Live cell imaging has been greatly advanced by the recent development of new fluorescence microscopy-based methods such as multiphoton laser-scanning microscopy, which can noninvasively image deep into live specimens and generate images of extrinsic and intrinsic signals. Of recent interest has been the development of techniques that can harness properties of a fluorescence, other than intensity, such as the emission spectrum and ezcited state lifetime of a fluorophore. Spectra can be used to discriminate between fluorophores, and lifetime can be used to report on the microenvironment of fluorophores. We describe a novel technique—combined spectral and lifetime imaging—which combines the benefits of multiphoton microscopy, spectral discrimination, and lifetime analysis and allows for the simultaneous collection of all three dimensions of data along with spatial and temporal information.

Introduction

All living organisms are cells or ensembles of cells. This insight (1,2), arguably the most significant in all of biology, was made possible by the development of the light microscope (3). One hundred and sixty-seven years later, the microscope is still at the forefront of biomedical research into the structure and dynamics of subcellular components in living tissue. The remarkable developments in molecular biology and genomics during the past few years have provided DNA sequences of many organisms, from which the complete complement of proteins making up these organisms can be deduced. However, knowledge of a comprehensive “parts list” of an organism does little in and of itself to explain the workings of the dynamic machinery that underlies the ability of a cell to divide or differentiate. It is light microscopy that is providing the key insights into subcellular dynamics, a task being greatly facilitated by technical developments in optical probes and instrumentation. Chimeric fluorescent protein reporters (4,5,6,7,8,9,10) allow virtually any protein to be labeled and thereby visualized in a cell, tissue, or organism. Fluorescent molecules have been engineered to reveal key intracellular physiological parameters, such as free calcium levels (11,12,13). Along with these developments in fluorescence probe technology, new optical techniques, such as fluorescence resonance energy transfer (FRET) (14,15,16,17), multiphoton laser-scanning microscopy (MPLSM) (18,19,20,21), second harmonic generation (SHG) imaging (22,23,24,25), and fluorescence lifetime imaging (FLIM) (26,27,28,29,30), are revealing how individual cellular components are assembled into cytoplasmic machinery and how this machinery functions. In this review, we will describe a relatively new technique, spectral lifetime imaging microscopy (SLIM), that simultaneously measures fluorescence lifetime and spectra for intrinsic and extrinsic fluorophores. This technique can utilize the benefits of MPSLM and has great promise in revealing more information about intrinsic and extrinsic fluorophores and their interactions with each other and with their microenvironment.

MPLSM has proven to be a powerful tool for imaging cancer and cell biology phenomena (19,20)(31,32,33,34) by allowing scientists to image noninvasively deep into biological tissue and collect four-dimensional data [three-dimensional (3-D) spatial dimensions plus time] of fluorescently labeled proteins. Recently, there has been a growing awareness that there are properties of fluorescence other than intensity, such as lifetime (how long a photon stays in the excited state) and spectra. These properties can be used to separate out fluorophores of interest (via spectra) and to reveal critical information about the microenvironment (via lifetime), as well as about the abundance and bound state of key endogenous fluorophores, such as nicotinamide adenine dinucleotide (NADH) (29,35) and flavin adenine dinucleotide (FAD). Until recently, all these dimensions of data have been collected separately, but now our group and others have developed instrumentation for simultaneous collection of intensity, space, time, spectra, and lifetime (36,37,38). This combined information can allow for the determination of the “fingerprint” of an intrinsic or extrinsic fluorophore in space and time, which allows investigators to track fluorophores accurately and determine their role in key processes.

Fluorescence Microscopy

Fluorescence microscopy has become the foremost tool in the study of the structure and dynamics of cellular machinery in living cells and tissue because of the high signalto-background ratio that may be obtained by observing fluorescent objects against a black background, together with the ability to spectrally discriminate between multiple fluorophores, These fluorophores can be endogenous metabolites (e.g., reduced NADH), genetically engineered proteins [e.g., green fluorescent protein (GFP)::tubulin], or exogenous probes [e.g., fluorescence microscopy (FM) series lipid probes or the Ca²⁺ indicator Fura]. Each fluorophore has a characteristic emission spectrum that may be used for identification purposes. In addition, fluorophores have characteristic excited state lifetimes, which can be used also to aid identification (28). Excited state lifetimes—and, to a lesser extent, fluorescence spectra—can be modified by the microenvironment of the fluorophore and therefore can be used to report on it. By utilizing photon-counting detectors, the fluorescence lifetime of individual fluorophores, such as the bound and free forms of NADH, can be tracked and recorded for metabolic mapping in breast cancer (29).