Time-resolved Hadamard fluorescence imaging

We present a new concept for fluorescence lifetime imaging (FLIM) based on time-resolved Hadamard imaging (HI). HI allows image acquisition by use of one single-point detector without requiring a moving scanning stage. Moreover, it reduces the influence of detector noise compared with raster scanning. By use of Monte Carlo simulations it could be confirmed that Hadamard transformation may decrease the error in lifetime estimation and in general in fluorescence parameter estimation when the signal-to-noise ratio is low and detector dark noise is high. This concept may find applications whenever the performance of FLIM or similar methods is limited by high dark-count rates and when the use of a single-point detector is preferable.     

Time-resolved fluorescence detection of mosaic DNA chip

We demonstrated a time-resolved fluorescence (TRF) label and detection of mosaic DNA chip in this paper. We synthesized oligonucleotide sequences in situ on glass slides directly, and then sliced them up into small pieces and patched up the pieces bearing different sequences to generate a mosaic DNA chip. With multiple 4, 7-bis(chlorosulfophenyl)-1, 10-phenanthroline-2, 9-dicarboxylic acid (BCPDA, abbreviated as BCPDA) labeling method based on avidin-biotin amplification, we established a TRF detection format on the mosaic DNA chip. The detection method allows discriminatory signals for perfect match, one-base mismatch, two-base mismatch, and three-base mismatch by TRF labeled DNA hybridization, whereby Europium (III, Eu3+) was captured and released on the principle of complexation and dissociation interaction between BCPDA and Eu3+ solution when the BCPDA-tagged avidin and biotin-capped oligonucleotide sequence linked. The fluorescence spectra and related lifetimes were determined. We also compared the TRF detection mode with the conventional fluorescence one. These results showed the former is a potential alternative replacement of the latter, especially for labeling the mosaic DNA chip. The discovery is of fundamental interest and has significant implications to biochips and biosensors based on time-resolved-fluorescence detection.     

Referencing techniques for the analog mean-delay method in fluorescence lifetime imaging

The analog mean-delay (AMD) method is a new powerful alternative method in determining the lifetime of a fluorescence molecule for high-speed confocal fluorescence lifetime imaging microscopy. Even though the photon economy and the lifetime precision of the AMD method are proven to be as good as those of the state-of-the-art time-correlated single photon counting method, there have been some speculations and concerns about the accuracy of this method with respect to the absolute lifetime value of a fluorescence probe. In the AMD method, the temporal waveform of an emitted fluorescence signal is directly recorded with a slow digitizer whose bandwidth is much lower than the temporal resolution of the lifetime to be measured. We have found that the drifts and the fluctuations of the absolute zero position in a measured temporal waveform are the major problems in the AMD method. We have proposed electrical and optical referencing techniques that may suppress these errors. It is shown that there may exist more than 2 ns drift in a measured temporal waveform during the period of the first 12 min after electronic components are turned on. The standard deviation of a measured lifetime after this warm-up period can be as large as 51 ps without any referencing technique. We have shown that this error can be reduced to 9 ps with our electronic referencing technique. It is demonstrated that this can be further reduced to 4 ps by the optical referencing technique we have introduced.     

Theoretical investigation of the signal-to-noise ratio in fluorescence lifetime imaging

We deduce the signal-to-noise ratio for fluorescence lifetime imaging when using frequency-domain methods. We assume mono-exponential decay and quantum-noise-limited performance. The results are compared with Monte Carlo simulations with good agreement. We also compare our results with previous investigations of time-domain methods for fluorescence lifetime imaging. For a given number of detected photons, we find that frequency-domain and time-domain methods are equally good. The correct choice of detection technique and its parameters is important for obtaining good results.     

Subnanosecond-resolution phase-resolved fluorescence imaging technique for biomedical applications

Characterization of fluorescence emissions from cells often leads to conclusive results in the early detection of cellular abnormalities. Cellular abnormalities can be characterized by their difference in the fluorescence lifetime, which may be less than nanoseconds. A sensitive frequency domain technique, also called a phase-resolved fluorescence imaging technique, is proposed in which fluorescence emissions at the same wavelengths can more effectively be separated with subnanosecond resolution in their lifetime difference. The system configuration is optimized by incorporating even-step phase shifting in the homodyne-assisted signal-processing concept along with the phase-resolved fluorescence technique to eliminate the dc offsets of emission. Experiments are carried out with simulated samples composed of two fluorescence emissions of the same wavelength but with different lifetime values. Suppression of either of the fluorescence emissions by selective imaging of the other validates the superiority of the proposed technique. Hence, this technique can potentially be applied in the early detection of cellular abnormalities.     

Time-resolved fluorescence spectroscopy for chemical sensors

A family of sensors is presented with fluorescence decay-time measurements used as the sensing technique. The concept is to take a single fluorophore with a suitably long fluorescence decay time as the basic building block for numerous different sensors. Analyte recognition can be performed by different functional groups that are necessary for selective interaction with the analyte. To achieve this, the principle of excited-state electron transfer is applied with pyrene as the fluorophore. Therefore the same instrumentation based on a small, ambient air-nitrogen laser and solid-state electronics can be used to measure different analytes, for example, oxygen, pH, carbon dioxide, potassium, ammonium, lead, cadmium, zinc, and phosphate.     

Multispectral fluorescence lifetime imaging of feces-contaminated apples by time-resolved laser-induced fluorescence imaging system with tunable excitation wavelengths

We recently developed a time-resolved multispectral laser-induced fluorescence (LIF) imaging system capable of tunable wavelengths in the visible region for sample excitation and nanosecond-scale characterizations of fluorescence responses (lifetime imaging). Time-dependent fluorescence decay characteristics and fluorescence lifetime imaging of apples artificially contaminated with a range of diluted cow feces were investigated at 670 and 685 nm emission bands obtained by 418, 530, and 630 nm excitations. The results demonstrated that a 670 nm emission with a 418 nm excitation provided the greatest difference in time-dependent fluorescence responses between the apples and feces-treated spots. The versatilities of the time-resolved LIF imaging system, including fluorescence lifetime imaging of a relatively large biological object in a multispectral excitation-emission wavelength domain, were demonstrated.     

Theoretical investigation of the photon efficiency in frequency-domain fluorescence lifetime imaging microscopy

We investigate the photon efficiency of frequency-domain fluorescence lifetime imaging microscopy, using both theoretical and Monte Carlo methods. Our analysis differs from previous work in that it incorporates the data fitting process used in real experiments, allows for the arbitrary choice of excitation and gain waveforms, and calculates lifetimes as well as associated F-values from higher harmonics in the data. Using our analysis, we found different photon efficiencies to those previously reported and were able to propose optimal excitation and gain waveforms. Additionally, we suggest measurement protocols that lead to further improvement in photon efficiency. We compare our results to other techniques for lifetime imaging and consider the implications of our higher-harmonic analysis for multi-exponential lifetime determination.     

Digital micromirror device as a spatial illuminator for fluorescence lifetime and hyperspectral imaging

Time-domain fluorescence lifetime imaging (FLIM) and hyper-spectral imaging (HSI) are two advanced microscopy techniques widely used in biological studies. Typically both FLIM and HSI are performed with either a whole-field or raster-scanning approach, which often prove to be technically complex and expensive, requiring the user to accept a compromise among precision, speed, and spatial resolution. We propose the use of a digital micromirror device (DMD) as a spatial illuminator for time-domain FLIM and HSI with a laser diode excitation source. The rather unique features of the DMD allow both random and parallel access to regions of interest (ROIs) on the sample, in a very rapid and repeatable fashion. As a consequence both spectral and lifetime images can be acquired with a precision normally associated with single-point systems but with a high degree of flexibility in their spatial construction. In addition, the DMD system offers a very efficient way of implementing a global analysis approach for FLIM, where average fluorescence decay parameters are first acquired for a ROI and then used as initial estimates in determining their spatial distribution within the ROI. Experimental results obtained on phantoms employing fluorescent dyes clearly show how the DMD method supports both spectral and temporal separation for target identification in HSI and FLIM, respectively.     

High-resolution colocalization of single dye molecules by fluorescence lifetime imaging microscopy

Conventional fluorescence microscopy can be used to determine the positions of objects in space when those objects are separated by distances greater than several hundred nanometers, as restricted by the diffraction limit of light. Fluorescence microscopy/spectroscopy based on fluorescence resonance energy-transfer techniques can be used to measure separation distances below approximately 10 nm. To fill the gap between these fundamental limits, we have developed an alternative technique for high-resolution colocalization of fluorescent dyes. The technique is based on fluorescence lifetime imaging. Under favorable conditions, the method can be used to distinguish, and to measure the distance between, two dye molecules that are less than 30 nm apart. To demonstrate the method, lifetime images of a mixture of Cy5 and JF9 (rhodamine derivative) molecules statistically adsorbed on a glass surface were acquired and analyzed. Since these two molecular species differ in fluorescence lifetime (for Cy5, tau(f) = 2.0 ns, and for JF9, tau(f) = 4.0 ns), it is possible to assign the contribution of fluorescence of the two dye types to each image pixel using a pattern recognition technique. Since both dye types can be excited using the same laser wavelength, the measurement is free of chromatic aberrations. The results presented demonstrate the first high-precision distance measurements between single conventional fluorescent dyes based solely on fluorescence lifetime.     

Optimizing frequency-domain fluorescence lifetime sensing for high-throughput applications: photon economy and acquisition speed

The signal-to-noise ratio of a measurement is determined by the photon economy of the detection technique and the available photons that are emitted by the sample. We investigate the efficiency of various frequency-domain lifetime detection techniques also in relation to time-domain detection. Nonlinear effects are discussed that are introduced by the use of image intensifiers and by fluorophore saturation. The efficiency of fluorescence lifetime imaging microscopy setups is connected to the speed of acquisition and thus to the imaging throughput. We report on the optimal conditions for balancing signal-to-noise ratio and acquisition speed in fluorescence lifetime sensing.     

Two-dimensional direct-reading fluorescence spectrograph for DNA sequencing by capillary array electrophoresis

We report a compact, two-dimensional direct-reading fluorescence spectrograph and demonstrate its application to DNA sequencing by capillary array electrophoresis. The detection cuvette is based on sheath flow, wherein the capillaries terminate in a two-dimensional array in a fluid-filled chamber that is pressurized with buffer. A thin metal plate is located downstream from the capillaries. This barrier plate has an array of holes that precisely matches the location of the capillaries. Buffer flows through the holes, drawing analyte from the capillaries in a well-defined array of thin filaments. Fluorescence is excited in the upper chamber with an elliptically shaped laser beam. The bottom chamber is sealed with a glass window and drained from the side. Fluorescence is detected by imaging the illuminated sample streams through the holes in the barrier plate. A prism is used to disperse fluorescence from each sample across a CCD camera so that the emission spectrum is monitored simultaneously from each capillary. The instrument is demonstrated in a 32-capillary configuration but can be scaled to several thousand capillaries.     

Quantitative kinetic analysis in a microfluidic device using frequency-domain fluorescence lifetime imaging

A novel microfluidic approach for the quantification of reaction kinetics is presented. A three-dimensional finite difference numerical simulation was developed in order to extract quantitative kinetic information from fluorescence lifetime imaging experimental data. This approach was first utilized for the study of a fluorescence quenching reaction within a microchannel; the lifetime of a fluorophore was used to map the diffusion of a quencher across the microchannel. The approach was then applied to a more complex chemical reaction between a fluorescent amine and an acid chloride, via numerical simulation the bimolecular rate constant for this reaction was obtained.     

Analytical solutions for time-resolved fluorescence lifetime imaging in a turbid medium such as tissue.

An analytical solution is developed to quantify a site-specific fluorophore lifetime perturbation that occurs, for example, when the local metabolic status is different from that of surrounding tissue. This solution may be used when fluorophores are distributed throughout a highly turbid media and the site of interest is embedded many mean scattering distances from the source and the detector. The perturbation in lifetime is differentiated from photon transit delays by random walk theory. This analytical solution requires a priori knowledge of the tissue-scattering and absorption properties at the excitation and emission wavelengths that may be obtained from concurrent time-resolved reflection measurements. Additionally, the solution has been compared with the exact, numerically solved solution. Thus the presented solution forms the basis for practical lifetime imaging in turbid media such as tissue.     

Calibration approach for fluorescence lifetime determination for applications using time-gated detection and finite pulse width excitation

Time-gated techniques are useful for the rapid sampling of excited-state (fluorescence) emission decays in the time domain. Gated detectors coupled with bright, economical, nanosecond-pulsed light sources like flashlamps and nitrogen lasers are an attractive combination for bioanalytical and biomedical applications. Here we present a calibration approach for lifetime determination that is noniterative and that does not assume a negligible instrument response function (i.e., a negligible excitation pulse width) as does most current rapid lifetime determination approaches. Analogous to a transducer-based sensor, signals from fluorophores of known lifetime (0.5-12 ns) serve as calibration references. A fast avalanche photodiode and a GHz-bandwidth digital oscilloscope is used to detect transient emission from reference samples excited using a nitrogen laser. We find that the normalized time-integrated emission signal is proportional to the lifetime, which can be determined with good reproducibility (typically <100 ps) even for data with poor signal-to-noise ratios ( approximately 20). Results are in good agreement with simulations. Additionally, a new time-gating scheme for fluorescence lifetime imaging applications is proposed. In conclusion, a calibration-based approach is a valuable analysis tool for the rapid determination of lifetime in applications using time-gated detection and finite pulse width excitation.     

DNA sequencing by capillary electrophoresis with four-decay fluorescence detection

A scheme for multiplex detection of dye-labeled DNA fragments in DNA sequencing is described in which on-the-fly, frequency-domain fluorescence lifetime detection is used to discriminate among the dye-labeled fragments of the four terminal bases in a single-lane CE separation. Two four-dye systems were evaluated, one excited at 488 nm and the other, at 514 nm. The 488 nm system proved successful for four-decay detection. Base calling was achieved either directly from on-the-fly lifetimes or from lifetime-resolved electropherograms recovered for each base from the electropherogram of the mixture of sequencing reaction products. The latter method was found to be more accurate (99% for two bases and 98.5% for three bases) and could achieve longer read lengths, but it was unsuccessful for sequencing of all four bases. The first method gave a base-calling accuracy of 96% for four-base sequencing over the fragment length range of 41-220 bases.     

Noninvasive imaging of nanogram quantities of DNA in agarose electrophoresis gels.

Image processing techniques which separate the DNA Kerr effect and induced electrokinetic distortion contributions to electric birefringence images of agarose nucleic acid electrophoresis gels are described. Under standard electrophoresis conditions, detection limits of 10 ng of DNA per well are obtained in hydroxyethylated agarose without signal averaging or 7.5 ng with averaging of four measurements. Maintaining constant gel temperature is shown to improve the quality of the images. Monochromatic light (589 nm) is shown to give a small increase in sensitivity compared to polychromatic (650 +/- 20 nm) light.     

Time-resolved imaging of solid phantoms for optical mammography

We have recorded time-resolved transillumination images of solid phantoms with objects embedded that differ in their scattering and absorption coefficients from those of the bulk material, simulating a compressed human breast with a tumor inside. Employing time-correlated single photon counting at rates of up to 1 MHz, we recorded distributions of times of flight of photons at 1369 scan positions within 2.5 min. Several quantities, such as fractional transmittance, first moments, Fourier amplitudes, phase shifts, and frequency-dependent effective transport scattering and absorption coefficients, have been derived from experimental data to form two-dimensional images. By recording such images at a selected total number of photons detected, we have determined the contrast and effective signal-to-noise ratio in each case.