Simultaneous time- and wavelength-resolved fluorescence spectroscopy for near real-time tissue diagnosis

Sun, Yinghua; Liu, Rui; Elson, Daniel S.; Hollars, Christopher W.; Jo, Javier A.; Park, Jesung; Sun, Yang; Marcu, Laura

doi:10.1364/ol.33.000630

Cited by 58 publications

(65 citation statements)

References 11 publications

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“…Instruments developed to monitor both fluorescence lifetime and spectra of tissue include the work of Glanzmann et al [40], Pitts et al [41], de Beule et al [42] and Sun et al [43]. There are also systems capable of measuring other optical properties, such as diffuse reflectance, in addition to the fluorescence, e.g.…”

Section: Introductionmentioning

confidence: 99%

In vivo measurements of diffuse reflectance and time‐resolved autofluorescence emission spectra of basal cell carcinomas

Thompson

Coda

Sørensen

et al. 2012

Journal of Biophotonics

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Journal of BIOPHOTONICSWe present a clinical investigation of diffuse reflectance and time-resolved autofluorescence spectra of skin cancer with an emphasis on basal cell carcinoma. A total of 25 patients were measured using a compact steady-state diffuse reflectance/fluorescence spectrometer and a fibre-optic-coupled multispectral time-resolved spectrofluorometer. Measurements were performed in vivo prior to surgical excision of the investigated region. Singular value decomposition was used to reduce the dimensionality of steady state diffuse reflectance and fluorescence spectra. Linear discriminant analysis was then applied to the measurements of basal cell carcinomas (BCCs) and used to predict the tissue disease state with a leave-one-out methodology. This approach was able to correctly diagnose 87% of the BCCs. With 445 nm excitation a decrease in the spectrally averaged fluorescence lifetime was observed between normal tissue and BCC lesions with a mean value of 886 ps. Furthermore, the fluorescence lifetime for BCCs was lower than that of the surrounding healthy tissue in all cases and statistical analysis of the data revealed that this decrease was significant (p ¼ 0.002).Schematic diagrams of the two spectrometers showing the steady state spectrometer measurement head (top) and the optical layout of the time-resolved system (bottom).

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Section: Introductionmentioning

confidence: 99%

In vivo measurements of diffuse reflectance and time‐resolved autofluorescence emission spectra of basal cell carcinomas

Thompson

Coda

Sørensen

et al. 2012

Journal of Biophotonics

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“…Obtaining reliable results from biological tissues with MSP approach is then greatly simplified and becomes accessible to nonexperts without using the complex and time consuming fitting procedure generally associated with SLIM image analysis. It is thus conceivable to apply our MSP approach to facilitate tumor detections for instance, which are performed until now with simultaneous time-and wavelength-resolved spectroscopy due to the complexity of the data analysis (28,29). Thanks to the spectrally and temporally resolved visual segmentation of our MSP approach, we might indeed envisage to realize a fast and visual diagnostic of cancerous tumor imaging without risk of error.…”

Section: Discussionmentioning

confidence: 99%

Three‐dimensional polar representation for multispectral fluorescence lifetime imaging microscopy

et al. 2009

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Multispectral fluorescence lifetime imaging microscopy is a promising and powerful technique for discriminating multiply labeled samples and for detecting molecular interactions inside thick, heterogeneous, and light-scattering milieu such as tissue. The fast and correct analysis of the spectral and lifetime images constitutes a major challenge, which requires a high level of expertise. We present here a new approach that considerably simplifies this analysis avoiding complex fitting algorithm strategies and permitting a fast and visual graphical representation of the fluorescence lifetimes. By transforming the experimental data from time domain to frequency domain for each spectral channel, we calculate the multispectral polar representation and demonstrate its interest on multiply fluorescent labeled sample. We further apply it on Förster resonance energy transfer (FRET) experiments and demonstrate that FRET measurements with a high level of precision can be performed. With addition of emission wavelength as third dimension in the polar representation, autofluorescence emitted by the sample is thus clearly identified. Analysis artifacts induced by the sample or by fitting algorithm choice become then totally inexistent. ' 2009 International Society for Advancement of Cytometry Key terms multispectral FLIM; SLIM; biological tissue; molecular interactions; FRET; phasor IN addition to intensity and wavelength, lifetime is a supplementary source of contrast in fluorescence microscopy, which greatly increases access to the biological information in living cells. Fluorescence Lifetime Imaging Microscopy (FLIM) has indeed been successfully applied to differentiate spectrally undistinguishable molecular species (1) and to map local modifications in labeled samples in terms of ion concentration (2), pH (3), and oxygen (4). FLIM has also been widely used to explore conformational change of proteins (5) or to separate interacting and non-interacting molecular fractions in Förster Resonance Energy Transfer (FRET) experiments (6,7).However, because of the low number of collected photons per pixel and the variability of the molecular environment inside a living cell, FLIM is usually limited to two lifetime components in the same pixel. This restriction makes it difficult to extract biologically relevant information from autofluorescent, heterogeneous, lightscattering samples such as biological tissue. Because of optical properties of tissue, a dedicated temporally and spectrally resolved system (called SLIM for Spectral and Lifetime Imaging Microscopy) is necessary for example to be able to explore molecular interactions on a nanometric scale (with FRET experiments).Temporal and spectral measurements can be performed using two approaches, either frequency domain (8,9) or time domain (10-12) measurements. In both cases, the determination of lifetimes and contributions of each molecular species are mainly achieved by fitting the collected data at each pixel using equations with multiple

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“…Fluorescence Lifetime Imaging (FLIM) techniques can be divided into time-domain and frequency-domain techniques, 6-9 photon counting 10,11 and analog techniques, 6,[12][13][14][15] and point-scanning 7,16 and widefield imaging techniques. 6,12,15,17 It also matters whether a technique acquires the signal waveform in a few time gates 10 or in a large number of time channels 11,18 and whether this happens simultaneously 10,11,18 or sequentially.…”

Section: Introductionmentioning

confidence: 99%

A wide-field TCSPC FLIM system based on an MCP PMT with a delay-line anode

Becker

Hirvonen

Milnes

et al. 2016

Review of Scientific Instruments

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We report on the implementation of a wide-field time-correlated single photon counting (TCSPC) method for fluorescence lifetime imaging (FLIM). It is based on a 40 mm diameter crossed delay line anode detector, where the readout is performed by three standard TCSPC boards. Excitation is performed by a picosecond diode laser with 50 MHz repetition rate. The photon arrival timing is obtained directly from the microchannel plates, with an instrumental response of ∼190 to 230 ps full width at half maximum depending on the position on the photocathode. The position of the photon event is obtained from the pulse propagation time along the two delay lines, one in x and one in y. One end of a delay line is fed into the “start” input of the corresponding TCSPC board, and the other end is delayed by 40 ns and fed into the “stop” input. The time between start and stop is directly converted into position, with a resolution of 200–250 μm. The data acquisition software builds up the distribution of the photons over their spatial coordinates, x and y, and their times after the excitation pulses, typically into 512 × 512 pixels and 1024 time channels per pixel. We apply the system to fluorescence lifetime imaging of cells labelled with Alexa 488 phalloidin in an epi-fluorescence microscope and discuss the application of our approach to other fluorescence microscopy methods.

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Simultaneous time- and wavelength-resolved fluorescence spectroscopy for near real-time tissue diagnosis

Cited by 58 publications

References 11 publications

In vivo measurements of diffuse reflectance and time‐resolved autofluorescence emission spectra of basal cell carcinomas

In vivo measurements of diffuse reflectance and time‐resolved autofluorescence emission spectra of basal cell carcinomas

Three‐dimensional polar representation for multispectral fluorescence lifetime imaging microscopy

A wide-field TCSPC FLIM system based on an MCP PMT with a delay-line anode

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