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&Geissinger2012).Theyarethemethodsofenergy-selectionspectroscopy,
suchashole-burninginabsorbance(Kharlamovetal.1974,Rebaneetal.1982)
andline-narrowinginfluorescence(Personovetal.1972b),seeref.(Wagie
&Geissinger2012)forrecentreview.Theyweredesignedtocircumventthe
problemoflargeinhomogeneouslinewidthexistingincommoncondensed
mediabyselectinganarrowpackageofmoleculesabsorbinglightaround
acertainfrequencywithintheinhomogeneousbandviaanarrowband-
widthlaserexcitation.Ultrafastdynamichole-burningandhole-fillinghas
beenrealized(Murakamietal.1992).Thoughtechnicallycomplicated,these
methodshavefounddifferentapplicationsinchemistryandbiology
(Friedrich&Haarer1984,Jankowiaketal.1993,Personov1983).Meantime,
featuringahighlyimprovedresolutionofspectratheyarelimitedtocryo-
genictemperaturesorultra-shortobservationtimes.
Byultimatedilutionofdyesolutions,illuminationofultra-smallsample
volumesandapplicationofconfocalortwo-photonmicroscopytheabsolute
limitofsensitivityinfluorescencespectroscopywasachieved–thelimitof
individualmolecules(Ambrose&Moerner1991,Moerner&Kador1989).In
thiswaytheindividualmoleculescanbeselectedfromtheensemble.They
canbeidenticalchemicalspecies,whichdifferininteractionswiththeenvi-
ronment,andaninhomogeneouscontouroftheensembleofmoleculescan
bebuiltupfromtheseindividualcontributions.However,thesenewdevel-
opmentscannotsubstituteinfullthe‘classical’observationsofinhomogene-
ousbroadeningandmolecularrelaxationsbasedontheRed-Edgeeffects,
sinceinrealsystemsphysicalbehaviorandchemicalreactivityistheproper-
tyofwholemolecularensembles.
Thesenewparadigmschangingthephysicsofcondensedmatterhave
foundimportantimplicationsforcharacterizingthesystemswithunknown
molecularorderanddynamics.TheDemchenko’slaboratorywasthefirstto
observetheRed-Edgeeffectstothestudiesofproteinmacromoleculesinso-
lutions.Atthattime,despitemanyeffortswithapplicationofdifferentre-
searchmethodsthemicroscopicheterogeneityanddynamicsinthesesys-
temsremainedunclear.ApplicationofmethodologyoftheRed-Edgeeffects
intheirsteady-stateversionandusingtryptophanasanintrinsicfluorescent
probe(Demchenko1981,1988a)orapplyingorganicfluorophoresadsorbed
inproteinbindingsites(Demchenko1982)havebroughtveryunexpected
results.Theextentoftheirmoleculardisorderonthelevelofinteractionsof
atomgroupswassimilartothatofamorphoussolidsthusallowingobserva-
tionofRed-Edgeeffectsandtheratesoflow-scaledynamicsaveragingthe
proteinsub-stateswasfoundslowerthanthatinsurroundingwaterbysev-
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