Class #lecturenumber – Prof. – Lecture Title Page 1 of 9 Histology&Embriology #lecture1 Introduction lesson Prof.Massimiani – 01/march/2021 Benedetta Pascucci – Andrea Di Napoli Practical advices and informations The course is divided in three parts: - Cytology --> prof. Lacconi -embriology--> prof. Massimiani -histology --> prof. Campagnolo Books of the course: -Histology a text and atlas - Pawlina -Larsen's Human Embriology- Shoenwolf (these are the books suggested and that the teachers will follow) The course will have both classes and practical histology. Informations about the exam: -written test: multiple choice questions, true/false questions and associations and ONLY for the students that will pass the written test there's an obligatory oral test where there will be: -identification of one/two histological slides and questions on cytology, histology and embriology. (There are a lot of calls programmed in June, July, September, December/January and February.) 1.Overview of the first lesson These first two lessons are a sort of introduction of the entire course; they're both based on the methods that are commonly used in histology, embriology and citology; It’s given a presentation of the tools that are fundamental to study cells and tissues’ structures. 2. Histology: introduction The classical histology is the scientific discipline that studies the morphology of plant and animal tissues and the cells that compose them both at a morphological and functional level. This is only the definition of the classical, traditional histology, but actually there's also a modern histology, which is not only a descriptive science but it’ is based on a series of techniques on molecular and cell biology level; a lot of techniques important to study the organization of cell’s tissues and their functions. 1 Class #lecturenumber – Prof. – Lecture Title Page 2 of 9 These methods are: -Morphological, classical methods based on the use of microscope, that allow variable observations of these tissues and cells; -biochemical, biological and molecular methods that are currently used in the histology lab. Data that we can obtain from these methods allow us to acquire and better understand not only the morphology but also the function of the cell’s tissues. We will go over first on morphological methods, that are based on the use of microscope. We will overview all the different types of microscopes: -Optical microscope, the most commonly used; -Electronical microscope, that allow us to see more in detail the structure of tissues and cells. (Image Diagnostic Technology is essential to study the morphology of tissues and cells) Modern histology is also based on other methods which are biochemically and biochemical biological important in order to study the structure and the regulation of genes, gene expression and how it is regulated, the structure and function of proteins and the interactions between biomolecules. In particular , let’s introduce briefly these methods: -dna sequencing that allow to sequence the genome -pcr (Polymeraze Chain Reaction), a technique important to study the genotide expression in particulat cells and tissues; -northern e southern blotting; -DNA micror, RNA sequencing; All important to understand what genes are expressed in a particular kind of cell or tissue and how they are regulated. Then there are these other methods like: -western blotting, important to see if a protein is expressed in a cell or tissue; -chromography; -eliza, which is important to verify if a protein is expressed in a serum or plasma in a cell culture; And then other techniques that are important to see if there are interactions between biomolecules: -Coimmunoprecipitation, to see if there’s interaction between protein and another protein (protein-protein); 2 Class #lecturenumber – Prof. – Lecture Title Page 3 of 9 -chromatin immuno precipitation (ChiP), used to see if there is interaction between protein and dna (protein-DNA); -electrophoretic mobility shift assay , to see if there’s interaction between protein and DNA or protein and RNA (protein-DNA/RNA); -Southwestern blotting (DNA-protein); Currently in histology labs are used animal models and cells culture to study in vivo e in vitro cells and tissues. The use of animal models (mammals) to study for example drugs or toxicity of different compounds, is possible because there are a lot of similarities between animals and humans’ cells; so animal models (mammals) can be used to test novel therapies and drugs before applying these discoveries on humans. In parallel to animal models is possible to use also cells culture. In fact, it is possible to culture in our labs, cells ; it’s then possible to remove and isolate animal cells and put them in culture in very controlled settings; so the cells put in culture are used in research and for diagnosis. The cell culture in vitro is an important and powerful method to study cells types isolated from the body. How it is possible to isolate these cells ? We can start from a piece of tissue taken from an animal for example and then is important to break this tissue , to mechanically or enzymatically digest the tissue in order to obtain a suspension of individual cells; after this fragmentation is possible to obtain a singular cell. The cells that we obtain are put in a culture dish with an appropriate nutrient medium, considering that arrays of particular type of cells posses their specific kind of medium. In this way we obtain a primary culture that means that is a culture obtained directly from the animal. Then is possible to culture the cells in appropriate conditions and expand them and obtain confluent culture that will be placed again at lower confluency, obtaining a secondary culture; in this way we maintain the cells in culture and we will be able to perform several and different types of experiments also studying these cells morphologically. [-Movie of cells putted in culture where you can see that they’re alive, they move, migrate and proliferate up until they reach the confluency after which they split at lower confluency in order to maintain in culture, so in this way we can functionally and morphologically study them. Cells move and proliferate , duplicate them through mitosis until they reach the confluency in culture plate , so now we can split them and propagate to study them.] In every cells’ culture, starting from a tissue digest, we’re able to obtain a population of cells and other elements like organelles for example that we can study. 3 Class #lecturenumber – Prof. – Lecture Title Page 4 of 9 To study the nucleus, the organelles, the plasma membrane of the cells we can use this technique called: -differential centrifugation that allows us to obtain the different population, organelles, nuclei (and so on), in order that we can study singularly these elements. When we have this suspenction of cells we have to break them and we have to perform several centrifugation at different speed to obtain different parts of the cells. After the centrifugation we can study the nuclei; then we have to take the supernatant so the upper part that we obtained centrifugate again and then we obtain lysosomes peroxisomes mitochondria and the rest that we can study; then we centrifugate again and we obtain the plasma membrane and the endoplasmic reticulum; in this way we can study the different elements of the cell culture, because they stratify differently after the centrifugation. 2.1 The importance of microscope Methods classically used in histology are: -morphological methods, based on the use of microscopes; The importance of the use of microscopes in histology is due to the fact that the cells, organelles and molecules are too small and lack of contrast to be visible to human eye. So we have to magnify these elements to make them visible to our eyes. The essential instrument of histology is the Optical microscope, which magnify the image and the different objects allowing us to visualize all the little details that would be impossible to study with only the human eye. The importance to use the different types of microscopes depending of what we need to study, is linked to concept of resolution limit or resolving power, that is the distance by which two object must be separated to be seen as two objects. The resolution limit of human eye is equal to 0,2 mm; with this resolution limit we are able to see up to the human egg, that possess a dimension of about this resolution limit. If we want to study a smaller object we need to use an optical microscope that possess a resolution limit of 0.2 micrometers or 200 nanometers. If we want to go down and study smaller object like proteins or amino acids, we need to use electron microscope, which possess a resolution limit of about 0.25 nanometers. (We rarely talk about mm in cytology and histology, but we usually talk about micrometers (= mmX10^-3) and nanometers (=micrometersX10^-3); sometime we also talk about Armstrom (micrometersX10^-4). 2.2 Overview of different kinds of optical and electron microscopes It’s important to consider the resolution limit of the human eye 0.2mm, that is determined by specific photoreceptors cells in the retina. These optical tools (electron and optical microscopes) allow to improve the limit of the human eye. 4 Class #lecturenumber – Prof. – Lecture Title Page 5 of 9 The fundamental parameter to describe a microscope is the: resolution or resolving power (and not its magnification), that is the minimal distance at which 2 dots are resolved as separated. ( for the human eye the resolving power is 0.2mm; for the optical microscopes 0.2micrometers; for the electron microscope 0.25nanometers).So when we define a microscope is important to define mainly its resolving power. Magnification is the process of enlarging the apparent size (not the physical size) of something; so by enlarging an object we will obtain an enlarged effect and image of the object, initially completely out of focus. Increasing the resolution power we can see and magnify the image but also increasing the resolution power; this allows us to obtain all the possible details; so magnification is related to the scanning up images to be able to see more details and this is due to an increase resolution. 2.3The resolution limit of a microscope can be calculated by the Abbe equation: where: -𝝀 is the wavelength of the light source; -N is the refractive index of the medium that is placed between the objective lens and the specimen; it can varie depending if between the specimen and the lens there’s air; it can be increased of 1.51 if we use immersion oil between the speciment and the objective.; so if we want to increase the resolution limit of the microcope we can use immersion oil; -𝐬𝐢𝐧 𝜶 represents the lens’ aperture; For example, considering the wavelength of light source as 400nanometers we can obtaining the maximum solving power for an optical microscope that is 0,2 micrometers; the maximum magnification that we can obtain is 1500times that is possible to obtain using the immersion oil and using as objective lens 100X so the magnification of the optical microscope is the product of the magnification of the eyepiece that is usually 10X and the objective lens (say 100X). In this case the maximum that we can use is 100X and if we put the immersion oil between the objective lens and the specimen we can obtain a maximum magnification of 1500X. The maximum resolving power 0,2 micrometers. While on the contrary, using an electron microscope we can obtain a maximum resolving power of 0.25nanometers with an higher magnification and the increase of the resolving power is due to the different wavelength of the electrons, which possess lower, smaller wavelength so for this reason if we put the wavelength of the electrons in this equation we will obtain this higher value of 0,25 nanometers . This demonstrates why the electron microscope (which uses electrons instead of light) and optical microscope possess different resolution limits. 5 Class #lecturenumber – Prof. – Lecture Title Page 6 of 9 2.4 Optical Microscopes -Brightfield microscope= the most used in histology and commonly used for routne histological examination; -Stereomicroscope= commonly used in histology for dissection and microsurgery; -Phase contrast microscope= commonly used to study living cells, so for the cells that are kept in culture in lab; -Dark field microscope= used to detect smaller individual particles; -Interference microscope= used to study also the surface of cells and tissues; -Polarized microscope= used to study the tissues (for example the striate muscles) exhibiting birefrangence; -Florescent microscope= based on the possibility to observe molecules that emit fluorescence; -Confocal microscope= that allow to see and study cells and tissues in three dimension. The first optical microscope that we will discuss today is the brightfield microscope that is also the easier microscope that for histological examinations that is also the most commonly used. This is the scheme that allows to see it’s structure so essentially it consists of a light source. So basically a lamp that allows the illumination of the specimen. So under the specimen is present a condenser lens that allows to focus the bi modular level of the specimen that is present here on the stage. On the stage are present different objective lenses with different magnifications (10x – 100x) that is the objective lens that is commonly used with an oil immersion. Then there is a pair of ocular lenses that allows to examine directly the specimen so the magnification is equal to the magnification of the objective lens so for example 40x for the magnification of the ocular lenses so for example 10x. So observing the specimen on the brightfield microscope you will observe picture like a cross section of a kidney that we will go over also the staining that is used in this case to visualize the different structure this the hematoxylin and eosin staining that is used in histology. 6 Class #lecturenumber – Prof. – Lecture Title Page 7 of 9 So when you observe a histological specimen under the microscope you will observe obviously a 2D object so here you have two images that we are talking about are obtained from 3D objects. So imagine the 3D structure of the object that we are examining. The second optical microscope that we will see is the stereo or dissecting microscope. First I would like to highlight the difference with the previous one that is essentially the space between the stage that is important to perform microsurgery and microdissection on an organ or a tissue. Under the microscope I magnify the sample using light reflected from the surface of an object rather than transmitted through it. So in the previously microscope the light was transmitted through the object here the light is reflected from the surface of the object that we want to study. Two separate optical paths with two objectives and eyepieces to provide different angles to create a 3D of the object that we are studying. The main difference between the microscope with just one ocular lens and the microscope with two ocular lens is that looking with both the eyes I can have a 3D of the object so it is always important we I look at the microscope to look with both the eyes and have a 3D that is completely absent If I look with just one eye or I use a microscope with just one ocular lens The phase contrast microscope is currently used in laboratory to study cell culture and to control it. It is based on the fact that there are differences in the refractive index in different parts of the cells or a tissue. So, the lighter and less dense area while the light will be deflected from the denser area so the dark portions of the image correspond to denser portions and the lighter portions to the less dense portions of the specimen. So, looking at cell culture under the microscope we observe image like this: 7 Class #lecturenumber – Prof. – Lecture Title Page 8 of 9 So, you can appreciate the nuclei for each cell. In each cell In the nucleus there is for example the nucleolus that appears like a dark area. Also, some other organelles that appear dark ,while the less dense area appear light. It is commonly used to see the cells that are in culture vision. Differential interference is essentially a modification of the phase contrast microscope that it is commonly used to study the surface properties of cells, it is based on the presence of a polarizer that is located between the light and the specimen that is always on the stage and it introduces a polarized light for interference imaging then there is the Nomarski condenser prism that is involved in beam splitting, so it is a prism that splits the light so it separates the polarized light into two components. Then there is the objective prism that is a second beam splitting that recombines the light. On the top there is an analyzer that is a second polarizer and it is located between the objective lenses and it always remains here so the ocular. In this manner it generates the interference contrast image. So here you can appreciate the surface of the cells that are in culture. 8 Class #lecturenumber – Prof. – Lecture Title Page 9 of 9 The first one on the left is a Phase contrast optical microscope in which you can see the different parts of the cells that are dark or light depending on the density. In the interference microscope you can see the surface property and in particular the dense particles show like an elevation: nuclei, mitochondria and lysosomes appear like hilltops while the inclusions with a lower refractive index like vesicles and vacuoles appear like depressions. The phase contrast allows to see the cells increasing their contrast and the interference microscope allows also to increase the resolution. This is a fibroblast under different types of microscopes: 1.The first one is a brightfield microscope, in which essentially the cells appear 2D and there is no contrast between the different areas of the cell. 2. The second picture is the same cell under the phase contrast microscope there it increased the contrast and I can see the different areas with different density. Under the differential interference microscope, it’s also focused on the surface of the cell so you can see an increase 3D. So, you can see how different microscopes allows to see different details with the same preparation. 9
