TEMPORALLY AND SPECTRALLY RESOLVED IMAGING MICROSCOPY OF LANTHANIDE CHELATES

The combination of temporal and spectral resolution in fluorescence mi croscopy based on long-lived luminescent labels offers a dramatic incr ease in contrast and probe selectivity due to the suppression of scatt ered light and short-lived autofluorescence. We describe various confi gurations of a fluorescence microscope integrating spectral and micros econd temporal resolution with conventional digital imaging based on C CD cameras. The high-power, broad spectral distribution and microsecon d time resolution provided by microsecond xenon flashlamps offers incr eased luminosity with recently developed fluorophores with lifetimes i n the submicrosecond to microsecond range. On the detection side, a ga ted microchannel plate intensifier provides the required time resoluti on and amplification of the signal, Spectral resolution is achieved wi th a dual grating stigmatic spectrograph and has been applied to the a nalysis of luminescent markers of cytochemical specimens in situ and o f very small volume elements in microchambers. The additional introduc tion of polarization optics enables the determination of emission pola rization; this parameter reflects molecular orientation and relational mobility and, consequently, the nature of the microenvironment. The d ual spectral and temporal resolution modes of acquisition complemented by a posteriori image analysis gated on the spatial, spectral, and te mporal dimensions lead to a very flexible and versatile tool. We have used a newly developed lanthanide chelate, Eu-DTPA-cs124, to demonstra te these capabilities. Such compounds are good labels for time-resolve d imaging microscopy and for the estimation of molecular proximity in the microscope by fluorescence (luminescence) resonance energy transfe r and of molecular rotation via fluorescence depolarization. We descri be the spectral distribution, polarization states, and excited-state l ifetimes of the lanthanide chelate crystals imaged in the microscope.