Recently, far-field optical imaging with a resolution significantly beyond diffraction limit has attracted tremendous attention allowing for high resolution imaging in living objects. Various methods have been proposed that are divided in to two basic approaches; deterministic super-resolution like STED or RESOLFT and stochastic super-resolution like PALM or STORM. We propose to achieve super-resolution in far-field fluorescence imaging by the use of controllable (on-demand) bursts of pulses that can change the fluorescence signal of long-lived component over one order of magnitude. We demonstrate that two beads, one labeled with a long-lived dye and another with a short-lived dye, separated by a distance lower than 100 nm can be easily resolved in a single experiment. The proposed method can be used to separate two biological structures in a cell by targeting them with two antibodies labeled with long-lived and short-lived fluorophores.
Typically the signal-to-background ratio is the limiting aspect of fluorescence-based detecting and imaging. The background signal can be composed of a variety of sources-excitation scattering, contaminants, and autofluorescence from cellular constituents. Most of these sources have a short-lived lifetime (ps to ns range). In order to increase the signal-to-background ratio, fluorophores with high brightness or in large concentrations are typically used along with time-gated detection. This unfortunately sacrifices the probe’s signal unless it has a very long lifetime. Herein we are presenting a simple method to enhance the detection of widely available and well-known mid-range lifetime (~20 ns) fluorophores’ signal against short-lived backgrounds. This requires a repetition rate of ~300 MHz to pump a 20 ns probe sufficiently. Typical laser sources today are not equipped with repetition rates above 80 MHz. However, this multipulse method allows these rates to be attainable for nearly any pulsed laser source. Multiple pulses of excitation are separated by a variable temporal length, which is short compared to the lifetime of the long-lived fluorophore, to increase the excited state population of a long-lived fluorophore, while the short-lived background decays almost completely between pulses. This is accomplished by simply redirecting the pulsed excitation beam through glass and then a delay length any number of times and lengths as desired to control the number of pulses and separation times.
Fluorescent nanodiamonds (NDs) are new and emerging nanomaterials that have potential to be used as fluorescence imaging agents and also as a highly versatile platform for the controlled functionalization and delivery of a wide spectrum of therapeutic agents. We will utilize two experimental methods, TIRF, a relatively simple method based on total internal reflection fluorescence and SPRF, fluorescence enhanced by resonance coupling with surface plasmons. We estimate that the SPRF method will be 100 times sensitive than currently available similar detectors based on detectors. The ultimate goal of this research is to develop microarray platforms that could be used for sensitive, fast and inexpensive gene sequencing and protein detection.