Module 2 Introduction and Lesson 1 - 3.pdf

Module 2: MICROSCOPIC CALCULATIONS PRPM 110L Pharmaceutical Botany & Taxonomy Prepared by: Mary Ruth Manansala, RPh Introduction What we are commonly using in the undergraduate college laboratory are light microscopes though in advanced science courses and program...

Module 2: MICROSCOPIC CALCULATIONS PRPM 110L Pharmaceutical Botany & Taxonomy Prepared by: Mary Ruth Manansala, RPh Introduction What we are commonly using in the undergraduate college laboratory are light microscopes though in advanced science courses and programs other sophisticated forms of microscopes are housed in the laboratory/instrumentation room and utilized. Most light microscopes used in our college Bio-sci lab can magnify cells up to approximately 400 times and have a resolution of about 200 nanometers. Introduction Two important parameters in microscopy are magnification and resolving power. Changes in the orientation of the image that you see owe it to the optical system of the microscope. Learning Outcomes At the end of this module, you will learn to: 1. Calculate the field of view in relation to magnification of lenses of the microscope; 2. Calculate the object size from the measured field of view; 3. Calculate the total magnification from the determined object size. Basic Metric System  Module 2 Lesson 1: Field of View PRPM 110L Pharmaceutical Botany & Taxonomy Definition of terms: Power of resolution: the measure for the ability to tell two points apart. It describes whether two adjoining points can still be perceived as separate. Definition of terms: Magnification of a microscope: is the product of Vobjective X Vocular Objectives: Scanning: 4x (40x) Low Power Objective: 10x (100x) High Power Objective: 40x (400x) Oil Immersion Objective: 100x (1000x) Definition of terms: Numerical aperture is the sine of half the angle of the cone of light from each point of the object that can be accepted by the objective multiplied by the index of refraction of the medium in which the object is immersed. The N.A. represents a performance number that can be compared to the relative aperture (f-number) of a camera lens. The N.A. values can be used for directly comparing the resolving powers of all types of objectives. The larger the N.A., the higher the resolving power is. Definition of terms: Working distance: refers to the distance from the cover glass to the nearest point of the objective. Definition of terms: Focal depth - refers to the distance between the upper and lower limits of sharpness in the image formed by an optical system. As you stop down the aperture iris diaphragm, the focal depth becomes larger. The larger the N.A. of an objective the shallower the focal depth is. FOCAL DEPTH Definition of terms: Field number – This is a number that represents the diameter in mm of the image of the field diaphragm that is formed by the lens in front of it. Definition of terms: Field of view diameter -The actual size of the field of view in mm on the object surface. Diameter of Field of View Enlargement or Magnification of a specimen is the function of a two-lens system: ocular lens is found in the eyepiece objective lens is situated in a revolving nose-piece The objective lens is nearer the specimen and magnifies it, producing the real image that is projected up into the focal plane and then magnified by the ocular lens to produce the final image. Overall Linear Magnification Magnification Magnification is the number of times an object is enlarged by a lens system or enlarged in the drawing or illustration The microscope’s objective lens which lies closest to the specimen focuses on an image of the object high up in the tube. The ocular or eyepiece at the upper end of the tube collects the light rays from the first image, produces a further medication of the image, and focuses a virtual inverted image of the object onto the retina of the observer’s eye. The combined action of the objective and eyepiece lenses forms the final magnified image seen through the microscope. Magnification The linear magnification of a microscope can be defined as the ratio of the length of the final image to the length of the original object. Linear magnification can be calculated by multiplying the magnification of the eyepiece by the magnification of the objective being used to view the specimen. Magnification Magnifying an object without good resolution is called empty magnification, as the image appears larger but no greater detail can be seen. Microscope magnification = Vobjective X Vocular For example, the magnification of the ocular lens is 10X with the magnification of lenses of objectives as follows: 4X, 10X, 40X, and 100X. So what will be the magnifications at low, medium, high power, and oil immersion? magnification at low power = 10 x 4 = 40 times magnification at medium power = 10 x 10 = 100 times magnification at high power = 10 x 40 = 400 times magnification at oil immersion type of power = 10 x 100 = 1,000 times  How do you now calculate the field of view in your microscope? Calculate the field of view in your microscope: If using an eyepiece only? Please check the specifications of the eyepiece, let's say you have WF 10X/20. The magnification of the eyepiece is 10X with a field number of 20. Therefore the FOV here is 10 divided by 20 is 0.5 or a FOV diameter of 0.5 millimeters. Calculate the field of view in your microscope: If eyepiece and objective lens are used? Let us use the specification of the eyepiece in the example above, WF 10X/20 and you set the objective lens at 40. Multiply 10 (Vocular ) by 40 (Vobjective ) to get 400. A FOV diameter of 0.05 mm will be obtained by dividing 20 by 400. If you know the dFOV for low power lens, the FOV of higher power lenses can be calculated using the ratio as follows and vice versa: Low Power Magnification × Low Power Field of View = Higher Power Magnification × Higher Power Field of View Example: The Vocular of 10× with the Vobjective of 10× produced a field of view of 800 µm. Compute for the field of view when the Vocular is 40× and the Vobjective is 100×. Solution: The magnification at lower power is 10 × 10 = 100 The magnification at higher power is 40 × 100 = 4000 Field of view = 100 × 800 (FOV) / 4000 = 20 µm Reduction Even the lowest power objectives such as x4 (Scanning) provide too much magnification for some specimens which cannot me encompassed within the field of view. This scenario happened to us all simultaneously, so we need to reduce the magnification so magnification will be at ease and less strain to our eyes. Use of an instrument such as a reduction image scale would be very convenient.  Lesson 2: Object (Actual) Size PRPM110L Pharmaceutical Botany & Taxonomy Prepared by: Mary Ruth Manansala, RPh Using medium power (10X objective), the diameter of the field of view is measured as 2 mm. Looking across the diameter are cells aligned. Counting it gives you 8 cells. Therefore there are 8 cells visible across the dFOV of 2 mm. You are determining the approximate size of each cell, dividing the dFOV by the number of cells (2 mm / 8 ) will give you 0.25 mm. The size of each cell is therefore 0.25 mm or 250 µm.  Module 2 Lesson 3: Drawing Magnification PRPM110L Pharmaceutical Botany & Taxonomy Below is the equation for calculating the drawing magnification: size of the drawn object in μm Magnification of drawing = ----------------------------------- object’s actual size in μm Let us say the drawing is roughly 7 cm long. This equates to 70 mm or 70,000µm. The actual size of the specimen is about 500 µm. What is the drawing magnification? Drawing magnification = drawing size / actual size. 70,000 µm / 500 µm = 140 X Drawing Magnification = 140 X How do you calculate the magnification of an image using a scale bar?  Plant Samples PRPM110L Pharmaceutical Botany & Taxonomy Plant Sample Common Name: Gumamela Scientific Name: Hibiscus rosa-sinensis Family Name: Malvaceae Plant Sample Common Name: Sesame seeds Scientific Name: Sesamum indicum Family Name: Pedaliaceae Plant Sample Common Name: Indian Rubber Tree Scientific Name: Ficus elastica Family Name: Moraceae  Any questions? END OF DISCUSSION