Using a novel imaging device—NCam—fluorophore lifetime measurements can be captured simultaneously with wide-field microscopy methods. Because NCam records single-photon events with spatial and temporal information, the localization precision can be improved compared to camera-based imaging or scanning confocal instruments. We demonstrate this new imaging capability by examining the fluorescence behavior of quantum dots.
Next generation single-photon detectors are becoming available. Research grade SPAD arrays promise camera-like detectors with dozens to hundreds of thousands of pixels, providing both spatial and temporal information about single photon events. Concurrently, other detector technologies are under development, such as crossed-delay line detectors. We evaluate the application of SPAD arrays and NCam, a crossed-delay line detector, towards quantum ghost imaging applications. We discuss implementation challenges and performance differences in the context of ghost imaging.
The near infrared (NIR) and mid-infrared (MIR) spectral regions contain absorption features that can identify specific molecular bonds and chemical species in a sample. For example, lignan and proteins in plants have specific absorption signatures in the NIR. However, because detectors are inefficient in the NIR and MIR, infrared spectroscopy requires high light levels to overcome detector limitations. Cameras in particular do not perform well in this spectral range, and microscopy methods such as Fourier transform infrared spectroscopy (FT-IR) typically rely on scanning confocal arrangements with single-element detectors to spatially map chemical information. To overcome these limitations, we have developed and exploited a new quantum ghost imaging microscope for obtaining absorption measurements in the NIR without the need of scanning or high light intensities. We report on the use of a novel detector–NCam–in quantum ghost imaging using non-degenerate photon pairs generated by spontaneous parametric down conversion (SPDC). NCam records single-photon arrival events with ∼100 ps resolution, enhancing the correlation window of SPDC pairs over previous wide-field ghost imaging by 30-fold. This permits ghost imaging of living and intact plant samples at light levels lower than what the plants would experience from starlight. For photosynthesizing organisms, this low-light imaging method enables the study of plants without disturbing or eliciting responses from the plant due to the measurement itself.
KEYWORDS: Photons, Quantum emitters, Pulsed laser operation, Point spread functions, Super resolution, Semiconductors, Photoluminescence, Super resolution microscopy, Nanostructures, Time correlated single photon counting
A time-dependent likelihood distribution for analyzing time correlated single photon counting data from a four-pixel time-resolved single molecule localization microscopy experiment is discussed. It is generated by accounting for the probabilities to record photons from two emitters, background counts, and dark counts during two different time channels relative to each incident laser pulse in the experiment. Maximizing the distribution enables localization of each emitter in a dual emitting nanostructure based on the disparate photoluminescence lifetimes of the emitters, even when both emitters are simultaneously in an emissive state. The technique is demonstrated using simulated photon counting data from a hypothetical non-blinking dual-emitter nanostructure in which the distance between the two emitters is less than 10-nm.
A dual-color super-resolution microscope with polarization and orientation-resolving capabilities is presented. Combining single-molecule localization methods with simultaneous polarization measurements enables the determination of the orientation of single emitters, such as quantum dot nanocrystals, with sub-10 nm precision. Additional simultaneous spectral characterization of particle emission allows the capture of multiple optical properties that impact energy transfer. We report on the instrumentation development and the results from coupled quantum dot clusters.
NIR imaging of biological samples can reveal details about the chemical makeup of the sample. To overcome the difficulties of current imaging devices, which are most sensitive in the visible region of the spectrum, we use ghost imaging with quantum entangled photons. A non-degenerate photon pair is used to probe a sample, revealing structures with fewer photons per second than starlight.
Modern instruments for imaging biological samples often use high-power lasers or fluorescent dyes that can disturb sensitive processes within living organisms such as plants. Additionally, many interesting plant processes have absorption bands within the near-infrared (NIR), a spectral region hard to efficiently and cost-effectively detect using current camera technology. We present a quantum ghost imaging (QGI) protocol using a proprietary time and space-resolving photon-counting visible camera, NCam, and a highly nondegenerate source of entangled photon pairs. The combination of these two technologies allows for low-noise, high-resolution non-destructive imaging in the NIR, while using a camera sensitive for visible wavelengths.
Semiconductor quantum dots (QDs) in small clusters can exchange excited state energy via various transfer mechanisms such as F¨orster resonant energy transfer (FRET). Such energy transfer enables excitons to move from larger bandgap donors to smaller bandgap acceptors. Clusters of mixed donor/acceptor QD species consequently have a spectral signature that is dependent on which QDs in the clusters are responsible for the emission. Using a dual-color super-resolution imaging approach, we report on the spectral characteristics of interacting QDs in clusters with nanometer spatial resolution. Higher emission intensities from clusters are shown to emanate from sub-regions of the clusters and have spectral signatures that indicate the emission is dominated by the acceptor region of the spectrum. Thus, energy transferring interactions among QDs in clusters funnel excitons primarily to acceptor particles. Acceptor particles are responsible for the majority of the emission from the clusters with an emission spectra corresponding to the spectral profiles of the acceptor species.
KEYWORDS: Sensors, Point spread functions, Monte Carlo methods, Optical fibers, Avalanche photodetectors, Avalanche photodiodes, Luminescence, Molecules, Time correlated photon counting, Super resolution microscopy, Fluorescence lifetime imaging, Single photon detectors, Single molecule spectroscopy, Quantum dots
Photoluminescence images can be acquired with detection schemes that have both single-photon sensitivity and nanosecond scale temporal resolution, enabling the study of possible structural bases for photoluminescence lifetimes and other features of the photon arrival statistics. Within the context of super-resolution (SR) imaging, this has been demonstrated with detection schemes that collect images with a bundle of optical fibers that are coupled to individual single-photon counting avalanche photodiode detectors. Recently, our group used a bundle of four optical fibers to collect these “time-resolved photon arrival” images. Despite the paucity of information contained in a four-pixel image, we precisely located the emission centroid of quantum dots (QDs) and observed correlations between centroid location, photoluminescence lifetime, and intensity within clusters of QDs that were suggestive of electronic interactions among them. This proceedings paper details the approach that we used to locate the emission centroid based on the counts in the four detectors.
We have developed a light-sheet microscope that uses confocal scanning of dual-Bessel beams for illumination. A digital micromirror device (DMD) is placed in the intermediate image plane of the objective used to collect fluorescence and is programmed with two lines of pixels in the “on” state such that the DMD functions as a spatial filter to reject the out-of-focus background generated by the side-lobes of the Bessel beams. The optical sectioning and out-of-focus background rejection capabilities of this microscope were demonstrated by imaging of fluorescently stained actin in human A431 cells. The dual-Bessel beam system enables twice as many photons to be detected per imaging scan, which is useful for low light applications (e.g., single-molecule localization) or imaging at high speed with a superior signal to noise. While demonstrated for two Bessel beams, this approach is scalable to a larger number of beams.
Single particle tracking has provided a wealth of information about biophysical processes such as motor protein transport and diffusion in cell membranes. However, motion out of the plane of the microscope or blinking of the fluorescent probe used as a label generally limits observation times to several seconds. Here, we overcome these limitations by using novel non-blinking quantum dots as probes and employing a custom 3D tracking microscope to actively follow motion in three dimensions (3D) in live cells. Signal-to-noise is improved in the cellular milieu through the use of pulsed excitation and time-gated detection.
Applications of conjugated polymers in photovoltaics and displays drive the need to understand how morphology affects
emission and charge migration. Due to the inherent complexity of polymers, parallel studies of oligomer aggregates are
required to ‘build-up' an understanding of the polymer features. Fluorescence lifetime imaging microscopy (FLIM) is
used to probe variations in vibronic patterns and emission lifetime between individual aggregates and trends in these
properties as a function of aggregate size. This technique yields insight into the structure and packing properties of these
materials in the aggregated state.
We developed a light-sheet illumination microscope that can perform fast 3D imaging of transparent biological samples. The light-sheet is created by raster scanning of a Bessel Beam with one galvo-mirror. Fluorescence excited from the thin layer of the sample is de-scanned by the same galvo-mirror, and then spatially filtered by a slit so that out-of-focus fluorescence generated by the side lobes of the Bessel beam is rejected. The spatially filtered fluorescence is returned by a series of optics and is re-scanned by the same galvo-mirror across the chip of a camera such that the fluorescence image is constructed in real-time. Compared to two-photon Bessel beam excitation or other confocal line scanning approaches, our method is of lower cost, simpler, and doesn’t require calibration and synchronization of multiple galvo mirrors. We demonstrated the capability of fast 3D imaging and background rejection capabilities of this microscope with fluorescent beads embedded in PDMS.
Here we present an automated microscope capable of 3D multi-color single molecule localization of individual messenger RNA molecules in a wide range of cell types. We have implemented astigmatic imaging with a cylindrical lens to improve z-localization, and a maximum likelihood estimator on a graphics processing unit to improve localization precision and speed. This microscope will aid in gene expression analysis by its capability to perform high throughput imaging of thick cells and tissues while still maintaining sufficient z resolution to resolve single RNA transcripts in three dimensions. Enhanced z-localization allows for resolving membrane localized and co-localized transcripts.
We have used super-resolution optical microscopy and confocal microscopy to visualize the cytoskeletal restructuring of HeLa cells that accompanies and enables Salmonella typhimurium internalization. Herein, we report the use of confocal microscopy to verify and explore infection conditions that would be compatible with super-resolution optical microscopy, using Alexa-488 labeled phalloidin to stain the actin cytoskeletal network. While it is well known that actin restructuring and cytoskeletal rearrangements often accompany and assist in bacterial infection, most studies have employed conventional diffraction-limited fluorescence microscopy to explore these changes. Here we show that the superior spatial resolution provided by single-molecule localization methods (such as direct stochastic optical reconstruction microscopy) enables more precise visualization of the nanoscale changes in the actin cytoskeleton that accompany bacterial infection. In particular, we found that a thin (100-nm) ring of actin often surrounds an invading bacteria 10 to 20 min postinfection, with this ring being transitory in nature. We estimate that a few hundred monofilaments of actin surround the S. typhimurium in this heretofore unreported bacterial internalization intermediate.
We have used single molecule fluorescence in situ hybridization (smFISH) to study cell-to-cell heterogeneity of
messenger RNA (mRNA) copy numbers for human host cells subject to a variety of external stimuli. In order to study
the effect of various stimuli and genetic modifications on mRNA copy number, we have constructed an automated highthroughput
multiplexed imaging system and data analysis package capable of localizing large numbers of individual
mRNA transcripts in three dimensions. These experimental distributions of mRNA are used to refine and down-select
regulatory models. Here we present a case example of Interleukin 1 alpha mRNA production in response to immune
system stimulation. We propose a methodology for extending these methods to study the effect of small RNA on genetic
expression by combining multiplexed imaging and numerical modeling at the system-level.
Fluorescent proteins are invaluable molecules in fluorescence microscopy and spectroscopy. The size and brightness of
fluorescent proteins often dictates the application they may be used for. While a monomeric protein may be the least
perturbative structure for labeling a protein in a cell, often oligomers (dimers and tetramers) of fluorescent proteins can
be more stable. However, from a quantitative microscopy standpoint, it is important to realize the photophysical
properties of monomers do not necessarily multiply by their number when they form oligomers. In this work we studied
oligomerization states of the Azami Green (AG) protein with fluorescence correlation spectroscopy (FCS) and photon
antibunching or photon pair correlation spectroscopy (PPCS). FCS was used to measure the hydrodynamic size of the
oligomers, whereas antibunching was used to count the number of fluorescent emitters in the oligomers. The results
exhibited that the dimers of AG were single emitters and the tetramers were dual-emitters, indicative of dipole-dipole
interactions and energy transfer between the monomeric units. We also used these methods to estimate the number of
fluorescent proteins displayed on T7 phage molecules.
The size transition from bulk conducting metals to insulating nanoparticles and eventually to single atoms passes through
the relatively unexplored few-atom nanocluster region. With dimensions close to the Fermi wavelength, these
nanoclusters demonstrate molecule-like properties distinct from bulk metals or atoms, such as discrete and size-tunable
electronic transitions which lead to photoluminescence. Current research aims to elucidate the fundamental
photophysical properties of metal nanoclusters made by different means and based on different encapsulation agents.
Here, we report the study of the photophysical properties, including quantum yields, lifetimes, extinction coefficients,
blinking dynamics and sizes, of silver and gold nanoclusters synthesized using oligonucleotides, a protein (bovine serum
albumin) and a Good's buffer molecule (MES, 2-(N-morpholino) ethanesulfonic acid) as encapsulation agents. We also
investigate the change of photoluminescence as a function of temperature. Furthermore, we show that the fluorescent
metal clusters can be used as a donor in forming a resonance energy transfer pair with a commercial organic quencher.
These new fluorophores have great potential as versatile tools for a broad range of applications in biological and
chemical detection.
The ability to follow and observe single molecules as they function in live cells represents a major milestone
for molecular-cellular biology. Here we present a tracking microscope that is able to track quantum dots in
three dimensions and simultaneously record time-resolved emission statistics from a single dot. This innovative
microscopy approach is based on four spatial filters and closed loop feedback to constantly keep a single quantum
dot in the focal spot. Using this microscope, we demonstrate the ability to follow quantum dot labeled IgE
antibodies bound to FcεRI membrane receptors in live RBL-2H3 cells. The results are consistent with prior
studies of two dimensional membrane diffusion (Andrews et al., Nat. Cell Biol., 10, 955, 2008). In addition, the
microscope captures motion in the axial (Z) direction, which permits tracking of diffusing receptors relative to
the "hills and valleys" of the dynamically changing membrane landscape. This approach is uniquely capable of
following single molecule dynamics on live cells with three dimensional spatial resolution.
Proteins from Anthozoa species are homologous to the green fluorescent protein (GFP) from Aequorea victoria but with absorption/emission properties extended to longer wavelengths. HcRed is a far-red fluorescent protein originating from the sea anemone Heteractis crispa with absorption and emission maxima at 590 and 650 nm, respectively. We use ultrasensitive fluorescence spectroscopic methods to demonstrate that HcRed occurs as a dimer in solution and to explore the interaction between chromophores within such a dimer. We show that red chromophores within a dimer interact through a Förster-type fluorescence resonance energy transfer (FRET) mechanism. We present spectroscopic evidence for the presence of a yellow chromophore, an immature form of HcRed. This yellow chromophore is involved in directional FRET with the red chromophore when both types of chromophores are part of one dimer. We show that by combining ensemble and single molecule methods in the investigation of HcRed, we are able to sort out subpopulations of chromophores with different photophysical properties and to understand the mechanism of interaction between such chromophores. This study will help in future quantitative microscopy investigations that use HcRed as a fluorescent marker.
Single molecule measurements are generally made in conditions that depart from physiological conditions, such as with molecules excised from cells or even immobilized on surfaces. Such departures can easily cause measurements on biomolecules to be inexact. A tracking instrument to follow a single molecule's path in three dimensions inside a living cell would be a major step towards enabling single-molecule observations in physiological conditions. We describe an instrument that will extend the state of the art in single-molecule
tracking technology, allowing extended observations of single particles as they diffuse and are transported. Computations show that our approach should be capable of tracking a protein-sized object diffusing at intracellular speeds for average times of over two seconds - long enough to track a typical fluorescent molecule
from capture to photobleaching.
In this work we report Single Molecule (SM) microscopy studies of binding events between individual surface immobilized antibodies and fluorescent antigens using Total Internal Reflection (TIR) microscopy. Specific binding events of single biotin-conjugated 1quantum dots to anti-biotin antibodies, immobilized on an amine-coated cover glass using a heterobifunctional photo-reactive cross-linker were observed. The methodology of calculating the binding affinity (dissociation constant) from the time series of 2-D images is described.
We use time-resolved single molecule fluorescence detection (MSMD) to investigate the fluorescence dynamics of a mutant of the wild-type Green Fluorescent Protein (GFP) from Aequorea victoria, the folding enhanced GFP (FEGFP). The folding enhanced GFP is a novel and robust variant designed for in vivo high-throughput screening of protein expression levels. This variant shows increased thermal stability and the ability to retain its fluorescence when fused to poorly folding proteins. Here we apply one- (OPE) and two- (TPE) photon excitation on freely diffusing FEGFP molecules. Under OPE, single FEGFP molecules undergo fluorescence flickering in the time scale of μs and tens of μs due to triplet formation and ground-state protonation-deprotonation, respectively. OPE fluorescence lifetimes of single FEGFP molecules show evidence for the presence of different emitting species, the I and B forms of FEGFP chromophore. TPE single FEGFP molecules flicker in fluorescence in the time scale of μs due to singlet-triplet transitions of the chromophore. Two-photon excitation of single FEGFP molecules results in the creation of a photoconverted species with a fluorescence lifetime of 2.5 ns, a species which is bright enough to be detected at the single molecule level. Our results indicate FEGFP is a promising fusion reporter for intracellular applications when
using OPE and TPE microscopy with single molecule sensitivity.
We report on the photophysical properties of a far-red intrinsic fluorescent protein by means of single molecule and ensemble spectroscopic methods. The green fluorescent protein (GFP) from Aequorea victoria is a popular fluorescent marker with genetically encoded fluorescence and which can be fused to any biological structure without affecting its function. GFP and its variants provide emission colors from blue to yellowish green. Red intrinsic fluorescent proteins from Anthozoa species represent a recent addition to the emission color palette provided by GFPs. Red intrinsic fluorescent markers are on high demand in protein-protein interaction studies based on fluorescence-resonance energy transfer or in multicolor tracking studies or in cellular investigations where autofluorescence possesses a problem. Here we address the photophysical properties of a far-red fluorescent protein (HcRed), a mutant engineered from a chromoprotein cloned from the sea anemone Heteractis crispa, by using a combination of ensemble and single molecule spectroscopic methods. We show evidence for the presence of HcRed protein as an oligomer and for incomplete maturation of its chromophore. Incomplete maturation results in the presence of an immature (yellow) species absorbing/fluorescing at 490/530-nm. This yellow chromophore is involved in a fast resonance-energy transfer with the mature (purple) chromophore. The mature chromophore of HcRed is found to adopt two conformations, a Transoriented form absorbing and 565-nm and non-fluorescent in solution and a Cis-oriented form absorbing at 590-nm and emitting at 645-nm. These two forms co-exist in solution in thermal equilibrium. Excitation-power dependence fluorescence correlation spectroscopy of HcRed shows evidence for singlet-triplet transitions in the microseconds time scale and for cis-trans isomerization occurring in a time scale of tens of microseconds. Single molecule fluorescence data recorded from immobilized HcRed proteins, all point to the presence of two classes of molecules: proteins with Cis and Trans-oriented chromophores. Immobilization of HcRed in water-filled pores of polyvinyl alcohol leads to a polymer matrix - protein barrel interaction which results in a 'freezing' of the chromophore in a stable conformation for which non-radiative deactivation pathways are either suppressed or reduced. As a result, proteins with both Cis- and Trans-oriented chromophores can be detected at the single molecule level. Polymer chain motion is suggested as a mediator for an eventual cis-trans isomerization of the chromophore in the case of single immobilized proteins.
Our current experiments further the development of a laser- based technique capable of sequencing an individual strand of DNA. We report the detection and identification of fluorescently labeled nucleotides enzymatically cleaved from DNA strands suspended in flow. We used fluorescence lifetime, fluorescence intensity, or a correlated measure of the intensity and lifetime to identify each individual tagged base traversing the detection region with high accuracy. DNA strands containing a single tetramethylrhodamine labeled uracil and/or a single Rhodamine 6G labeled cytosine were attached to polystyrene microspheres. An optical trap was used to capture and hold a single DNA-laden microsphere nominally 20 microns upstream of the detection region of an ultra- sensitive flow cytometer. The addition of an exonuclease cleaved bases from the 3' end of the fluorescently labeled strand. The cleaved, labeled nucleotides were carried by the flow downstream and detected and identified one-at-a-time with high efficiency by laser-induced fluorescence.
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