Microscopic Observation of the Cell (2023-2024) PDF

1st Year SNL (2023-2024) / Cell biology module / Tutorials / Tut - 3 Microscopic observation of the cell: Principles of operation of the optical and electronic microscope The limited resolution of the human eye does not allow observing cells with their small...

1st Year SNL (2023-2024) / Cell biology module / Tutorials / Tut - 3 Microscopic observation of the cell: Principles of operation of the optical and electronic microscope The limited resolution of the human eye does not allow observing cells with their small size in micrometers. Indeed, the structural study of the cell can only be carried out using magnifying optical instruments: light microscopes (light microscope, fluorescence light microscope, X-ray light microscope and phase contrast light microscope, etc.) el electron microscopes (transmission electron microscope or TEM and scanning ME or SEM). The term microscope comes from the Greek “mikros” which means “small” and “scope” which means “to observe”. In 1667, the English microscopist Robert Hooke invented a microscope equipped with three lenses. With this device he observed the regular, air-filled cells of the cork. By giving them the name "air cells", he was the first to use the word "cell". Since then, scientific advances have continued to improve the capabilities of microscopes (magnification and resolution); and in 1939 the first electron microscopes were developed. I. Light microscope (LM) = (Optical microscope= Photonic microscope) I.1. Description - The photonic microscope uses light (photon = elementary particle of light). - It allows you to observe objects or living beings whose size is between 1 μm and 1 mm. - It is an optical instrument that allows observing very thin objects (2 to 10 μm) by magnifying them (15 to 1800 times). The object to be observed is called a preparation; it is placed between a slide and a glass slide. This preparation must be thin to allow light to pass through during observation. - The light microscope includes: a stand (foot) which ensures the stability of the device; an optical tube along which there is a system of glass lenses and comprising at these ends an eyepiece allowing the image to be collected, and objectives used to magnify the image of the preparation a certain number of times; an object holder plate pierced with a hole and fitted with clamps to immobilize the slide; a light source illuminating the preparation. - The magnification of the eyepiece (ME) multiplied by that of the objective (MO) gives the total magnification (M) of the image by the microscope. - Resolving power: the smallest distance between two points that can be distinguished using the microscope. The resolving power of the best microscopes is around 0.2 μ. 1  1st Year SNL (2023-2024) / Cell biology module / Tutorials / Tut - 3 I.2. Physical principle of Light microscope (LM) A classic microscope consists of the following main elements: - A source of light radiation and a device for lighting the object; - An optic made up of several lenses providing the magnification function; - A detector allowing observation or recording of the image (eye, camera and TV monitor). The light microscope uses a wave light flow of uncharged particles (PB), photons, through a system of lenses (L1, L2 and L3) so as to form an object (O) to be studied (AB) into a enlarged image (A'B') on a screen S seen by the user (Figure 1). The objective gives of the object AB observed a real image A1B1 inverted and very enlarged which plays the role of a real object for the eyepiece. The eyepiece gives an enlarged virtual A2B2 image of this object, seen by the observer, and then the eyepiece plays the role of a magnifying glass. Figure 1: Optical composition and light trajectory in a LM II. Electron microscope (EM) I.1. Description The transmission electron microscope has a number of advantages over the optical microscope. - Image magnification can reach 100,000 times and more. - The resolution is close to 0.2 nm - Thin sample with a thickness of 300 to 500A° 2  1st Year SNL (2023-2024) / Cell biology module / Tutorials / Tut - 3 In the case of the transmission electron microscope, the object to be observed is penetrated by radiation. Here, we no longer use light but a beam of electrons which will be more or less transmitted by the sample, as is the case for light in the optical microscope. I.2. Physical principle of EM Indeed, the two microscopes (LM and EM) are based on the same principle. This is also shown in the diagram in Figure 2. However, the operation of EM is based on the use of electrons and not photons and requires a trajectory and conditions different from that of LM: -Electrons are easily absorbed by matter. It is therefore essential to create a vacuum in the device. - Glass lenses have no effect on an electron beam. To deviate them, we uses electromagnets which act as electromagnetic lenses. - Preparations for transmission electron microscopy must still be thinner than for optical microscopy. - Our eye does not perceive electrons. It is therefore necessary to project the image obtained on a fluorescent screen. An additional lens is therefore necessary, this is the projection lens. Figure2: Comparison between LM and EM 3  1st Year SNL (2023-2024) / Cell biology module / Tutorials / Tut - 3 I.3. Types of EM Two types of EM are used to study the ultrastructure of cells and their components (Figure 3): The transmission electron microscope (TEM) includes: an electron gun (tungsten filament raised to a high voltage of 200,000 V); a vacuum tube (vacuum pump) having a series of electromagnetic lenses; an object holder grid; a fluorescent screen connected to a photographic plate. - In the TEM technique, the electrons transmitted through the sample form the image of the sample and are those observed on the screen. The scanning electron microscope (SEM) includes: an electron gun; a vacuum tube (vacuum pump); an object holder grid; a scanning circuit scanning the surface of the object with a very fine beam of electrons and a screen. - In the SEM technique, electrons backscattered by the observed sample are collected rather than transmitted electrons. - The sample to be observed must be treated in a special way, covered by a layer of metal. We therefore only observe the metallic layer which covers the sample. - The magnifications obtained are generally lower than in transmission microscopy. But the images obtained show the observed object in relief. Figure 3: Comparison between TEM and SEM 4