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Introduction to Fluorescence Imaging ofLive Cells: An Annotated Checklist

Yu-li Wang

It is becoming clear that m ost biological m oleculesn living cells are in a highly dynamic state, chang-ng their interactions and spatial organizations in re-ponse to signals. With recent advances in optics,probe design, and photon detection, there arc fewapproaches as su itable as fluorescence imaging fornvestigating dynam ic events in living cells. However,uccessful execution of fluorescence imaging relicsheavily on proper setup of facilities. The purpose ofhis article is to provide a comprehensive list of thequipment, w ith associated notes, for those setting upnew imaging facil itie s.

There arc m any options in setting up a fluorescencemaging facility, each with its advantages and disad~vantages. The decision is usually dictated by a combi-nation o f experimental goals and personal prefe rence s.While this article attempts to provide some usefulguidelines, it is important not to equip much beyondwhat is necessary, as excess equipment wastes money,reates confusion, and often becomes obsolete whenone finally finds the opportunity to use it.

Microscope S tand

An inverted m icroscope generally allow s more flex-bility and workspace for the culture (see later) andmanipulation of live cells than an upright m icroscope.n addition, w hile inverted stands by major manufac-urers give comparable performances, it is im portanto take into con sidera tion th e feasibility, acce ssibility,nd convenience for special third-party accessories,uch as the culture chamber, micromanipulator, filterwheels, and confocal optics to be used. Therefore,hoice of the stand should not be made until the designf the rest of the system becomes largely clear.One aspect particularly relevant to living cell

m aging is the stability of the m icroscope stand. Some

old m icroscopes are prone to stage drift, particularlyat an elevated temperature. They require either exten-sive manual input or an autofocusing mechanism fortime-lapse imaging. However, it is often difficult toobtain reliable information on the stability throughmanufacturers or on-site demonstration, and experi-ence of colleagues is often the most reliable source.

Several current microscope designs incorporateuseful automatic features, such as lamp shutters (seelater) and motorized magnification and stage controls,which may alleviate the need to incorporate third-party components and facilitate automated multimode

t ime-lapse imaging.Objective Lenses and Contrasting Method

A basic set of objectives consists of lOx, 40x dry, 40ximmersion, lOOx immersion lenses and possibly a 60or 63x immersion lens. Dry lenses are used primarilyfor scanning the samples and do not have to be expen-sive. However, immersion lenses should have as higha numerical aperture and Jight transm ission efficiencyas possible . Becau se images are typica lly co llected nearthe center of the field, lenses highly corrected for flatfield usually provide no detectable benefit and aremore costly and less light efficient than sim pler lensessuch as Fluar lenses. A ll lenses for fluorescence im ag-ing should be checked upon delivery for the qualityof point-spread function, using fluorescent beads asthe sample (see later).

F or most applications, phase-contrast optics shouldsuffice for scanning the sample and for collectingpaired fluorescence and transmission images. Thepresence of quarter-wave plate in the phase lens doescause some (-

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the repeated insertion and removal of an analyzer inthe optical path when one shifts between DIC and flu-orescence optics. This, and the higher cost, makes D ICoptics less desirable in most cases.

COndenser

Unless the experiment involves high-resolutiontransmission optics or dark-field optics, a condenserwith a long working distance should be used in con-junction w ith am inverted stand.

Control o f Pro jection Magnification

It is critical to choose an optimal magnification forlive cell imaging, balancing between signal intensity(favored by low magnification) and reso lu tion (faavoredby high magnification). It is particularly important tomatch the final magnification with the pixel size ofthe detector (see later). Projection magnification may

be controlled conveniently in some microscopes byswitching the tube lens or by adding additional lenses(referred to as Optovar for Zeiss microscopes). Newstands allow changes of magnification to be contrnlledau tomatically in time-lap se imag ing.

Epi-illuminator and F luorescence F ilter. S ets

Some new epi-illumination systems provide a lighttrap, which reduces the background stray light, andstandard Kohler illumination with both field and aper-ture diaphragms. The aperture diaphragm may beused to control the lamp intensity as well as the

angular span and. depth of illum ination. The light trapis useful for single molecular imaging, which is typi-cally lim ited by the background.

There are several commercial sources of high-quality fluorescence filter sets. A handbook on theselection of filter sets may be found at the Web site ofChroma Optics. The main consideration is to balancesignal strength (favored by cuton/ cutoff filters or w ideband-pass filters) against the reduction of signa[crossover from probes of different colors (favored bynarrow band-pass filters). The latter consideration isparticularly important when an intense, long wave-

length probe is used in conjunction with a weak, shortwavelength probe. 1n addition to standard filter sets,mu]tiband fi]ters are now readily available that allowsimultaneous illum ination and/or detection of multi-pIe fluorophores.

Heater Filter or Heat M irror

When imaging living cells it is critical to remove theinfrared component from the light source, as the fluo-rescence filter set may not be able to block infraredlight. Failure to do so may cause not only heat damageto the cell, but also high background with some

infrared-sensitive cameras. The filter (e.g., BG38) orheat-reflecting mirror may be placed either in front ofthe lamp or in the epi-illuminator. Attention should bepaid to the UV transmission of these filters if UV exci-tation is to be used for imaging.

Lamps and Lamp Power SuppliesThe system shou ld include bo th a mercury arc lamp

and a lOO-W quartz-ha logen lamp fOf epi- illumination,coupled through a selection mirror to the m icroscope.Contrary to common practice, the most suitable lampfor fluorescence imaging of Jive cells is often a lOo-Wquartz-halogen lamp. Unless the experim ent involvessingle molecule or speckle im aging, quartz-halogenlamps are much more cost effective and arc sufficientlyintense for imaging most cellular structures whileminimizing radiation damage. They also allow easyadjustment of the light intensity, using a va riable, sta-

bilized DC power supply.The mercury arc lamp should consist of a well-

shielded housing, power supply, and power supplycable to minimize the potentially damaging electro-magnetic w ave during ignition. Although some powersupplies allow adjustment of the light intensity, therange of adjustment is limited. Therefore, it is oftennecessary to attenuate the light from mercury arclamps using a set of neutral density fi]ters to avoidradiation damage. A lternatively, the intensity m ay becontrolled using the aperture diaphragm in the epi-illum inator as mentioned earlier.

Shutters

Electronic shutters should be used to control fluo-rescence excitation. These shutters should be used asmuch as possible to minimize the duration of illumi-nation and should have the interface for computercontrol during automated time-lapse recurding.

Cameras

Cooled CCO cameras are used for most fluorescentimaging applications. In choosing a camera, importantparameters include quantum efficiency, noise level,

pixel size and full-well capacity, and scanning fre-quency. In order to minimize the excitation light forimaging live cells, the camera should be as "sensitive"as possible, which generally means a high quantumefficiency, low noise, large pixel size, and slow scanrate. The sensitivity requirement must therefore bebalanced against the required resolution (favored by a]arger number of small pixels) and imaging rate(favored by a higher scanning frequency).

Slow-scan CCD cameras are generally limited intheir frame rate. In addition, unless the sample is veryintense, the signal-to-noise ratio is poor under short

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exposures. This limits both their use for high-speedimaging and the ease in focusing the images. Focusingis facilitated w ith cameras using the shutterless, frame-transfer or interline CCDs, which are able to provide acontinuous stream of images at video rate in additionto s low-scan d igital s ignals.

Intensified CCO cameras are generally more suit-able for high-speed imaging, although usually with acompromised quantum efficiency. Of particular inter-est arc cameras that use CCD chips with the new elec-tron amplification technology (e.g., photometricCascade and Andor Ixon cameras), which allow bothlong-exposure and high-speed imaging at a highquantum eff ic iency.

Optical Coupling of Cameras

Coupling vw liththe detector should be achieved w ithas few lens elements as possible. It is recommended to

have several couplers with different magnificationfactors. In conjunction with different objective lensesand projection magnifications mentioned earlier, theyallow a wide range of magnifications for both light-lim iting and high-resolution applications. The Nyquistresolution criterion should be considered when choos-ing the magnification: each pixel should correspond tono more than half the required resolution limit on thesample. Due to the diffraction limit of the microscope,this distance needs not be smaller than 50nm for mostapplications. The actual area imaged onto each pixelmay be determined easily by taking an image of a scale

standard (see later).Vibr ation Isola tionl Table

A full-fledged vibration isolation table is necessaryin adverse environment, e.g., in areas of heavy trafficor in high-rise buildings, or for demanding experi-ments of micromanipulations or single molecularimaging. Simple isolation measures may suffice other-wise. These include inner tires under the table orrubber isolation pads (Edmond Scientific) or tennisballs under a slab tabletop.

Motorized Stage and FocI/sing Control

A motorized XY stage is optional. It allows one tomonitor multiple cells in separate regions and mayincrease the output greatly in time-lapse experiments.A motorized focusing control is required for opticalse ction ing , th ree-d imensional imag ing , and automaticfocusing. However, many simple imaging experimentsmay be better served without the complications ofmoto rized s tage contro ls.

Computer Hardware mnd Software

A high-end personal computer is required not only

for image acquisition, but also for device control anddata analysis. The system should have a high-capacityhard disk, a recordable CD/DVD drive for archivingand porting data, and double monitors to accommo-date all the images and control windows. Beforemaking a decision on the software package, it is advis-

able to prepare a list of application requirements, asmany features seen in soflw~are demonstrations arev isually dazzling but practically use le ss. The efficiencyof a package should be judged by counting the numberof mouse clicks or key strokes for setting up and trig-gering the most frequently used functions.

As a minimum for live cell imaging, the programshould be able to control the camera, shutters, andmotorized devices, to perform time-lapse recording,and to allow changes of recording param eters withoutstopping the recording. In addition, the user should beable to review dynamic processes as movies even

during the recording. The program should also be ableto perform automatic contrast enhancement (withoutlosing the original intensity values) and to save imagesin a non lossy file format w ith automatically generatedor manually entered file names.

Microscope Incubators

There are a number of options available throughmicroscope manufacturers and independent compa-nies such as Bioptechs. Discussions of cell culture onm icroscopes maY be found at the Web site of Bioptechsand in McKenna and Wang (1989).

There is no "ideal" culture device for all the appli-cations. The range of possible devices varies from aheated stage, a heating collar for the objective lens, asmall heated culture chamber, to a large enclosure thatfits over the entire microscope. A heated stage is themost convenient but the least functional, as the pointof observation is over an open area far away from theheat source. A heating collar for the objective lensapplies heat much closer to the sample. However, itworks only in conjunction with immersion lenses andhas a limited area of heating. It is usually used in con-junction with an additional chamber or enclosure.

Large microscope enclosures provide the most stabletemperature; however, it must be designed carefullyto provide convenient access. There are also a numberof perfusion and heated culture chambers formicroscopy. These chambers generally provide excel-lent optical and culture conditions; however, the asso-ciated wires and tubing may add to the inconvenience,and the sealed environment may not be compatiblew ith microman ipu lation experimen ts.

It is im portant to m aintain the temperature stabilityto within a fraction of a degree, as even minor drifts intemperature can cause severe drifts in focusing and

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sample positioning. In addition to heating, it is impor-tant to maintain the pH and osmolarity of the culturemedium. While this should not be a problem withsealed chambers or perfusion chambers, open cham-bers should be used in conjunction with either a CO2-independent medium (e.g., 1.-15) or injection of C02into the incubator. It should be noted that HEPES-buffered media only slow down pH drift and are notsuitable for long-term cultures by themselves. Osmo-larity may also be a serio us problem with open dishesin a heated environment. It may be controlled byreplacing the medium periodically or by covering themedium with a layer of mineral oil (Sigma).

Testing Samples

Several testing samples should be prepared forcharacterizing the imaging system. First, a micro me-

tern scale is essential for all microscopy laboratoriesfor determ ining the final magnification. Second, a "flatfield" sample is prepared by spreading a drop ofappropriate fluorophores in 50%~0glycerol under acoverslip. It is useful for checking the uniformity ofepi-fluorescence illumination and for setting the fielddiaphragm. Also essential is a sample of fluorescentbeads. it is prepared by diluting O.l-um-diameter flu-orescent latex beads (Molecula r Probes) by~ 10' intomelt 1%~~,garose, and mounting a sma]] volume(-20-50 Ul) of the suspension on a heated glass slideunder a coverslip, before letting the sample cool down.

The bead sample is used for checking the combinedoptical quality of the imaging system. Defects notreadily visible with cell samples are often recognizedeasily when one examines the images of single beads.Oefocused beads should appear as radially symmetricdisks or concentric rings.

Additional Equipmen t

Additional equipment may be required for specialpurposes. Multiw avelength imaging is often achievedwith filter wheels at the illumination and/or detectionoptical path. In addition, devices such as Dual-Viewand Quad-View from Optical Insights generate com-posite images at different wavelengths. Spinning diskconfocal heads have been used extensively for imagingfine structures in single cells. Its balance between lightefficiency and resolution proves particularly suitable

fo r cu ltured cells. Finally, to tal in ternal reflection fluo -rescence optics is being used extensively for imagingstructures near the cell-glass interface, such as focaladhesions. Details of these approaches and devices arebeyond the scope of this article; however, their poten-tial use should be considered when designing theimaging sys tem.

Reference

McKenna, N. M ., and Wang, Y.-L. (1989). Culturing cells on thetTIicroScopC stage. Met/hods Cell Biol. 29, 295-305.