Based on W.H. Heidcamp Edited by C. Antonescu, R. Botelho and L. Victorio 1. 2. 3. 4. 5. 6. 7. 8. Laboratory 1 The Light Microscope and Image Analysis Learning Objectives Properly care for and confidently use a bright field microscope. Identify parts of a bright field microscope and understand their function. Set up the microscope. Focus on a specimen at each magnification: 4x, 10X, 40X and 100X. Use proper technique with oil immersion lens. 9. BLG411 Cell Biology II Lab Capture digital images Employ software (ImageJ) to analyse image data. Employ numerical analysis software to obtain statistical relevant information and build graphs representing data (eg. Excel, Prism) Employ multimedia software to generate a figure comparing data (eg. Powerpoint, Adobe Illustrator). 10. Learn the basics of micropipetting Experimental Objectives 1. Use a light microscope to observe fixed cells. 2. Develop micropipetting technique 3. Compare and identify differences between wild-type yeast and fab1▲ mutant yeast. 4. Use simple tools in image analysis software to quantify cellular features Laboratory Exercises Exercise 1.1 The Bright Field Microscope Exercise 1.2 Measurements of Cell Organelles Exercise 1.3 Micropipetting Exercise 1.4 Use of Oil Immersion (100x) Lens 1 Based on W.H. Heidcamp Edited by C. Antonescu, R. Botelho and L. Victorio NOTE: ************ ****** 1. 2. 1. All experiments are to be done in pairs. 2. All observations, drawings, and calculations are to be recorded in your lab notebook and must be completed during lab period. Your TA will need to sign your notebook at the end of the day. You will NOT receive marks if this is not done. 3. 3. You will need to acquire image data and import into an approved digital device for future analysis (USB, laptop, phone, etc.). You may need to upload these images as proof that you acquired them. BLG411 Cell Biology II Lab ******** Your lab notebook In this laboratory series, you are required to use a laboratory notebook to annotate your observations and answer questions. You may use a partitioned lab notebook for BLG411 if it is clearly labeled which section belongs to BGL411. A few comments about the notebook: ************* You should number your pages, starting with page 1 for the beginning of the partition dedicated to BLG411. For each experimental lab (Lab 1, Lab 2, etc.), you need to give: a. A lab title (as above) b. Complete date of your lab c. Use subheading like 1.1, 1.2, etc. with a title for each exercise. d. Answer the relevant questions and make the relevant observations within the relevant subheading. e. At the end of the day, your TA will need to review and initial your lab book at the last page of your annotations for each specific day. 2 f. If there are calculations, please do them all ahead of time in the flowchart and/or lab notebook You must not lose the lab notebook. Based on W.H. Heidcamp Edited by C. Antonescu, R. Botelho and L. Victorio Introduction to the Light Microscope The light microscope is a valuable tool in the development of scientific theory by enabling visualization of tiny objects through the use of magnifying lenses. Modern light microscopes are called compound microscopes because they have two lens sets; a primary magnifying lens and a secondary lens system. Light is made to pass through an object and is then focused by the primary and secondary lens. - Using these magnifying lenses, microscopes do two things: magnify and enhance resolution. Magnification is the visual enlargement of a small object (Figure 1.1). However, one can enlarge the image of an object, and obtain a highly pixelated and blurry display (i.e compare the small top panel and its progressive magnification of the left panels of Figure 1.1 — the final 100x magnified image is pixelated though it is bigger see left panels of Figure 1.1). In order for magnification to be useful, microscopes must "fill in" the extra pixels with new information that was previously condensed into the few pixels that made up the small image (compare the small top panel and its progressive magnification of the right panels of Figure 1.1 – the final 100x magnified image displays a lot of new information and structures that were not visible in the top, small image). When this happens, microscopes enhance resolution, in other words we can now observe the details and components of what looked like a small, simple object. For the human eye to see two objects as two, it is necessary that the two objects be about 0.1 mm apart when held 25 cm from the face. If they are closer than 0.1 mm, we will perceive them as a single object. Thus, if two objects are 0.01 mm apart, we cannot detect them unless we magnify an image of them by 10X. What has happened is that we have effectively altered our resolution ability from 0.1 mm to 0.01 mm through the use of a magnifying lens. We would say that our limit of resolution has changed from 0.1 mm to 0.01 mm, or inversely, our resolving power (resolution) has increased by a factor of 10. Magnification with Resolution Magnification Alone. 45X BLG411 Cell Biology II Lab 10X Figure 1.1. Magnification vs. Resolution. 3 Based on W.H. Heidcamp Edited by C. Antonescu, R. Botelho and L. Victorio If an image of a cell is magnified from 10X to 45X, the image gets larger, but not necessarily any clearer. The image on the left, in Figure 1.1, is magnified with no increase in resolution. The image on the right is magnified the same, but with increasing resolution. Note that by the time the image is magnified 10X (from 10X to 100X), the image on the left is completely unusable. The image on the right, however, presents more detailed information. Without resolution, no matter how much the image is magnified, the amount of observable detail is fixed, and regardless of how much you increase the size of the image, no more detail can be seen. At this point, you will have reached the limit of resolution of the lens. This property of the lens is fixed by the design and construction of the lens. As mentioned, the value for resolution may be determined in one of two ways. It can be measured as the smallest distance between two points, which allows us to see the points as distinct. With this measurement, resolution increases as the distance decreases - that is, there is an inverse correlation between the limit of resolution and what you actually resolve. Equation 1.1a Limit of Resolution= Equation 1.2 0.61 χλ BLG411 Cell Biology II Lab The resolution of a lens is a property of its physical properties and of the wavelength of light that is passed through the lens. The physical properties are summed up in a value known as the numerical aperture (N.A.) while the wavelength ( is determined by the color of light (also see your book, Chapter 9 for a review). N.A. = N sin 0 N.A. 4 The numerical aperture of a lens is dependent upon two parameters: i) the angle at which the light hits the lens at (the angle of incidence, also known as the cone angle) and ii) the refractive index of the glass of which the lens (see Figure 1.2). In equation 1.2, 0 is equal to half of the angle of incidence or cone angle. The cone angle can be altered by a sub-stage condenser. If the condenser is moveable, then the cone angle can be adjusted; the closer the sub-stage condenser is to the object, the greater is the cone angle. Thus, one can improve the numerical aperture (NA) and resolution by equipping microscopes with a sub-stage condenser. Based on W.H. Heidcamp Edited by C. Antonescu, R. Botelho and L. Victorio Lower magnification Ⓒ = 32⁰ Commediamark N.A. = <1.0 45 X High Dry Power Higher magnification Ⓒ = 58⁰ Cone Angle 0 100 X Oil Immersion Cone Angle N.A. = 1.3 5 BLG411 Cell Biology II Lab Immersion oil film Figure 1.2. Cone angle and numerical aperture. The ottom part is a sub-stage condenser - by moving it closer to the sample, one can increase cone angle and resolution (Source: http://www.healthtard.com/journey-to-optical-light- microscopy). The refractive properties of a lens are summed up in a measurement known as the refractive index (R.I. or N). The refractive index is a function of the bending of light from air through glass and back again. In a microscope, the glass of the lens is specially formulated to increase its refractive index. Once manufactured, however, this property cannot be changed. However, by adding oil between objective and slide, this can further improve the total refractive index (light bending) of the light path further boosting the resolution. Putting all of this to practical use, resolution can be increased in four ways: 1) increase the angle of light incidence through a sub-stage condenser; 2) the refractive index can be maximized by using specially manufactured lenses; 3) by controlling the medium through which the light travels, i.e. using oil; 4) by decreasing the wavelength of light used. For maximum resolution, all four properties must be optimized.