoming familiar with the appearance of a cross-sected peripheral nerve in an organ. 23

An organ showing several types of cells and tissues. BACK 3.2 Tissue Types – Nervous Nervous cells specialize in generation and conduction of electrical impulses. (We will cover nervous tissue in detail in Block 5.) Nervous tissue has abundant cells of different types and virtually NO ECM. For our purposes (before Block 5), we will identify nerves in terms of the structure highlighted below. This is a small peripheral nerve showing the long neuronal axons, as they appear in a cross-section. It looks like a bundle of cross-sected wires. In this particular section, some of the axons are running obliquely – appearing oval in shape (vs. perfect circles). An organ showing several types of cells and tissues. The purpose of this slide is to start becoming familiar with the appearance of a cross-sected peripheral nerve in an organ. 24

3.3 Tissue Types – Connective BACK 3.3 Tissue Types – Connective Connective tissue has much ECM and few cells . Thus, connective tissues are good “supporters” (since ECM is usually stronger than cells). They are found mainly forming the framework of organs or supporting other tissues. This week’s lab will cover connective tissue in detail. 25

3.3 Tissue Types – Connective BACK 3.3 Tissue Types – Connective Connective tissue has much ECM and few cells . Thus, connective tissues are good “supporters” (since ECM is usually stronger than cells). They are found mainly forming the framework of organs or supporting other tissues. This week’s lab will cover connective tissue in detail. 26

3.4 Tissue Types – Epithelial BACK 3.4 Tissue Types – Epithelial Epithelia have many cells and little ECM . Epithelia are found mainly covering and lining organs. This week’s lab will cover epithelia in detail. Ignore the i.ds in the image. 27

3.4 Tissue Types – Epithelial BACK 3.4 Tissue Types – Epithelial Epithelia have many cells and little ECM . Epithelia are found mainly covering and lining organs. This week’s lab will cover epithelia in detail. Ignore the i.ds in the image. 28