Multiphoton fluorescence lifetime imaging microscopy (FLIM) using a streak camera

Krishnan, Ramanujan Venkata; Saitoh, H.; Terada, Hirotoshi; Centonze, V.; Herman, Brian

doi:10.1117/12.478001

Cited by 7 publications

(6 citation statements)

References 18 publications

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“…Also streak cameras have been used to record FLIM in combination with laser scanning microscopes. The capability of the streak camera to resolve an optical signal both in x and t has been used to record multispectral FLIM data (Biskup et al ., 2007) or to record a full line of the scan before the data are read out (Krishnan et al ., 2003). The performance of such systems strongly depend on the parameters of the streak camera, especially the excitation pulse rate the camera can handle, and the speed the data can be read out.…”

Section: Other Flim Techniquesmentioning

confidence: 99%

Fluorescence lifetime imaging – techniques and applications

Becker¹

2012

Journal of Microscopy

732

553

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SummaryFluorescence lifetime imaging (FLIM) uses the fact that the fluorescence lifetime of a fluorophore depends on its molecular environment but not on its concentration. Molecular effects in a sample can therefore be investigated independently of the variable, and usually unknown concentration of the fluorophore. There is a variety of technical solutions of lifetime imaging in microscopy. The technical part of this paper focuses on time-domain FLIM by multidimensional time-correlated single photon counting, time-domain FLIM by gated image intensifiers, frequency-domain FLIM by gainmodulated image intensifiers, and frequency-domain FLIM by gain-modulated photomultipliers. The application part describes the most frequent FLIM applications: Measurement of molecular environment parameters, protein-interaction measurements by Förster resonance energy transfer (FRET), and measurements of the metabolic state of cells and tissue via their autofluorescence. Measurements of local environment parameters are based on lifetime changes induced by fluorescence quenching or conformation changes of the fluorophores. The advantage over intensity-based measurements is that no special ratiometric fluorophores are needed. Therefore, a much wider selection of fluorescence markers can be used, and a wider range of cell parameters is accessible. FLIM-FRET measures the change in the decay function of the FRET donor on interaction with an acceptor. FLIM-based FRET measurement does not have to cope with problems like donor bleedthrough or directly excited acceptor fluorescence. This relaxes the requirements to the absorption and emission spectra of the donors and acceptors used. Moreover, FLIM-FRET measurements are able to distinguish interacting and noninteracting fractions of the donor, and thus obtain independent information about distances and interacting and noninteracting protein fractions. This is information not accessible by steady-state FRET techniques. Autofluorescence FLIM exploits changes in the decay parameters of endogenous fluorophores with the Correspondence to:

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Section: Other Flim Techniquesmentioning

confidence: 99%

Fluorescence lifetime imaging – techniques and applications

Becker¹

2012

Journal of Microscopy

732

553

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“…A streak camera operates by transforming the temporal profile of a light pulse into a spatial profile on a detector, by causing a time‐varying deflection of the light across the width of the detector (Krishnan et al ., 2003a, b). The resulting image forms a ‘streak’ of light, from which the duration of the light pulse can be inferred.…”

Section: Methodsmentioning

confidence: 99%

Spatially resolved fluorescence lifetime mapping of enzyme kinetics in living cells

Ramanujan¹,

Jo²,

Cantù³

et al. 2008

Journal of Microscopy

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SummaryTraditional cuvette-based enzyme studies lack spatial information and do not allow real-time monitoring of the effects of modulating enzyme functions in vivo. In order to probe the realistic timescales of steric modifications in enzymesubstrate complexes and functional binding-unbinding kinetics in living cells without losing spatial information, it is imperative to develop sensitive imaging strategies that can report enzyme kinetics in real time over a wide dynamic range of timescales. Here we present a multi-photon excitationbased, ultra-fast photon detection using a streak camera and Laguerre expansion-based fast deconvolution approach for achieving high spatio-temporal resolution in monitoring realtime enzyme kinetics in single cells. In particular, we report spatially resolved, nanosecond-scale fluorescence dynamics associated with binding-unbinding kinetics of endogenous metabolic co-factor nicotinamide adenine dinucleotide with enzymes in intact living cells. By monitoring real-time kinetics of NAD(P)H-enzyme kinetics in primary hepatocytes isolated from young and aged mouse models, we observed that the mechanism of inhibition of mitochondrial respiration at complex I site is mediated by redistribution of free and protein-bound nicotinamide adenine dinucleotide pools and that this equilibrium redistribution is affected by agerelated modifications in mitochondrial function. We describe unique advantages of Laguerre deconvolution algorithm in comparison with conventional lifetime analysis approaches. §These authors contributed equally to this work.Correspondence to: V Krishnan Ramanujan. Tel: (+1) 310-423-7785; fax: (+1) 310-423-7707; e-mail: Ramanujanv@cshs.org Non-invasive monitoring of metabolic dysfunctions in intact animal models is an attractive strategy for gaining insight into the dynamics of tissue metabolism in health and in various metabolic syndromes such as cancer, diabetes and aging-induced metabolic dysfunctions. Besides the example demonstrated above, we envisage that the proposed method can find applications in a variety of other situations where intensity-based approaches fall short owing to spectroscopic artefacts.

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“…4 This system employs a 'streak camera' which has the best time-resolution (~2-20 ps) among fast photon detectors (fast photomultiplier tube ~ 300-500 ps and microchannel plate ~ 50-100ps). This is the first time where a streak camera has been incorporated in a multiphoton microscope for biological imaging.…”

Section: Multiphoton Fluorescence Lifetime Imaging Systemmentioning

confidence: 99%

“…In order to utilize the available mouse models employing GFP reporters and to alleviate the problems due to 1Mi$P ubiquitous autofluorescence, it is necessary to realize the insufficiency of the existing intensity-based approaches and to address these issues in a fundamental way. Since the photophysical schemes of the GFP fluorophore and autofluorescence contributions are distinct, fluorescence lifetime (τ) can be reliably employed as an 'intrinsic contrast' parameter in obtaining information from cells and tissues without compromising the signal-to-noise ratio (3)(4)(5)(6)(7). Fluorescence lifetime (τ) defines the duration of molecular state in which an excited fluorophore resides before returning to the ground state and is of the order a few nanoseconds (10 -9 sec) for most of the biologically relevant fluorophores (3).…”

Section: Introductionmentioning

confidence: 99%

Fluorescence lifetime contrast in small animal imaging

et al. 2007

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Early detection of primary tumors is the key for effective therapeutic intervention and successful patient survival. Small animal models emulating human diseases are powerful tools for our comprehensive understanding of the pathophysiology of tumor formation and metastasis to distant sites. Our long-term goal is to develop a non-invasive, multiphoton-fluorescence lifetime imaging (MP-FLIM) modality that can precisely quantify these steps in animal tumor models at a very early stage. The specific hypothesis is that fluorescence lifetime can be employed as reliable contrast parameter for providing higher detection sensitivity as compared with conventional intensity-based tumor imaging approaches and therefore it is possible to detect smaller tumor volumes (early detection) than those achieved by other prevailing methods. We base this hypothesis on our recent observations that (1) fluorescence lifetime is "intrinsic" to the fluorophore and its measurement is not affected by concentration and/or spectral artifacts as in intensity-based methods, (2) multiphoton excitation can enable increased tissue penetrability and reduced phototoxicity and (3) MP-FLIM approach can discriminate background autofluorescence from the fluorescent proteins in thick tissues thereby achieving a ten-fold increase in signal-to-background ratio over the intensity-based approaches. We present our preliminary data to support this hypothesis in primary tumor detection in nu/nu athymic mouse models. .

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Multiphoton fluorescence lifetime imaging microscopy (FLIM) using a streak camera

Cited by 7 publications

References 18 publications

Fluorescence lifetime imaging – techniques and applications

Fluorescence lifetime imaging – techniques and applications

Spatially resolved fluorescence lifetime mapping of enzyme kinetics in living cells

Fluorescence lifetime contrast in small animal imaging

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