THE MICROSCOPE BIO 1101L LEC 1 AUG 9 2021 Interpupillar distance scale Eyepieces Observation Tube (Fixed at 100x) Binocular Tube: CX31RBSF microscope frame Binocular Tube: CX31RBSFA microscope frame Tilting tube: CX31RTBISF microscope frame clamping TubeTube clamping screwscrew (Use the provided Allen wrench) Interpupillar distance scale Interpupillar distance scale Simplified pre-focusing dial Diopter adjustment ring Microscope Frame Revolving Nosepiece CX31RBSF CX31RBSFA CX31RTBISF Objective Transport lock pin* Interpupillar distance scale Specimen holder Main switch Transport lock pin* Transport lock pin* Transport Aperturelock iris pin* diaphragm knob Light intensity control knob Transport lock pin* Condenser centering screws Stage Condenser Fine focus adjustment knob Transport lock pin* Filter holder Coarse focus adjustment knob Place a 45 mm filter on this Transport lock pin* Field iris diaphragm ring Interpupillar X-axis knob distance scale Interpupillar Y-axis knob distance scale I. PARTS AND FUNCTION OF A MICROSCOPE 1. Eyepiece: The lens the viewer looks through to see the specimen. The eyepiece usually contains a 10X or 15X power lens. 2. Diopter Adjustment: A means to change focus on one eyepiece to correct for any difference in vision between your two eyes. 3. Body tube (Head): The body tube connects the eyepiece to the objective lenses. 4. Arm: The arm connects the body tube to the base of the microscope. 5. Coarse adjustment: Brings the specimen into general focus. 6. Fine adjustment: Fine tunes the focus and increases the detail of the specimen 7. Nosepiece: A rotating turret that houses the objective lenses. The viewer spins the nosepiece to select different objective lenses. 8. Objective lenses: One of the most important parts of a compound microscope, as they are the lenses closest to the specimen. 1 A standard microscope has three, four, or five objective lenses that range in power from 4X to 100X. When focusing the microscope, be careful that the objective lens doesn’t touch the slide, as it could break the slide and destroy the specimen. 9. Specimen or slide: The specimen is the object being examined. Most specimens are mounted on slides, flat rectangles of thin glass. The specimen is placed on the glass and a cover slip is placed over the specimen. This allows the slide to be easily inserted or removed from the microscope. It also allows the specimen to be labeled, transported, and stored without damage. 10. Stage: The flat platform where the slide is placed. 11. Stage clips: Metal clips that hold the slide in place. 12. Stage height adjustment (Stage Control): These knobs move the stage left and right or up and down. 13. Aperture: The hole in the middle of the stage that allows light from the illuminator to reach the specimen. 14. On/off switch: This switch on the base of the microscope turns the illuminator off and on. 15. Illumination: The light source for a microscope. Older microscopes used mirrors to reflect light from an external source up through the bottom of the stage; however, most microscopes now use a low-voltage bulb. 16. Iris diaphragm: Adjusts the amount of light that reaches the specimen. 17. Condenser: Gathers and focuses light from the illuminator onto the specimen being viewed. 18. Base: The base supports the microscope and it’s where illuminator is located. II. GUIDE QUESTIONS 1. What is a monocular compound microscope? In terms of the eyepiece and lens system, what type of microscope did you see on the on this activity? - Monocular microscope is a single tube with interchangeable eye pieces and objects subjected under the monocular microscope will always look flat and without depth. After conducting the activity, the observed type of microscope is a compound microscope. Compound microscope in an upright kind of microscope that utilize a two-lens system, first in its ocular lens and the other is the objective lenses. 2. Define the following terms: - Total magnification - The total magnification of the microscope is the product of the objective and ocular lens. - Resolving power - Resolving power is defined as the minimum distance between two objects at which the objects can just be distinguished as separate and is a function of the wavelength of light used and the quality of the optics. It is this effect of diffraction that limits a microscope’s ability to resolve fine details. - Magnification - magnification is the ratio of the object as viewed to its real size. It is the process of magnifying or enlarging something only in its appearance and not it’s physical and actual size. - Resolution - the resolving power of the microscope is the minimum distance two point in the specimen can be separated and still be seen as two points. - Numerical aperture- The numerical aperture of a microscope objective is a measure of its ability to gather light and resolve fine specimen detail at a fixed object distance. Image-forming light waves pass through the specimen and enter the objective in an inverted cone. - Refractive index- is defined as the relative speed at which light moves through a material with respect to its speed in a vacuum. By convention, the refractive index of a vacuum is defined as having a value of 1.0. Refraction is an important characteristic of lenses, allowing them to focus a beam of light onto a single point, and is also responsible for a variety of familiar phenomena, such as the apparent distortion of objects partially submerged in water. - Working distance - Working distance is the distance between the objective lens and the specimen. At low magnification the working distance is relatively long. As you increase the magnification the working distance decreases dramatically. The working distance is the distance between the specimen and objective lens. The working distance decreases as you increase magnification. 2 - Field of vision - Microscope field of view (FOV) is the maximum area visible when looking through the microscope eyepiece (eyepiece FOV) or scientific camera (camera FOV), usually quoted as a “diameter measurement.” - Depth of focus - is the axial depth of the space on both sides of the image plane within which image appears acceptably sharp while position of the object plane and of the objective are maintained. 3. When you moved the slide on the stage up and down, and from left to right, to what direction did the image of the object move in each case? - Any movement of the object also shows up in the opposite way. When you move an object to the right, it appears to move to the left, and when you move it up, its image moves down. The optics of a microscope’s lenses change the orientation of the image that the user sees. A specimen that is right-side up and facing right on the microscope slide will appear upside-down and facing left when viewed through a microscope, and vice versa. 4. When you changed from LPO to HPO, a) was there an increase or decrease in the area of the image in the field? o In instances when you change the objective lens from low power to high power, the high-power objective lens moves directly over the specimen, and the low-power objective lens rotates away from the specimen. Therefore, there is a decrease in area because it was zoomed in. b) was there a change in the orientation of the image? o The image moves in the direction opposite to the movement of the slide. Refraction happens as light passes through the stage aperture, specimen, objective, and the eyepiece in which there’s another lens system that causes the ima5ge to appear inverted. If the slide is moved left while looking through the microscope, it will appear to move right, and if moved down, it will seem to move up. This occurs because microscopes use two sets of lenses to magnify the image. Because of how light travels through the lenses, this system of two lenses produces an inverted image. 5. How do you check if the dot or line you see on the eyepiece is dirt? - It must be noted that, any dirt in the eyepiece/ocular lens or the objectives to the microscope would affect its integrity. Firstly, try to locate where the dirt is whether in the ocular or in the objective lens. Once you’ve located the dirt, Use a squeeze bulb or one of the camera lens cleaner bulbs with the camel hairbrush on the end. If you need more, you can use the compressed air cans that are sometimes used to clean computer keyboards. Do not use any sprays with cleaners. Once blown clean, lightly wipe the lens with Kimwipes or another approved lens cloth. 6. What is an electron microscope? What are its advantages over a light microscope? Enumerate and describe the different types of electron microscope. - Use a beam of electrons instead of a beam of light. Not only does this allow for higher magnification (up to 5 million times with some specialized but also provides higher resolving power. The method used to prepare the specimen for viewing with an electron microscope kills the specimen. Electrons have short wavelengths (shorter than photons) that move best in a vacuum, so living cells cannot be viewed with most electron microscopes. Advantages of Electron microscope over Light Microscope according to Davey (2019) 1. Diverse applications – Electron microscopy has a diverse range of applications in many different fields of research including technology, industry, biomedical science, and chemistry. Examples of applications include semiconductor inspection, computer chip manufacture, quality control and assurance, analysis of atomic structures, and drug development. 2. High-quality images – With proper training, an electron microscope operator can use the system to produce highly detailed images of structures which are of a high quality, revealing complex and delicate structures. 3 3. Magnification and higher resolution – as electrons rather than light waves are used, it can be used to analyze structures which cannot otherwise be seen. The resolution of electron microscopy images is in the range of up to 0.2 nm, which is 1000x more detailed than light microscopy. Types of Electron Microscope There are two main types of electron microscope: the Transmission Electron Microscope and Scanning Electron Microscope. 1. Transmission Election Microscope (TEM) The transmission electron microscope is used to view thin specimens (tissue sections, molecules, etc.) through which electrons can pass generating a projection image. The TEM is analogous in many ways to the conventional (compound) light microscope. o TEM is used, among other things, to image the interior of cells (within sections), the structure of protein molecules (contrasted by metal shadowing), the organization of molecules in viruses and cytoskeletal filaments (prepared by the negative staining technique), and the arrangement of protein molecules in cell membranes (by freeze-fracture). 2. Scanning Electron Microscope (SEM) Scanning electron microscopy depends on the emission of secondary electrons from the surface of a specimen. It provides detailed images of the surfaces of cells and whole organisms that are not possible by TEM. o It can also be used for particle counting and size determination, and for process control. It is termed a scanning electron microscope because the image is formed by scanning a focused electron beam onto the surface of the specimen in a raster pattern. Enumerate other types of microscopes. Describe each. 1. Compound Light Microscopes - type of microscope that uses visible light and magnifying lenses. It is called ‘compound’ microscope with more than one lens and its own light source. 2. Electron Microscopes - uses beam of accelerated electrons as a source of illumination and gives high resolution of 3. Transmission Electron Microscope – most ideal microscope for visualizing structures within the specimen 4. Scanning Electron Microscope – ideal for visualizing 3D surfaces 5. Stereo/Dissecting Microscope – provides a 3D image and typically both transmitted and reflected illumination 6. Inverted Microscopes - used to view living samples that are in a petri dish 7. Polarizing Microscopes- use polarized light along with transmitted and/or reflected illumination to examine 8. Confocal Microscope- uses a laser light to scan samples that have been dyed images chemicals, rocks, and minerals 4
