B I O G R A P H YElias Horn received hisdiploma in biotechnol-ogy from the Universityof Applied Sciences, Mit-tweida, Germany. One ofhis principal researchactivities is chemotaxis.Elias is studing slowly migrating cells in lin-ear concentration gradients over long peri-ods of time and is also interested in thekinetics of fast cells such as Dictyosteliumdiscoideum. He is now developing chemo-tactic assays in microfluidic devices.

A B S T R A C TPhase contrast, one of the most commonlyused contrasting techniques in lightmicroscopy, has crucial drawbacks whenused to image cells in small culture wells dueto the formation of a meniscus at the air-water interface. In this study we demon-strate how it is possible to perform phase-contrast microscopy on living cells in vol-umes of less than 30 µl. Adherent cells werecultured in the 25 µl channels of microslides,fluorescently stained, and observed byphase-contrast and fluorescence micro-scopy. 95% of the cells were accessible byphase contrast in the channels, whereas inthe 96-well plates only 1% of the culturearea showed satisfactory contrast. In orderto explain this finding we have analysed theoptical basis of phase-contrast microscopyto elucidate the disturbing influence of ameniscus on the optical light path.

K E Y W O R D Slight microscopy, phase-contrast microscopy,fluorescence microscopy, cell culture, menis-cus formation

A C K N O W L E D G E M E N T SThis work was supported by Ludwig-Maxim-ilians-Universität in Munich and BMBF Live-Cell Screening FKZ:13N8777.

A U T H O R D E TA I L SElias Horn, Geschwister-Scholl-Platz 1, 80539 Munich, GermanyTel: +49 89 2180 1480Email: ehorn@ibidi.de

Microscopy and Analysis 20(3): 5-7 (UK), 2006

PHASE CONTRAST

MI C R O S C O P Y A N D AN A LY S I S • MAY 2006 5

I N T R O D U C T I O NPhase-contrast light microscopy is a well-established imaging technique in cell biology.It is a powerful tool for taking high-resolutionimages of living cells. It is so common thatmost scientists don’t even mention phase-con-trast microscopy in their materials and meth-ods. The technique is an indispensable tool toexamine cell morphology [1] and to distin-guish or identify different kinds of cells [2].Nearly all types of cells in culture can beobserved with this inexpensive, standardmethod. Bacterial cells [3], stem cells [4], andneuronal cells [5] are only a few examples ofthe wide-spread, in-vitro use of this technique.Furthermore, phase contrast is often used to‘optically counterstain’ subcellular organellessuch as the nucleus in combination with fluo-rescence microscopy techniques [6].

The phase-contrast technique was firstdescribed by Frits Zernike in 1934 [7]. The firstprototype phase-contrast microscope wasbuilt by Zeiss (Figure 1a) in 1936. Within a fewyears the method became an indispensabletool in medical research as structures such asthe chromosomes of living cells could beimaged (Figure 1b). In 1943, phase contrastwas used when cell mitosis was observed bytime-lapse microscopy for the first time [8].

Today, the phase-contrast technique isnearly unchanged in its optics, but over theyears the requirements have changed. Nowa-days cell biologists and biomedical researchersneed low-volume observations since antibod-ies and staining solutions are limited andexpensive. Therefore, most assays are con-ducted in multiwell systems such as 96-wellplates since coverslips are not suitable for live-cell microscopy. These multiwell tools fulfil theneeds for small observation volumes but theyfail when it comes to phase-contrastmicroscopy. Everyone working with small,open wells knows the problem of low contrastnear the walls of the well. Before explainingthe disturbing influence of the meniscus onphase-contrast imaging we will review theprinciple of phase-contrast microscopy. Thenwe show that culturing and observing cells insmall channels in a specially designedmicroslide is a solution to the problem of per-forming phase contrast in small volumes.

M AT E R I A L S A N D M E T H O D S

Cell culturesRat fibroblasts from the cell line Rat1 were cul-tivated in Dulbecco´s Modified Eagle´sMedium (PromoCell, Germany) with 10% fetalcalf serum. After trypsinization, the cells wereseeded into 96-well plates (Nunc, Denmark)

and into microslides (µ-Slide VI, ibidi, Ger-many). Both plastic surfaces have a similar tis-sue-culture treated surface. Due to the differ-ent geometry, different cell densities wereused to obtain comparable cell monolayers.96-well plates were used with 200 µl of a 0.25�105 cells ml-1 suspension. The cell culturechannels of the µ-Slide VI were filled with 30 µlof a 3�105 cells ml-1 suspension. After 30 min.adhesion time, the reservoirs were filled with60 µl DMEM for long-term cultivation.

Live cell stainingCellTracker Green CMFDA (Molecular Probes,Invitrogen Inc., USA) is a nonfluorescent dyeand passes freely into living cells. In the cytosolthe dye starts to exhibit a bright fluorescenceby cytosolic esterase cleavage. The signal is sta-ble for at least a few hours. After 24 hours ofcultivation Rat1 cells were stained for 20 min-utes with CellTracker green CMFDA at a work-ing concentration of 1 µM in serum-freeDMEM. Phase-contrast and epifluorescenceobservations were conducted in DMEM with10% fetal calf serum.

MicroscopyLive-cell microscopy at low magnifications wasperformed using a Zeiss Axiovert 25 micro-scope (Carl Zeiss, Germany) equipped with anA-Plan 5� 0.12 NA Ph0 objective. Images weretaken using a Canon PowerShot A80 digitalcamera. Large field of view images were com-bined with a tool in Canon PhotoStitch 3.1software. High-magnification phase- contrastand fluorescence images were taken using aZeiss Axiovert 100 microscope equipped with aPlan-Neofluar 100� 1.3 NA Oil Ph3 objectiveand a cooled CCD camera (Hamamatsu Pho-tonics, Japan). Both microscopes wereequipped with heat-controlled stages toobserve cells under 37°C conditions for shortperiods of time.

Phase-Contrast Light Microscopy of LivingCells Cultured in Small VolumesE. Horn1,2, R. Zantl 2 1. Ludwig-Maximilians University, and 2. Integrated BioDiagnostics GmbH, Munich, Germany

Figure 1: (a) First prototype of a phase-contrast microscope, the Zeiss L-Stativ1936.(b) The first phase-contrast photomicrograph of a living nucleus taken in1941 shows giant chromosomes of Chironomus sp. (Courtesy of Carl Zeiss, Germany.)

M I C R O S C O P Y A N D A N A LY S I S ¥ M A Y 2 0 0 66

H O W D O E S P H A S E C O N T R A S TW O R K ?Objects that directly change the amplitude oftransmitted light by absorption are calledamplitude objects. Those objects can be seenusing simple brightf ield microscopy. But smallobjects without pigments or dyes change theamplitude of the scattered light only veryslightly. They can hardly be observed bybrightfield microscopy at all. But cell structuressuch as the cell membrane and organelleshave slightly different refractive indices to thesurrounding medium and consequentlychange the phase of the transmitted light. Thissmall change of the incident light vector bythe object can be described by the sum of twovectors. One having nearly the same ampli-tude and exactly the same direction as the inci-dent light, and the other a slightly differentvector with a phase shift of roughly 90¡ (= /2)with respect to the incident light vector. In Fig-ure 3 the vector of the incident light wave isdrawn dark blue, the diffracted wave vector isred and the difference light vector is green.The principle of phase contrast is based onbringing the incident light from the sourceinto the same phase as this small differencevector (see Figure 3b). In this way the phaseshift is translated into an amplitudedifference.

A simplif ied sketch of a phase-contrast setupis shown in Figure 2a. In practice the phaseshift of the incident light with respect to thediffracted light is achieved by passing the inci-dent beam through the circular phase plate.Therefore, the annular ring and the phaseplate have to be aligned in the back focalplane of the objective.

I M A G E D I S T O R T I O N I N S M A L LO P E N C A V I T I E SProper alignment of the annular aperturewith the phase plate cannot only be affectedby an incorrect lateral position of the annularring but also by other components disturbingthe light path. As seen in Figure 2b, an inclinedsurface of the specimen diffracts the incident

light from the condenser which is seen by adisplacement of the annular ring relative tothe phase plate. Therefore, by not passing thephase ring the phase of the incident light is nolonger changed. As a result the phase contrasteffect is overlaid by common brightf ield illu-mination and is strongly reduced. This effect isa major drawback in cell microscopy whenphase contrast is performed in multiwell platesor chambered coverslips. The protein-richmedia have a high aff inity for the hydrophilicplastic walls which results in the formation ofa meniscus at the air-water interface. As Figure4a indicates, the local curved surface of themedium is similar to the wedge drawn in Fig-ure 2b and therefore affects the phase con-trast. The steeper the water surface, thestronger is the visible displacement of theannular ring relative to the phase plate. Infact, by moving the well with the meniscus in

Figure 2:Simplified beam path for phase-contrast microscopy. (a) The light from the annular ring illuminates the speci-men and is partly diffracted. The non- diffracted fraction passes through the phase plate and undergoes a phaseshift of /2 which is necessary for phase contrast. Therefore the annular ring must be aligned with the phaseplate in the back focal plane. (b) A locally inclined water surface disturbs the alignment of annular ring and phaseplate. Common brightfield illumination overlays the phase contrast effect.

Figure 3:The blue vector and the blue wave in (a) represent the incident light. The red vector is slightly changed in phaseby the specimen. The green vector is the difference between the incident and diffracted vectors. Passing thephase plate the incident light is phase shifted by /2 and is therefore in phase with the difference wave as seenin (b).

Figure 4:Beam pathways in small cavities and resulting phase-contrast images in the centre of the multiwell dishes. (a) Meniscus wall effects and condensationdisturb incident light in a small open well. An optical water wedge is formed near the walls. Only the centre provides a nearly planar air-water surface.Additionally, condensation water on the lid lowers contrast by light scattering. (b) As a result, phase contrast is possible only in a small area (1% ) of thetotal area of the 96-well plates. The phase-contrast area is indicated by the dashed circle. (c) Undisturbed parallel beam path in a channel. (d) The wholeculture area provides excellent phase contrast independent of the location. Scale bars = 100 µm.

Table 1:Characteristics of the multiwell and microslide systems used for theculture of adherent Rat1 fibroblast cells.

96-w ell plate µ-Slide VI

grow th areaper w ell

0.38 cm2 0.60 cm2

volume perw ell

200 µl 30 µl

cellconcentration

0.25 x 105

per ml3 x 105

per ml

cells per w ell 5,000 9,000

cells per cm2 13,158 15,000

a b

c d

PHASE CONTRAST

MI C R O S C O P Y A N D AN A LY S I S • MAY 2006 7

the optical path, this displacement can easilybe seen in the back focal plane.

T H E S O L U T I O NFor microscopy reasons only, there is nothingbetter than mounting the sample between aclassical microscope slide and a coverslip. Thesample medium fills out the total volumebetween the glass plates and no meniscus canbe formed. From a cell culture point of view,the slide system has to provide the cells withmedium. Also, the exchange of medium forstaining and other reasons must be possible,but this is not an option in the classical micro-scope slide system.

The solution to this problem is a small chan-nel like that in the µ-Slide VI. As shown in Fig-ure 4c, the channel defines a small fluid reser-voir for culturing cells between the top andthe coverslip-like plastic bottom. Just like thetraditional microscope slide system, the chan-nel volume is totally filled with medium avoid-ing meniscus formation due to the lack of anair-water interface. The liquid can easily beexchanged using standard pipettes for drugscreening and staining steps while the adher-ent cells remain inside the channel.

R E S U LT SWe found that Rat1 cells grew with identicalmorphology in 96-well plates and the chan-nels of the microslides (Table 1). Under bothcultivation conditions, confluency wasreached after 3 days.

Fluorescence and phase-contrast imageswere taken and overlaid as shown in Figure 6.Phase-contrast microscopy was possible over alarge area inside the microslides (Figure 5).Low-magnification phase-contrast imagesrevealed a total phase-contrast area of 95%.Figure 5b shows the full width of a cell culturechannel in phase contrast assembled from 15separate images. In 96-well plates the phase-contrast area was about 1% of the growtharea located in the centre of the well. Figures4b and 4d show the centres of the cultivationsystems in phase contrast.

During the cultivation of Rat1 cells wefound 110 dividing cells out of a total of 4952cells total (Figure 5b). These 2.2% mitotic cellscorresponded very well with our recent find-ings: i.e. Rat1 cells have a doubling time ofapprox. 16 hours [9] where one mitosis takesapprox. 20 min (unpublished data). This givesthe expected value of 2.1 % for all cells show-ing mitosis at the same time.

Additionally, we found that there was nocondensation water disturbing the phase-con-trast effect, unlike in the 96-well plate wherecondensation effects lowered the quality ofthe microscopic image. Even in the goodphase-contrast area of the 96-well plate thequality still lagged behind due to the scatter-ing effects of condensation water on the lid(Figure 4b).

D I S C U S S I O NIt is often the case with multiwell plates thatthe nicest cell is always near the wall andtherefore not accessible to phase contrastmicroscopy. In 96-well plates only 1% of the

total growth area can be observed properly byphase-contrast microscopy. However, with themicrochannel slides more than 90% of thegrowth area can be observed. This means hav-ing better statistics, having a higher chance ofobserving the expected event and thereforesaving time, cells and reagents.

Due to the incompatibility of phase contrastand low-volume open wells many micro-scopists were switching from phase contrastto differential interference contrast (DIC)microscopy. But DIC causes new problems suchas low image quality because of the high bire-fringence shown by most plastic materialsused for cell culture.

Micro channels offer a good solution forphase contrast microscopy in low-volume cellculture. Also, the contamination risk andevaporation are lowered, which is especiallyimportant using very small volumes. Togetherwith recent findings that cell homogeneity isalso remarkably improved [9], we see highpotential for cell culture microchannels in lab-oratory use and for automated screenings.

Figure 5: Adherent Rat1 cells growing in a low-volume cell culture channel allowing optimum quality in phase-contrast imaging. (a) The microslide with six par-allel 30-µl volume channels with cells and cell medium. (b) Montaged picture of the whole channel width assembled from from 5x3 single frames usingan automated software. The upper part shows that phase contrast is possible inside the channel geometry only. Scale bar = 500 µm.

Figure 6: Live cell imaging of Rat1 cells after24 h culture using fluorescence (a)and phase-contrast (b) modes,and as an overlay (c). The cytosolwas stained with CellTracker greenCMFDA. Scale bars = 10 µm.

R E F E R E N C E S1. Leonardi, A. et al. In vitro effects of fluoroquinolone and

aminoglycoside antibiotics on human keratocytes. Cornea25(1):85-90, 2006.

2. Lanfranco, G. and Segoloni, G.P. ‘Decoy cells’ in urine.Transplant Proc. 37(10):4309-4310, 2005.

3. Dworkin, J., Losick, R. Developmental commitment in abacterium. Cell 121(3):401-409, 2005.

4. Kadivar, M. et al. In-vitro cardiomyogenic potential ofhuman umbilical vein-derived mesenchymal stem cells.Biochem. Biophys. Res. Commun. 340(2):639-647, 2006.

5. Pujol, F. et al. The chemokine SDF-1 differentially regulatesaxonal elongation and branching in hippocampal neurons.J. Cell Sci. 118(5):1071-1080, 2005.

6. Brero, A. et al. Methyl CpG-binding proteins induce large-scale chromatin reorganization during terminaldifferentiation. J. Cell Biol. 169(5):733-743, 2005.

7. Zernike, F. Beugungstheorie des Schneidenverfahrens undseiner verbesserten Version der Phasenkontrastmethode.Physica 1:689-704, 1934.

8. Michel, K. Die Kern- und Zellteilung im Zeitrafferfilm. Diemeiotischen Teilungen bei der Spermatogenese derSchnarrheuschrecke Psophus stridulus L. Zeiss Nachrichten4(9):236-251, 1943.

9. Horn, E. et al. Homogeneous cell distribution in cell cultureµ-channels. Bio Tech International (Submitted) 2006.

©2006 John Wiley & Sons, Ltd

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