Typesof microscopeobjectives

Multilayer Coatings - Quality microscope objectives are protected and enhanced by unique high-transmission anti-reflective multilayer coatings that are applied to the lens air-interface surfaces to reduce flare and ghosts and ensure high-contrast images. These specialized coatings are also used on the phase plates in phase contrast objectives to maximize contrast.

To attain higher working numerical apertures, many objectives are designed to image the specimen through another medium that reduces refractive index differences between glass and the imaging medium. High-resolution plan apochromat objectives can achieve numerical apertures up to 1.40 when the immersion medium is special oil with a refractive index of 1.51. Other common immersion media are water and glycerin. Objectives designed for special immersion media usually have a color-coded ring inscribed around the circumference of the objective barrel as listed in Table 3 and described below. Common abbreviations are: Oil, Oel (oil immersion), HI (homogeneous immersion), W, Water, Wasser (water immersion), and Gly (glycerol immersion).

In the tutorial questions we make use of more substantial simulations, which are available for your use in our PC Classroom. The tutorial questions printed in this handout are also reproduced electronically on the World-Wide-Web. These pages are accesible from networked computers within the st-andrews.ac.uk domain. But to gain full benefit from them, the PC Classroom here is best, as there are links from the web-pages to the simulation programmes that are loaded on the PC Classroom network. To get to the pages associated with this (and other) courses within the School, follow the links from the University’s page => Academic Schools => Physics and Astronomy. Once there, follow the links on Teaching and Courseware. Special sessions will be run in first week to introduce students to the PC Classroom, Windows NT, the Web, and the use of the simulations. As well as this being useful for your study of physics, experience of computers is now almost a pre-requisite for many careers.

Objective microscopefunction

Red sunrise/sunset - Blue light (short wavelength) is scattered more strongly than red (longer wavelength), as the amount of scattering depends on l-4. This is why the sky looks blue to us. Also, when the sun's rays propagate through a long path in the atmosphere more blue light has been scattered out of the beam than red, and so more red than blue remains (Rayleigh scattering).

Microscopeparts

Blue skies - Some of the sunlight travelling through the atmosphere is scattered towards the earth; blue light is scattered more strongly than red, therefore ...

Some objectives specifically designed for transmitted light fluorescence and darkfield imaging are equipped with an internal iris diaphragm that allows for adjustment of the effective numerical aperture. Abbreviations inscribed on the barrel for these objectives include I, Iris, and W/Iris. The 60x apochromat objective illustrated above has a numerical aperture of 1.4, one of the highest attainable in modern microscopes using immersion oil as an imaging medium.

The compact disc player - Our prime example of the modern relevance of optics - and the excuse to get some musical backing.

Quantum Waves - We will leave a good treatment of quantum mechanics to Physics 1B, but let us note that many of the ideas of classical waves transfer to the complexities of the quantum world.

Most of the fundamental ideas in waves optics are well covered in the general textbooks that have been recommended to you (Halliday, Resnick, & Walker; Ohanian). Books specifically on optics that you may find useful reading include

Colours in oil spills and mega-bubbles - Where do these colours appear from? We will need to use ideas about light as a wave, and how waves can interfere with each other, in order to explain the origin of these patterns.

Microscope manufacturers offer a wide range of objective designs to meet the performance needs of specialized imaging methods, to compensate for cover glass thickness variations, and to increase the effective working distance of the objective. Often, the function of a particular objective is not obvious simply by looking at the construction of the objective. Finite microscope objectives are designed to project a diffraction-limited image at a fixed plane (the intermediate image plane), which is dictated by the microscope tube length and located at a pre-specified distance from the rear focal plane of the objective. Microscope objectives are usually designed to be used with a specific group of oculars and/or tube lenses strategically placed to assist in the removal of residual optical errors. As an example, older Nikon and Olympus compensating eyepieces were used with high numerical aperture fluorite and apochromatic objectives to eliminate lateral chromatic aberration and improve flatness of field. Newer microscopes (from Nikon and Olympus) have objectives that are fully corrected and do not require additional corrections from the eyepieces or tube lenses.

The interactive tutorial above allows the visitor to adjust the correction collar on a microscope objective. There are some applications that do not require objectives to be corrected for cover glass thickness. These include objectives designed for reflected light metallurgical specimens, tissue culture, integrated circuit inspection, and many other applications that require observation with no compensation for a cover glass.

Various colours - What is "colour"? How much is physics, and how much is psychology and physiology? The colours of the spectrum are directly related to the wavelength of the light, red at ~630 nm, orange as in sodium street lights at 590 nm, yellow around 570 nm, green around 530 nm, blue around 480 nm. Other colours such as brown, grey, purple, etc, are as much psychological and physiological in origin as physical.

Sirens - The shift in frequency that we hear as it goes past is due to the Doppler effect. We will come back to the same ideas later using light.

What did we see in the introductory slide show? This list is intended to indicate some examples of ideas in optics and waves, and to describe the path we will be taking in the subject during the forthcoming lectures .

Stagemicroscopefunction

I positively welcome interruptions from any of you during the lecture if you have not understood a point that I have tried to put across - you will probably not be the only one. Likewise, you are welcome to try picking my brains at the end of the lecture if there are any remaining problems. Your tutors can also be a great help, but remember that you will get the most out of your tutorials if you prepare beforehand, and, in particular, if you have attempted the questions on the tutorial sheets. You are welcome to ask me questions at other times of the week if you can find me (room 214, across the corridor from the school office and down a wee bit).

Compoundmicroscope

Optics is the study of light and its uses. "Light" is electromagnetic radiation which can be detected by our eyes, ie electromagnetic radiation with wavelengths in the range ~400 nm - 700 nm (though often very similar ideas apply beyond both ends of this wavelength range). In this lecture course we will look at basic ideas of light propagation, geometrical optics (imaging, etc), interference and diffraction of light, and some of the many uses to which light is put. Far from being just an "old" subject, optics is becoming increasingly important as more and more use is being made of lasers and optoelectronics in industry and society. We will look at various examples including laser-based remote sensing, optical communications, laser-based length measurement, and optical data storage (the CD). The last topic is the one which has brought lasers, precision optics, and optoelectronics into many households; we will be using this example throughout the course to illustrate the various optical phenomena that we shall be attempting to explain.

World-class Nikon objectives, including renowned CFI60 infinity optics, deliver brilliant images of breathtaking sharpness and clarity, from ultra-low to the highest magnifications.

Lasers - A flourishing research field in this school, and a technologically important topic. The laser is a prime example of the modern use of optics, with applications in material processing, optical communications, optical data storage, medicine, warfare, science, metrology, and reprographics.

There is a wealth of information inscribed on the barrel of each objective, which can be broken down into several categories. These include the linear magnification, numerical aperture value, optical corrections, microscope body tube length, the type of medium the objective is designed for, and other critical factors in deciding if the objective will perform as needed. A more detailed discussion of these properties is provided below and in links to other pages dealing with specific issues.

Cats eyes - What is happening in our cat and on the road to give strong reflections back towards us? Some interesting ideas in refraction and reflection here. Timepieces - The pendulum clock and the "quartz" watch both function due the well-defined period associated with an oscillator. Although the sizes are very different, much of the physics is the same.

Glass Design - The quality of glass formulations has been paramount in the evolution of modern microscope optics. Numerous designs have been implemented by a variety of manufacturers, but we will limit this discussion to a specialized low dispersion glass formulation. Extra Low Dispersion (ED) glass was introduced as a major advancement in lens design with optical qualities similar to the mineral fluorite but without its mechanical and optical demerits. This glass has allowed manufacturers to create higher quality objectives with lens elements that have superior corrections and performance.

Aimsof microscopepractical

Identification of the properties of individual objectives is usually very easy because important parameters are often inscribed on the outer housing (or barrel) of the objective itself as illustrated in Figure 1. This figure depicts a typical 60x plan apochromat objective, including common engravings that contain all of the specifications necessary to determine what the objective is designed for and the conditions necessary for proper use.

Rainbows - You have all seen them, but how is the white light split up into the arcs of different colours that we see in the rainbow?  See the material on rainbows in the ray optics section to learn more.

Other features found on specialized objectives are variable working distance (LWD) and numerical aperture settings that are adjustable by turning the correction collar on the body of the objective as illustrated in Figure 2. The plan fluor objective on the left has a variable immersion medium/numerical aperture setting that allows the objective to be used with multiple different immersion media, including oil, water, and glycerin. The plan apo objective on the right has an adjustable working distance control (termed a "correction collar") that allows the objective to image specimens through glass coverslips of variable thickness. This is especially important in dry objectives with high numerical aperture that are particularly susceptible to spherical and other aberrations that can impair resolution and contrast when used with a cover glass whose thickness differs from the specified design value.

Optical Fibres - Application of basic ideas of reflection and refraction show that light can be guided along transparent fibres. This may be used for illumination or for optical communications. Optical communications? If you telephone anyone more than a few tens of miles away you are likely to be using optical fibres, and if you use the University computers, that whole network is linked together using pulses of light speeding through a loop of fibres around St Andrews. The "light" that is chosen for use has a long wavelength - due to Rayleigh scattering again.  This School is currently leading a 10.5 million pound research collaboration looking at how to get even faster data transfer across optical communication networks.

I will be using a number of computer-based simulations in this course to try to explain what is going on.  I believe that you will find them useful to explore. In the web pages of lecture summaries you will find a number of "Java applets" that should be able to run on most computers.  These are small programs that allow us to get you exploring some of the optics ideas.  Most are from other sites, and are acknowledged as such.  Those from Lightlink have a <=Back button at the foot of their pages - this is not a back button in the correct sense; clicking on this will take you to the Lightlink index.  Please use your browser's back button instead.

From the discussion above it is apparent that objectives are the single most important element of a microscope. It is for this reason that so much effort is invested in making sure that they are well-labeled and suited for the task at hand.

All are on reserve in the departmental library. Longhurst is my favourite, but Hecht is the one that is currently recommended for purchase for the Honours course on Optics.

Special Features - Objectives often have additional special features that are specific to a particular manufacturer and type of objective. The plan apochromat objective illustrated in Figure 1 has a spring-loaded front lens to prevent damage when the objective is accidentally driven onto the surface of a microscope slide.

Investigate how internal lens elements in a high numerical aperture dry objective may be adjusted to correct for fluctuations in coverslip thickness.

Although not common today, other types of adjustable objectives have been manufactured in the past. Perhaps the most interesting example is the compound "zoom" objective that has a variable magnification, usually from about 4x to 15x. These objectives have a short barrel with poorly designed optics that have significant aberration problems and are not very practical for photomicrography or serious quantitative microscopy.

Prism and Refraction - "White" light is composed of many different wavelengths, which we can separate by using, for example, a prism. Spectacles, cameras, - The idea of the lens and image formation are important for telescopes, binoculars many optical instruments, and we shall spend some time developing the theory needed to predict the behaviour of light and lenses.

The objective depicted on the left in Figure 3 has a parfocal distance of 45mm and is labeled with an immersion medium color code in addition to the magnification color code. Parfocal distance is measured from the nosepiece objective mounting hole to the point of focus on the specimen as illustrated in the figure. The objective on the right in Figure 3 has a longer parfocal distance of 60 millimeters, which is the result of its being produced to the Nikon CFI60 200/60/25 Specification, again deviating from the practice of other manufacturers such as Olympus and Zeiss, who still produce objectives with a 45mm parfocal distance. Most manufacturers also make their objective nosepieces parcentric, meaning that when a specimen is centered in the field of view for one objective, it remains centered when the nosepiece is rotated to bring another objective into use.

MicroscopeObjectives magnification

What isobjectivelens inmicroscope

Hovering spectra - Produced by shining a spotlight through a special effects filter - a diffraction grating in fact. We will need to look at light as a wave again here, and will show how these effects can be used to separate light of different wavelengths in spectroscopy (terrestrial or astronomical), and to colour certain insects.

The physics and mathematics of wave motion underlie many important phenomena. The water wave on the sea, the vibration of a violin string, and the quantum mechanical wave associated with an electron can all be described in a similar way. Light too, often displays properties that are wave-like. We will start the course looking at "ray" optics, but then pause for a general treatment of waves of all types. We will start this waves section by reviewing ideas of oscillations and simple harmonic motion, and go on to look at the physics of travelling and standing waves. We will apply these ideas to various types of wave, and see how all-pervading this topic is in physics. We will then be in a position to consider a number of phenomena in which the wave properties of light are important.

Parfocal Distance - This is another specification that can often vary by manufacturer. Most companies produce objectives that have a 45 millimeter parfocal distance, which is designed to minimize refocusing when magnifications are changed.

Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.

Most manufacturers have now transitioned to infinity-corrected objectives that project emerging rays in parallel bundles from every azimuth to infinity. These objectives require a tube lens in the light path to bring the image into focus at the intermediate image plane. Infinity-corrected and finite-tube length microscope objectives are not interchangeable and must be matched not only to a specific type of microscope, but often to a particular microscope from a single manufacturer. For example, Nikon infinity-corrected objectives arenot interchangeable with Olympus infinity-corrected objectives, not only because of tube length differences, but also because the mounting threads are not the same pitch or diameter. Objectives usually contain an inscription denoting the tube focal length correction as will be discussed.