Fluorescence lifetime imaging measurements were made on intact avocado leaves (Persea americana Mill.) during the slow part of chlorophyll (Chl) a fluorescence transient, the P to S and the S to M phase. Contributions of lutein-epoxide and violaxanthin cycles operating in parallel on the ΔpH-dependent (transthylakoid H+ concentration gradient) thermal energy dissipation (qE) and slowly reversible ApH-independent fluorescence quenching (qI) were studied. A polar plot analysis of the lifetime data revealed three major chlorophyll a fluorescence lifetime pools for photosystem II. The longest lifetime pool (centered at 2 ns) was observed when linear electron transport and the resulting ΔpH build-up were inhibited in leaves. The other two lifetime pools (1.5 and 0.5 ns) were observed during ΔpH build-up under illumination. Interconversion between these two lifetime pools took place during the slow part of the chlorophyll a fluorescence transient. Formation of the 0.5 ns pool upon illumination was correlated with dark-retention of antheraxanthin and photo-converted lutein in leaves. In the absence of ΔpH, neither the intensity nor the lifetimes of fluorescence were affected by the presence of antheraxanthin and photo-converted lutein. We conclude that both antheraxanthin and photo-converted lutein are able to enhance ΔpH-dependent qE processes associated with the 0.5 ns lifetime pool.

Measuring changes in a molecule's fluorescence emission is a common technique to study complex biological systems such as cells and tissues. Although the steady-state fluorescence intensity is frequently used, measuring the average amount of time that a molecule spends in the excited state (the fluorescence lifetime) reveals more detailed information about its local environment. The lifetime is measured in the time domain by detecting directly the decay of fluorescence following excitation by short pulse of light. The lifetime can also be measured in the frequency domain by recording the phase and amplitude of oscillation in the emitted fluorescence of the sample in response to repetitively modulated excitation light. In either the time or frequency domain, the analysis of data to extract lifetimes can be computationally intensive. For example, a variety of iterative fitting algorithms already exist to determine lifetimes from samples that contain multiple fluorescing species. However, recently a method of analysis referred to as the polar plot (or phasor plot) is a graphical tool that projects the time-dependent features of the sample's fluorescence in either the time or frequency domain into the Cartesian plane to characterize the sample's lifetime. The coordinate transformations of the polar plot require only the raw data, and hence, there are no uncertainties from extensive corrections or time-consuming fitting in this analysis. In this chapter, the history and mathematical background of the polar plot will be presented along with examples that highlight how it can be used in both cuvette-based and imaging applications.

The Journal of Biomedical Optics (JBO) is a Gold Open Access journal that publishes peer-reviewed papers on the use of novel optical systems and techniques for improved health care and biomedical research.

We report the fluorescence lifetime imaging and quantum yield measurement of five different fluorescence dyes spanning different quantum yield and excitation wavelength ranges in solution as well as on irregular nanoplasmonic substrate surface. Due to a distribution of dye molecules at random distances and orientation to the metal nanoplasmonic structure, the dyes showed multi-component lifetime decays on the surface. We have simulated the distribution of lifetime on the surface based on fractional intensity relative to steady-state value and derived an average lifetime with species fraction. From the quantum yield and fluorescence lifetime measurements we calculated the modified radiative and non-radiative decay rates for the dyes due to energy coupling on the substrate. We measured up to 100 fold fluorescence enhancement on nanoplasmonic substrate, and all molecule fluorescence showed not only considerably higher radiative decay rate but also higher non-radiative decay rate.

Fluorescence lifetime imaging (FLI) has been used widely for measuring biomedical samples. Practical guidelines on taking successful FLI data are provided to avoid common errors that arise during the measurement. Several methods for analyzing and interpreting FLI results are also introduced; e.g., a model-free data analysis method called the polar plot allows visualization and analysis of FLI data without iterative fitting, and an image denoising algorithm called variance-stabilizing-transform TI Haar helps to elucidate the information of a complex biomedical sample. The instrument considerations and data analysis of Spectral-FLI are also discussed.

This chapter is an introduction to some basic theoretical aspects of fluorescence and Förster resonance energy transfer (FRET). The similarities between fluorescence and FRET phenomena are discussed. Although the physical process of FRET has nothing directly to do with emitted photons, fluorescence is the usual experimental method for measuring FRET. The attempt is made to present the reader with sufficient background to be able to explore deeper into the different theoretical and experimental aspects of FRET and to be able to understand some of the new developments and applications. Short historical introductions are given for both fluorescence and FRET.

In January of 2011, the Biomedical Engineering Society (BMES) and the Society for Physical Regulation in Biology and Medicine (SPRBM) held its inaugural Cellular and Molecular Bioengineering (CMBE) conference. The CMBE conference assembled worldwide leaders in the field of CMBE and held a very successful Round Table discussion among leaders. One of the action items was to collectively construct a white paper regarding the future of CMBE. Thus, the goal of this report is to emphasize the impact of CMBE as an emerging field, identify critical gaps in research that may be answered by the expertise of CMBE, and provide perspectives on enabling CMBE to address challenges in improving human health. Our goal is to provide constructive guidelines in shaping the future of CMBE.

In this chapter, I have provided a complete list of contributions of Govindjee that covers the period 2000-2011, his retirement life thus far. I have provided information on: Research publications in photosynthesis; contributions of historical interest; contributions of educational interest; and recent recognition in the form of three prestigious Awards. He has been as active as when he was in service. He has served the International Society of Photosynthesis, and the wider scientific community, well, and is an example of intense dedication to the field of photosynthesis and its practitioners.

SummaryMembrane type 1 matrix metalloproteinase (MT1‐MMP) is a membrane‐tethered collagenase primarily involved in the mechanical destruction of extracellular matrix proteins. MT1‐MMP has also been shown to be upregulated in several types of cancers. Many coordinated functions of MT1‐MMP during migration and invasion remain to be determined. In this paper, live cells from the invasive cell line HT‐1080 were imaged using an intracellular Förster resonance energy transfer‐based biosensor specific for MT1‐MMP; a substrate specific for MT1‐MMP was hybridized with the mOrange2 and mCherry fluorescent proteins to form the Förster resonance energy transfer‐based sensor. The configuration of the biosensor was determined with fluorescence lifetime‐resolved imaging microscopy using both a polar plot‐based analysis and a rapid data acquisition modality of fluorescence lifetime‐resolved imaging microscopy known as phase suppression. Both configurations of the biosensor (with or without cleavage by MT1‐MMP) were clearly resolvable in the same cell. Changes in the configuration of the MT1‐MMP biosensor were observed primarily along the edge of the cell following the removal of the MMP inhibitor GM6001. The intensities highlighted by phase suppression correlated well with the fractional intensities derived from the polar plot.

With the goal of improving photo-absorption of photovoltaic device and for plasmonic application we have fabricated nanopillar black silicon devices through etching-passivation technique which does not require any photomask and whole wafer scale uniformity is achieved at room temperature in a short time. We have carried out thorough optical characterization for nanopillar black silicon devices to be used for solar cell and plasmonic applications. Cathodoluminescence (CL), current dependent CL spectroscopy, photoluminescence (at room temperature and 77 K), Raman spectroscopy, reflectance and absorption measurement have been performed on the device. A thin layer of Ag is deposited to render with plasmonic property and the plasmonic effect is probed using surface plasmon enhanced fluorescence, angle dependent reflectance measurements, high resolution cathodoluminescence (CL), surface enhanced Raman spectroscopy (SERS) measurement and Fluorescence Lifetime Imaging Microscopy (FLIM) experiment. We obtained reduction in optical reflection of similar to 12 times on b-Si substrate from UV to NIR range, the nanostructured fluorescence enhancement of similar to 40 times and the Raman scattering enhancement factor of 6.4x10(7).

Lifetime-resolved imaging measurements of chlorophyll a fluorescence were made on leaves of avocado plants to study whether rapidly reversible ΔpH-dependent (transthylakoid H+ concentration gradient) thermal energy dissipation (qE) and slowly reversible ΔpH-independent fluorescence quenching (qI) are modulated by lutein-epoxide and violaxanthin cycles operating in parallel. Under normal conditions (without inhibitors), analysis of the chlorophyll a fluorescence lifetime data revealed two major lifetime pools (1.5 and 0.5 ns) for photosystem II during the ΔpH build-up under illumination. Formation of the 0.5-ns pool upon illumination was correlated with dark-retention of antheraxanthin and photo-converted lutein in leaves. Interconversion between the 1.5- and 0.5-ns lifetime pools took place during the slow part of the chlorophyll a fluorescence transient: first from 1.5 ns to 0.5 ns in the P-to-S phase, then back from 0.5 ns to 1.5 ns in the S-to-M phase. When linear electron transport and the resulting ΔpH build-up were inhibited by treatment with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), the major fluorescence intensity was due to a 2.2-ns lifetime pool with a minor faster contribution of approximately 0.7 ns. In the presence of DCMU, neither the intensity nor the lifetimes of fluorescence were affected by antheraxanthin and photo-converted lutein. Thus, we conclude that both antheraxanthin and photo-converted lutein are able to enhance ΔpH-dependent qE processes that are associated with the 0.5-ns lifetime pool. However, unlike zeaxanthin, retention of antheraxanthin and photo-converted lutein may not by itself stabilize quenching or cause qI.

Fluorescence lifetime-resolved imaging microscopy (FLIM) has been used to monitor the enzymatic activity of a proteolytic enzyme, Membrane Type 1 Matrix Metalloproteinase (MT1-MMP), with a recently developed FRET-based biosensor in vitro and in live HeLa and HT1080 cells. MT1-MMP is a collagenaise that is involved in the destruction of extra-cellular matrix (ECM) proteins, as well as in various cellular functions including migration. The increased expression of MT1-MMP has been positively correlated with the invasive potential of tumor cells. However, the precise spatiotemporal activation patterns of MT1-MMP in live cells are still not well-established. The activity of MT1-MMP was examined with our biosensor in live cells. Imaging of live cells was performed with full-field frequency-domain FLIM. Image analysis was carried out both with polar plots and phase differential enhancement. Phase differential enhancement, which is similar to phase suppression, is shown to facilitate the differentiation between different conformations of the MT1-MMP biosensor in live cells when the lifetime differences are small. FLIM carried out in differential enhancement or phase suppression modes, requires only two acquired phase images, and permits rapid imaging of the activity of MT1-MMP in live cells.

We report the optimization of a novel redox-sensitive probe with enhanced dynamic range and an exceptionally well-positioned oxidative midpoint redox potential. The present work characterizes factors that contribute to the improved Förster resonance energy transfer (FRET) performance of this green fluorescent protein (GFP)-based redox sensor. The α-helical linker, which separates the FRET donor and acceptor, has been extended in the new probe and leads to a decreased FRET efficiency in the linker's reduced, 'FRET-off' state. Unexpectedly, the FRET efficiency is increased in the new linker's oxidized, 'FRET-on' state compared with the parent probe, in spite of the longer linker sequence. The combination of a lowered baseline 'FRET-off' and an increased 'FRET-on' signal significantly improves the dynamic range of the probe for a more robust discrimination of its reduced and oxidized linker states. Mutagenesis of the cysteine residues within the α-helix linker reveals the importance of the fourth, C-terminal cysteine and the relative insignificance of the second cysteine in forming the disulfide bridge to clamp the linker into the high-FRET, oxidized state. To further optimize the performance of the redox probe, various cyan fluorescent protein (CFP)/yellow fluorescent protein (YFP) FRET pairs, placed at opposite ends of the improved redox linker (RL7), were quantitatively compared and exchanged. We found that the CyPet/YPet and ECFP/YPet FRET pairs when attached to RL7 do not function well as sensitive redox probes due to a strong tendency to form heterodimers, which disrupt the α-helix. However, monomeric versions of CyPet and YPet (mCyPet and mYPet) eliminate dimerization and restore redox sensitivity of the probe. The best performing probe, ECFP-RL7-EYFP, exhibits an approximately six-fold increase in FRET efficiency in vitro when passing from the oxidized to the reduced state. We determined the midpoint redox potential of the probe to be -143 ± 6 mV, which is ideal for measuring glutathione (GSH/GSSG) redox potentials in oxidative compartments of mammalian cells (e.g. the endoplasmic reticulum).

A spectrograph with continuous wavelength resolution has been integrated into a frequency-domain fluorescence lifetime-resolved imaging microscope (FLIM). The spectral information assists in the separation of multiple lifetime components, and helps resolve signal cross-talking that can interfere with an accurate analysis of multiple lifetime processes. This extends the number of different dyes that can be measured simultaneously in a FLIM measurement. Spectrally resolved FLIM (spectral-FLIM) also provides a means to measure more accurately the lifetime of a dim fluorescence component (as low as 2% of the total intensity) in the presence of another fluorescence component with a much higher intensity. A more reliable separation of the donor and acceptor fluorescence signals are possible for Forster resonance energy transfer (FRET) measurements; this allows more accurate determinations of both donor and acceptor lifetimes. By combining the polar plot analysis with spectral-FLIM data, the spectral dispersion of the acceptor signal can be used to derive the donor lifetime - and thereby the FRET efficiency - without iterative fitting. The lifetime relation between the donor and acceptor, in conjunction with spectral dispersion, is also used to separate the FRET pair signals from the donor alone signal. This method can be applied further to quantify the signals from separate FRET pairs, and provide information on the dynamics of the FRET pair between different states.

Photosynthesis converts solar energy into chemical energy. It provides food and oxygen; and, in the future, it could directly provide bioenergy or renewable energy sources, such as bio-alcohol or hydrogen. To exploit such a highly efficient capture of energy requires an understanding of the fundamental physics. The process is initiated by photon absorption, followed by highly efficient and extremely rapid transfer and trapping of the excitation energy. We first review early fluorescence experiments on in vivo energy transfer, which were undertaken to understand the mechanism of such efficient energy capture. A historical synopsis is given of experiments and interpretations by others that dealt with the question of how energy is transferred from the original location of photon absorption in the photosynthetic antenna system into the reaction centers, where it is converted into useful chemical energy. We conclude by examining the physical basis of some current models concerning the roles of coherent excitons and incoherent hopping in the exceptionally efficient transfer of energy into the reaction center.

Fluorescence measurements have been an established mainstay of photosynthesis experiments for many decades. Because in the photosynthesis literature the basics of excited states and their fates are not usually described, we have presented here an easily understandable text for biology students in the style of a chapter in a text book. In this review we give an educational overview of fundamental physical principles of fluorescence, with emphasis on the temporal response of emission. Escape from the excited state of a molecule is a dynamic event, and the fluorescence emission is in direct kinetic competition with several other pathways of de-excitation. It is essentially through a kinetic competition between all the pathways of de-excitation that we gain information about the fluorescent sample on the molecular scale. A simple probability allegory is presented that illustrates the basic ideas that are important for understanding and interpreting most fluorescence experiments. We also briefly point out challenges that confront the experimenter when interpreting time-resolved fluorescence responses.

The applications of Forster resonance energy transfer (FRET) have expanded tremendously in the last 25 years, and the technique has become a staple technique in many biological and biophysical fields. Many publications appear weekly using FRET and most of the applications use FRET as a spectroscopic research tool. In this chapter, we have examined some general salient features of resonance energy transfer by stressing the kinetic competition of the FRET pathway with all other pathways of de-excitation. This approach emphasizes many of the biotechnological and biophysical uses of FRET, as well as emphasizing the important competing processes and biological functions of FRET in photosynthesis.