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Lifetime and spectral imaging are complementary techniques that offer a non-invasive solution for monitoring metabolic processes, identifying biochemical compounds, and characterizing their interactions in biological tissues, among other tasks. Newly developed instruments that perform time-resolved spectral imaging can provide even more information and reach higher sensitivity than either modality alone. Here we report a multispectral lifetime imaging system based on a field-programmable gate array (FPGA), capable of operating at high photon count rates (12 MHz) per spectral detection channel, and with time resolution of 200 ps. We performed error analyses to investigate the effect of gate width and spectral-channel width on the accuracy of estimated lifetimes and spectral widths. Temporal and spectral phasors were used for analysis of recorded data, and we demonstrated blind un-mixing of the fluorescent components using information from both modalities. Fractional intensities, spectra, and decay curves of components were extracted without need for prior information. We further tested this approach with fluorescently doubly-labeled DNA, and demonstrated its suitability for accurately estimating FRET efficiency in the presence of either non-interacting or interacting donor molecules.

Optical tweezers have become an important tool for the manipulation of single biomolecules. However, their application to stretching biopolymers is usually limited to molecules that are several microns in length because conventional optical tweezers manipulate molecules laterally in the focal plane of the microscope objective, a mode in which steric hindrances from the attached microsphere and the surface are substantial. In order to study the properties of short DNA fragments that are typically 1000 bp long, we used optical tweezers in the axial direction to pull microsphere away from the cover glass surface. The microsphere was held in the linear region of the optical potential where the optical force is least sensitive to the bead position. By varying the laser intensity, different stretching forces were applied to the DNA molecule, and the axial position of the tethered microsphere was obtained from its diffraction pattern. The results indicate that the wormlike chain model is still valid for such short DNA fragments.

We developed a photothermal method based on scattering around a nano-absorber that allows for the unprecedented detection of individual nano-objects such as gold nanoparticles with diameter down to 1.4 nm as well as CdSe nanocrystals. This method relies on the absorptive properties of the nano-object and does not suffer from the drawbacks of luminescence-based methods. We present here two different applications of this versatile detection method. First, we performed absorption spectroscopy of individual gold nanoparticles as small as 5nm and CdSe nanocrystals in the multiexcitonic regime. Second, we show the applicability of our method for new types of gold nanoparticles based DNA microarrays. In addition to the intrinsic signal stability due to the use of gold labelling, our technique does not require silver staining enhancement and permits to push the signal dynamics of such microarrays from the single nanoparticle detection to almost the full surface coverage.