Temporally and Spectrally Resolved Imaging Microscopy of Lanthanide Chelates

Abstract

The combination of temporal and spectral resolution in fluorescence microscopy based on long-lived luminescent labels offers a dramatic increase in contrast and probe selectivity due to the suppression of scattered light and short-lived autofluorescence. We describe various configurations of a fluorescence microscope integrating spectral and microsecond temporal resolution with conventional digital imaging based on CCD cameras. The high-power, broad spectral distribution and microsecond time resolution provided by microsecond xenon flashlamps offers increased luminosity with recently developed fluorophores with lifetimes in the submicrosecond to microsecond range. On the detection side, a gated microchannel plate intensifier provides the required time resolution and amplification of the signal. Spectral resolution is achieved with a dual grating stigmatic spectrograph and has been applied to the analysis of luminescent markers of cytochemical specimens in situ and of very small volume elements in microchambers. The additional introduction of polarization optics enables the determination of emission polarization; this parameter reflects molecular orientation and rotational mobility and, consequently, the nature of the microenvironment. The dual spectral and temporal resolution modes of acquisition complemented by a posteriori image analysis gated on the spatial, spectral, and temporal dimensions lead to a very flexible and versatile tool. We have used a newly developed lanthanide chelate, Eu-DTPA-cs124, to demonstrate these capabilities. Such compounds are good labels for time-resolved imaging microscopy and for the estimation of molecular proximity in the microscope by fluorescence (luminescence) resonance energy transfer and of molecular rotation via fluorescence depolarization. We describe the spectral distribution, polarization states, and excited-state lifetimes of the lanthanide chelate crystals imaged in the microscope.

Spectrally resolved fluorescent lifetime imaging

“…The modulation depth and phase are predictable for a mixture of multiple fluorophores that do not undergo energy transfer or excited-state reactions (Weber 1981 Figure 1. The first known SFLIM system as reported by Vereb et al (1998). The SFLIM system was implemented with a grating-based imaging spectrograph, image intensifier and Xe flash lamp.…”

“…The first spectrally resolved FLIM (SFLIM) system (figure 1; Vereb et al 1998) was a time-domain instrument designed to measure lifetimes on the millisecond time scale and applied to the analysis of lanthanide chelate crystals. It operated as a detection system for a single line within the sample corresponding to the location, where the sample image coincided with the entrance slit of the spectrograph.…”

High intensity solid‐state UV source for time‐gated luminescence microscopy

“…Flashlamps require the period following the primary excitation pulse to be extended far longer than that required for autofluorescence decay. This is necessary to allow the glowing flashlamp plasma to fade to an acceptably low level that typically takes more than 50 ls (15,20). As a consequence of this relatively long gate-delay, only fluorophores with lifetimes of hundreds of microseconds are suitable as probes for flashlamp-excited time-resolved luminescence.…”

“…Early luminescence time-gated instruments employed chopper wheels as inexpensive pulsed excitation sources; however, the pulse profile was characterized by a slow rising and falling edge that imposed limits on resolution and sensitivity (15)(16)(17). Other limitations of chopper wheels include the inflexible nature of the pulse regime, the inefficient use of light energy, and the risk of image-blur arising from drive motor vibration.…”

Development of Responsive Lanthanide-Based Magnetic Resonance Imaging and Luminescent Probes for Biological Applications

“…12). [66][67][68][69][70][71][72][73][74][75] Thus, this TRLLM imaging has very different characteristics from conventional timeresolved fluorescence imaging. A schematic of the TRLLM system, which was newly developed by us, is shown in Fig.…”