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Optics and Microscopy by David Wu1 IntroductionMicroscopy is one of the main tools for visualization in the biological sciences. In order tokeep a record of what we see, we must transfer an image of an object to film or hard disk -to that end, we interpose and need optics. Thus, we seek a grasp of what our microscopesare doing, and how to get the most out of them. This short write up is meant to accompanypower point slides, available in the form of a ‘pdf’ on the course website. Most of the materialwas taken and modified from either the Olympus microscopy website, the Newport tutorials,CVI Laser tutorials, Fourier Optics by Goodman, and especially, Optics by Hecht.1Following the power point presentation, we’ll start with a few quick pointers on how toclean optics you will encounter in Aph162 lab, a brief review of geometric optics, aberrationsupon those optics, Fourier optics, and finally talk about some of the microscopy configurationsin the lab, if I can get it done.2 How to handle and clean opticsWe will b e dealing with lasers, halogen or mercury lamps, objectives of various sorts, andbare lenses, all, perhaps, with coatings. But we want clean optics because high power lasersor high power arc sources can interact with contaminants on the optics and cause damageto the various coatings on the substrate, or ignite due to the intense heat. Moreover, dust,fingerprints, etc, w ill inevitably reduce the quality of our images.That being said, the first rule of cleaning optics is don’t clean optics unless one absolutelyneeds to. Any amount of cleaning will inevitable introduce microscopic scratches in the optic;multiple cleaning rounds will reduce the quality of the optic multiplicitally. But if one needsto, then the first thing to do is to lightly blow off the dust - that way, when lens tissueis applied to the surface, small particles won’t be dragged around and scratch the surface.When blowing off dust or lint, one should generally use air sources without propellant, andwith low particle number, such as purified nitrogen sources, a bulb blower, or camel hair lintbrushes. It is generally discouraged to use office cleaning blowers, such as THF, since theyhave large diameter particles, and if one is not careful, prop ellant comes out and ends upcovering the optic. Since THF is organic, it just might dissolve any organic coatings. Hence,if one must use office cleaning supplies, then light pressure and keeping the canister upright- so no propellant comes out - will do.After removing as much lint as possible, there are three main techniques used to cleanoptics. The first thing to mention is the necessity of wearing gloves while handling bare optics.This is es se ntial for avoiding getting fingerprints on the optics. The first method is drag anddrop. A drop of methanol is placed on lens tissue (Kodak - not VWR!!), and the wet portionof the wipe is dragged across the surface, exerting no pressure.The wipe method is used for harder s tains, and involves folding the lens tissue until a singleuntouched c orner is available; the tisse is then handled by a pair of hemostats or tweezers,1For example, see the Olympus microscopy website, http://www.olympusmicro.com, or the wiki-bookhttp://en.wikibo oks.org/wiki/Optics, or chapters 5 and 6 in Hecht. The first 100 or so pages in Goodman’sFourier Optics suffices.1solution is applied to the wipe and excess shaken off. The wipe is then applied from the centerof the optic outwards. The tissue is then discarded, regardless if another wiping must be used.The last method is called the immers ion technique, and is used for most metallic coatings.After the surface dust is removed, the optics can be immersed in acetone and rinsed in freshsolvent until clean. In general, bare metal coating and filters should not be touched in anyway.In the case of removing immersion oil from our microsc ope objectives, we fold a lens tissueand using a hemostat or tweezers, apply it to the surface with the oil on it, and let the oilsoak into the lens tissue - we do this without moving the tissue at all. Scratching the surfaceof an objective is bad news. We then proc ee d by using the wipe method as described above.Again, once we move out of the region of the objective lens, we don’t go back!There are, of course, other more specialized ways of cleaning optics, but these are themethods that are appropriate for our use. So what solvents should we use for which surfaces?For our mirrors (non-metallic) or lenses, we use isopropyl alcohol, methanol or acetone ora combination thereof. Acetone plus alcohol forms an azeotrope, meaning that the mixturewill not separate into its constituent components before evaporation; acetone increases thevolatility of the solved, thus allowing for faster evaporation and less streaking. It is also moreorganic than either methanol or isopropyl alcohol. For the filters we have (including dichroics),methanol is generally okay (especially if the optic was made from a reputable company) butoften times, we are just not sure (sometimes the coating is organic, meaning we don’t useorganize, otherwise the coating will be dissolved!). In this case, we need to call the companyto ask. And again, for metal-coated optics, we only use the immersion technique, as describedabove.3 Geometric OpticsWe can think of objects in space as collec tions of point sources that em anate spherical wavesupon illumination of light. A lens, therefore, acts as a collector of light by reshaping thosespherical waves and imaging at the focal point of the lens. Since the lense will be inevitablyimperfect, and can never collect all the light emitted, the image of the object will be blurredsomewhat from the original - that is, the image is diffraction limited. We will return to thispoint in the Fourier optics section.But for now, let’s ignore any talk of waves or wavelength, and keep only the interpositionof refracting objects in our light (ray) path. This limit is the geometric optics limit, andsuffices for many practical aspects of optics.Now, in order to form an image, the refracting surface must be shaped such that all lightrays arrive at the focal point at the same time. So, if we consider a point emitter of light rays,and interposed in the path of the rays, an object of different refractive index but spherical inshape, it can be shown that for a certain ray that arrives at the spherical surface at height h,thatn1lo+n2li=1Rn2sili−n1solo(1)where the notation can


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