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Chapter 3: A Primer How Lenses "Image" and Microscopes Magnify! Copyright James Pawley, 2000 Introduction For over a century and a half, spherical aberration has been recognized as characteristic of lenses made having spherical surfaces. Basically, refraction occurring near the periphery of such a lenses produces a shorter focal length then that near the axis. However, as early as 1830 Joseph Jackson Lister showed that the judicious coupling of lenses of different shapes and glasses could reduce the blurring produced by spherical aberration to below that caused by diffraction. That was all most biologists needed to know: Spherical aberration had been conquered! They should have read the fine print. In fact, spherical aberration can only be compensated if the refractive index (RI) of every segment of the optical path (not just the lenses!) is known and unchanging. Although this condition is substantially met when thin, embedded sections are viewed with a properly oiled, immersion objective, success with even this limited class of specimens can collapse if the specimen is heated or cooled Because the refractive index (IR) of the oil varies with temperature. The purpose of this article is to summarize the causes and effects of spherical aberration, to explain how wavelength, working distance, RI and temperature can constrain efforts to correct it, and to describe both experimental techniques that allow these conditions to be met and new accessories and techniques that can compensate it. Along the way, we will also have to sort out basic microscope optics. The problem of spheres

optics that can be understood with reference to Fig. 1. Because it is thin, the lens can be represented as if all of its focussing action occurs at a central plane delineated by the vertical line. (Most microscope optics are not actually 'thin' in this regard.) 1. Any ray that passes through the center of a thin lens is unaffected by it and can therefore be drawn as a straight line. Both the optical axis and the heavy line joining the two vertical arrow-heads, representing the object and the image, are such rays. 2. Any ray that is parallel to the optical axis on one side of the lens, will pass through a point called the focal point of the other side of the lens. 3. The distance between this point and the center of the lens is the most important characteristic of a lens: the focal length, f. Every lens has two focal points that are defined, one focal length along the optical axis on either side of the central plane. (Thick lenses are just thin ones, except that instead of a single, central plane, they have two principle planes with a variable space in between. Optically they act as if these principle planes coincide and the optical axis is shortened by the distance between the two planes.) Unfortunately, the only type of surfaces that we know how to make with optical accuracy are spherical ones and, and as noted above, ideal lenses cannot be made with spherical surfaces. Therefore, simple lenses always have aberrations and lenses with enough elements to correct the aberrations are not thin. Simple glass converging lenses with spherical surfaces always have some spherical aberration because the effective focal length of the light passing through the edges of the lens (the peripheral rays) is always shorter than that for rays nearer to the axis (paraxial rays). The curvature near the edge of a spherical lens is just too steep. Fig 2. The result is that the image is not well focused and the blurriness is proportional to the amount of misfocus, !fsph.

.
f Object distance f

.
Image distance

Optical axis

Fig. 1. Lenses are made by rubbing one circular piece of glass over another, with abrasive in between. If the moving piece rotates and slides over the other in a close-to-random manner, the force concentrates at the edges of the fixed piece whenever the upper block extends past its edge. This causes the edge to erode preferentially, making the upper surface of the lower piece convex. The mating surface of the moving piece becomes concave with a similar curvature. For over 300 years, this simple process has allowed craftsmen to fashion surfaces that are perfectly spherical to within a fraction of the wavelength of light. Such surfaces are the functional components of almost all lenses. The manner in which an ideal thin lens forms an image can be described by 3 simple statements about geometrical

fperi

!f sph fpar

Figure 2. On the other hand, because the focal length of a refracting lens depends on the focussing properties of the both surfaces, lenses can be made with a variety of shapes, or "form factors" and still have the same focal length. The amount of inherent spherical aberration varies with the form factor.

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Form factors

Figure 3. Lenses with concave surfaces cause parallel light to diverge. Axial parallel light striking such a lens diverges in such a way that it appears to come from a point on the same side as the incoming parallel beam. This point is still called the focal point but the focal length is now considered to be negative. Such lenses also have negative values for !fsph. By combining a strong (i.e., short focal length) positive lens having a low spherical aberration form factor and a weaker, negative lens having a relatively strong (but negative) spherical aberration form factor, it is possible to make a reasonably strong converging lens having almost no spherical aberration. This system works for light of a single wavelength but as the focal length of any refracting lens depends for the RI of the glass from which it is made, and as the RI of glass drops as the wavelength increases (a phenomenon called dispersion), it follows that f increases with wavelength. This is called chromatic aberration (Fig. 4) and the amount of misfocus (fch) is proportional to the dispersion of the glasses used.

tional elements can increase the number of wavelengths at which the focal length will be the same: 3 for apochromat and 4 for plan-apochromat. The phenomenon of dispersion that creates chromatic aberration also limits efforts to correct spherical aberration because the compensating aberrations of the various elements only have the desired effects at specific wavelengths. In general, spherical aberration in an objective can be corrected at about the same number of wavelengths as chromatic aberration, although the specific wavelengths may not be identical. In practice, the system works reasonably well at nearby wavelengths as well. In fact, as long as the blurriness caused by an aberration remains less than that caused by diffraction, the optic is said to be diffraction-limited. The working microscopist must spend considerable time and effort to insure that the optic performance stays this way! From the forgoing, it is clear that if one only uses spherical surfaces to create a simple lens, having a single focal length for all wavelengths and for both peripheral and paraxial rays, one has to use a large number of elements. Although such an assemblage is unlikely to be thin in terms of its actual shape, it is the only type of lens for which Fig. 1 is an adequate description. Tube length and magnification Figure 5 shows the geometry of a simple lens being used to form a magnified image of an arrow. It is easy to see, by similar triangles, that for any lens, the ratio of the size of the Image arrow divided by the size of the Object arrow (i.e., the magnification, m) is the same as the ratio of the object distance to the image distance. Magnification (3.1) = m = Image distance Object distance

lens of low dispersion glass

..
!f ch

a.

fblue fred

By similar triangles:

.
lens of high dispersion glass fblue fred

Magnification = image = image distance object object distance

Optical axis

. .
!f ch Achromatic doublet made with positive element of low-dispersion glass and weaker, negative element out of high-dispersion glass

b.

f Object distance

f Image distance

Figure 5

fblue fred

c.

Figure 4 As with spherical aberration, chromatic aberration is at least partially correctable by pairing short focal length lenses of low dispersion glass with weaker negative lenses of high dispersion glass (Fig. 4c). Such a design allows chromatic aberrations to cancel out at 2 specific wavelengths, producing an achromat. Addi-

Theoretically, any lens can be used to obtain an arbitrarily high magnification, just by increasing the image distance and then changing the optic distance slightly just to maintain focus. However, this can become inconvenient in terms of size. For the eye to be able to see the features that are a fraction of a micrometer in size, it needs a total magnification of 1,000 to 2,000x. To obtain this much magnification from a single lens with a focal length of 2mm (equivalent to a 100x objective), would require an object distance of 4 meters: and very long arms to move the specimen! More to the point, lenses can only be designed to work in a diffraction limited manner for one set of object and image planes. Therefore, all modern light microscopes are compound, in that the magnified image is produced by the action of two groups of optical elements: the objective and

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the ocular, each of which acts as a simple lens (Fig. 5.5).

Figure 5.5 The purpose of the objective is to focus an enlarged image of the structure present in the focus plane onto the intermediate image plane, 10 mm below the edge of the tube that the ocular slips into. The ocular then focuses this image, through the lens of the eye, and onto the retina. Alternatively, a photo-ocular may focus an enlarged version of the intermediate image onto the surface of a piece of photographic film or a CCD sensor. The light-tight structure connecting these two lenses is called the tube and below we will discuss how the length of this structure effects the performance of the entire ensemble. Because the ocular operates on an image that has already been magnified, its optical quality is less important to the accuracy of the final image than is that of the objective. As should be clear from the preceding discussion, spherical aberration is more severe for rays going through the edges of a lens: those leaving the specimen at close to the angle, !, as shown in Fig. 6.
Lens aperture stop Acceptance angle Radius, r Convergence angle = ! mag

.
Figure 6

Object distance = o.

Image distance = i.

To a reasonable approximation, the angle at which these peripheral rays converge at the intermediate image plane (and therefore the angle that must be accepted by the next optical element: the ocular) is smaller than ! by a factor equal to the magnification of the system.1 Consequently, the optics of oculars need only work at low acceptance angles. In the following discussion we will assume that the blurring caused by aberrations in the ocular is small compared to the smallest features present in the intermediate image and confine our attention to the optical characteristics of the objective. How do we change magnification? Referring to Fig 7a, we see that the size of the image (right arrow) is about 2 times as large as the object (left arrow).

By moving the object closer to the focal point of the 2x objective, it is possible to use the same lens to produce a more highly magnified image, (i.e., 4x, Fig. 7b). However, this image will now occur at a plane located !zI behind that at which the 2x image was focused (i.e., the plane on which the ocular is normally focused and which you can see with your eye.). In other words, if you were willing to make the tube length longer and refocus the objective slightly, you could use the same objective to produce twice the magnification. The reverse is also true: if you moved the ocular closer to the objective, the in-focus plane would be farther from the focal point and the magnification of the system would be lower. The fact that each objective has a specific magnification engraved on the barrel is a direct consequence of the fact that, in modern instruments, the tube length is not a variable but is fixed by the construction of the microscope. As we will see below, there are other reasons why this dimension must be fixed. To produce the same increase in magnification without moving the image plane, one must reduce the focal length of the objective by a factor of ~2, (Fig 7c). Now, however, the plane in the specimen that is focused onto the intermediate image plane will be one that is about 2x closer to the central plane of objective than that focused on in the 2x case (Fig. 7a). Were we to implement this sort of system in a microscope, we would have to move the specimen a long way (millimeters) toward the objective, !zob, whenever we increased magnification. As this is impractical, an alternative strategy is used. Today, microscopes are designed so that the position of the focus plane in the object is held at a fixed distance ("parfocal") from the intermediate image plane, (Fig. 7d). In older microscopes with a fixed tube length of 160 or 170 mm, and objectives mounted in barrel 45 mm long, the distance between these two planes was usually either 205 mm or 215 mm (resp). With modern infinity conjugate systems, the tube length is somewhat more variable but, in general, this dimension is the same for each manufacturer, to allow interchange of objectives. In the end, it is constrained by the space available between the top of a table and the eye level of a seated observer! To summarize: for microscopes with 205mm between the focus plane in the specimen to the intermediate image plane: - To increase magnification, the focal length of the objective must be reduced and its optical center must be moved towards the object plane. - The magnification is approximately inversely proportional to the focal length of the objective (only approximately because as its focal length becomes shorter its center must move towards the object so the image distance becomes longer). - With 160 mm tube length objectives, the focused plane in the object will be slightly past the focal plane. With infinity conjugate objectives, these two planes coincide. - Therefore, the optical center of any high-magnification objective will be closer to the focus plane in this specimen than that of a lower magnification objective.

This inverse relationship between NA and image size is important in many areas of microscopy, particularly the optics used to collect the light from the source.

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.
!z ob

2x magnification, reference image and object position a.

image in the intermediate image plane that was (186.5/18.5) = ~10x larger than the object. - An objective with a focal length of 2 mm, located about 2 mm above the focus plane provides an image magnification of (205-2)/2 = ~100x. In all cases, the optical center of the objective is about one focal length above the focus plane. It follows then, that the back focal plane (BFP) of the objective (which will be discussed below) is always about 2 focal lengths above the focus plane, i.e., that the BFP position varies with objective magnification. This is why you must refocus the Bertrand lens or phase telescope in order to obtain a sharp image of the BFP of different objectives. For microscopes with infinity objectives, the actual dimensions may be different than the examples above, but the distance between the tube lens and the intermediate image plane is still fixed and the ratio between this distance and the focal length of the objective still determines the magnification in a straightforward manner.3 I have gone over these calculations in such tedious detail for a reason: by defining BOTH the object and the image distances, we have described an optical system that has what are called fixed conjugates. For each objective, the exact locations of the object and image planes are known and fixed. The specific term, fixed conjugates, is less important than the idea that it is ONLY because microscopes use fixed conjugates that it is possible to design their optical systems to be diffraction-limited (i.e., free from aberrations). Fixed conjugates are the sine qua non for effectively eliminating spherical, chromatic and other aberrations. It is important to recognize that the object and image distances are not defined simply in terms of geometrical dimensions but in terms of optical distance: the geometrical dimension multiplied by the RI of each segment along the path. Normally we think of the space between lenses as being filled with air. However, in our everyday world, we have become used to the idea that we can look through a sheet of glass without this effecting the sharpness of the image that we see. Incautious microscopists have followed this logic to assume that the flat components in a microscopic imaging system (such as filters inserted to change color balance, the slide, the coverslip, and any immersion liquids that at may be constrained by them) play no part in the image formation process. Nothing could be farther from the case! We see little effect when a window is interposed between us and the view because the lens in our eye operates at a very low numerical aperture. (If the pupil diameter is 2 mm and the view is only 2 meters away, the NA is ~0.001). Microscopical imaging systems work at much higher NA, a situation that make the refractive index of every segment of the optical path a potential source of spherical aberration in the final image. If a flat plate of glass, 1 mm thick and with an RI of 1.51 (i.e., a microscope slide), is inserted into the path behind the objective, it will increase the optical path by 0.51
3

!z im 4x magnification, w/same f, larger image distance, shorter object distance.

b.

!z ob 4x magnification, w/shorter f, same image distance but shorter object distance. c.

.
!z lens

.
Figure 7

4x magnification, w/shorter f, same image position AND same object position as reference. d.

Rays from the focus/focal plane in the objective leave any 'infinity focus' objective traveling parallel to the axis. A "tube lens" mounted between the objective and the ocular is needed to bring these rays into focus at the intermediate image plane. Together, the objective plus the tube lens forms this image at a fixed distance behind the tube lens.2 Some examples: - The optical center of an objective with f = ~18.5 is a little more than 18.5 mm above the focus plane, or 205 18.5 = 186.5 mm below the intermediate image plane. According to Eq. 3.1, this arrangement would produce an
2

Perhaps this is a good place to consider what happens to light that emerges from specimen planes other than the focus plane. Of course, such light is a common feature of 3D specimens and the fraction of it that reaches the objective will be focused by it on to some conjugate plane determined by geometric optics, as shown in Fig. 1. As this plane will NOT be the one viewed by the ocular, this light will not be brought to a sharp focus in the observer's eye or the camera. Even if the ocular is moved so as to image this formerly out-of-focus plane, the image quality will not generally be optimal because the objective is only corrected to work optimally between the normal planes. In addition, the magnification will be incorrect. So why worry about it? Because much of 3D LM is concerned with how to eliminate the trouble caused by out-of-focus light.

In fact, the magnification relationship given here, while simple to grasp is only approximate and works best for objectives of more than 10x in power.

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mm. As this error is only ~0.3% of the (160 mm) distance involved, the effect is not very severe. However, the presence or absence of a 0.17 mm coverslip can make a very significant difference on the specimen side of the objective where the distances involved are smaller and, more to the point, the angles involved are much larger. Fig. 8a shows a dry (non-immersion) optical system that is free from spherical aberration. Fig. 8b shows that, if a coverslip is introduced between this objective and its focus plane, the peripheral rays will be offset by an amount, ", as they traverse the coverslip and now fail to reach the focal spot formed by the paraxial rays. Clearly, the size of " will increase with both the RI and thickness of the coverslip. On the other hand, the paraxial rays are affected less because they strike the coverslip at almost normal incidence. By introducing this flat optical element, we have effectively created spherical aberration. (and, of course, as RI is always a function of wavelength, you are also introducing a new source of chromatic aberration. Though this may be important when looking deep into tissue with confocal fluorescence, it is not a factor with non-descanned multi-photon imaging as this uses only a single wavelength.)

Correction

Correction Object distance Coverlip

in the objective. As one focuses farther into the specimen, the reduction in the thickness of the immersion oil layer would be exactly compensated by the increase in the thickness of the amount of specimen that is being looked through. As both these layers have the same RI, there is no change in the optical arrangement, and so an objective could be designed that would produce diffraction-limited performance throughout the focus range. On the other hand, serious problems occur if the specimen is mounted in water (RI = 1.33) rather than immersion oil (RI = 1.515) (ref) or even if the oil is used at the incorrect temperature (because the RI of some oils is a strong function of temperature). When a water lens (i.e., a lens designed to perform properly with water as both the embedding and the immersion medium) is used with a coverslip, there is again a large difference its RI and that of the surrounding water. Under these conditions, the exact thickness and RI of the coverslip becomes very important. Having a coverslip that is only 160 !m rather than 170 !m can reduce the effective spatial resolution of an NA 1.2 water lens by a factor of 2. (Handbook, pp. 115, Fig. 9). Therefore, as with high-NA dry objectives, high-NA water objectives often come with correction collars to compensate for variation in coverslip thickness. Although water lenses that are corrected for use without a coverslip (so called dipping-cone lenses) avoid problems with coverslip thickness, they cannot overcome the fact that the RI of cells is substantially different from that of water.

Paraxial focus Peripheral focus Offset, " Objective designed for use without coverslip a. Introduction of coverslip produces spherical aberration b. Objective redesigned for presence of coverslip c.

Figure 8 The problem can be overcome by changing the design of the objective to make the peripheral rays over-converge enough to compensate for the offset ", Fig 8c. However, as we have corrected a problem of lateral displacement by changing an angle, the correction will only work properly for a specific coverslip thickness and RI. The peripheral misfocus shown in Fig. 8b is large because the RI of the coverslip is very different from that of the air for which the lens shown in 8a and 8b was designed and also because the acceptance angle of the objective, !, is quite large. Clearly the offset becomes larger with !. This is the reason that most manufacturers incorporate correction collars to compensate for variations in coverslip thickness in dry (air) objectives that have large ! (i.e., high numerical aperture, usually above NA 0.75). The opposite situation occurs with oil immersion objectives. Oil immersion is usually used primarily because it allows the maximum NA to be increased from 0.95 to about 1.4 thereby providing better diffraction-limited spatial resolution. However, a practical advantage that is probably more important is that homogeneous immersion reduces the chance of spherical aberration. Consider Figure 9. If the front element of the objective, the immersion oil, the coverslip and the specimen all had the same RI, light rays would be undeflected until they reached the far surface of the first element

Figure 9 What to do? Another side of the fixed conjugate condition is that, if the RI conditions along the actual optical axis do not match those for which the optical components were designed (improper oil, wrong coverslip thickness etc.), it is theoretically possible to vary the image distance (i.e., tube length) in order to compensate for the spherical aberration produced by this mismatch. In an earlier time, it was common to change the length of the tube between the objective and the ocular specifically for this reason. However, because such an adjust-

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ment also changes the magnification and complicates the attachment of heavy photographic or video recording systems, no commercial microscopes currently offer this facility. More recently, Sedat and Agard and also Wan et al. have shown that one can substantially compensate for the spherical aberration caused by using an oil-corrected objective on an aqueous specimen by a higher RI immersion oil between the lens and the coverslip (ref). However, as the thickness of this compensation layer changes depending on the distance between the objective and the coverslip and also on the distance that the focus plane is below the coverslip, the use of oil of any specific RI is successful only over a limited range of focus planes. Intermediate summary Spherical aberration occurs when the focal length of different rays in a bundle of light varies with the distance away from the optical axis that the ray passes through the lens. It can be corrected by careful design of the optics BUT only if the RI of all the components outside the lens are known and vary only with z-position along the optical axis (in other words, any changes that occur, are in flat, homogeneous slabs oriented perpendicular to the axis). Components likely to effect the RI are the immersion liquid (if any), the coverslip, and the embedded specimen itself: all must have a known thickness and RI. As we see below, it is possible to design and build objectives in which the displacement of some elements can change the spherical aberration correction of the entire lens to compensate for changes in these external factors. In addition, spherical aberration can be partially corrected by changing the tube length, by using an immersion oil with a non-standard RI or by adding elements specifically designed to change SA. Detecting spherical aberration Fig. 2, shows that spherical aberration blurs the sharpness of the focus spot. However, the blurring is not the same in all directions: it looks different at different focus distances. Let us consider the imaging of a point object. In the absence of aberration, the in-focus image of this point will be an Airy Disk image (Figure 10b). The Airy Disk is actually a horizontal section through the center of a symmetrical, threedimensional pattern called the Airy figure (Figure 10a). Therefore, if we focus above and below the plane of the point object, we will see the corresponding section of the 3D Airy figure. As the figure is symmetrical around the focus plane, the way in which the image goes out of focus above the plane of best focus, will be the same as when it does below the plane of best focus. (Figure 11a). In the presence of spherical aberration, this defocus is not symmetrical. In Fig, 2, one can easily see that the rays at high NA (i.e., those that should carry high resolution information) will form a focus at a different height than light at lower angles. As one moves the point object above the peripheral focus, it will appear as an expanding ring, while as one moves it below the paraxial focus, the central spot will become a larger, fainter blur. Should the optical conditions

change so that the spherical aberration is negative and the paraxial focus is nearer to the lens, then the relative focus positions at which the ring and the blur are seen to interchange. It is easiest to see this effect when looking at light emerging from a small (<0.2 "m) hole in a metal film or when viewing a small fluorescence bead because, unlike phase or DIC contrast, these conditions meet the requirements of incoherent imaging and one can neglect phase effects. In the absence of spherical aberration, the in-focus image will look like Fig 11a, while the best-focus image in the presence of spherical aberration will look like Fig 11b.

b. Airy Disk

a. 3D Airy disk

c. Profile through center

Figure 10

Figure 10(a-c from left to right) The most important aspect of these representations can only be appreciated by comparing intensity plots across the center of each. The relevant feature is that the blurring spreads out the same number of photons over a larger area.4 This has the effect that, in the presence of spherical aberration, the central peak is not only larger but also less intense. This is often a major effect: if spherical aberration increases the diameter at best focus by 2x, the peak intensity will be reduced to <25% of what it should be. In the low signal level conditions that characterize fluorescence microscopy, one may be able to distinguish the signal from some point object above the background stain level only if your image is free from spherical aberration: i.e., the major effect of spherical aberration can be loss of signal rather than resolution pure and simple. In a confocal microscope used with a small pinhole, SA reduces intensity in both the excitation and the detection paths: In the example above, the excitation intensity may drop to 25% but the detection signal drops to 6.25%. In fact, it is probable that spherical aberration is usually THE major cause of signal loss as the focus plane of a confocal microscope moves farther into the specimen. Attempting to interpret the image of a point object that

The intensity effects have been suppressed in Fig 11 by using different exposure times for each image. This was done in order to make the patterns more easily visible.

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Figure 11 is above or below the plane of best focus in the presence of spherical aberration is complex. The best approach is first to detect the presence of the aberration and then take steps to eliminate it. As discussed above, spherical aberration is easiest to detect when imaging a point object. Once such an object is in view, (and you may need to use a higher power ocular in order to see it clearly), focus above and below to ascertain if the in-focus image goes out of focus in a symmetrical manner. If it does not do so, spherical aberration is present. To remove it, you can apply one of the corrective measures described in the next section. This detection procedure is harder to implement in the confocal microscope because whenever the point object is moved out of focus, the signal disappears altogether! There are two approaches. One is to base your adjustment of the correction system on the intensity of the signal from a small fluorescent bead with the idea that the signal will increase as the aberration is corrected. This is easier to say than to do because all of the corrective measures described below also change the focus plane and, in confocal, going out of focus also affects the brightness of the image of a point! The better approach is to open the detector pinhole as much as possible so as to approximate the widefield imaging situation. In this case, you should be able to detect asymmetry above and below the focus plane after collecting a zseries image of a point object or a plane. How do you correct Spherical Aberration? Assuming that, to the best of your ability, you have already selected a coverslip of the correct thickness (a #1.5 coverslip, 0.17 mm or 170 !m thick. You can easily measure this dimension 1 !m using a metric digital micrometer.) and applied the proper immersion and embedding media, residual spherical aberration can be corrected in four ways: Adjust the coverslip correction collar incorporated into your objective. Such collars work well but they can usually only compensate for small amounts of aberration. If your objective doesnt have such a collar, one can introduce an accessory spherical aberration corrector between the objective and the ocular. (InFocus, Infin-

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ity Photo-optical, Boulder, CO) This new device performs essentially the same function as a correction collar but will do so for any objective used and in a location away from the objective, where its adjustment can be controlled by a computer-driven stepper motor Change the immersion liquid to partially compensate for refraction error. Make arrangements to change the tube length of your microscope by increasing or decreasing the optical distance between the objective (or the tube lens) and the ocular (or confocal scan lens). The operation of these techniques is covered in more detail below. Correction collars Spherical aberration becomes a severe limitation whenever high NA is used and the pattern of RI along the optical axis is other than that for which the objective was designed. Practically speaking, the two situations most likely to meet these criteria are: high-NA dry and water-immersion objectives used with coverslipped specimens. In the first case, it is clear that the specimen must necessarily have an RI that is higher than that of the air between the objective and the coverslip. It is possible to correct a lens for a proper operation at some specific distance below the top surface of the coverslip because this condition also specifies the thickness of the layer of air between the front of the objective and the top of the coverslip. However, this correction will fail if the coverslip is the wrong thickness or as soon as the plane of focus is moved farther into the specimen (making the layer of air thinner). To accommodate this, some manufacturers provide their high-NA dry objectives with a coverslip correction collar. This collar allows the axial position of a subset of the elements of the objective to change with respect to the remainder of the lens. Although various mechanisms are used to accomplish this change, Fig. 11 shows one possibility. This design includes behind the main lens, a meniscus element in which the two curved surfaces are almost parallel. Because the surfaces are almost parallel, this element has very low power (long focal length) and its presence doesnt affect the overall focal length very much. Axial rays that intersect the meniscus nearer to its center, where it is almost plan/perpendicular to the axis, are not deflected. Peripheral rays are deflected slightly, as they would be by a coverslip (Figure 11a). However, as the meniscus moves towards from the main group of elements, the peripheral rays strike it where its slope is higher, causing the peripheral rays to focus at correspondingly longer focal lengths. This action provides a mechanism whereby the focal length of the peripheral rays can be changed compared to that of the paraxial rays: ergo it is a spherical aberration corrector. As the adjustment to change spherical aberration correction also slightly changes the focal length, and therby the magnification, in some cases, objectives contain two sets of moveable elements: one to change the aberration correction and the other to make a compensating change in the focal length (and magnification). Because of the extreme mechanical precision needed to maintain the proper alignment of all the elements in a high-NA, diffraction-limited objective,

correction collars greatly increase the manufacturing cost of the lens. ($12,000 vs. $3,000) It is possible to make a general purpose spherical aberration corrector. The InFocus is such a device. (Zam, et al., Bioimaging, 5:40-49, 1997) The InFocus consists of a set of optical components mounted in a barrel fitted with to fit into the ocular tube. (Fig 13)

.
a.

.
!f sph motion

!f sph

.
b.

Figure 12: Spherical aberration correction collar

Figure 12 Spherical Aberration Correctors. (Images of InFocus can be found at: http://www.infinityusa.com/products.asp?pg=Insert ) Figure 13 As with the correcting collar, the focus position of the peripheral rays can be altered compared with that of the paraxial rays by rotating a moveable ring (Kam et al, 1997). The advantage is that this device can be used to adjust spherical aberration with any objective, not just those that have collars already fitted. The disadvantage is that use of such a corrector involves the addition of 4-5 optical elements to the column with all the potential for chromatic aberration and signal loss through reflection, that this implies. Initially, different implementations of the InFocus technology had to be designed for each model of microscope because of variations in the tube length standards and other optical parameters among the different manufacturers. More recently however, a single system has been introduced which is capable of working properly with any infinity-focus. This makes it suitable for use as part of the optical coupling interfacing a confocal scanner to a microscope body. As with normal correction collars, the adjustment of the corrector produces a small shift in the focus plane and therefore a slight change in magnification. This focus shift can now be compensated by using a computer to adjust both a motor-driven version of the corrector and the focus motor. Using a non-standard immersion liquid or incorrect coverslip thickness. When discussing Figure 8, we assumed that 8a represented a dry lens (one used without immersion liquid). We stated that we could compensate for the offset produced by the flat plate, by changing the angle at which the peripheral

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ray exited the objective.We assumed that this needed change in angle could be produced by changing the internal optical characteristics of the objective. Figure 14 shows a similar setup except now the lens is an oil immersion lens that is assumed to provide proper correction as long as the specimen has the same RI as that of the immersion oil, the coverslip, and the front element of the objective (Fig. 14a). Unfortunately, many cells seem to have an RI closer to 1.35 than the 1.518 of most immersion oil. Therefore, focusing some distance into the cell produces severe spherical aberration (Fig. 14b). This can be partially compensated for by using an oil of higher RI to compensate for the lower RI of the specimen. The compensation is partial for two reasons. As is obvious from Fig. 14b, the amount of correction produced by the special oil is proportional to the thickness of the oil layer, but this thickness (and hence the correction) must change as the lens moves towards or away from the specimen. In addition, it is clear from Fig. 14b that the distance between the peripheral and paraxial focus planes will increase as one moves farther into the cell (more RI 1.40, less RI 1.518). For these two reasons, the use of a highRI oil can correct for spherical aberration over only a limited range of focus planes: say from 15 to 20 !m or 30 to 35 !m below the coverslip. If one accepts that the RI of tissues may be close to 1.4, then it immediately becomes evident that if one views such a specimen using a water-immersion lens designed for use with RI = 1.33, then, we will have the opposite situation compared to Figure 14. The peripheral rays will now focus below the paraxial rays. By analogy, one might imagine that one could overcome this problem by replacing the water between the lens and the coverslip with a liquid having an RI that is less than 1.33. In fact such liquids exist but they are unlikely to be viscous enough to make good immersion liquids.

problem. Assume that the RI of cells is 1.40. If the layer between the coverslip and the objective is also filled with a liquid having an RI = 1.4, then moving the focus plane no longer changes the optical system and there will be no change in performance with depth. The problem now is that, although the performance wont change, performance will be bad everywhere because there are no objectives designed to work properly with RI 1.40 oils and specimens. Tube length adjustment As mentioned above, it has long been possible to change the spherical aberration correction of an optical system by changing the tube length. The problem is how do adjust this parameter, given the fixed shapes of the body section of modern microscopes. Although it might seem possible to insert a variable offset such as that shown in Fig. 15, this is by no means a trivial operation. The prisms must be large enough to transmit the entire intermediate image (up to 25 mm in diameter) and modern instruments already contain a plethora of other prisms for shifting the image to various cameras and the oculars. In addition, although light from the focus plane passes through all the prism surfaces at normal incidence and the total internal reflections are perfect, it does not follow that such a system has no diffraction/dispersion effects. As the path length inside the prisms will be their geometrical size times the RI, and the RI is always a function of wavelength, the tube length of such a system will vary with wavelength unless it uses mirrors rather than prisms. And mirrors collect dust. Perhaps this is why there are no modern commercial instruments in which the tube length can be adjusted to change spherical aberration correction.

Oil Coverlip

Oil

High Oil Coverlip Correction

! Oil

Object distance

Cell Peripheral focus Paraxial focus Cell

Offset, " a: Oil objective: Corrected Figure 14 b: Looking into cell with RI = 1.4 produces spherical aberration

Offset, " c: Using immersion oil with higher RI can correct this offset at a certain depth

Figure 14 Alternatively, you may note that this water immersion lens is designed for use with a coverslip. One should be able to correct at least some of the spherical aberration produced by the RI 1.4 specimen by using a coverslip that is thinner than that for which the objective is designed (or the correction collar is set.) As with the case of using high-RI oil with a watery specimen, this will only work for a narrow range of focus depths. To see how we might solve the limited focus range

Figure 15 It is likely that only those with home-made microscopes built on optical tables will be able to benefit from such a system. Upon Reflection. Traditionally, microscopy texts discuss optical components as if all they did was refract. In fact, whenever light travels from a medium having one RI to one of different RI some light is reflected. The amount reflected depends on the

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ratio of the squares of the two RIs and, even more strongly, on the angle at which the light strikes the surface (light at glancing incidence may be totally reflected.) In microscopy, reflected light is generally just a nuisance, and the walls surrounding the optical axis are coated with matte black paint in an effort to absorb as much of it as possible. In addition, all air-glass surfaces are coated with multi-layer, broadband anti-reflection coatings. Although this helps, one must remember that the number of fluorescent photons coming up through the objective from the specimen is probably at least a million times less than the number of photons in the exciting beam going down through this same lens. An optical system having only 10 glass-air interfaces, each with only 1% reflectivity can easily produce the amount of reflected light 100,000x greater than the fluorescent signal. In fluorescence imaging, most of this reflected light can be separated on the basis of its wavelength and excluded by the dichroic and barrier filters. The same cannot be said for epi-illuminated darkfield imaging or imaging with backscattered (reflected) light in the confocal microscope. In transmitted light techniques, such as polarization microscopy and Nomarski-DI, where high intensity polarized light can reflect off optical surfaces while the final image is formed by only a weak differential beam, double reflections can be a problem. The first reflection goes back towards the source and is of little consequence. However, a portion of this reflected light can reflect again on its way out of the system and this second reflection will be going in the right direction to cause trouble.

them slightly tilted. (Slightly! <1) In the special case of reflections from the glass wall of the cell chamber, one should be aware that light really is being reflected, whether or not it is reaching your detector. This reflected light can sometimes be sufficiently intense to elicit measurable fluorescence. For instance, consider a living cell in a 20 micron aqueous layer between coverslip and slide. When the beam of a confocal microscope is focused a few micrometers into the slide, light reflected at the water / slide interface will form a focus somewhere in the cell chamber. If fluorescent dyes are presents at this location, they will fluoresce and, depending on the size of your detector pinhole, this light may be detected to form a ghost image. Beyond this, the interference of the exciting light with light reflected from a surface of this type can produce a weak standing wave in which the effective excitation intensity varies with the distance from the interface. In general, there is not a lot one can do about this other than use coverslips and slides made out of a plastic such as CYTOP, that has an RI close to that of water (See: http://www.agc.co.jp/english/chemicals/shinsei/cytop/cytop. htm .**) One should remain aware of this problem, however, and use the knowledge that much of the light is not going where it is supposed to go, as brake against rash extrapolation of weak data. When checking to see that the light hitting the specimen, we all have seen the light emerging from the edges of a slide. If the trajectories of all of the photons in the microscopy obeyed only the laws of refraction as laid out in most microscopy books, this light would not be scattered into the room for our eyes to pick up. Reflections from spherical surfaces Axial, parallel, light reflecting from a spherical surface seems to emerge from a point on the far side of the surface (Fig. 16). This is good news if the other optical components defocus light from this virtual source before it reaches the detector. The defocused light hits the blackened walls of the tube and is removed. It is bad news if the optical system does the opposite and focuses the reflector near to the plane of the detector pinhole (Fig. 17). Consequently, it is not uncommon that only a small fraction of the suspect optical surfaces actually produce damaging results. In any case, most of the problem is caused by light reflecting very near to the optical axis. The presence of more than one axial blob in an image is an indication that some of the offending optical components are improperly centered. If the offending components are essential and cannot be replaced by something similar but producing reflections with different virtual sources, the only solution is to apply the anti-flex techniques common in metallurgical microscopy where epi-illumination for reflection imaging, is normal. These techniques separate light reflecting from the optics from that scattered back from the specimen on the basis of the effect each has on the polarizatoin of incoming light. In some cases, a quarter-wave plate is incorporated as part of

Figure 16 Reflections from flat surfaces. Reflections from flat optical surfaces such as coverslips and specimen slides, filters, graticles, 1/4-wave plates, polarizers, analysers and Wollaston prisms, can often be eliminated by slightly tilting the component involved with respect to the optical axis. If you notice features in your image that do not seem to be related to the specimen (i.e., dont move when you move the specimen or dont go out of focus when they should) try to move all the flat components in turn and see if any are responsible for the problem. Then try to mount

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the front element of the objective, to improve this separation. (see Handbook: Chapter 2)

Figure 17. To be continued Additional reading Hiraoka, Y, JW Sedat and DA Agard: Determination of three-dimentional imaging properties of a light microscope system. Biophysical J. 1990, 57:325-333. Wan, D.-S, Rajadhyaksha, M. Webb, RH, Analysis of spherical aberration of a water immersion objective: application to specimens with refractive indexes 1.33-1.40, J. Microsc. 197:274-284, 2000. Kam, Z, Agard, DA, and Sedat, JW, 1997, Threedimensional microscopy in thick biological samples: a fresh approach by adjusting focus and correcting spherical aberration Bioimaging. 5:40-49. Scalettar, Swedlow, Sedat & Agard. 1996. Dispersion, abberation and deconvolution in multi-wavelength fluorescence images. Journal of Microscopy. 182:1, 50-60.