How to Choose the Optimal Objective?
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Resolving Power
The 5 I’s of Culturing Microbes • Inoculation • Isolation • Incubation • Inspection • Identification 8/18/12 MDufilho 1 Table 4.1 Metric Units of Length 8/18/12 MDufilho 2 Microscopy • General Principles of Microscopy – Wavelength of radiation – Magnification – Resolution – Contrast 8/18/12 MDufilho 3 Figure 4.1 The electromagnetic spectrum 400 nm 700 nm Visible light X UV Infra- Micro- Radio waves and Gamma rays rays light red wave Television Increasing wavelength 10–12m 10–8m 10–4m 100m 103m Crest One wavelength Trough Increasing resolving power 8/18/12 MDufilho 4 Figure 4.2 Light refraction and image magnification by a convex glass lens-overview Light Air Glass Focal point Specimen Convex Inverted, lens reversed, and enlarged 8/18/12 MDufilho image 5 Principles of Light Microscopy • Magnification occurs in two phases – – The objective lens forms the magnified real image – The real image is projected to the ocular where it is magnified again to form the virtual image • Total magnification of the final image is a product of the separate magnifying powers of the two lenses power of objective x power of ocular = total magnification 8/18/12 MDufilho 6 8/18/12 MDufilho 7 Resolution Resolution defines the capacity to distinguish or separate two adjacent objects – resolving power – Function of wavelength of light that forms the image along with characteristics of objectives • Visible light wavelength is 400 nm–750 nm • Numerical aperture of lens ranges from 0.1 to 1.25 • Oil immersion lens requires the use of oil to prevent refractive loss -
Sky & Telescope
S&T Test Report by Rod Mollise Meade’s 115-Millimeter ED Triplet This 4.5-inch apochromat packs a lot of bang for the buck. 115mm Series 6000 THIS IS A GREAT TIME to be in the ice and was eager to see how others market for a premium refracting tele- performed. So when I was approached ED Triplet APO scope. The price for high-quality refrac- about evaluating Meade’s 115mm Series U.S. Price: $1,899 tors has fallen dramatically in recent 6000 ED Triplet APO, I was certainly up meade.com years, and you can now purchase a for the task. 4- to-5-inch extra-low dispersion (ED) First impressions are important, and What We Like apochromatic (APO) telescope that’s when I unboxed the scope on the day Sharp, well-corrected optics almost entirely free of the false color it arrived, I lit up when I saw it. This is Color-free views that plagues achromats for a fraction of Attractive fi nish the cost commonly seen a decade ago. I’ve had a ball with my own recently q The Meade 115mm Series 6000 ED Triplet What We Don’t Like purchased apochromat after using APO ready for a night’s activity, shown with an optional 2-inch mirror diagonal. The scope Visual back locking almost nothing but Schmidt-Cassegrain also accepts fi nderscopes that attach using a system can be awkward telescopes for many years. However, I standardized dovetail system commonly found Focuser backlash still consider myself a refractor nov- on small refractors. -
Practical Tips for Two-Photon Microscopy
Appendix 1 Practical Tips for Two-Photon Microscopy Mark B. Cannell, Angus McMorland, and Christian Soeller INTRODUCTION blue and green diode lasers. To provide an alignment beam to which the external laser can be aligned, light from this reference As is clear from a number of the chapters in this volume, 2-photon laser needs to be bounced back through the microscope optical microscopy offers many advantages, especially for living-cell train and out through the external coupling port: studies of thick specimens such as brain slices and embryos. CAUTION: Before you switch on the reference laser in this However, these advantages must be balanced against the fact that configuration make sure that all PMTs are protected and/or commercial multiphoton instrumentation is much more costly than turned off. the equipment used for confocal or widefield/deconvolution. Given Place a front-surface mirror on the stage of the microscope and these two facts, it is not surprising that, to an extent much greater focus onto the reflective surface using an air objective for conve- than is true of confocal, many researchers have decided to add a nience (at sharp focus, you should be able to see scratches or other femtosecond (fs) pulsed near-IR laser to a scanner and a micro- mirror defects through the eyepieces). The idea of this method is scope to make their own system (Soeller and Cannell, 1996; Tsai to cause the reference laser beam to bounce back through the et al., 2002; Potter, 2005). Even those who purchase a commercial optical train and emerge from the other laser port. -
Extension of Solid Immersion Lens Technology to Super-Resolution Raman Microscopy
Nanospectroscopy 2014; 1: 1–11 Research Article Open Access Edwin Ostertag*, Anita Lorenz, Karsten Rebner, Rudolf W. Kessler, Alfred J. Meixner Extension of solid immersion lens technology to super-resolution Raman microscopy Abstract: Scanning Near-Field Optical Microscopy 1 The near-field accessed with solid (SNOM) has developed during recent decades into a valuable tool to optically image the surface topology of immersion lens technology materials with super-resolution. With aperture-based The main reason for using solid immersion lenses is SNOM systems, the resolution scales with the size of the their capability to enhance the spatial resolution. Solids aperture, but also limits the sensitivity of the detection have higher refractive indices than air. When used as the and thus the application for spectroscopic techniques immersion medium, light travels more slowly and the like Raman SNOM. In this paper we report the extension wavelength is shorter in the optically denser medium of solid immersion lens (SIL) technology to Raman SNOM. which results in an improvement in resolution. Mansfield The hemispherical SIL with a tip on the bottom acts as and Kino were the first to recommend the SIL for an apertureless dielectric nanoprobe for simultaneously increasing the spatial resolution of the optical microscope acquiring topographic and spectroscopic information. [1]. A SIL is a spherical lens (diameter 2r, refractive index The SIL is placed between the sample and the microscope n ) which is polished to a thickness of r (hemisphere objective of a confocal Raman microscope. The lateral SIL type) or (1+1/n )r (supersphere or Weierstrass type) [2,3]. -
31 the Superachromat
Lesson 31: The Superachromat This lesson will explore a unique feature of SYNOPSYS that can be helpful when you need exceptional color correction, better even than an apochromat. Lesson 8 in this Online Tutorial showed how to select three glass types that make it possible to correct axial color at three wavelengths. For many tasks, that is as good as you will need. But not always. Suppose you are designing a lens to be used over the range 0.4 to 0.9 um. Can you do it with an apochromat? Let’s find out. Here is the RLE file for a starting system, where all surfaces are flat except for the last, which will give us an F/8 telescope objective of 6-inch aperture. (Copy these lines and paste them into the MACro editor.) RLE ID WIDE SPECTRAL RANGE EXAMPLE OBB 0 .25 3 UNITS INCH 1 GLM 1.6 50 3 GLM 1.6 50 5 GLM 1.6 50 6 UMC -0.0625 YMT 7 1 TH .6 2 TH .1 3 TH .6 4 TH .1 5 TH .6 END We did not specify the wavelengths yet, so we get the default CdF lines. We need to change this. Open the Spectrum Wizard (MSW), and change the points indicated. 1 After clicking the Get Spectrum button, click the Apply to lens button. Our lens now has a wider spectrum. Here is our starting lens, in the SketchPad display Uggh! Yes, it’s really awful. Let’s optimize it, varying the glass models. Make a MACro: LOG STO 9 PANT VLIST RAD 1 2 3 4 5 VLIST TH ALL AIR VLIST GLM ALL END AANT END SNAP SYNOPSYS 50 Now put the cursor on the blank line after the AANT command, and click the button . -
A New Refracting Telescope Optical Design in This Article
THE MELIOR A NePw ROefracCtingH TeleRscopOe OMptical ADesT ign By Joseph Bietry Refracting telescopes have de - veloped over centuries, from the humble beginnings of the Galilean telescope to the modern apochromats of today. Advances in astronomical refractors often came about through improvements to optical materials that offered better control of color er - rors (chromatic aberration). Occa - sionally, a different optical design offered improvements as well. This article will describe a new optical-de - sign form that offers improvements to chromatic aberration, as well as field of view beyond that of current refracting apochromatic telescopes. The name, the Melior Apochro - mat, was chosen for two reasons. First, the English translation for the Latin “melior” is “better” or “im - Figure 1: A simple lens with spherical aberration. proved,” and this is indeed a per - formance improvement over current apochromats. But of more impor - tance to me is that “Meliora” is the motto of my alma mater, the Univer - sity of Rochester. I was lucky enough to have studied lens design at the UR’s Institute of Optics under such greats as Rudolf Kingslake and Robert Hopkins. By using the root of the motto for the naming of this de - sign, I am honoring the University Figure 2: The upper half of a simple lens with spherical aberration. The plot at the right shows and the professors who gave me the the focus error with respect to the position of the ray within the aperture. Astronomy TECHNOLOGY TODAY 69 THE MELIOR APOCHROMAT parallel rays of light entering from the left of the lens represent an infinitely distant point of light (i.e. -
CCAM Objectives.Pdf
CCAM’s Selection of Zeiss Microscope Objectives Things to consider when selecting an objective 1. Magnification Image scale 2. Resolution The minimum separation distance between two points that are clearly resolved. The resolution of an objective is limited due to diffraction and the nature of light Defined by Abbe’s formula d= l /2NA (l = wavelength of light used, NA = the numerical aperture of the objective) Things to consider when selecting an objective 3. Numerical Aperture (NA) Objective’s ability to collect light and resolve specimen detail at a fixed distance. n = refractive index of medium between front lens element and cover slip. m = ½ the angular aperture (A) (https://micro.magnet.fsu.edu/primer/anatomy/numaperture.html) Things to consider when selecting an objective 3. Numerical Aperture/Refractive index (cont.) • The refractive index is the limiting factor in achieving numerical apertures greater than 1.0. • To obtain a higher numerical aperture, a medium with a higher refractive index must be used. • Highly corrected lenses are designed with higher numerical apertures. Things to consider when selecting an objective 4. Working Distance Distance between the front lens of the objective and the cover glass of the specimen. Note the working distance is reduced with the increase in numerical aperture and magnification. Things to consider when selecting an objective 4. Flatness of Field Correction of field curvature Objectives provide a common focus through the field of view. Such objectives are traditionally named as “plan” Edges in focus Entire field in focus Center in focus https://micro.magnet.fsu.edu/primer/anatomy/fieldcurvature.html Things to consider when selecting an objective 6. -
Oil Immersion Condensers Robert R
Oil Immersion Condensers Robert R. Pavlis One of the most important equations in optics is the law of refraction, often called Snell's law. sin θ1 v1 n2 = = sin θ2 v2 n1 The θ's are the angles from the normal to the light path. The v's are the velocities of light, and the n's are the indices of refraction. We can also write Snell's law thus: n2 sin θi = sin θr n1 Where θi is the angle of incidence, and where θr is the angle after refraction. When sin θi is equal to 1.0 we have the maximum angle of incidence, often called the critical angle. Thus n2 θc = arcsin n1 In microscopy numerical aperture, often abbreviated \na", is defined as na = n sin θ .(θ is the angular radius of the cone of light that is taken in by the lens. n is the index of refraction of the medium between the lens and the object.) The angular aperture is the angle across the cone of light taken in by an objective. It is commonly α denoted by α. This is twice the angular radius of the cone of light. Thus θ = 2 For water at 20C n is 1.3330, and for the glasses used for slides, those used for most \outer" microscope glass lens surfaces, and standard microscope immersion oils it is 1.515. Thus the critical angle from water to air is 48.607o. The critical angle from immersion oil or glass to air is 41.305o. The critical angle from immersion oil or glass to water is 61.626o. -
IMMERSION OIL and the MICROSCOPE John J
IMMERSION OIL AND THE MICROSCOPE John J. Cargille New York Microscopical Society Yearbook, 1964. Second Edition ©1985, John J. Cargille - All rights reserved Since the microscopist’s major field of interest is the application of microscopes and related equipment, the fields in which the instruments are used are, in a sense, secondary. However, many scientists, having selected a field, find the use of a microscope a necessity but secondary to their major field of interest. Therefore, the microscope is thrust upon them as an essential tool. Often, basic background necessary for proper use of the “tool” is lacking or inadequate, having been picked up on the job so they can “get by.” Considering the number of microscopes being used in all types of laboratories and the number of scientists and technicians using these instruments, from reports and requests we gather that they have learned to use them in what might be referred to as “on the job” training to the “get by” level of proficiency. This paper will attempt to broaden the understanding of the “business area” of the microscope, between the condenser and the objective, as it is affected by the use of oil immersion objectives, and also expand on properties of immersion oils and how they can be more fully utilized. THE FUNCTION OF IMMERSION OIL Immersion Oil contributes to two characteristics of the image viewed through the microscope: finer resolution and brightness. These characteristics are most critical under high magnification; so it is only the higher power, short focus, objectives that are usually designed for oil immersion. -
Solid Immersion Microscopy Readily and Inexpensively Enables 12 Nm Resolution on Plunge-Frozen Cells
bioRxiv preprint doi: https://doi.org/10.1101/373647; this version posted July 20, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Solid immersion microscopy readily and inexpensively enables 12 nm resolution on plunge-frozen cells Lin Wanga,1, Benji Batemana,1, Laura C. Zanetti-Dominguesa,1, Amy N. Mooresa, Sam Astburya, Christopher Spindloea, Michele C. Darrowb, Maria Romanoc,d, Sarah R. Needhama, Konstantinos Beisc,d, Daniel J. Rolfea, David T. Clarkea and Marisa L. Martin-Fernandeza,2 aScience and Technology Facilities Council, Central Laser Facility, Rutherford Appleton Laboratory, Didcot OX11 0QX, United Kingdom bDiamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE, United Kingdom cDepartment of Life Sciences, Imperial College London, London SW7 2AZ, United Kingdom dResearch Complex at Harwell, Rutherford Appleton Laboratory, Didcot OX11 0FA, United Kingdom 1These authors contributed equally to the work 2 To whom correspondence should be addressed. Email: [email protected] Keywords: Cryo-fluorescence microscopy, solid immersion lens, STORM, super-resolution, cell imaging Running title: Low-cost/tech STORM for 12 nm resolution Abstract Super-resolution fluorescence microscopy achieves 20-30 nm resolution by using liquid-immersion objectives to optimize light collection and chemical sample fixation to minimize image blurring. It is known that fluorophore brightness increases substantially under cryogenic conditions and that cryo- fixation is far superior in preserving ultrastructure. However, cryogenic conditions have not been exploited to improve resolution or sample quality because liquid immersion media freezes at the objective, losing its optical properties. -
Oil-Immersion Objective Design and Use
EVOS® Cell Imaging Systems APPLICATION NOTE EVOS® Objectives Oil-immersion objective design and use Introduction Oil-immersion objectives are designed to be used with a coverslipped sample. This is usually a 25 x 75 x 1 mm thick glass slide, with the sample mounted between the slide and a thin glass coverslip. The sample is often thinly sliced fixed tissue that has been stained with a dye or treated with fluorescently labeled antibodies for visualization of cellular details. The slide is oriented with the coverslip facing the objective, and a small drop of oil is placed between the front lens of the objective and the coverslip. The oil “links” the two glass surfaces with a similar refractive index, and this optical link greatly improves the amount of light and detail obtained in the image. Immersion oil should only be used with objectives labeled Oil – if used on other (i.e., “dry”) objectives it may harm them. Live cell imaging using an oil-immersion objective is slightly different from fixed- tissue imaging. For live-cell experiments, cells are commonly grown on a coverslip For best results, coverslip- and then viewed in a chamber containing a cell culture medium or buffered saline. corrected objectives are designed to The coverslip forms the bottom of the chamber, and an inverted microscope is used be used with a “#1.5” coverslip. This to focus through the coverslip and see the cells. Microtiter plates with coverslip- corresponds to a glass thickness of thick glass bottoms are also available for high-resolution imaging of live cells. 0.170 mm, or 170 micrometers. -
Chapter 7 Lenses
Chapter 7 Lenses Chapter 7 Lenses © C. Robert Bagnell, Jr., Ph.D., 2012 Lenses are the microscope’s jewels. Understanding their properties is critical in understanding the microscope. Objective, condenser, and eyepiece lenses have information about their properties inscribed on their housings. Table 7.1 is an example from objective lenses. This chapter will unfold the meaning and significance of these symbols. The three main categories of information are Numerical Aperture, Magnification / Tube Length, and Aberration Corrections. Zeiss Leitz Plan-Apochromat 170/0.17 63X/1.40 Oil Pl 40 / 0.65 ∞/0.17 Nikon Olympus Fluor 20 Ph3 ULWD CDPlan 40PL 0.75 0.50 160/0.17 160/0-2 Table 7.1 – Common Objective Lens Inscriptions Numerical Aperture & Resolution Figure 7.1 Inscribed on every objective lens and most 3 X condenser lenses is a number that indicates the lenses N.A. 0.12 resolving power – its numerical aperture or NA. For the Zeiss lens in table 7.1 it is 1.40. The larger the NA the better the resolving power. Ernst Abbe invented the concept of numerical aperture in 1873. However, prior to Abbe’s quantitative formulation of resolving power other people, such as Charles Spencer, intuitively understood the underlying principles and had used them to produce 1 4˚ superior lenses. Spencer and Angular Aperture 95 X So, here is a bit about Charles Spencer. In the mid N.A. 1.25 1850's microscopists debated the relationship of angular aperture to resolution. Angular aperture is illustrated in 110˚ Pathology 464 Light Microscopy 1 Chapter 7 Lenses figure 7.1.