Ultraviolet confocal fluorescence microscopy ofthe in vitro cornea: redox metabolic imaging

Barry R. Masters, Andres Kriete, and Jorg Kukulies

A laser scanning microscope was fitted with two argon-ion lasers that provided wavelengths in the regionsof 364, 488, and 514 nm. A Zeiss water objective of 25 x, with a numerical aperture of 0.8, corrected forthe UV, was used to measure the fluorescence from optical sections of freshly enucleated rabbiteyes. The confocal microscope was used in both the reflected and fluorescent modes to image in situepithelial and endothelial cells. An excitation wavelength of 364 nm and emission at 400-500 nm wereused to image the fluorescence from reduced pyridine nucleotides. We demonstrate the feasibility oftwo-dimensional fluorescent confocal imaging of reduced pyridine nucleotides in corneal epithelial andendothelial cells.

Key words: Confocal laser scanning microscope, cornea, epithelium, endothelium, reduced pyridinenucleotides, redox fluorometry.

Introduction

The application of confocal fluorescence microscopicimaging to noninvasively monitor cellular hypoxia inthe limiting layers of the in vitro cornea is demon-strated. The cornea is an avascular, thick (400-jim),semitransparent, living optical element in the frontregion of the eye. The corneal epithelium and endo-thelium comprise the limiting cell layers on theanterior and posterior surfaces of the cornea. Anoninvasive optical technique is described and demon-strated for two-dimensional metabolic imaging cellu-lar hypoxia in these limiting cell layers of the cornea.

The fluorescence from the naturally occurring re-duced pyridine nucleotides in cells is an indicator ofcellular respiration.' The fluorescence of reducedpyridine nucleotides [NAD(P)H] is excited with364-nm light; the fluorescence emission is in therange of 400-500 nm. Cellular hypoxia is associatedwith an increased ratio of reduced to oxidized pyri-dine nucleotides and therefore increased fluorescenceintensity in the region of 400-500 nm. Since thequantum efficiency, and thus the fluorescence inten-

B. R. Masters is with the Department of Anatomy and CellBiology, Uniformed Services University of the Health Sciences,Bethesda, Maryland 20814-4799. A. Kriete is with the Institutefor Anatomy and Cytobiology, University of Giessen, Aulweg 123,D-6300, Giessen, Germany. J. Kukulies is with the Division ofApplied Microscopy, Carl Zeiss, D-7802, Oberkochen, Germany.

Received 30 January 1992.0003-6935/93/040592-05$05.00/0.© 1993 Optical Society of America.

sity, of the reduced pyridine nucleotides is signifi-cantly greater than that of the oxidized pyridinenucleotides, the fluorescence intensity monitors thedegree of cellular hypoxia. This noninvasive opticaltechnique is called redox fluorometry.1 We havedemonstrated that the 400-500-nm fluorescence ex-cited at 364 nm is due to the reduced pyridinenucleotides.24 We have investigated the fluores-cence intensity of the cornea by using an opticallysectioning microscope to monitor the degree of cellu-lar hypoxia.5-10 These studies demonstrated andcharacterized the NAD(P)H fluorescence and showedthe alteration of NAD(P)H fluorescence by chemicalinhibitors of cellular respiration as well as contact-lens-induced cellular hypoxia. We have not previ-ously investigated the possibility of two-dimensionalimaging of reduced pyridine nucleotide fluorescencein the cornea because of the low levels of fluorescence.Recently two-dimensional redox imaging was demon-strated by other investigators for isolated cardiacmyocytes in tissue culture." Therefore we initiatedthis study to obtain two-dimensional images of cor-neal cellular metabolism with a special confocal micro-scope.

The confocal microscope serves as an optical devicefor observing a single focal plane of thick objects withhigh resolution and contrast compared with standardmicroscopes.'2 The confocal microscope when usedin the fluorescent mode has one excitation wave-length, and the fluorescent image is formed at alonger wavelength. This differs from the reflectedlight mode in which the confocal image is formed at

592 APPLIED OPTICS / Vol. 32, No. 4 / 1 February 1993

the same wavelength as that of the laser illumination.The depth resolution of fluorescent confocal scanningoptical microscopes is reduced compared with thereflected imaging mode.13-'5 The advantages of UVconfocal microscopy include increased resolution anda reduced depth of focus compared with visible lightconfocal microscopy.161 7 These advantages are bestrealized with a microscope objective, which is cor-rected for the UV.

The use of the confocal microscope to section thecornea optically was shown by Lemp et al. 18 The finestructure of the in vitro cornea has been shown withboth the one-sided Nipkow disk confocal microscopeand with the laser scanning confocal microscope.19-22

The three-dimensional volume reconstruction fromserial confocal optical sections of the in vitro corneahas been demonstrated.2 324

The laser scanning confocal microscope (a Zeiss UVconfocal laser scanning microscope) permits two-dimensional redox confocal imaging of the NAD(P)Hfluorescence intensity of corneal cells. Thus a two-dimensional image or map of cellular metabolism canbe obtained. A combination of both the reflectedlight images of cell morphology and the redox fluores-cence images of cellular metabolism may be used toconstruct a multimodality three-dimensional imageof cell structure and function. The three-dimen-sional images can be visualized by volume-renderingcomputer-assisted reconstruction methods.2 4

Methods

Biological Material

Eyes were obtained from male New Zealand whiterabbits weighing 2.5 kg. The rabbits were main-tained and handled in accordance with the Associa-tion for Research in Vision and Ophthalmology Reso-lution on the Use of Animals in Research. Therabbits were euthanized with an overdose of pentabar-bital. After death the eyes were removed and freedof adhering tissue. The intact eyes were placed in acontainer filled with a bicarbonate Ringer solution9

that contained 5 mM of glucose and 2 mM of calciumat 250C. The corneas of the in situ eyes were imagedwithin 5 min, and the duration of the experimentalimaging was 30 min. The corneal endothelial cellswere imaged, and then the corneal epithelial cellswere imaged. The cells were in a normoxic stateduring the time of the experiments as a result of theaerated Ringer solution.

Reflected and Fluorescence Light Confocal Microscopy

A Zeiss confocal scanning laser microscope (10 UV,Carl Zeiss, Oberkochen, Germany) was adapted forUV-fluorescence confocal microscopy.2 52 6 In addi-tion to the argon-ion laser (488 and 514 nm) and theHe-Ne laser (543 nm) another argon laser (364 nm)was added to the microscope. The exact wavelengthwas 363.6 nm. The optical and mechanical arrange-ment of the scanning laser confocal microscope isshown in Fig. 1. The UV argon-ion laser was aSpectra-Physics type 2016 with a variable output

Fig. 1. Optical system of the Zeiss laser scanning microscope.The schematic laser beam paths of the laser confocal microscopeare shown. The standard Zeiss laser scanning microscope hasbeen fitted with two external lasers: an external argon-ion laser(488-nm, 514-nm laser lines) and an external UV argon-ion laser (a364-nm line). There is an internal He-Ne laser (543- and 633-nmlaser lines). Switchable mirrors and barrier filters permit UVfluorescence or reflectance measurements. The various barrierfilters are located between the confocal variable pinhole and thebeam splitter that is adjacent to the two photomultiplier detectors(PMT1, PMT2). For confocal UV operation the UV laser and anexternal beam expansion unit are added to the laser scanningconfocal microscope. A Zeiss water objective of 25 x, with an NAof 0.8, corrected for the UV, was used for fluorescence andreflected-mode confocal microscopy. FT, filters.

power of 20-100 mW. In addition to the third laserthere were other changes to the confocal microscope;the antireflection coatings on the x-y scanner wereoptimized for the UV light.

A Zeiss water objective of 25 x, numerical aperture(NA) of 0.8, corrected for the UV, was used tomeasure the fluorescence from optical sections of afreshly enucleated rabbit eye. The microscope objec-tive was able to focus across the full 400-,um thick-ness of the in vitro rabbit cornea. The laser scan-ning confocal microscope produced digital images of256 x 265 pixels and 256 gray levels. To obtain thefluorescence image we first used the reflected lightmode of the confocal laser scanning microscope at awavelength of 364 nm to locate the endothelial cells,which are situated 400 m below the anteriorsurface of the cornea. We focused the microscope

2 ,m into the corneal endothelial cells and formed areflected light confocal image. Then the laser scan-ning fluorescence confocal microscope was switchedto the fluorescence mode. The excitation wave-length was 364 nm, and the emission filters collectedlight in the region of 400-500 nm. Eight imageswere averaged to improve the signal-to-noise ratio ofthe final image. A similar procedure was used toimage the corneal epithelial cells at the surface of thecornea. The wavelengths used for the laser excita-tion (364 nm) and the fluorescent emission (400-500)correspond to the excitation and emission of thereduced pyridine nucleotides. 3 6 9 Several confocalimages of the in vitro cornea were produced by usingthe reflected light mode with the laser source at 488

1 February 1993 / Vol. 32, No. 4 / APPLIED OPTICS 593

nm to demonstrate the optical resolution and con-trast of the modified laser scanning confocal micro-scope with the corrected UV microscope objective(Figs. 2 and 3).

To demonstrate the optical quality of the laserscanning microscope, we produced images of the invitro cornea that were both perpendicular to thecorneal surface (Fig. 2) and in the plane of the cornea(Fig. 3) by using 488-nm laser light in the reflectedlight confocal mode. The small fibers shown in Fig.3 are -1 jIm in diameter and represent thin fiberswithin the plane of focus of stromal fibroblast cells.The images show the cell nuclei.

The image quality at the anterior surface of thecornea is shown in Fig. 4. The reflected light imageformed at 364 nm of the superficial corneal epithelialcells is shown in Fig. 4(a). A similar focal plane isshown imaged in the fluorescent mode in Fig. 4(b).The fluorescence of the reduced pyridine nucleotidesis shown in Fig. 4(b). The cells that are fluorescentare those that are in the focal plane of the confocalmicroscope; the darker cells are not in the focal plane.

The image quality at the posterior corneal surface,which is made through 400 jim, is shown in Fig. 5.Figure 5(a) shows reflected light images produced at364 nm within the corneal endothelium. Figure 5(b)shows the corresponding fluorescent images resultingfrom NAD(P)H. This image represents a two-dimensional map of cellular metabolic function.

Discussion

We have demonstrated the ability to obtain confocalmicroscopic fluorescent images of pyridine nucleotideNAD(P)H fluorescence in cells on the surface of thecornea (corneal epithelium) and cells 400 jim belowthe surface of the cornea (corneal endothelium).What do the images represent and why are the imagesof the surface cells different from those images of the

Fig. 2. Z-scan confocal microscopic image of the anterior region ofthe cornea from an in vitro rabbit eye. The image is producedwith backscattered light (488 nm). The uppermost bright regionis the highly reflective superficial epithelial cells adjacent to thetear film. A bright line, 50 m below, represents the highlyreflective basal lamina. Directly above the basal lamina are thebasal epithelial cells with their nuclei. Below the basal lamina areshort horizontal lines that are the nuclei of the stromal fibroblasts.The 50-,um scale bar is shown.

Fig. 3. Confocal microscopic image of the anterior region of thecornea from an in vitro rabbit eye. The image is produced withbackscattered light (488 nm). The image plane is perpendicular tothe optic axis of the eye. The nuclei of stromal fibroblasts cells areshown together with submicrometer filaments that interconnectthe cells. The cell bodies are not visible.

deeper cells? A comparison of Figs. 4(a) (364-nmreflected light) and 4(b) (364-nm excitation, 400-500-nm emission) with Figs. 5(a) (364-nm reflectedlight) and 5(b) (364-nm excitation, 400-500-nm emis-sion) shows the following differences. First, theimages of the cells on the corneal surface have ahigher contrast and resolution compared with those400 jim below the surface. This was observed forboth the reflected light modes and the fluorescentmodes. Second, the resolution of the confocal im-ages is decreased compared with the image made inreflected light. This is consistent with calculationsof the microscope point spread function for reflectedlight imaging and fluorescence light imaging; thelatter shows a wider point spread function. 4

15

Several factors contribute to the loss of imagequality in the fluorescence imaging mode. One fac-tor is the loss of signal for the images made at a400-,um depth. The laser light must pass through400 jim of scattering and absorbing corneal tissue,and the emission light passes through this tissue onthe return to the microscope. This results in a lossof light on each pass, and therefore a reduction ofsignal intensity is observed. For example, scatteringwould reduce the intensity of the incident radiationreaching the endothelium by 10-15%, and thefluorescent radiation reaching the detector would bereduced somewhat less because of its longer wave-length. These losses in intensity are not significantfor the technique of redox imaging, since the intensi-ties can be corrected and the important considerationis the difference in fluorescence intensity betweenindividual cells.

A second factor that contributes to image degrada-tion is the sample thickness. Most microscope objec-tives are designed to work with a cover slip of knownrefractive index and thickness. The refractive indexof the optical coupling fluid is usually defined in thecalculation of the microscope objective lens design.When we image the surface cell on the cornea, the

594 APPLIED OPTICS / Vol. 32, No. 4 / 1 February 1993

-11 ......... - ,--

(a)

(D)Fig. 4. (a) Confocal microscopic image of the anterior region of thecornea from an in vitro rabbit eye. The image is produced withbackscattered light of 364 nm. The superficial epithelial cells andtheir nuclei are shown. (b) Similar focal plane view imaged withUW 364-nm light in the fluorescent mode. The image shows thefluorescence from the pyridine nucleotides in the corneal cellsEmission is 400-500 nm.

refractive index of the Ringer solution is not signifi-cantly different from water. However, when weimage across 400 m of cornea the refractive index isboth variable and unknown. The average refractiveindex of the cornea is known, not the profile of therefractive index along the optic axis of the cornea.

Furthermore, when we image in the fluorescentmode, the depth of focus of the excitation light (364nm) differs from the depth of focus for the 400-500-nm emission light of the sample fluorescence.This reduced the image quality in the confocal micro-scope.' 4 "15

The fields of view are slightly different for theimages in reflected light and in fluorescent light.When attempting to compensate for the difference inthe focus depth at the different wavelengths the eyeshifted slightly. The redox fluorescent image of thecorneal epithelium shows the bright fluorescence ofthe cells in the focal plane of the microscope. Thenuclei appear dark because of their lack of reducedpyridine nucleotides. The center of the nuclei showsfluorescence that may be fluorescence from the nucle-oli [Fig. 4(b)].

(a)

(b)Fig. 5. (a) Confocal microscopic image of the endothelial cells ofthe cornea from an in vitro rabbit eye. The image is producedwith backscattered light of 364 nm. (b) Confocal microscopicimage of the endothelial cells of the cornea from an in vitro rabbiteye. The reduced pyridine nucleotide fluorescence is shown in thebright regions; the cell nuclei are the dark regions. The imagesare made through 400 pLm of corneal tissue. The laser excitationwas 364 nm, and the emission was 400-500 nm. The dark regionsin the image are the nuclei of the endothelial cells. The brightregions show the fluorescence from the reduced pyridine nucleo-tides.

The redox fluorescent image from the cornealendothelial cells [Fig. 5(b)] shows the bright fluores-cence from the reduced pyridine nucleotides. Thedarker regions are where the large cell nuclei arepresent. The cornea endothelial cells contain a largenucleus that occupies most of the cell volume.Endothelial cells contain highly packed mitochondriasituated around the central nucleus. However, thesurface epithelial cells contain a much smaller nu-cleus and fewer mitochondria. In Fig. 5(b) the un-even illumination of the microscope results in the leftside of the figure showing a higher intensity. This isnot due to different metabolic states of the endothe-lial cells as observed by redox metabolic imaging butis due to the lack of even illumination over the field.This could be corrected with computer methods.In this study no attempt was made to quantitate thefluorescence intensity of the images, and histogramsof the NAD(P)H fluorescence from individual cellswere not provided.

1 February 1993 / Vol. 32, No. 4 / APPLIED OPTICS 595

All the previous studies using both biochemicalmicroanalysis and optical fluorescence measurementshave verified that the fluorescent signal from thecorneal epithelial and endothelial cells (excitation,364 nm; emission, 400-500 nm) is due to the reducedpyridine nucleotides in these cells.--6 These previ-ous measurements obtained an average value of thefluorescence intensity over several square millime-ters of cornea.

The current study demonstrates for the first timewe believe the feasibility of UV confocal fluorescencemicroscopy for imaging the intrinsic-reduced pyri-dine nucleotides in cells of the corneal epithelium andendothelium in an in vitro rabbit cornea. No at-tempt was made to obtain redox images of the wingcells or the basal cells in the corneal epithelium.These two-dimensional redox fluorescent images aremetabolic maps of cellular metabolism. In spite ofthe difficulties of imaging the fluorescence across 400[Lm of living tissue, it is possible to map out theindividual cellular fluorescence resulting from theintrinsic fluorescence from the reduced pyridine nucle-otides.

The two-dimensional imaging of the corneal endo-thelial cells (fluorescence from the reduced pyridinenucleotides) presents a new technique for investigat-ing the heterogeneity of the cellular respiration in thecorneal cell layers. The significance of this work isthat multimodality three-dimensional images24 of thecell structure and function could be made by combin-ing three-dimensional reconstructions of cell morphol-ogy and cell metabolism through the use of noninva-sive redox metabolic imaging.

This work was supported by a grant (EY-06958) toB. R. Masters from the National Institutes of Health,National Eye Institute.

References1. B. Chance and B. Thorell, "Localization and kinetics of

reduced pyridine nucleotide in living cells by micro-fluorometry," J. Biol. Chem. 234,3044-3050 (1959).

2. B. Chance and M. Lieberman, "Intrinsic fluorescence emissionfrom the cornea at low temperatures: evidence of mitochon-drial signals and their differing redox states in epithelial andendothelial sides," Exp. Eye Res. 26, 111-117 (1978).

3. B. R. Masters, "Noninvasive redox fluorometry: how lightcan be used to monitor alterations of corneal mitochondrialfunction," Curr. Eye Res. 3, 23-26 (1984).

4. B. R. Masters, "In vitro corneal redox fluorometry," in Nonin-vasive Diagnostic Techniques in Ophthalmology, B. R. Mas-ters, ed. (Springer-Verlag, New York, 1990), pp. 223-247.

5. B. R. Masters, S. Falk, and B. Chance, "In vivo flavoproteinredox measurements of rabbit corneal normoxic-anoxictransitions," Curr. Eye Res. 1, 623-627 (1982).

6. B. R. Masters, "Noninvasive corneal redox fluorometry," inCurrent Topics in Eye Research, J. Zadunaisky and H. Davson,eds. (Academic, London, 1984), pp. 139-200.

7. B. R. Masters, "Confocal microscopy of ocular tissue," inConfocal Microscopy, T. Wilson, ed. (Academic, London, 1990,pp. 305-324.

8. B. R. Masters, "Effects of contact lenses on the oxygen

concentration and epithelial mitochondrial redox state ofrabbit cornea measured noninvasively with an optically section-ing redox fluorometer microscope," in The Cornea: Transac-tions of the World Congress on the Cornea III, H. D. Cavanagh,ed. (Raven, New York, 1988), pp. 2810-3860.

9. B. R. Masters, M. V. Riley, J. Fischbarg, and B. Chance,"Pyridine nucleotides of rabbit cornea with histotoxic anoxia:chemical analysis, noninvasive fluorometry and physiologicalcorrelates," Exp. Eye Res. 36, 1-9 (1983).

10. B. R. Masters, "An optical method for the determination ofoxygen concentration in the tear film," in The Precorneal TearFilm in Health, Disease and Contact Lens Wear, F. Holly, ed.(Dry Eye Institute, Lubbock, Tex. 1986), pp. 966-970.

11. J. Eng, R. M. Lynch, and R. S. Balaban, "Nicotinamideadenine dinucleotide fluorescence spectroscopy and imaging ofisolated cardiac myocytes," Biophys. J. 55, 621-630 (1989).

12. T. Wilson, "Optical aspects of confocal microscopy," in Confo-cal Microscopy, T. Wilson, ed. (Academic, London, 1990), pp.93-141.

13. T. Wilson, "Optical sectioning in confocal fluorescentmicroscopes," J. Microsc. 154, 143-156 (1989).

14. S. Kimura and C. Munakata, "Depth resolution of the fluores-cent confocal scanning optical microscope," Appl. Opt. 29,489-494 (1990).

15. S. Kimura and C. Munakata, "Calculation of three-dimen-sional optical transfer function for a confocal scanning fluores-cent microscope," J. Opt. Soc. Am. 6, 1015-1019 (1989).

16. S. D. Bennet, E. A. Peltzer, and I. R. Smith, "Ultra-violetconfocal metrology," in Scanning Microscopy Technologiesand Applications, E. C. Teague, ed., Proc. Soc. Photo-Opt.Instrum. Eng. 897, 75-82 (1988).

17. P. A. Heimann and R. Urstadt, "Deep ultraviolet microscope,"Appl. Opt. 29, 495-501 (1990).

18. M. A. Lemp, P. N. Dilly, and A. Boyde, "Tandem-scanning(confocal) microscope for optically sectioning the living cornea,"Cornea 4, 205-209 (1986).

19. B. R. Masters and S. W. Paddock, "In vitro confocal imaging ofthe rabbit cornea," J. Microsc. 158, 267-275 (1990).

20. B. R. Masters and G. S. Kino, "Confocal microscopy of theeye," in Noninvasive Diagnostic Techniques in Ophthalmol-ogy, B. R. Masters, ed. (Springer-Verlag, New York, 1990), pp.152-171.

21. B. R. Masters and G. S. Kino, "Real-time confocal scanningimaging of the eye: instrument performance of reflection andfluorescence imaging," Proc. Inst. Phys. (UK) 98, 625-628(1989).

22. G. Q. Xiao, G. S. Kino, and B. R. Masters, "Observation of therabbit cornea and lens with a new real-time confocal scanningoptical microscope," Scanning 12, 161-166 (1990).

23. A. Kriete and B. R. Masters, "Three-dimensional visualizationof the living cornea," in Transactions of the Royal Microscopi-cal Society, MICRO 90, V. Howard, ed. (Institute of Physics,Bristol, UK, 1990), Vol. 1, pp. 401-404.

24. B. R. Masters and S. W. Paddock, "Three-dimensional recon-struction of the rabbit cornea by confocal scanning opticalmicroscopy and volume rendering," Appl. Opt. 29, 3816-3822(1990).

25. M. Montag, J. Kukulies, R. Jorgens, H. Gundlach, M. F.Trendelenburg, and H. Spring, "Working with the confocalUV-laser scanning microscope: specific DNA localization athigh sensitivity and multiple parameter fluorescence," J.Microsc. 163, 201-210 (1991).

26. H. G. Kapitza and V. Wilke, "Applications of the microscopesystem LSM," in Scanning Imaging, T. Wilson, ed., Proc. Soc.Photo-Opt. Instrum. Eng. 1028, 173-179 (1988).

596 APPLIED OPTICS / Vol. 32, No. 4 / 1 February 1993