quantitative analysis of reduced nicotinamide adenine dinucleotide

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Biophysical Journal Volume 72 April 1997 1849-1860 1849
(Semi-)Quantitative Analysis of Reduced Nicotinamide Adenine
Dinucleotide Fluorescence Images of Blood-Perfused Rat Heart
J. M. C. C. Coremans,* C. Ince,*# H. A. Bruining,* and G. J. Puppels*
*Department ofGeneral Surgery, Erasmus University Rotterdam, Rotterdam, and #Department ofAnaesthesiology, University of
Amsterdam, Amsterdam, The Netherlands
ABSTRACT Invivoanalysis ofthemetabolicstateoftissue bymeansofreduced nicotinamide adeninedinucleotide(NADH)
fluorimetry isdisturbed bytissuemovementsand byhemodynamic andoximetric effects.Thesefactorscausechangesinthe
absorption of ultraviolet (UV) excitation light by the tissue. Many different methods have been used in the literature to
compensate measured NADHfluorescence intensitiesfortheseeffects. Inthispaperweshowontheoretical groundsthatthe
ratio of NADH fluorescence intensity and UV diffuse reflectance intensity provides a (semi-)quantitative measure of tissue
NADH concentrations. This result is corroborated by experiments with tissue phantoms in which absorption and back-
scattering properties were varied. Furthermore, we have verified the validity of this compensation method in isolated
Langendorff-perfused rat heart preparations. In this preparation oximetric effects (of blood and tissue) are the major
determinants of the metabolism-dependent UV diffuse reflectance change. Hemodynamic effects accompanying compen-
satory vasodilation are negligible. Movement artifacts were eliminated by simultaneously recording fluorescence and reflec-
tanceimages, usingaCCDcamerawithabiprismconfiguration. TheresultsshowthattheNADHfluorescence/UVreflectance
ratio can be used to monitor the mitochondrial redox state of the surface of intact blood-perfused myocardium.
INTRODUCTION
Intraoperative assessment ofhypoxic areas in myocardium nucleotide in mitochondria of heart (Klingenberg et al.,
can yield important information during, e.g., by-pass sur- 1959) and the fluorescent yield of NADH is three to four
gery. In general, knowledge ofthe adequacy Of02 delivery times higher than that of reduced nicotineamide adenine
by the circulation to the mitochondria in tissue is crucial in dinucleotide phosphate (NADPH) (Avi-Dor et al., 1962;
clinical diagnosis. In mitochondria, the major net energy Estabrook, 1962; Jobsis and Duffield, 1967), the contribu-
conversion is based on electron transport from reduced tion of NADPH to the measured blue autofluorescence of
nicotinamide adenine dinucleotide (NADH) to molecular heart tissue is negligible. The UV-induced blue fluores-
02 via a series of catabolic reactions in the respiratory cence of collagen and fat is independent of the tissular
chain. Hence the NADH/NAD+ redox couple reflects the redox/oxygenation state and therefore does not disturb the
mitochondrial redox state and cellular hypoxia, when oxi- evaluationofthemetabolic stateoftissue (Deyletal., 1969;
dative phosphorylation is curtailed by insufficient 02. Pappajohn et al., 1972; Schomacker et al., 1992).
NADH, unlike its conjugated electron acceptor NAD+, In spite of the potential of NADH fluorimetry for a
fluoresces intheblue (broad-band emissioncentered around noninvasive diagnostic approach, clinical application ofthe
460 nm) upon excitation with ultraviolet (UV) light. There-
technique has been limited because of the problems in-
fore the fluorescent property of NADH may be used to volved in quantitative analysis of the NADH redox state
monitor cellular metabolism, as was first pointed out by (Duboc etal., 1986, 1987; Renaultetal., 1987; Austinetal.,
Chance et al. (Chance and Thorell, 1959).
1978). Several physiological factors thataffectfluorescence
Theblue autofluorescence ofdifferentcell types canonly
intensity must be taken into account (Ince et al., 1992). In
be used as an indicator ofcellular hypoxia when mitochon-
organsandtissues withan intactbloodsupply, absorptionof
drial NADH is the dominant intrinsic fluorescent probe
fluorescence excitation and emission light by blood pig-
(Chance etal., 1962). Forhearttissueithasbeen shownthat
ments (hemoglobin) results in a hemoglobin concentration-
the blue autofluorescence is primarily of mitochondrial or-
dependent reduction of NADH fluorescence intensity (he-
igin, with insignificant contributions of cytoplasmic frac-
modynamic effect). The absorption spectra of hemoglobin
tions (Jobsis andDuffield, 1967; Nuutinen, 1984; Engetal.,
and tissue pigments (e.g. myoglobin, cytochromes) depend
1989). Because NADH is thepredominantreducedpyridine
on their oxygenation/redox state. Hence NADH fluores-
cence intensity is also affected by metabolic state-related
changes in the spectral characteristics of these pigments
Receivedforpublication 24 June 1996 and infinalform 23 December (oximetric effect) (Coremans et al., 1993). In addition,
1996.
tissue movement can easily alter the amount of specular
Address reprint requests to Dr. J. M. C. C. Coremans, Department of
reflection of UV excitation light at the air-tissue interface
General Surgery, University Hospital Rotterdam, dr Molewaterplein 40,
3015 GD Rotterdam, The Netherlands. Tel.: [phone]; Fax: 31-10- and thereby the effective UV excitation intensity in the
4369140; E-mail: [email protected]. tissue. The extent to which these effects interfere with
( 1997 by the Biophysical Society NADH fluorescence measurements is dependent on the
0006-3495/97/04/1849/12 $2.00 organ under investigation.
1850 Biophysical Journal Volume 72 April 1997
Over the past 25 years several methods have been pro- ofsimultaneously measured NADH fluorescence image in-
posed to correct measured NADH fluorescence intensities tensities and UV reflectance image intensities provides an
for light absorption by the tissue that is investigated, to adequate correction for the influence of hemodynamic ef-
obtain (semi-)quantitative information onthe mitochondrial fects, oximetric effects, and tissue movements on measured
redox state (Ince etal., 1992). Mostmethodsutilized alight NADH fluorescence intensities. Therefore this ratio can be
reflectance measurement at acertain wavelength tomonitor used to obtain semiquantitative information about the mi-
changes in optical properties ofthe tissue surface (Dora et tochondrial redox state of surface layers of intact myocar-
al., 1984; Harbig et al., 1976; Ji et al., 1977, 1979; Jobsis et dium in vivo.
al., 1971; Kobayashi et al., 1971; Kovach et al., 1977;
Mayevsky andChance, 1974, 1982; Mayevsky et al., 1987;
MATERIALS AND METHODS
Millsetal., 1977; Osbakken, 1994; Renaultetal., 1984),but
fluorescencemeasurements have alsobeenused (Heineman All experiments described in this paper were performed according to
and Balaban, 1990; Koretsky et al., 1987; Kramer and experimentalprotocols,whichwereapprovedbytheInternalAnimalCare
Pearlstein, 1979; Vein et al., 1975). When the oximetric andUseComittee oftheErasmus University Rotterdam.
effectwas regardedtobe ofminorimportance, theintensity
of the reflected excitation light (365 nm) was subtracted Optics
from the measured fluorescence signal to obtain corrected
NADHfluorescenceintensities (Doraetal., 1984; Harbiget Similar to the fluorimeter described by Vemn et al. (1975), a biprism (30
wedge, fused silica; Melles Griot B.V., Zevenaar, The Netherlands) was
al., 1976; Ji et al., 1977; Jobsis et al., 1971; Mayevsky and
used for simultaneous projection ofNADH fluorescence and UV reflec-
Chance, 1974, 1982; Mayevsky et al., 1987; Kovach et al., tance images ontheCCD chipofavideocamera(Fig. 1).Wehaveused
1977; Osbakken, 1994). In case the oximetric effect of asecond-generationCCDvideocamera(MXRi;AdimecAdvancedImage
hemoglobin was considered to significantly disturb the flu- SystemsB.V., Eindhoven, TheNetherlands), equippedwithanultraviolet
blue-sensitive S20 photocathode (Philips, Eindhoven, The Netherlands)
orescence measurement, reflectance measurements were
andaMicro-Nikkor 105-mmmacrolens.Thevideofluorimeterwithwhich
carried out at isosbestic wavelengths of hemoglobin (e.g.,
theNADHfluorescence imageinFig. 4wasrecordedhasbeendescribed
586 nm (Renault et al., 1984), 720 nm (Kobayashi et al., indetail (Coremans etal., 1993; Inceetal., 1993).
1971). OthershaverecordedtheblueNADHfluorescenceat UVexcitationlightof365nmwasprovidedbya200-Wxenonmercury
isosbestic wavelengths ofhemoglobin (e.g.,445 nm, Harbig lamp (Universal arc lamp source 66006, F/1.5 singleelementfused silica
condenser; Oriel, Stratford, CT) and selected by means ofa UG-1 color
et al., 1976; 448 nm, Kramer and Pearlstein, 1979).
glassfilter(Oriel).Theintensityoftheexcitationlight('1 mW/cm2)was
TosuppresstheeffectofmotiononNADHfluorimetryof
notabletoexcitealloftheNADHmoleculesinthesurfacelayersofintact
heart, fluorescence emission ratios (Brandes et al., 1992) myocardium. A linear relationship was found between the intensities of
and fluorescence excitation ratios (Scott et al., 1994) have NADH fluorescence excitation and emission light, when the intensity of
been suggested. These methods, however, were not devel-
oped to account for hemodynamic and oximetric effects on
the absorption ofNADH fluorescence excitation and emis-
sion light by the tissue.
Por rat heart, the relative difference in NADH fluores-
cence intensity between hypoxic and normoxic tissue in
vivo is smaller than the corresponding difference in fluo-
rescence intensity found in vitro (see, e.g., below, Fig. 7).
Nevertheless, enhanced NADH fluorescence intensity dur-
ing hypoxia remains clear (see below, Fig. 6, and Ince and
Bruining, 1991), despite compensatory vasodilation and
oximetric effects of blood and tissue in blood-perfused
heart. Earlierinvestigations, in whichNADHvideofluorim-
etry was used in combination with diffusereflectance spec-
troscopy (400-700 nm), have shown that the reduction of HEART
470 NM
the NADH fluorescence intensity difference between hy-
poxic and normoxic tissue in vivo is not due to increased FIGURE 1 Schematic representation ofthe experimental set-up for si-
absorptionofthefluorescence light(Coremans etal., 1993). multaneous measurement of NADH fluorescence (±470 nm) and UV
reflectance (365 nm) images. Images arerecorded with a second-genera-
This impliesthatthedecrease inNADHfluorescence inten-
tionimage-intensifiedCCDvideocameraequippedwitha 105-mmmacro
sity difference in vivo is caused by reduced NADH fluo- lens. Excitation light at 365 nm, which is provided by a 200-W xenon
rescence excitation efficiency under hypoxic conditions. mercury lamp, is selectedbymeansofanUG-1 colorglassfilter. NADH
We have investigated the use ofdiffuse reflectance mea- fluorescence is selectedfromtheemittedtissuefluorescencebya470-nm
surements at the excitation wavelength (365 nm) for the narrow-band interference filter. A biprism (30 wedge) is used to enable
simultaneous measurementofNADHfluorescenceanddiffuseUVreflec-
correction ofmeasuredNADHfluorescence intensities. Itis
tanceimagesofratheart. Topreventcontributions ofspecularlyreflected
shown, both on theoretical grounds and by experiments on UV light to the diffuse reflectance image, a crossed polarizer/analyzer
tissuephantoms and freshly excisedrathearts, thatthe ratio combination is used.
Coremans etal. NADH Fluorescence Imaging 1851
UV excitation light was varied (0-100%) with a set of neutral density A'10
1.0
filters.
One half of the biprism was covered with an interference filter for
transmission of NADH fluorescence (470 nm, BWHM 10 nm; Melles 8 0.8
T T T
fGroirott)r.anTshmeisostihoenrohaflfreoffletchteedbiUprVismexwciatsatcioovnerleigdhtw.itRhefalneoctthaenrceUGm-ea1sfuirltee-r - 6 H 0j[pM 0 0 0.6
ments ofa spherical and beating heart are complicated by the presence of
signal contributions due to specularly reflected UV light. We have elimi- U.
natedthis unwantedcontribution ofspecularly reflectedexcitation lightto - 4 *0 0.4
the UV reflectance image by means ofa crossed polarizer/analyzer com- 0
bination (PolaroidTypeHNP'B Linearpolarizer; PolarizerDivision, Hert- 2 0.2
fordshire,England). Specularlyreflected,linearlypolarizedexcitationlight
preserves its polarization and is blocked by the analyzer. Diffusely re- 0 0.0
flected excitation lightbecomes depolarizedbecause ofmultiple scattering
0 100 200 300 400 500
ofphotonsinsidethetissue.Partofthediffuselyreflecteddepolarizedlight
willbetransmitted bytheanalyzerin front ofthe camera lens andis used
[NADH] [pM]
to form the reflectance image.
In someexperimentsreflectance measurements werecarriedoutatboth B
365 nm and 586 nm. In those cases the biprism was removed and NADH 10 1.0
fluorescence, 365 nm reflectance, and 586 nm reflectance were measured
consecutively. Appropriatefiltercombinations wereusedinexcitation and 8 0.8
emission paths. Light at586nmwas selected by means ofan interference 0
filter (586 nm, BWHM 20 nm; Chroma Technology Corp., Brattleboro,
6 0.6
VT).
00
Images were video recorded (Panasonic S-VHS professional video LI-
recordermodelAG-7330;MatsushitaElectricIndustrialCo.,Osaka,Japan) 4 0.4
and computer analyzed off-line. With image-processing software (TCL -0, 0
Image,Multihouse, Amsterdam, TheNetherlands; Olivetti M380-XP5-386 2 0.2
personal computer; digitizer, Digital Production Mixer type WJ-MX10, 0
Panasonic; framegrabber, DataTranslation typeDT2801, Marlboro, MA),
identical tissue areas in the simultaneously recorded NADH fluorescence 0 512n_n
anddiffuse UV reflectance images could be selected. The averaged inten- 50 75 100 125 150
sities ofthese areas are displayed in Figs. 2 and 5-7.
A small circular piece of uranyl fluorescence calibration glass was [Hbj [pM]
placed next to the heart in the lower left corner of the excitation field
(Ploem, 1970).NADH fluorescence andUVreflectance intensities ofheart FIGURE 2 FNADH intensity (0), R365 intensity (L]), and FNADH/R365
were related to the fluorescence intensity ofthis calibration glass and are ratio (O) as (A) a function of NADH concentration in a tissue phantom
expressed in arbitrary units (A.U.). The fluorescence calibration glass composed of50,uMhemoglobin andIntralipid-3%. Thepositive intercept
served as a marker for the image-processing software to select identical of the fluorescence axis is caused by Intralipid, which also fluoresces
tissue areas inthe reflectance andfluorescence image ofone video frame. around470nmuponexcitationwithUVlight. (B)Afunctionofincreasing
Itwas alsousedtomonitorfluctuations intheintensityofthelightsource. hemoglobin concentration with 400,uM NADH added to the tissue phan-
tom. A.U., Arbitrary units.
Preparation of hearts
Perfusion system
Perfusion experiments were performed on hearts of male Wistar rats
weighing 275-375 g. Rats were anesthetized by ether inhalation. After Theperfusionsystemofferedthepossibility toswitchrapidlybetweenfour
heparinization of the animal (200 IU/kg body weight iv, Heparin; Leo available perfusates: both oxygenated and deoxygenated Tyrode solution
Pharmaceutical Products B.V., Weesp, The Netherlands), the thoracic and oxygenated and deoxygenated suspensions ofwashed raterythrocytes
in Tyrode solution were used.
cavitywas openedby stemotomy. Hearts were excised, chilled in ice-cold
Tyrode, andimmediatelytransferredtoaLangendorffset-up. InaLangen- The Tyrode solution consisted of 128 mM NaCl, 4.7 mM KCI, 1 mM
MgCl2, 0.4mMNaH2PO4* H20, 1.2mMNa2SO4, 20.2mMNaHCO3, 1.3
dorffpreparation the perfusate leaves thecoronary vascular system at the
coronary sinus and the opened right atrium and drips offthe apex ofthe mM CaCl2, and 11.0 mM glucose, supplemented with 1% bovine serum
albumin (BSA). This solution wasfiltered through a 1.2-,um filter(Schlei-
heart. In blood perfusion experiments a film of perfusate on the heart
cher and Schuell, Dassel, Germany).
surface would interfere with our measurements. In that case, all efferent
Rat erythrocytes were obtained from fresh blood from five to seven
vessels were closed, except for one pulmonary artery, which was cannu-
anesthetized male Wistar rats, weighing 300-400 g. Anesthesia was in-
lated with fine-bore polythene tubing fordrainage (800/100/240/100: Por-
ducedby ether inhalation. After heparinization ofthe donor animals (280
tex, Hythe, England). Hearts were allowed to beat freely. No special IU/kgbody weight, i.v.), bloodwas collected in syringes containing 50IU
precautions were taken to control the oxygenation of the ambient gas heparin via punction of the abdominal aorta. Blood was centrifuged at
phase.
1500 X g for 10 min to remove plasma and buffy coats. The remaining
Forinvivomeasurementsratswereanesthetizedbyetherinhalationand cells were washed three times with Tyrode equilibrated with 95% 02/5%
heparinized. After tracheotomy the rats were mechanically ventilated CO2 and supplemented with 0.1% BSA. Finally, the erythrocytes were
(Loosco, Amsterdam MK2 infant ventilator, The Netherlands) with 65% suspended up to a hematocrit of9-10% in Tyrode containing 1% BSA.
02, 32.5% N20, and 2.5% of the anesthetic ethrane. To create hypoxic Before transfer ofthe erythrocyte suspension to the perfusion system,
hypoxia, N2 was substituted for 02 in the respiratory gas. The rats were theentire flow circuit hadbeencoated withBSAtoprevent damagetothe
kept on aheated mat (37°C) topreventhypothermia. erythrocytes by shear forces. To each reservoir containing erythrocyte
1852 Biophysical Journal Volume 72 April 1997
suspension, 170 IU heparin was added. Occasional cell aggregates were The addition ofhemoglobin was used to mimick changing tissue absor-
removed from the suspension by an extracorporealbloodfilter(peadiatric bance. VaryingconcentrationsofNADH and/orbovinehemoglobin were
size LPE 1440; Pall Biochemical Limited, Portsmouth, England). The added to stirred 3-ml quartz cuvettes (1-cm path length), filled with
erythrocyte suspension was checked regularly for hemolysis and changes Intralipid 3% or Intralipid 6% in saline. NADH and hemoglobin were
in composition with a Sysmex microcell counter F-800 (TOA Medical prepared as concentrated stock solutions in saline. With this method the
Electronics Co., LTO, Kobe, Japan). The oxygenated erythrocyte suspen- final dilution of the Intralipid suspension by NADH and/or hemoglobin
sion was equilibrated with 95% air/5% CO2,and oxygenated Tyrode was addition never exceeded 3.5%.
equilibrated with 95% 02/5% CO2. In the following, the erythrocyte
suspension will be referred to as blood. Deoxygenated perfusates were
equilibrated with 95% N2/5% CO2.pH, P02,and pCO2 ofthe perfusates Optical parameters of tissue phantoms and
were monitored by an AVL 945 automatic blood gas system (AVL LIST rat heart
GmbH, Medizintechnik, Graz,Austria).Whennecessary,pHwasadjusted
by bicarbonateaddition. Perfusates werethermostattedat37°C,andaortic To verify the suitability of hemoglobin/Intralipid suspensions as tissue
pressure was setat 60mm Hg. Meancoronaryflow wasmeasuredjust in phantoms for rat heart, the absorption coefficient ,Ua and the reduced
front of the aortic cannula with an in-line flow probe, operating on the scatteringcoefficientA.' = As(1 - g)weremeasuredatroomtemperature
ultrasonic transit-time principle (T106; Transonic Systems, New York). in the UV-spectral region (331, 351, 364 nm) with a double-integrating
sphere system as described by Pickeringet al. (1993).
Reflectanceanddiffusetransmittanceofthesample,whichwasplaced
Experimental protocols between the two integrating spheres, were measured by photodiodes
(EG&G Princeton Applied Research UV1OOBQ). A photomultiplier tube
Although hemodynamic and oximetric changes are linked physiological (Hamamatsu, R928) was used to collect the collimated transmittance. A
responses, experimental protocols were designed to separate the contribu- 1-mmcollimatedbeamoftheUVmultiline(331,351,364nm)ofanargon
tionsofhemodynamic andoximetricchangesandtodiscriminatebetween ionlaser(SpectraPhysics model20)wasusedtoirradiatethesample. To
blood and tissue oxygenation changes. Four distinct protocols wereused: avoidcontributionsofUV-inducedfluorescence,UG-1 filterswereplaced
I, II, andIII(seebelow) wereperformedonisolatedratheart;protocol IV in frontofeach detector. Theuse ofpolychromatic lightprevents separa-
was performed on rat heart in vivo. tionofthereducedscatteringcoefficientintothe scatteringcoefficient/is
After arrangement forLangendorffperfusion with oxygenatedTyrode, andthe anisotropy ofscattering g. An inverse addingdoubling algorithm
hearts were allowed to stabilize for30 min. In the bloodperfusion proto- was usedto solve the radiative transferequation (Prahletal., 1993).
cols II and III, hearts were immediately perfused with oxygenated eryth- Freshlyexcisedrathearts(n = 9)wereflushedwithsalineandcutinto
rocyte suspension afterattachmenttothe aortic cannula. Allotherprepar- slices. For each heart several slabs were positioned in a glass sample
ative steps wereperformedontheblood-perfusedorgan. Aftercompletion holder, ensuring that the sample port was completely covered (average
of the cannulation procedure, hearts were allowed to adjust to perfusion sample thickness 427 + 45 ,Am, n = 9). Refractive index mismatch
conditions for at least 30 min. between sample and glass was minimized by a drop ofsaline. With this
Protocol I was used to assess the metabolic state-related change in typeofpreparationitwasnotpossibletopoisetheoxygenation/redoxstate
absorption of heart tissue (oximetric effect of tissue) at the excitation ofthe examined hearttissue.
wavelength(365 nm)andatanisosbesticwavelengthforhemoglobin(586 Aflow cuvette was used (samplethickness 500 ,um) formeasurement
nm). InTyrode-perfusedoxygenatedhearts, hypoxichypoxiawasinduced oftheopticalpropertiesofbothoxygenatedanddeoxygenatedblood.The
by perfusion with deoxygenated Tyrode for 2.5 min. Upon return to flow circuit, which consisted ofthe cuvette, a silicon membrane oxygen-
perfusion with oxygenated Tyrode, the normoxic state was recovered. ator(Alexanderetal., 1984;Hamiltonetal., 1974),andabubbletrap,was
Perfusion with oxygenatedTyrode was maintained for 10min. Thiscycle coated with BSA through circulation ofa Tyrode solution supplemented
of hypoxia and normoxia was performed for the NADH fluorescence with 1% BSA. Justbeforethe additionoffreshlycollectedratblood, 170
measurement and the reflectance measurements at 365 nm and 586 nm IUheparin wasaddedtotheflowcircuit.Toallowtransmissionmeasure-
each. ments,blood wasdilutedto ahematocritofeither5.4% or2.5%. Oxygen-
Protocol II was used to investigate the effectofabsorptionand scatter- ated blood was equilibrated with 95% 02/5% C02; deoxygenated blood
ing properties oferythrocyteson NADHfluorescence andUVreflectance was equilibratedwith95% N2/5% CO2.
intensities under normoxic and ischemic conditions. To this end oxygen- Optical properties ofIntralipid (3% or 6%) with 50, 100, or 200 ,uM
ated blood was replaced with oxygenatedTyrode for 30 s. Threeconsec- hemoglobin were determined for both 300-,m and 400-,m sample
utiveTyrodeflushes,spaced 1 minapart,wereperformed.AftertheTyrode thicknesses.
flushes and a stabilization interval of 6 min, the supply of oxygenated NADH (grade II) was obtained from Boehringer (Mannheim, Ger-
bloodwas interruptedfor 1 min.Reoxygenation withbloodwas sustained many), Intralipid 10% from Kabi Pharmacia AB (Sweden). Hemoglobin
for 5 min before a second ischemia/recovery cycle was performed. Sub- andBSA(fractionV) wereobtainedfromSigmaChemicalCompany (St.
sequently, the perfusate was temporarily switched to oxygenated Tyrode Louis, MO), and the constituents of Tyrode were obtained from Merck
for another ischemialreperfusion cycle. (Darmstadt, Germany).
Protocol III served to estimate the relative contribution of oximetric
changes and hemodynamic changes accompanying compensatory vasodi-
lationtothefluorescenceandreflectancemeasurements.Inblood-perfused RESULTS
hearts hypoxic hypoxia was induced by replacing oxygenated perfusate
with deoxygenated perfusate for 10 min. 02 supply was restored by Theory
perfusion with oxygenated bloodfor 10 min.
ProtocolIVwasusedtoestablishNADHfluorescenceand365-nmand For description ofthe radiation transfer involved in NADH
586-nm reflectance changes during hemodynamic and oximetric changes fluorimetry of rat heart, the theories of front-face fluorim-
in vivo. To this end, three consecutive cycles of hypoxic hypoxia were etry (Eisinger and Flores, 1979) and Kubelka and Munk
createdby replacing 02 with N2 in the respiratory mixture for 2 min.
(Kubelka, 1948, 1954; Kubelka and Munk, 1931) were
combined.
Tissue phantom experiments Front-face fluorimetry refers to the geometry that is used
primarily in studies ofsolid samples in whichtheexcitation
A mixture of Intralipid and soluble absorbers was used to simulate the
opticalpropertiesofhearttissue(Flocketal., 1992;Staverenetal., 1991). lightandemittedfluorescencelightenterandleavethesame
Coremans et al. NADH Fluorescence Imaging 1853
sample face. Regarding the NADH fluorescence measure- UV source instability ortissue movements do not affect the
ment of myocardial tissue, this requires that the UV exci- determination ofrelative NADH concentration changes.
tationlight(intensity, Iex)isabsorbedwithinasampledepth
less than the thickness of the myocardial wall (d). The
EXPERIMENTAL
optical penetration depth for UV excitation light (6) is
defined as the distance over which the total fluence rate Tissue phantom
(W mm-') drops to e-1 (Svaasand and Ellingsen, 1983).
- To verify the insensitivity of the FNADH/R365 ratio to total
The condition that must be fulfilled is therefore
sample absorbance (hemodynamic changes), experiments
d/>»> 1. (la) were carried out with tissue phantoms consisting of mix-
tures of hemoglobin in Intralipid. To determine phantom
In terms ofoptical properties:
compositions withopticalproperties intherangeofthoseof
freshly excised rat heart, absorption and back-scattering
d/6 = d*{3pa(pa + (1-g))}2>> 1, (lb)
properties of the phantoms were varied. The optical prop-
where 6 is given by {3p5(p5a + (1- g) tLS)I-2, ga is the erties in the UV spectral region were determined by means
absorption coefficient (mm-1), .s is the scattering coeffi- of a double-integrating sphere system (Table 1).
cient (mm-1), and g is the anisotropy factor (Ishimaru, A good correlation was found between Pta and the hemo-
1978). globin concentration in the tissue phantoms (Table 1). ta
Under this condition, NADH fluorescence intensity of didnotdependonsamplethickness (resultsnotshown). The
myocardial tissue (FNADH) is proportional to the ratio fact that a UV multiline (331, 351, 364 nm) was used may
ANADH/AT, where AT is total absorbance and ANADH is explain why jt' did not exactly multiply with doubling
absorbance by NADH (Eisinger and Flores, 1979): Intralipid concentration, and why jtk was found to decrease
slightly (6 ± 3%) with increasing sample thickness (300-
FNADH = G#(ANADH/AT)eX' (2) 400 ,gm).
The uta of 100 ,uM hemoglobin in Intralipid (3% or 6%)
where Gis ageometrical factorcharacteristic forthe instru-
ment, and 4) is the fluorescence quantum yield of NADH. corresponds tothe ptaofratheart slabs. Hearttissue is more
forward scattering than Intralipid suspensions (,u' = 1.6
From the theory of Kubelka and Munk (Kubelka, 1948,
1954), arelationship can be derived between the total sam- mm1 versus I-L > 6 mm-1). For our purposes, however,
differences in ji' are not important as long as criterion lb
ple absorbanceAT, scattering S, and diffuse UV reflectance
(see above) is fulfilled (for heart tissue: dla ' 10.4).
R365 (Kessler and Frank, 1992):
Fig. 2A demonstrates the linearity between NADH con-
AT/S = 0.5 R365/Iex + Iex/R365 - 1- (3) centration and FNADH/R365 ratio over the physiological
NADHconcentrationrangeintissuephantomscomposedof
Atsufficiently highabsorbance, theright-hand sideofEq. 3
Intralipid 3% and 50 ,uM hemoglobin. The positive inter-
will be dominated by the second term, and the ratio of
cept ofthe fluorescence axis is caused by Intralipid, which
absorption to scattering becomes inversely proportional to
the reflectance intensity:
TABLE I Absorption coefficient Izua and reduced scattering
AT/S o2 Iex/R365 (4)
coefficient ,u4 = ttj(1 - g) inthe UVspectral region (331, 351,
364 nm) determined with a double-integrating sphere system
Underthe assumption thatthe basic scattering properties of
foran intervening sample ofthickness t(see Materials and
the tissue are constant (Heinrich et al., 1987; Hoffmann et Methods)
al., 1983, 1984;KesslerandFrank, 1992), Eq. 4implies that
the diffuse reflectance intensity can be used to monitor Hemoglobin /-a (1- g) A.,
Preparation t(,um) (p,M) (mm-') (mm-')
changes in tissue absorbance.
Combination ofexpressions 2 and 4 leads to Heart(n = 9) 427 ±45 - 2.3±0.5 1.6±0.2
Bloodhematocrit 2.5%*
HbO2# 500 - 2.1 ±0.1 0.27 ± 0.01
FNADH/R365 'CANADH, (5)
Hb§ 500 3.2 ± 0.1 0.18 ± 0.01
with S, G, and 4 incorporated into the constant C . Bloodhematocrit 5.4%*
HbO2# 500 4.6 ± 0.01 0.31 ± 0.08
ANADH is proportional to the NADH concentration. Ex- Hb§ 500 6.4 ± 0.1 0.26 ± 0.01
pression 5 therefore gives a linear relationship between Intralipid 3% 400 50 1.10 ±0.01 6.93 ± 0.08
NADH concentration and fluorescence intensity. This 100 2.23 ± 0.02 6.86 ± 0.04
makes the FNADH/R365 ratio a highly suitable measure for 200 4.49 ±0.06 6.82 ± 0.07
semiquantitative fluorimetry. Intralipid 6% 400 50 1.15 ± 0.02 12.77 ±0.12
100 2.23 ±0.03 12.59 ±0.12
Moreover, becauseAT is eliminated inthe expression for
200 4.54±0.03 12.21 ±0.01
FNADH/R365, this ratio is independent ofhemodynamic and
*Ratblood was diluted inTyrode solution supplemented with 1% BSA.
oximetric changes. BecauseFNADH/R365 isalsoindependent #HbO2, oxygenated blood, equilibrated with 95% 02/5% Co2-
ofIex,fluctuations ineffectiveexcitationintensitycausedby §Hb, deoxygenated blood, equilibrated with 95% N2/5% CO2.
1854 Biophysical Journal Volume72 April 1997
also fluoresces around 470 nm upon excitation with UV
light.
FNADH/R365 as a function of total sample absorbance is
shown in Fig. 2B. As expected fromEqs. 3 and4, stability
of the FNADH/R365 ratio improves with increasing sample
absorbance. Only for tissue phantoms containing less than
65 AM hemoglobin, with absorption coefficients signifi-
cantly smaller than the /la of virtually bloodless rat heart,
does the FNADH/R365 ratio deviate more than 5% from the
stable value reached at higher values of tUa. Similar results
were obtained with phantoms containing Intralipid 6% and
with whole blood instead ofpurified hemoglobin.
Rat heart
The validity ofthe FNADH/R365 ratio, as a suitable measure
for semiquantitative fluorimetry, was tested for isolated rat
heartpreparations thatwere subjected tohemodynamic and
oximetric challenges. An example of NADH fluorescence
and UV reflectance images of a blood-perfused rat heart
obtained with the biprism configuration is shown in Fig. 3.
To illustrate the potential ofNADH fluorimetry and the
importance ofimaging, an example is given in which a rat FIGURE 4 NADHfluorescenceimageofablood-perfusedratheartwith
heart showing a heterogeneous fluorescence pattern was a heterogeneous fluorescence pattern recorded with the videofluorimeter
subjected to hemodynamic and oximetric challenges. Such describedby Coremans etal. (1993) andInce etal. (1993).
heterogeneity in NADH fluorescence intensity was occa-
sionaly observed in blood-perfused rat hearts, which other-
wise appeared normal. Fig. 4 shows the NADH fluores- tissue areas. Whereas NADH fluorescence intensities of
cence image of such a heart. In these particular heart weakly fluorescent, normoxic, tissue areas did reflect
preparations localdifferences incellularenergy metabolism changesinperfusionP02,NADHfluorescenceintensitiesof
had resulted in highly fluorescent and weakly fluorescent highly fluorescent tissue areas did not change when the 02
supply was interrupted (see below).
Using the biprism configuration of Fig. 1, changes in
NADH fluorescence and UV reflectance intensities of a
highlyfluorescent andaweaklyfluorescentnormoxic tissue
area have been measured in response to hemodynamic and
oximetric challenges according toprotocol II (seeMaterials
and Methods and Fig. 5).
When oxygenated blood is replaced by an oxygenated
Tyrode solution, amarkedandreproducibleincreaseinboth
fluorescence and UV reflectance intensity is observed due
todecreasedabsorptionoffluorescenceexcitationandemis-
sion light by blood pigments-a hemodynamic effect in
extremis. Comparison ofthe kinetics ofthe UV reflectance
change inweakly fluorescent tissue (Fig. 5 D) withthose in
relativelyhighlyfluorescenttissue(Fig. 5 E)doesnotreveal
any differences, indicating that the two tissue areas were
equally well perfused. During the Tyrode flushes the
FNADH/R365 ratioofhighlyfluorescenttissueremainedcon-
stant (Fig. 5 E). In normoxic tissue the FNADH/R365 ratio
remained unchanged during the first 15 s of the Tyrode
flush. Inthis time intervalmosterythrocytes arewashedout
of the microcirculation. Hereafter, apparently some meta-
bolic change occurred, because the FNADH/R365 ratio
dropped slightly; NADH fluorescence intensity declined,
FIGURE 3 AnexampleofUVreflectance(left)andNADHfluorescence whereas diffuse UV reflectance intensity remained virtually
(right) images ofblood-perfusedratheartobtained withthebiprismcon-
figuration. constant (Fig. 5 D). In any case, it is clear that the FNADH/
Coremans et al. NADH Fluorescence Imaging 1855
A 5
L12 12
4 10 10
-8
3
6 k. V\A y \ F r6
LI L.
.'2
'2- 4 - 2
4
j2 12 2
F/R F/R
0 - 0 04 0
40 50 60 70 80 42 43 44 45 46 47
B 5 12 E 5 12
4 10 -10
--8
A'
-
-6
R-44
1
,--2 1 2
.~F/R
O0 80 0 0 T
40 50 60 70 80 42 43 44 45 46 47
time [min] time [min]
C
F -...............
......, ..
.,. .Il.
40 50 60 70 80
time [min] 42 43 44 45 46 47
time [min]
]Blood-ox Tyrode-ox l Ischemia
]Blood-ox Tyrode-ox
FIGURE 5 Effectofhemodynamic and/oroximetricchangesonFNADH(toptrace),R365 (middletrace), andFNADH/R365 (bottomtrace) intensitiesofa
weakly fluorescent, normoxic, tissue area (A, D) and a highly fluorescent, relatively hypoxic, tissue area (B, E) ofablood-perfused ratheart showing a
heterogeneous NADH fluorescence image. Substitution ofoxygenatedTyrodeforbloodandinterruption ofcoronaryperfusionwereperformedaccording
toprotocolII(seeMaterialsandMethods). (A,B)Fulltrace; (D,E)detailsofTyrodeflushes; (C,F)schematicrepresentation ofrelevantpartsofprotocol
II. A.U., Arbitrary units.
R365 ratio provides adequate compensation for hemody- with oxygenated Tyrode for a longer time interval. As
namic changes. expected, the switch to Tyrode evoked an change in fluo-
AftertheTyrodeflushes, bloodperfusionoftheheartwas rescence and reflectance intensities identical to that of the
repeatedly interrupted, inducing both hemodynamic and earlierflushes. Forthe highly fluorescent tissue area, occlu-
oximetric changes. In weakly fluorescent normoxic tissue, sion of the salt perfusion resulted in a decrease in both
interruption of the blood supply resulted in NADH accu- NADH fluorescence and UV reflectance intensities, which
mulation as a consequence of insufficient 02 supply to the is mainly caused by shrinkage of the heart. Despite the
mitochondria (Fig. 5A). Surface NADHfluorescence inten- marked changes in fluorescence and UV reflectance during
sities ofthe highly fluorescent tissue area did not vary with the protocol, the FNADH/R365 ratio ofthis highly fluorescent
changes in the perfusion P02, induced by occlusion of the tissue area remained practically constant during all inter-
coronary perfusion (Fig. 5 B). The absence of significant ventions (Fig. 5 B). In normoxic tissue, cessation of the
changes in the UV reflectance intensity, in both normoxic oxygenated Tyrode supply resulted in a distinct increase in
and highly fluorescent tissue areas, indicates that oximetric NADHfluorescence intensity andaconcomitantdecrease in
and hemodynamic effects cancelled out at the excitation UV reflectance intensity. During the anoxic periods, caused
wavelength. by interruption of either blood or Tyrode perfusion, the
After the Tyrode flushes and occlusion of the coronary FNADH/R365 ratio appeared to stabilize at the same value
perfusion, the blood perfusion was replaced by perfusion (Fig. 5 A).
1856 Biophysical Joumal Volume 72 April 1997
A:6 is induced (Fig. 6). To estimate the relative effects of
Blood-ox Blood-deox Blood-ox compensatory vasodilation and bloodoxygenation changes
on NADH fluorescence and UV reflectance intensities of
5 isolated rat heart, protocol III was performed. During per-
fusion with deoxygenated blood, accumulation ofreducing
7 4
equivalents at the high-energy side ofthe respiratory chain
a 3 J :is reflected by an increase in NADH fluorescence intensity
(Fig. 6A). Because ofcompensatory vasodilation, the flow
2 i; b S inthecoronarycirculationincreasedby 53% (Fig. 6C). The
1 absorption coefficient of deoxygenated hemoglobin (and
, a .......myoglobin) at the excitation wavelength exceeds that of
0
0 5 10 15 20 25 30 oxygenated hemoglobin (and myoglobin). In this case, both
hemodynamic and oximetric changes contributed to in-
time [min] creased absorption ofexcitation light, causing adrop inUV
B 7 reflectance intensity of approximately 18% (AR365 =
Blood-ox BBllooodd--odeox Blood-ox -17.8 ± 2.1%, n = 5) (Fig. 6 A).
6 Comparison of the kinetics of UV reflectance intensity
5 and coronary flow changes revealed that the oximetric ef-
fect on the UV reflectance change largely exceeded the
4
a hemodynamic effect. Upon substitution of deoxygenated
3 blood for oxygenated blood, UV reflectance intensity
LL dropped immediately to a new baseline, whereas the coro-
2
nary flow increase was relatively slow. Conversely, imme-
,
1 diate recovery of the reflectance intensity was observed
....,. upon reoxygenation, in spite of sustained increased coro-
0 ------ ..
0 5 10 15 20 25 30 nary flow.
Fig. 7 compares the NADH fluorescence and reflectance
15 ; . 1 changes ofrat heart accompanying hypoxic hypoxiaoftwo
Blood-ox Blood-deox Blood-ox extremes: invivoforhematocrit40.8% (protocol IV) andin
c
I0~.i A D
5.0
E 10 4.0
C
3.0 £
2.01 I*.
z 1.0 amF rf.w, z
IL 0.0 j U-
E
5 B 1.4
0 5 10 15 20 25 30
M 1.2
In
time [mln] I(n0 1I..0 (0
CI 0.8 Cf)
CC.
FIGURE 6 Effectofhemodynamicandoximetricchangeson(A)FNADH 0.6
(toptrace),R365(bottomtrace),and(B)FNADH/R365intensitiesofisolated F , 2.0 .
rat heart, when compensatory vasodilation is induced by perfusion with C 2.0 l,
deoxygenatedbloodaccordingtoprotocolIll(seeMaterialsandMethods). * 1.0 to 1.0 lJW" P1v
(C)Corresponding coronary flow. A.U., Arbitrary units.
0.0 c 0.0
0 2 4 6 8 10 0 2 4 6 8 10
t[min] t[min]
This exampleclearly showsthattheFNADH/R365ratiocan
be used to discriminate between tissue areas with different FIGURE 7 EffectofhypoxichypoxiaonFNADH(),R365 (),andR586
oxidative metabolism, in spite of disturbing hemodynamic (V)intensitiesof(A-C)ratheartinvivo(hematocrit40.8%), where02in
and oximetric effects. Changes in measured FNADH due to the respiratory mixture is replaced by N2 according to protocol IV (see
effects other than changes in NADH concentration are ad- Materials and Methods), and (D-F) Tyrode-perfused isolated rat heart,
whereoxygenatedTyrode isreplacedbydeoxygenatedTyrode according
equately compensated for by changes in R365. to protocol I (see Materials and Methods). The NADH fluorescence and
When oxygenated blood is replaced with deoxygenated reflectance traces are aligned such that the start ofeach metabolic chal-
blood, acombinationofhemodynamic andoximetriceffects lenge corresponds to t = 3 min on thetime axis. A.U., Arbitrary units.
Coremans et al. NADH Fluorescence Imaging 1857
vitro for Tyrode-perfused isolated heart (protocol I). The anaerobic suspensions of cardiac myocytes coincide with
screening effect of the blood pigments on the relative flu- the spectral features ofmyoglobin (Wittenberg and Witten-
orescence change in vivo is evident (Fig. 7, A and B). berg, 1985). Therefore, the observed increase in reflectance
Althoughthekineticsdiffered, theUVreflectancechangein ofhearttissueat586nmcausedbyhypoxic hypoxiaismost
blood-perfusedheart(-18%,Fig. 6A; -19%,Fig.7B)was likelyduetodeoxygenationofmyoglobin. Compensationof
comparable to the UV reflectance reduction of23% (Fig. 7 the blue fluorescence with red reflectance (720 nm: another
E) induced by substitution of deoxygenated Tyrode for isosbestic point hemoglobin), as proposed by Kobayashi et
oxygenated Tyrode. Because the UV reflectance signal of al. (1971), introduces another source of error by probing
cardiac tissue is largely determined by myoglobin, and different tissue volumes due to the wavelength dependence
hemoglobin and myoglobin have similar spectroscopic oflight propagation in tissue. Moreover, in blood-perfused
properties in the UV region, the presence oferythrocytes in organs, where metabolic changes can be accompanied by
the microcirculation has little effect on the relative UV appreciable changes in blood volume, the existence ofsuit-
reflectance change during hypoxia. able (stable) isosbestic wavelengths is unlikely. Therefore,
In Tyrode-perfused heart, diffuse reflectance measure- compensation methods that rely on the existence of isos-
ments carried out at 586 nm (isosbestic wavelength hemo- bestic wavelengths, whether in reflectance spectroscopy or
globin) showed increased reflection during hypoxic hyp- in isosbestic fluorimetry, are error prone.
oxia, whichismostlikelyduetoanoximetric effectofheart Use of365-nm diffuse reflectance as the reference signal
tissue (Fig. 7 F). Even in vivo, hypoxic hypoxia is accom- hasbeen subjecttodiscussionbecauseofthefactthatatthis
panied by increased 586-nm reflectance, despite a possible wavelength the absorption characteristics of pigments in
microcirculatory blood volume increase (Fig. 7 C). blood and tissue depend on the metabolic state (Antonini
and Brunori, 1971). However, it is irrelevant which effects
underly a change in tissue absorbance of UV excitation
light, as long as its effect on NADH fluorescence intensity
DISCUSSION
is amply compensated for.
In the literature, selection criteria for the most accurate The results presented in this paper show that the NADH
reflectance wavelength to compensate for hemodynamic fluorescence/UV reflectance ratio provides a good measure
and oximetric effects on the NADH fluorescence signal for (semi-)quantitative analysis oftissue NADH concentra-
have been discussed at length. Nevertheless, discrepancies tion, unaffected by oximetric or hemodynamic effects. In
in results and interpretation remain. This is mainly because heart tissue, the conditions that have to be fulfilled for
methods designed for a specific tissue type, focusing on validity ofthe theoretical approach ofthe FNADH/R365 ratio
compensation of either hemodynamic (Dora et al., 1984; are easily satisfied. Because of the strong absorption of
Harbig et al., 1976; Ji et al., 1977; Jobsis et al., 1971; blood and tissue in the near-UV region, the optical pene-
Mayevsky and Chance, 1974, 1982; Mayevsky et al., 1987) tration depth fordiffuse 365-nmlightis only afew hundred
oroximetric (Ji et al., 1979; Kobayashi et al., 1971) effects, microns (6 200 ,um) (Chance et al., 1965; Ji et al., 1977),
have been applied to experimental systems with properties andallexcitationlightisabsorbedorscatteredin superficial
different from those for which they were developed. In tissue layers ofthe myocardial wall (d 2 mm (Kanaide et
choosing 586 nm as the most suitable reflectance wave- al., 1982)). Because NADH fluorescence has alarge Stokes
length (isosbestic point of hemoglobin), Renault neglected shift (Lakowicz, 1983), NADH self-absorption within the
the oximetric effect of heart tissue, arguing that the spec- sample can be ignored. Incorporation ofthe unknown scat-
troscopic properties of hemoglobin and myoglobin are in- tering term S, the fluorescence quantum yield 4P, and instru-
distinguishable with the optical filters used in the laser ment characteristic G in constant C of Eq. 5 makes the
fluorimeter (Renaultetal., 1984). Wedidobserve,however, FNADH/R365 ratio a (semi-)quantitative measure for NADH
a significant contribution of an oximetric effect to the fluorimetry. This is shown for tissue phantoms in Fig. 2,
586-nm reflectance measurement (Fig. 7 C). Although the where a linear relationship between NADH concentration
absorption spectra of the oxygenated and deoxygenated and the FNADH/R365 ratio was found over the complete
derivatives ofhemoglobin andmyoglobinhave similarline- physiological NADH concentration range from 20-25 ,uM
shapes, the spectra differ significantly in the position of during normoxia to 300-450 ,uM during anoxia (Bessho et
absorption bands and isosbestic points (Antonini and al., 1989; Chance et al., 1965; Katz et al., 1987).
Brunori, 1971). A wavelength of 586 nm is approximately Subtraction of the UV reflectance signal from the fluo-
halfway between the maximum absorption difference ofthe rescence signal (FNADH- kR365) has frequently been used
oxygenated and deoxygenated forms of myoglobin (581/ to correct measured NADH fluorescence intensities of the
582 nm) and its isosbestic point (592 nm). Because of the cerebral cortex (Dora et al., 1984; Harbig et al., 1976; Ji et
steepness of the (MbO2-Mb) difference spectrum in this al., 1977, 1979; Jobsis et al., 1971; Kovach et al., 1977;
spectral region, small changes in monitoring wavelength Mayevsky andChance, 1974, 1982; Mayevskyetal., 1987),
result in large changes in the absorption difference. More- but application to in vivo heart models has also been re-
over, the maximum absorption difference andthe isosbestic ported (Osbakken, 1994). For compensation of hemody-
pointintheabsorptiondifference spectrumofaerobicminus namic effects on NADH fluorescence intensities, this
1858 Biophysical Journal Volume 72 April 1997
(FNADH- k *R365) correction was found to be suitable for In accordance with the tissue phantom data (Fig. 2), the
most brain preparations (Harbig et al., 1976; Jobsis et al., FNADH/R365 ratio in a metabolically stable heartis indepen-
1971; Mayevsky andChance, 1982). However, this method dent ofthe erythrocyte concentration in the perfusate (Fig.
provides inadequate correction for oximetric effects in ce- 5). The hemodynamic effect induced by autoregulation is
rebrocortical fluorimetry (Kovach et al., 1977). Although more difficult to control. Compensatory vasodilation is ac-
KramerandPearlstein (1979) arguedthatalgebraic subtrac- companied by pronounced coronary flow increase. The
tion of the reflectance from the NADH fluorescence is available literature, however, offers little information as to
inappropriate and a ratio technique should be used instead, how this flow control is linked to microcirculatory blood
studies based on fluctuations in the intensity ofUV reflec- volume (Ploeg, 1994). It is therefore unclearto what extent
tance of a small tissue area yielded inconsistent results. heart surface fluorescence measurements are affected by
Sometimes an increase in UV reflectance intensity was absorbance changes due to changes in microcirculatory
observed during hypoxia of brain cortex (Ji et al., 1979), blood volume. Nevertheless, itis clearthatsuch an effectis
whereas in similar experimental protocols the UV reflec- small compared to the oximetric effect of hemoglobin
tance decreased (Doraet al., 1984; Harbig et al., 1976; Ji et (Fig. 6).
al., 1977; Mayevsky and Chance, 1982). The issue is ob- The decrease in the FNADH/R365 ratio that was observed
scured by the fact that these investigators have all used when the perfusate of the isolated heart preparation was
microscopic and fiber optic techniques that suffer from an switched from oxygenated blood to oxygenated Tyrode is
unpredictable contribution ofspecularreflection andcannot probably related to a metabolic and vascular response (Fig.
discriminate between spatial heterogeneity and temporal 5). Although the 02-carrying capacity of an oxygenated
changes. Although the intensity ofspecularly reflected light erythrocyte suspension exceeds that of oxygenated Tyrode
is sensitive to tissue movement, it is not suitable to com- solution, myocardial perfusion with oxygenated Tyrode ap-
pensate movement artefacts in NADH fluorimetry. This is parently increases sufficiently in this non-working heart
due to the fact that specularly reflected light is reflected preparation to compensate for the decrease in arterial oxy-
directly atthe air-tissue interface, whereas fluorescent light gen content (Buckberg and Brazier, 1975; Olders et al.,
is only emitted after absorption- and scattering-induced 1990; Priebe, 1981; Race et al., 1967).
radiation transfer phenomena (Brandes et al., 1992). Be- In blood-perfused heart the absence of significant
cause specularly reflected light does not relate to hemody- changes in the UV reflectance intensity during ischemia
namic and/or oximetric changes, contamination of diffuse (Fig. 5,A andB)impliesthatthe 365-nmreflectancechange
reflectancemeasurements withspecularreflection shouldbe associated with a possible decrease in microcirculatory
avoided. In our optical configuration the problem of spec- bloodvolume andwithoximetriceffectsofbloodandtissue
ular reflection is avoided by using two crossed polarizers. (deoxygenation ofhemoglobin, myoglobin, reduction cyto-
The small (10°) angle between excitation and detection chromes) cancels out. Because the muscle tone ofthe myo-
beams (Fig. 1) ascertains thattotaltransmissionofpolarized cardiumincreaseswithincreasingviscosityoftheperfusate,
excitation light by the analyzer is constant over the filter ischemia-induced tissue shrinkage is more pronounced dur-
surface and never exceeds 0.005%. Application of "the ing interruption ofTyrodeperfusion. As couldbeexpected,
polarization principle" to obtain a true diffuse reflectance compression oftissue that is unresponsive to changes in 02
measurement has been recognized before (Brandes et al., supply decreases both NADH fluorescence and UV reflec-
1992), but has not been put into practice. tanceintensities (Fig. 5 B). Significantqualitativevariations
BecauseNADH fluorescence and UVreflectance images in UV reflectance changes during ischemia that have been
are measured simultaneously and image-processing soft- reported in the literature can probably be attributed to con-
ware allows identification and analysis of identical tissue tributions ofspecular reflection (Dora et al., 1984; Ji et al.,
areas inthefluorescence andreflectance image, theFNADH/ 1977, 1979; Mayevsky and Chance, 1982; Mills et al.,
R365 ratio is disturbed neither by motion nor by possible 1977).
spatial heterogeneity of the heart surface (Ashruf et al., In summary, the FNADH/R365 ratio provides a suitable
1995; Barlow and Chance, 1976; Ince et al., 1993; Steen- measure for (semi-)quantitative NADH fluorimetry under
bergen et al., 1977; Williamson et al., 1982). Moreover, the following conditions:
with a CCD imaging system, NADH fluorimetry is truly 1. The penetration depth of the UV excitation light is
noninvasive, because no direct contact between instrumen- small, so that remitted excitation light and evoked NADH
tation andtissue is needed. Anotherimportant advantage of fluorescence light leave the same organ face.
the FNADH/R365 ratio over other compensation methods is 2. The physiological phenomena accompanying meta-
that the FNADH/R365 ratio does not depend on instrument bolicchanges shouldnotsignificantlyaltertissueabsorption
settings and does not require internal calibration. Further- ofNADH fluorescence light.
more, the uranyl calibration glass, which served to enable The superfilcial nature of the fluorescence technique as
correction ofimages for fluctuations in the intensity ofthe dictated by condition 1 poses a disadvantage of NADH
light sourceintheexperimental set-upusedforFig. 4, is no fluorimetry as monitorofthe mitochondrial redox state. On
longer necessary when the FNADH/R365 ratio technique is the other hand, oxygenation at the tissue surface is often a
used. good indicator of the condition of an organ. We have