Innovating lifetime microscopy: a compact and simple tool for life sciences, screening, and diagnostics

Abstract

Fluorescence lifetime imaging microscopy (FLIM) allows the investigation of the physicochemical environment of fluorochromes and protein-protein interaction mapping by Forster resonance energy transfer (FRET) in living cells. However, simpler and cheaper solutions are required before this powerful analytical technique finds a broader application in the life sciences. Wide-field frequency-domain FLIM represents a solution whose application is currently limited by the need for multichannel-plate image intensifiers. We recently showed the feasibility of using a charge-coupled device/complementory metal-oxide semiconductor (CCD/CMOS) hybrid lock-in imager, originally developed for 3-D vision, as an add-on device for lifetime measurements on existing wide-field microscopes. In the present work, the performance of the setup is validated by comparison with well-established wide-field frequency-domain FLIM measurements. Furthermore, we combine the lock-in imager with solid-state light sources. This results in a simple, inexpensive, and compact FLIM system, operating at a video rate and capable of single-shot acquisition by virtue of the unique parallel retrieval of two phase-dependent images. This novel FLIM setup is used for cellular and FRET imaging, and for high-throughput and fast imaging applications. The all-solid-state design bridges the technological gap that limits the use of FLIM in areas such as drug discovery and medical diagnostics.

Beyond Range: Innovating Fluorescence Microscopy

“…This has changed rapidly in the past few years [7][8][9]11,15,24,25]. The only conceptual difference between ranging applications and fluorescence detection is that instead of detecting the light reflected by objects, a ToF sensor detects light emitted by fluorophores.…”

“…An on-board field-programmable gate array or a computer can process this information to provide distance measurements. ToF technologies can then provide single-shot real-time imaging systems for high speed FLIM, an application that is currently only available in specialist laboratories [8,33,34]. …”

“…Soon after, we flew to Zurich to secure an agreement with Centre Suisse d'Electronique et de Microtechnique to acquire one of their prototypes. Within a year, we generated the first FLIM image using ToF technology, and the first image with an all solid state system (source and detector) within a frequency range relevant to biology [7,8].…”

Advances in cellular, subcellular, and nanoscale imaging in vitro and in vivo

“…Recent developments in the use of allsolid-state CCD-based FLIM detectors have the potential to increase the use of lifetime information in both microscopic and macroscopic applications. Two major approaches are currently being followed: the utilization of the charge transfer properties of the CCD chip can be used to modulate the detection efficiency (52), and we recently introduced a specially designed chip that operates by the lock-in principle (53,54). The lock-in camera incorporates in its structure smart pixels that are composed of a light-sensitive area and two photoelectron collection bins that are modulated at the excitation frequency and at opposite phase.…”

Fluorescence lifetime imaging in biosciences: technologies and applications

“…interactions between proteins in living cells 48,[82][83][84][85][86] , photophysics of complexes involved in plant photosynthesis 87 or the redox metabolism 38,50,[88][89][90] . In the last years, FLIM gained on interest in the biomedicine, as well 27,49,78,[91][92][93][94][95][96] . For instance, a FLIM device for the clinical diagnosis of malign skin tissue is commercially available (DermaInspect, JenLab, Jena, Germany) 27 .…”