Photonic approaches to detect single molecule fluorescence at physiological concentration

Get Complete Project Material File(s) Now! »

Introduction: Single molecule fluorescence spectroscopy

Single molecule study has become a major involvement of research in modern biophysics and every year more researchers are getting attracted to it. The driving idea has been to understand the function of all constituent parts of living organisms. The single-molecule approach bears the intrinsic advantage to reveal information not normally accessible by ensemble measurements, such as sample heterogeneity, local concentration, and variances in kinetic rates. It does not require any perturbing synchronization of molecules to reach a sufficient ensemble-averaged signal, and it circumvents the need for 100% pure samples. Complex problems, such as protein structure folding, molecular motor operation or single-nucleotide polymorphism detection, are best studied at the single molecule level because of the molecular structure dispersion and the stochastic nature of the processes. Although modern molecular biology has made enormous progress in identifying single molecules and their functions, efficiently detecting a single molecule is still a major goal with applications in chemical, biochemical and biophysical analysis. Progress towards this goal crucially depends on the development of techniques that provide visualisation and imaging of processes down to the molecular scale in intact cells [15]. It is established that single molecule techniques have comparatively high vantage points and we have now advanced technology to perform these techniques. Even though there are key limitations of optical single-molecule techniques that have to be rectified in order to get the advantages of these techniques for various applications [10].
In order to find a general strategy to observe single molecule for a broader range of appli-cation, the biggest challenge is to overcome the limitation imposed by the diffraction. The microscopic observation volume must have only a single fluorescent molecule of interest during the measurement acquisition time to detect a single molecule. With the diffraction limited optics we get the focal volume of the order of 0.5 fL, which limits the concentration of the fluorescent species in the nanomolar range to get an isolated single molecule in the focal volume. Practically most biologically related processes involving binding or catalysis require the active molecule to be micro- to millimolar concentration regime as shown in Figure 1.1 [1,2]. The common strategies of optical single molecule fluorescence detection viz. Fluorescence Correlation spectroscopy (FCS), Forster fluorescence resonance energy transfer (FRET), based on confocal microscopy [3] or total internal reflection fluorescence microscopy (TIRF) [4–6] are restricted by experimental condition limited by diffraction. Hence to get the single molecule resolution the detection volume should be decreased by at least three orders of magnitude to reach the physiological condition compared to the confocal condition [1,7–11]. Besides this challenge, the diffraction phenomenon ultimately limits the amount of collected light from a single molecule and the achievable signal-to-background ratio which actually determines the maximum acquisition speed and temporal resolution of the experiments. As a consequence, single molecule detection can be performed only on fluorescent species which are relatively bright and have good photostability.
Figure 1.1: Histogram of Michaelis constant KM for 118,000 enzymes taken from the brenda database ( in November 2013. The top axis show the de-tection volume required to isolate a single molecule. The vertical bars indicate the effective concentration regime and detection volume reached by different techniques (TIRF: Total Internal Reflection fluorescence microscopy; ZMW: Zero mode waveguide).
To overcome this challenge, research have been going on to tailor the photonic environment surrounding the molecule that can affect the fluorescence emission in three ways: (i) by locally enhancing the excitation intensity, (ii) by increasing the emitter’s radiative rate and quantum efficiency, and (iii) by modifying its radiation pattern, towards a higher emission directionality to the detectors [9].
In this chapter we will be discussing briefly about well established strategies already applied to tackle the key parameters of volume reduction and fluorescence rate enhancement. These strategies can be divided into two main areas. The first one takes advantages of shaping the laser excitation beam and the second area covers photonic nanostructural approaches [9].

Improving single molecule fluorescence detection

Methods by structuring the laser excitation beam

Confining the laser beam spatially on the nanoscale using several optical methodologies provides significant improvements (Figure:1.2). Although in practice these methodologies encounter unavoidable difficulties due to optical alignment issues.
Figure 1.2: Different methods for improving single molecule fluorescence detection by taking advantages of structuring the laser excitation beam. Figure Courtesy [9].
TIRF microscopy setup uses a prism or objective to excite the fluorescent molecules diffusing above the upper interface as shown in figure 1.2. Total internal reflection at the solid/liquid interface generates the evanescent wave for the illumination of fluorophores [4]. The detection volume defined by the evanescent field is reduced along the longitudinal direction. It typically extends » ‚/6 offering a reduction of 10 compared to conventional confocal microscopes. A constraint of this technique is that it does not provide lateral confinement of the excitation profile. TIRF-FCS offers a solution for lateral confinement by using a pinhole conjugated to the object plane to reduce the lateral extension of the detection profile [5]. This poses a serious issue of out-of-focus photobleaching, leading to a depletion of fluorophores and limiting the accuracy of FCS measurement.

Fluorescence detection on a mirror

Single molecule detection in solution is tightly bound to the implementation of confocal microscopy. An elegant way to reduce the confocal analysis volume and enhance the fluores-cence rate emitted per molecule takes advantage of a dielectric mirror set at the focal point of the excitation beam as shown in figure 1.2. The mirror affects both the laser excitation intensity and pattern, and the collection of the emitted fluorescence. The coherent excitation beam, which is reflected, produces an interference pattern along the optical axis with an interfringe spacing of ‚/2n, where ‚ is the excitation wavelength and n is the medium refractive index.
Two important effects occur when the confocal detection volume is located on the mirrors sur-face [16,17]. First, interference fringes in the excitation beam give rise to a new characteristics time in the fluorescence correlation function. This new time is found to be independent of the transverse excitation fields beam waist and permits accurate measurement of diffusion coefficients without any a priori knowledge of the confocal volume geometry. Second, the count rate per emitter is significantly enhanced owing to control of spontaneous emission and enhancement of the excitation field, with a gain up to four times.

Pi Microscopy

4Pi microscopy takes advantage of two opposite microscope objectives with high numerical apertures [18]. Coherent light from a laser is split into two beams, which are focused at the same point onto a sample by two opposite objectives. Constructive interference of the two beams enhances the focusing of the light, and the illuminated region gets narrower along the optical axis than in the case of the common confocal microscope. In 4pi microscopy, various types of illumination and detection are utilized: type A corresponds to the illumination via two objectives with constructive interference and detection through one of the objectives in a confocal mode.
A different approach to overcome the diffraction barrier is to use stimulated emission depletion (STED) of the fluorescent molecular state (Fig. 1.2) [18]. STED is a far-field method bearing sub-diffraction analysis volumes suitable for FCS. In STED, a regular diffraction-limited focal spot (green) is used to excite the fluorescence, while a second laser beam (red) stimulates the excited molecules down to their ground state. The red laser beam is custom-tailored to feature a zero-intensity minimum at the center but high intensity in the focal periphery. This configuration ensures that fluorescence occurs only in the very center of the focal spots and is strongly suppressed in the spots periphery. An additional attractive feature of STED is that it allows to adjust the detection volume by increasing the power of the stimulating beam.
The first implementation of a STED experiment with FCS was shown by Kastrup et at. [19]. In a series of FCS measurements on a dilute solution of a red-fluorescing oxazine dye, the STED irradiance was successively increased yielding a 25-fold reduction of the axial diffu-sion time, equivalent to a 5-fold reduction of the focal volume. However, there is a chance to expect even stronger analysis volume reduction with that method. STED-FCS was also used to investigate the cell membrane architecture at the nanoscale [20]. Single diffusing lipid molecules were detected in nanosized areas in the plasma membrane of living cells. Tuning of the probed area 70-fold below the diffraction barrier reveals that sphingolipids and glycosylphosphatidylinositol-anchored proteins are transiently trapped in cholesterol-mediated molecular complexes dwelling within 20 nm diameter areas. This tunable noninva-sive optical recording combined to nanoscale imaging is a powerful new approach to study the dynamics of molecules in living cells.
Even though it provides potentially unlimited resolution, while used in combination with fluo-rescence correlation spectroscopy STED is not a common tool to study individual molecules at elevated concentrations due to the high laser intensities involved.

Methods using photonic structures

To further enhance the detection, nanofabricated photonic structures viz. Nanofluidic chan-nels and slits , Near-field Scanning optical microscopy (NSOM), zero mode wave guides have been used to perform single molecule experiments. Nanofluidic channels and slits provide moderate observation volume confinement of the order of tens of atto-liters requiring nano-to micromolar concentration for single molecule experiments [1]. Zero mode waveguides have been arguably the most prominent example in this course hence we have dedicated a section (section 1.3) on this to get an elaborated overview of the functioning and applications of ZMWs.
Near-field scanning optical microscopy (NSOM) is based on subwavelength-sized light source that is raster-scanned across a surface at a distance of a few nanometers to image the sam-ple. A standard approach to NSOM probes implements tapered optical signal-mode fibers that are coated with metal. At the apex of the tip, an aperture of nanometer dimension is opened by focused ion beam milling. The nanoaperture at the apex of the tip constrains the illumination along both lateral and longitudinal directions. The light confinement can be used to improve the optical resolution for bioimaging, reaching about 50 nm for imaging on cell membranes [21–23]. Measurements using aperture-based NSOM probes have been reported on lipid bilayers [24], single nuclear pore [25], and intact living cell membranes [26]. These dynamic measurements provide sub-millisecond temporal resolution at spatial resolutions below 100 nm. A bowtie aperture has been used to improve the light throughput, enabling even better spatial and temporal resolution [27]. Another approach uses gold nanoparticles attached to glass tips as NSOM probes [28,29]. Moving one step ahead, the aperture-based and nanoparticle-based NSOM approaches can be combined by carving a resonant antenna tip on the top of a nanoaperture NSOM probe (Fig. 1.3) [8,30,31]. The antenna tip provides a high local field enhancement that suppresses the background from the aperture-based NSOM. The antenna tip can be used to control the fluorescence emission polarization and direction [30]. Maria and group [31] fabricated a NSOM probe with monopole optical antenna tip (Fig.1.3b,c). These probes are reported to image individual antibodies with a resolution of 26 §4 nm as well as a resolution of 30 §6 nm is obtained to image intact cell membranes in physiological conditions.
NSOM has the drawback of unreliable probe fabrication and complex implementation [1]. The principle has been picked up and led to the development of nanophotonic structures that allow the fabrication and observation of ultra-small volumes in a parallel and reliable fashion [10].

READ  Workflow for South African translations

Plasmonic Nanoapertures: Zero Mode Waveguides (ZMWs)

In 2003, the groups of Harold Craighead and Watt Webb used single nanometric apertures milled in an opaque metallic films to surpass the diffraction limited detection volume barrier [32]. Since then these ZMWs have been fabricated and studied several times using gold, chromium, Aluminum films using different fabrications techniques viz e-beam lithography followed by dry etching or metal lift-off or by Focused Ion beam milling. Waveguides with metal clad has a cutoff wavelength ‚c above which no propagating modes exist inside the waveguide and an evanescent wave is generated at the aperture’s entrance. (Figure 1.4c). ‚c is related to the diameter of aperture with the waveguide theory relation: ‚c ˘ 1.7d, where d is the aperture diameter [1, 32]. Owing to the fact that there can not be any propagating mode existance inside these nanometric aperture these are termed as zero-mode waveguides (ZMWs) to emphasize the nature of evanescent wave. For the diameter of 100 nm, a single nanoaperture reduces the diffraction limited confocal volume by three orders of magnitude reaching the detection volume of about 2 attoliters (10¡18L) [33,34].
These nanoapertures acts as the pinhole directly milled in the sample plane (Figure 1.4a,b). A particularly simple implementation of zero-mode waveguides consists of small holes in a metal film deposited on a microscope cover-slip. In this case, the metal film acts as the cladding, and the contents of the hole compose the core of the waveguide. Millions of such holes can be made on a single coverslip, resulting in massive parallelism. For direct observation of single-molecule enzymatic activity, enzymes can be adsorbed onto the bottom of the (b) Scanning electron microscope image of a tip-on-aperture probe [31]. (c) Zoomed-in confocal microscopy image of LFA-1 at the cell surface of monocytes visualized by confocal microscopy (left). The right panel shows the NSOM imaging of the highlighted region in the confocal image [31].
waveguides in the presence of a solution containing the fluorescently tagged ligand molecules. Using a microscope objective the coverslip is illuminated from below and the fluorescence is collected back (Figure 1.4a). These sub-wavelength aperture significantly enhances the detected fluorescence rate per emitter, which increases the signal-to-noise ratio for single molecule detection. It has been shown that in isolated 150 nm diameter apertures milled in aluminum, a 6.5 fold enhancement of the fluorescence rate per molecule was obtained using single rhodamine 6G molecules [33]. Further enhancement up to 25-fold can be obtained by tuning the plasmon properties of nanoapertures [35–38].
Figure 1.4: Zero Mode waveguides to enhance single molecule fluorescence detection at physiological concentration. (a) Nanoaperture for enhanced single molecule enzymology [32].
(b) Electron Microscope images of 120 and 160 nm apertures milled in gold [36].(c) Field intensity distribution on a 120 nm water-filled gold aperture illuminated at 633 nm [36].(d) Comparison of normalized FCS correlation curves between confocal and nanoapertures configurations [39]. (e) Observation volumes measured for aluminum apertures. The right axis shows the corresponding concentration to ensure there is a single molecule in the observation volume. [33]. (f ) Fluorescence Enhancement factor for Alexa Fluor 647 molecules in apertures milled in gold and for Rhodamine 6G molecules in apertures milled in aluminum [35].
A large range of biological processes have been monitored with single molecule resolution at micromolar concentrations using ZMW nanoapertures. Most studies take advantages of fluorescence correlation spectroscopy (FCS) (see chapter 3 for detail of FCS)as a biophotonic method to analyse the fluorescence intensity trace from individual molecules diffusing inside and outside the nanoaperture. Levene and co-workers [32] have effectively shown that arrays of ZMWs provides a highly parallel means for studying single-molecule dynamics at micromo-lar concentrations with microsecond temporal resolution. They monitored DNA polymerase activity at 10 „M dye concentration with an average of 0.1 molecule inside a 43 nm diameter aperture. However, for the experiments conducted on ultrasmall structures, the signal to noise ratio comes close to one, as a consequence of quenching losses and increased background. This work has led to a number of other applications combining nanometric apertures with single molecule detection. Among them are oligomerization of the bacteriophage ‚-repressor protein [2], protein-protein interactions considering the GroEL-GroES complex [40,41], or ob-servation of flow mixing [42]. The applications can be extended to dual-color cross-correlation FCCS analysis to monitor DNA enzymatic cleavage at micromolar concentrations with im-proved accuracy. [43]. To avoid the use of fluorescent labelling, the fluorescence detection technique can be operated in reverse mode: the solvent solution filling the aperture is made highly fluorescent by using a millimolar concentration of small fluorescent molecules. Label-free(non-fluorescent) analytes diffusing into the aperture displace the fluorescent molecules in the solution, leading to a decrease of the detected fluorescence intensity, while analytes diffusing out of the aperture return the fluorescence level [44].
Real time DNA RNA sequencing A very promising application of nanometric apertures includes real-time single-molecule DNA and RNA sequencing [45–47]. The development of personalized quantitative genomics re-quires novel methods of DNA sequencing that meet the key requirements of high-throughput, high-accuracy and low operating costs simultaneously. To meet this goal, each nanoaperture forms a nano-observation chamber for watching the activity of a single DNA polymerase enzyme performing DNA sequencing by synthesis (Fig 1.5) [45]. The sequencing method records the temporal order of the enzymatic incorporation of the fluorescent nucleotides into a growing DNA strand replicate. Each nucleotide replication event lasts a few millisec-onds, and can be observed in real-time. Currently, over 3000 nanoapertures can be operated simultaneously, allowing massive parallelization.

Live cell membranes investigation at the nanometer scale with ZMWs

Plasma membrane are highly dynamic structures, with key molecular interactions underlying their functionality occurring at nanometre scale. At this resolution it gets challenging to observe these interactions in living cells [48], as standard optical microscopy does not provide enough spatial resolution while electron microscopy lacks temporal dynamics and can not be easily applied to live cells. ZMWs combines with FCS offer the advantages of both high spatial and temporal resolution together with a direct statistical analysis as shown by Moran-Mirbal et al. [48](Fig. 1.6a). They showed that fluorescence from actin-eGFP correlates well with DiI-C12 fluorescence from a cell incubated on ZMW structures, indicating cellular membrane penetra-tion into nanoscale apertures. The nanoaperture works as a pinhole directly located under the cell to restrict the illumination area (Fig. 1.6b). The fluorescent markers labeled into the cell membrane give the dynamic signal while they diffuse, which is analyzed by correlation spec-troscopy to extract information about membrane organization (Fig. 1.6c,d) [49–51]. Wenger et al. [52] provided more insight about the membrane organization by performing measurements with increasing diameters. It was shown that fluorescent chimeric ganglioside proteins parti-tion into 30 nm structures inside the cell membrane. Apart from the translational diffusion, the stoichiometry of nicotinic acetylcholine and P2X2 ATP receptors isolated in membrane portions inside zero-mode waveguides was analysed using single-step photobleaching of green fluorescent protein incorporated into individual subunits [53].
Figure 1.5: Application of zero-mode waveguides to single-molecule real-time DNA sequenc-ing.(a) Principle of the experiment: a single DNA polymerase is immobilized at the bottom of a ZMW, which enables detection of individual phospholinked nucleotide substrates against the bulk solution background as they are incorporated into the DNA strand by the polymerase.
(b) Schematic event sequence of the phospholinked dNTP incorporation cycle, the lower trace displays the temporal evolution of the fluorescence intensity. (c) Section of a fluorescence time trace showing 28 incorporations events with four color detection. Pulses correspond to the least-squares fitting decisions of the algorithm [45].
Performing live cell investigation requires cell membranes to adhere to the substrate. It depends on the membrane lipidic composition [50], and on actin filaments [48]. To further ease cell adhesion, and avoid membrane invagination issues, planarized 50 nm diameter apertures have been recently introduced [54]. The planarization procedure fills the aperture with fused silica, to achieve no height distinction between the aperture and the surrounding metal.

Plasmonic control of the fluorescence directivity

ZMWs provide a new pathway of directional control on the emitted light by adding concen-tric surface corrugations (or grooves) (Fig 1.7), while preserving the light localization inside the nanoaperture. Corrugated aperture have been reported to provide high fluorescence enhancement together with beaming of the fluorescence light into narrow cone [56,57]. The fluorescence light from single molecules can thus be efficiently collected with a low numerical aperture objective, releasing the need for complex high numerical aperture objectives. By tuning the geometrical properties of the corrugation design, the fluorescence directionality can be controlled, [55,58] which offers photon sorting abilities from nanoscale volumes. Also, Cell membranes have been outlined (lightgray), and aperture locations have been circled. Cell membrane spanning a nanoaperture dips down (arrow), suggesting membrane invagination. The scale bar is 500 nm [48]. (b) Fluorescence micrographs of cells labelled with DiI-C 12 membrane probe through 280 nm aluminum apertures [48]. (c) Normalized FCS correlation functions and numerical fits (thick lines) obtained for the FL-GM1 ganglioside lipid analog, demonstrating a significant diffusion time reduction in the nanoaperture [52].

Table of contents :

1 Photonic approaches to detect single molecule fluorescence at physiological concentration
1.1 Introduction: Single molecule fluorescence spectroscopy
1.2 Improving singlemolecule fluorescence detection
1.2.1 Methods by structuring the laser excitation beam
1.2.2 Methods using photonic structures
1.3 Plasmonic Nanoapertures: ZeroModeWaveguides (ZMWs)
1.4 Overview of plasmonic antennas under research
1.5 Conclusion
2 Light matter interaction at nanoscale 
2.1 Optical properties of bulkmetals
2.1.1 Dielectric functions of free electron metals
2.1.2 Interband transitions
2.1.3 Skin depth of metals
2.2 Localized surface plasmon polariton
2.3 Optical antennas
2.3.1 Field enhancement
2.3.2 Decay rates emission close to a nanoantenna
2.3.3 Optical antenna design rules
2.4 Applications of optical antennas
3 Experimental techniques 
3.1 Fluorescence Correlation Spectroscopy (FCS)
3.2 Time Correlated Single Photon counting (TCSPC)
3.2.1 Experimental Realization
3.3 Fluorescence characterization procedure in the vicinity of nanoantenna
3.4 Low quantumyield effect
4 NanoAntenna-in-box design to enhance single molecule fluorescence detection 
4.1 Fabrication of Nanoantenna-in-box
4.2 Numerical Simulations
4.3 Experimental Setup andMethodology
4.4 Experimental Results
4.5 Applications of Nanoantenna-in-box
4.6 Conclusion
5 Self Assembly of gold nanoparticles for enhanced single molecule detection 69
5.1 Individual Gold nanoparticles
5.1.1 Materials andMethods
5.1.2 FCS analysis in the near-field of a single metal nanoparticle
5.1.3 Results and Discussion
5.2 Gold nano-dimers and trimers
5.2.1 Sample Preparation
5.2.2 Numerical simulation and spectral analysis
5.2.3 FCS analysis in the near field of gold nano-dimers and trimers
5.2.4 Results and Discussion
5.3 Conclusion


Related Posts