B I O G R A P H Y Peter Drent obtained hisdegree in biology atUtrecht University in theNetherlands. He is nowworking as a manager formicroscopy products forNikon InstrumentsEurope BV.

A B S T R A C TMicroscopes have been traditionallydesigned to use the human eye to observethe image. But nowadays more and moredigital cameras are used as the imagingdevice in both standard and confocal microscopes. These new digital detectorsare more sensitive to intensity and flatnessdifferences than the human eye and requirenew strategies for objective lens design. This article describes the optical performance of modern objective lensesand discusses criteria for the selection of objectives for a variety of microscope applications including fluorescencemicroscopy, confocal microscopy, and livecell imaging.

K E Y W O R D Slight microscopy, confocal microscopy, digital imaging, objective lens, numericalaperture, oil immersion, water immersion

A U T H O R D E TA I L SPeter Drent, Product Manager Microscopy, Nikon Instruments Europe BV, Schipholweg 321, 1171PL Badhoevedorp, The Netherlands Tel: +31 20 4496258 Email: drent@nikonbv.nl

Microscopy and Analysis 19(6):5-7 (UK), 2005

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I N T R O D U C T I O NPrior to reviewing modern objective lens selec-tion, it is worthwhile reflecting on some keydrivers in microscope development. Ever sincethe days of Hooke and Leeuwenhoek, micro-scope lenses and optical systems have tradi-tionally been designed to provide optimumperformance when samples are viewed by thehuman eye. It is only relatively recently thatthe differing needs of the digital camera havehad to be taken into account. A key advance insample observation was the Koehler set-up(developed by August Koehler in1893), whichensured even illumination of the specimenplane. However, it was not until the 1950s thatmicroscopes were equipped with a trinoculareyepiece tube that used a photoeyepiece toenable connection of photographic equip-ment. At that time, bright field, dark field andphase contrast were the most common illumi-nation techniques; fluorescence was not yet inuse. In the 1960s film cameras began to beconnected to the microscope, again via thephotoeyepiece.

Nowadays, a multitude of imaging systemsare regularly added to the modern microscopeand fluorescence has become one of the mostpopular contrasting techniques. The latestadditions for improved specimen observation,documentation and analysis include digitalcameras, laser scanning confocal systems, andadvanced systems equipped with fluorescentlive-cell imaging capabilities and spectraldetector systems. In essence, the modernmicroscope has grown from being a mereobservation station into a complete imaginganalysis workstation.

Although the traditional microscope set-up,

with correct Koehler illumination, ensureseven illumination of the specimen plane, mod-ern sensors, such as those in digital cameras,are far more sensitive to differences in theevenness of illumination than the human eye.This has driven the development of a new illumination strategy in modern microscopes,in effect, a digital Koehler illumination equivalent.

OBJECTIVE LENS DEVELOPMENTSAlong with the progression of the microscopefrom an observation station into an analysisworkstation, the microscope objective lens hasalso evolved considerably. A key step in thisevolution was the transition from the achro-mat objective lens, which was adequate in theearly days of microscopy when the eye was theonly method of observation, to the planachro-mat objective lens, needed when photo-graphic systems arrived and image flatnessbecame an issue. Plan correction of the micro-scope objective lens ensured flat image repro-duction, resulting in uniformly focused pho-tographs. Later, when fluorescence becamepopular as a contrasting technique, plan fluorobjective lenses were introduced and, for spe-cial applications that needed ultraviolet exci-tation, super fluor objective lenses were developed.

Plan fluor objective lenses are essentiallyplanachromat-style lenses with respect to cor-rection but because of different glass selectionthey show higher transmission rates in theinfrared (IR) and blue violet (BV) parts of thespectrum. In addition, planapochromat objec-tive lenses are regarded as being superior inperformance with respect to resolution and

Properties and Selection of Objective Lensesfor Light Microscopy ApplicationsPeter Drent, Nikon Instruments Europe, Amsterdam, The Netherlands

Figure 1: Modern violet-corrected objectivelenses for light microscopy. From left to right: Plan Apo VC 60xwater immersion, numerical aperture1.2, working distance 0.27 mm, cover-glass thickness 0.15-0.18 mm; PlanApo VC 100x oil immersion, NA 1.4,WD 0.13 mm, CG 0.17 mm; Plan ApoVC 60x oil immersion, NA 1.4, WD0.13 mm, CG 0.17 mm.

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correction and are regularly selected forhigher-level photographic applications.

Typically, achromat objective lenses aredesigned to focus blue (about 486 nm, alsocalled the f line) and red (656 nm, c line) in thesame plane, providing axial chromatic aberra-tion correction. Furthermore, plan fluor objec-tive lenses are also designed to correct for thegreen (588 nm, also called the d line). In addi-tion to the above, planapochromat objectivelenses are corrected for the g line (436 nm).Just recently, a new type of objective lens hasbeen introduced — the planapochromat vio-let-corrected (VC) series (Figure 1), that coversthe h line (405 nm) as well, totally correctingaxial chromatic aberration for five lines, rang-ing from 405-656 nm (Figure 2).

Live cell imagingIn recent years, live cell imaging has becomeextremely popular. However, since live cells areoften kept in climatically controlled chambers,these impose special demands on the micro-scope objective lens, such as a long workingdistance. (Extra long working distance may beneeded for observing specimens that are con-tained in thick walled vessels.) Long workingdistance correction typically covers around 2mm, enough for Petri dishes or multiwellplates. If the cells are kept in more elaborateclimate control chambers, or cell culture bot-tles, a longer correction for working distanceof 4-6 mm is needed, which can normally befound with extra long working distance objec-tive lenses. Super long working distance lensesare also available.

Water immersionThe demands of high-resolution microscopyon live cells also resulted in the introduction oftwo other types of objective lenses: waterimmersion and water dipping. Water-immer-sion objective lenses use water instead of oil asthe immersion medium. The maximum numer-ical aperture (NA) of these water-immersionlenses (e.g. planapochromat 60� with NA 1.2)is lower than oil-immersion types (e.g.planapochromat 60� with NA 1.45) and thishas an important effect on resolution. This isbecause the resolution of an objective lensdepends directly on its NA, and can bedescribed as the wavelength divided by twicethe NA. When live cells or tissues need to beobserved deeper inside the specimen, water-immersion lenses show superior performancewith respect to resolution and aberration cor-rection. These lenses offer additional benefitssince most cells live in a watery environment.By using water as the immersion medium, theoptical system becomes symmetric. If we fol-low the optical axis from specimen to objectivelens, light passes through the following mate-rials: water (cell environment), glass (cover-glass), water (immersion medium) and glass(objective lens). As a rule of thumb, whenobserving up to 25 �m into the specimen,high-NA water-immersion lenses should beselected as these will give the brightest andbest resolved images.

Water-dipping lenses are designed to workwithout a cover glass, and can be submerged

directly into the Petri dish or a climate-controlled chamber. Typically, their NA isslightly lower than water-immersion lenses(e.g. plan fluor 100� NA 1.1 compared to planfluor 100� NA 1.3) but the working distance ismuch longer, possibly up to 2.5 mm (e.g. planfluor 100� NA 1.1) compared to 0.2 mm (e.g.plan fluor 100� NA 1.30). This combination ofwater-dipping and long working distanceenables micromanipulation to be undertakenat extremely high magnifications while thetissue being observed is kept in an optimalmicroenvironment. Because the NA of thewater-dipping lenses is still relatively high,this high-resolution micromanipulation canalso be combined with digital imaging or evenwith laser scanning confocal microscopy.

The relative intensity of the image in anoptical system is given by the expression:

NA4 /TM2

where NA is numerical aperture of the objec-tive lens and TM is total magnification. Usingthis formula, it can be shown that theplanapochromat 60� NA 1.45 is the highestresolution lens available, while the plan fluor40� NA 1.3 is the brightest.

The most flexible microscope objectiveslenses currently available are the multi-immersion type (plan 20� MI). These can beused dry or with water, glycerin or oil, offeringgreater flexibility, and are often used as alower magnification companion lens for ahigh-NA, high-resolution immersion lens,especially in confocal microscopy.

Correction collarsThere are several types of correction rings thatallow the performance of the microscopeobjective lens to be offset against certain para-meters, such as cover glass thickness, immer-sion medium refractive index, temperaturecorrection, or correction for depth penetra-

tion into tissues (refractive index). By correct-ing for these parameters, optimum perfor-mance can be readily achieved.

Physical dimensionsAlong with the increase in microscope objec-tive lens performance, the physical propertiesof the objective have also changed. The parfo-cal distance has grown from 33 mm to RMS 45mm and, with Nikon, to 60 mm. A similar evo-lution has occurred for the objective lensdiameter, which has grown from 20.3 mm to25 mm — a move which has given microscopedesigners morelatitude in their optical designs.

Zoom systemsHowever, it is not only the microscope opticsthat have evolved significantly, the entire optical system has had to adapt too. Whendigital sensors became the prime detector, the

Figure 3: Fly-eye lens for uniform specimen illumination.

Figure 2: Chromatic aberration correction of objective lenses. In the CFI60 planapochromat VC series the h line (405 nm) is focused in the same focal plane asthe g, f, d and c lines (right), whereas with traditional Plan Apochromat lenses the h line is focused in a different plane (left). CFI60 planapochromatsare fully corrected from 405 nm up to 656 nm.

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microscope needed to be equipped with someform of magnification changer. We alreadyknow (see above) that a microscope objectivelens has a resolving power that is determinedby its NA. This optical resolution thereforeneeds to be matched to the digital resolutionof the camera. To balance the output resolu-tion with the resolution of the digital sensorthe microscope needs to be equipped with azooming system. According to Nyquist, thesampling frequency needs to be 2-3 timeshigher than the object that is being sampled.As a result, the modern microscope isequipped with a system that allows zooming.When this is motorised it allows automatic set-ting of the correct zooming position for eachindividual microscope objective lens.

Uniformity of illuminationAs has been described, the modern microscopeobjective lens has been specifically designedto cover a broad range of corrections. How-ever, as these lenses are almost always used aspart of a high-level imaging system, the even-ness of the image they create has also had tobe redesigned. The traditional Koehler illumi-nation has had to go ‘digital'.

Uniformity of illumination is particularlyimportant for digital images as vignetting,which is sometimes not detected by film pho-tography or through the eyepieces, may havea deleterious influence on image quality.

Another factor that can compromise theuniformity of the image is uneven illumina-tion, which can be eliminated by introductionof a ‘fly-eye’ lens arrangement in the micro-scope illuminator (Figure 3). This is the digitalequivalent to the traditional Koehler illumination.

In traditional Koehler illumination the lampfilament is spread out by a diffuser and thenprojected onto the specimen. To the humaneye, and even for film photography, thisappears to be even, but digital cameras, withtheir high sensitivity, can easily detect anyunevenness. The fly-eye lens element multi-plies and projects the single illuminating fila-ment as more than 300 filaments, evenly dis-tributed across the view field. This approach isideal for the most sensitive digital cameras.

One choice for digital photomicrography,the planapochromat VC lens, not only shows acorrection for up to five lines, but is alsodesigned to be perfectly vignetting free. Thismeans that the resolution of the objective lensis even across the entire view field, as shown inFigure 4.

O B J E C T I V E S E L E C T I O NIn summary, selecting appropriate objectivelenses can be the most critical decision whensetting up a digital imaging system, or whenbuilding up a laser scanning confocal micro-scope. When using bright field applications ina digital microscope system, light intensity isalmost never a limiting factor. Evenness of illu-mination (fly-eye illumination) correction andvignetting-free objective lenses (planapochro-mat VC series) are desirable specifications.Zoom optics are very important to balance thedigital and optical resolution.

Figure 4: Comparison of resolving power of normal (top) and violet-corrected (bottom) planapochromat oil-immersion (H) objectives.

Fluorescence microscopyWhen using fluorescence applications in a dig-ital microscope system, a choice needs to bemade between maximum signal collection(plan fluor 40� NA 1.3), in case of photon lim-iting applications, or super fluor lenses whenusing dyes that work at UV or IR wavelengths.When the signal is not limited, correction, asoffered by planapochromat lenses, is the preferred option.

Confocal microscopy For laser scanning confocal microscope sys-

tems, objective lens selection is arguably themost critical selection choice in producingsuperior results. A laser scanning confocalmicroscope scans the view field point by pointand collects the signal. By adjusting the focusposition of the specimen, the next planewithin the specimen can be scanned in a simi-lar fashion, resulting in a digital 3D image ofthe signal distribution within the specimen.

Most laser scanning confocal microscopeswork with blue (488 nm), green (543 nm) orred (633 nm) excitation. At these wavelengthscorrection and transmission of the objectivelens is at its optimum (for plan fluor and planapochromat series microscope objectivelenses). When using infrared (IR) light to excitethe specimen, as in two-photon laser scanningmicroscopy, the IR transmission of a lens is crit-ical. Planapochromat VC lenses not only workwell in the violet part of the spectrum, but alsoperform extremely well in the IR. One of thebenefits of multiphoton microscopy is thedepth penetration into the specimen. Thelong working distance of the fluor water-dip-ping lenses (up to 2.5 mm) even permit imag-ing into larger tissues like mouse brain.

In summary, good lenses for laser scanningconfocal microscopy are: 1. For most general, highest resolution,vignetting-free operation: planapochromat60� VC, oil immersion, NA 1.4. 2. For living cell imaging deeper than 25 �m,vignetting free: planapochromat 60� VC,water immersion, NA 1.2. 3. For brightest imaging in cases of photon limitation: plan fluor 40�, oil immersion, NA 1.3.4. For living cell imaging deeper than 250 �m,vignetting free: fluor 60�, water dipping,NA 1.0.

C O N C L U S I O N SWith sophisticated optical design, the latest inillumination techniques and advanced detec-tors — all working together in harmony —modern microscopes are now capable of deliv-ering superior performance for anything fromobserving sections of tumours, single mole-cules or cells growing in culture dishes, toreconstructing sets of chromosomes in thenucleus.

However, even the performance of the mostflexible of microscopes is still dependent onthe design and correct selection of the partclosest to the specimen itself. Fortunately,objective lens have responded to the chal-lenge and now offer the high performanceneeded to meet the demands posed by eventhe very latest illumination and detectiontechniques.

©2005 John Wiley & Sons, Ltd