Robust single-particle tracking in live-cell time-lapse sequences

However, it was only demonstrated within fixed organoids and combined with direct Stochastic Optical Reconstruction Microscopy dSTORM [ 24 ] using AlexaFluor which possesses optimal blinking properties for this application [ 28 ]. Here, we demonstrate that SELFI can be used in combination with single quantum dots Qdot tracking or with the prevalent super-localization microscopy approaches developed to study live samples, using different fluorescent molecules.

Finally, we show that 3D single Qdot tracking can be performed at video rate with SELFI, several tenths of micrometers deep in organotypic brain slices. The SELFI module described in more details in the results section is used to record single fluorescent emitter images and includes a homemade diffraction grating based self-interferometer. Figure 1 shows a scheme of the setup and summarizes the different illumination possibilities. A homemade Labview program National Instrument is used to drive the acquisition setup, analyze the interferograms to retrieve the 3D position of each emitter, and render the reconstructed 3D super-resolved images.

Figure 1. DM, dichroic mirror; f 1 , mm achromatic doublet; f 2 , mm achromatic doublet. The cells were detached with 0. PALM imaging was performed with 60 ms integration time to ensure high single molecule localization precision during 15 min. After 1—3 days, cells were plated at low confluency on coverslips coated with poly-L-lysin with nm gold nanobead embedded inside Sigma-Aldrich for 3D microscope stabilization [ 30 ]. A second coating was then made with Fibronectin Sigma-Aldrich to optimize cell adhesion.

The fluorescence is excited under highly oblique illumination. Organotypic slice cultures were prepared as previously described [ 31 ]. After 25 min of incubation, slices were transferred on white FHLC membranes 0. The medium was changed every 2—3 days. Organotypic were incubated with unfunctionalized QDs 1 h before experiment. To measure the 3D position of a single emitter with nanometric precision, we use a self-referenced interferometer placed in the detection path of the microscope. The interferometer allows the simultaneous measurement of the phase and intensity of a fluorescent beam with negligible photon loss.

Briefly, the diffraction orders of the grating interfere after a small lateral shearing and the interference pattern of each single molecule image i. The light curvature will thus be encoded into interferogram frequency modulations. This curvature directly carries the distance between the light source here a single fluorescent emitter and the imaging plane where the interferometer registers the interference fringes.

Analysis of the interferogram in the frequency domain thus allows quantitative measurement of the axial localization of the emitter with respect to the imaging plane of the microscope. On the other hand, its lateral localization can be retrieved by applying a low pass filter on I z to remove the modulation component -keeping only the envelope PSF z - and then fitting to a Gaussian, as the PSF can be considered as free of aberration. In a standard experiment, a camera raw image contains one or multiple PSFs sub-structured by interferences see Figure 1.

By operating in the single molecule regime where each elemental PSF can be isolated from the others, one can independently retrieve the 3D localization of each molecule. They are activated at the cell-surface following binding with epidermal growth factors EGFs to their extracellular domain, inducing signaling pathways that are important e.

Indeed, upon EGF binding, EGFR dimerization, and tyrosine autophosphorylation occur, which activates intracellular signaling cascades that can lead to phenotypic changes such as increased proliferation and migration. EGFR distributions, activation and signaling pathways have been extensively studied using fluorescent microscopy at both ensemble [ 32 , 33 ], single-molecule levels [ 34 — 36 ], and also using super-resolution imaging [ 7 ]. These studies revealed for instance the transient distribution of EGFR dimers at the cellular membrane immediately after EGF binding and before their endocytosis.

Interestingly, using uPAINT microscopy, we were also able to image on live cells the super-resolved localization of panitumumab, a human monoclonal antibody highly specific to EGFRs and widely used in cancer treatments. This antibody impedes EGF binding and the subsequent signaling cascades. Indeed, uPAINT relies on stochastically imaging in real-time ligand binding with target receptors at the single molecule level using highly oblique illumination [ 37 ]. For this, we applied fluorescent panitumumab on A cells, an epidermoid carcinoma cell line which abnormally overexpresses EGFR.

We coupled panitumumab to AttoN, a red fluorescent dye with excellent photophysical properties well-established in single molecule research.

1. Introduction

In practice, immediately after the beginning of recording and while cells were illuminated with a nm laser beam, fluorescent panitumumab was introduced at low concentration 0. The super-resolved image presented in Figure 2a displays the 3D localizations of the EGFR population targeted by panitumumab at the membrane of live cells. For comparison, a diffraction limited image was generated in Figure 2b by pooling diffraction limited detections of fluorescent panitumumab.

Interestingly, local inhomogeneities in axial localizations can be seen and be related to local cell membrane topography. This observation indicates that panitumumab is able to efficiently target EGFR in different areas of the cell, including in highly mobile and dynamic membrane compartments. Figure 2. The nano-topography of the cell is clearly visible.

The white arrow indicates membrane ruffling linked to an increase in height at the edge of the cell. Cells adhere and generate forces on the extracellular matrix through integrin-dependent adhesion sites, in particular mature Focal adhesion FAs. While quite stable at the macroscale, those macromolecular assemblies are highly dynamic and well-organized at the molecular level [ 11 , 38 ]. Here, we wanted to determine whether SELFI is also able to quantify the height of different proteins constituting FAs with a much more convenient one-objective imaging scheme and importantly, within live cells.

For this purpose, we use fluorescent molecules adsorbed on the coverslip and detected outside of the cells. By subtracting the coverslip position to the raw protein localizations we could generate the absolute values of the localizations with respect to the coverslip position. Note that for durable determination of coverslip position, fluorescent particles can be embedded within the coating layer fibronectin. This analysis clearly shows that in live cells, Paxillin, and Talin-C are almost exclusively detected in FAs with Paxillin being closer to the surface than Talin-C.

Figure 3. Being able to study the dynamics of proteins in living samples is key to understand the complex molecular processes implicated in biological systems. In this quest, single particle tracking SPT has been largely performed on dissociated cells plated on coverslips [ 7 , 37 , 39 — 41 ], while SPT in intact living tissues is only emerging [ 10 , 42 , 43 ]. Sign in.

Get my own profile Cited by View all All Since Citations h-index 15 12 iindex 16 Peter K. Sorger Harvard Medical School Verified email at hms. Verified email at utsouthwestern. Articles Cited by Co-authors. Nature Reviews Molecular Cell Biology 7 11 , , Journal of structural biology 2 , , The Journal of cell biology 6 , , Cold Spring Harbor Protocols 12 , pdb. Journal of computational chemistry 23 4 , , The Journal of chemical physics 2 , , Biomedical Imaging: From Nano to Macro, The output is then sent through a pulse picker Conoptics to reduce the repetition rate by a factor of 9.

The sample dispersed on a microscope slide was placed on a piezo scanning stage Mad City Laboratories , where laser illumination was focused to a diffraction-limited spot on the slide through the microscope objective NA 1. A confocal ray path was created from a pinhole in the image plane where only the signal from the focal plane is transmitted.

Single QDs on the slide were located through point-by-point scanning of the laser beam across the sample by the piezo scanner. Photoluminescence spectra were acquired using a spectrofluorometer from Photon Technology International. Quantum yields were obtained by using a standard of fluorometric grade Rhodamine 6G R6G dye dissolved in methanol. The mean particle size was determined by inspecting different particles through the use of the analysis software ImageJ. Once the reaction is complete, the disulfide bond of the linker is reduced through adding an excess of DTT.

In a separate vial, to introduce thiol reactive groups of maleimido to the antibody, the amine groups of anti-EGFR Invitrogen 31G7 were activated by reacting with sulfo-SMCC at a ratio of 10 linking agents to 1 antibody. A region of interest was selected based on the bright field image of the cell.

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Once this region was chosen, the fluorescence image was then taken to observe the QDs present in the area. Once a well isolated QD had been spotted, real time fluorescence image recording was performed at 33 frames per second with a CCD camera Hamamatsu C The total duration of an imaging experiment did not exceed 15 min to ensure cell viability when tracking the surface receptors. Results and Discussion. The composition and structure of the multishell domains were chosen to simultaneously minimize interfacial lattice strain and to confine the charge carriers in a deep potential well.

However, due to a general trend of decreasing bond length with increasing bandgap, greater insulation also increases the lattice mismatch with the core material, leading to a compressive strain in the core and a net tensile strain in the shell. Beyond a critical shell thickness, the strain energy will become large enough to cause the formation of lattice defects such as loop dislocations that act as carrier traps and Auger recombination sites, leading to reduced radiative excitonic recombination. Core CdSe nanocrystals with a diameter of 2.

We used highly reactive shell precursors, diethylzinc, dimethylcadmium, and TMS 2 Se, to enable efficient shell growth at low temperatures that are needed to prevent Ostwald ripening of the small cores, to balance cation activity, and to promote isotropic growth. Commonly used metal-oleates and TOP-Se typically require temperatures higher than the temperature at which CdSe ripens, and the reactivities of zinc-oleate and cadmium-oleate are drastically different.

The major ligand during shell growth is initially the secondary amine DOA, a softer Lewis base than primary amines. DOA balances the reactivity between zinc and cadmium precursors at the initial stages of shell growth, as zinc has a higher affinity toward primary amines and only weakly binds to the QD surface atoms to allow efficient shell growth at low temperatures. As subsequent shells are deposited with increasing zinc content, more primary amine is introduced into the system, since the cation precursors are dissolved in oleylamine, which becomes the major ligand on the nanocrystal surface.

Primary amines are favored in the later stages of shell growth as they provide enhanced binding strength to the nanocrystal surface, affording greater colloidal stability beneficial for dispersion of larger particles with lower surface energy. Shell precursors were added to the reaction vessel via syringe pump, and reaction progress was monitored by UV—vis, emission spectrophotometry, and transmission electron microscopy. After 14 monolayers of growth, chemical composition was determined through X-ray photoelectron spectroscopy see Figure S1 of the Supporting Information.

The final graded alloy QDs were highly crystalline, nearly spherical with a size of Figure 2 c shows that there is decreasing emission energy as more monolayers of shell material were added, while there is an increase in the absorption at shorter wavelengths. This is an indication of the charge carrier wave functions increasing in volume less confined , along with an increase in the absorption cross section due to larger particle size more oscillators.

The energy profile with monolayer additions is qualitatively similar to that of what would be expected from theoretical calculations based on the effective mass approximation see Figure 2 d. Particles solvated in hexane solution were spun onto glass microscope slides and were observed under ambient conditions. Single QDs on the slide were located through point-by-point scanning of a focused laser beam and were further verified with atomic force microscopy see Figure S3 of the Supporting Information.

As shown in Figure 3 panels a and b , there is a drastic change in total time spent between emissive and nonemissive states between the two samples. The cores without a shell are mostly in the off state during illumination, with few brief on states, whereas after shell growth the particles spend most of the time in the on state with few short off states.

In addition, the infrequent off times were of short durations, typically less than ms see Figure 3 f. Such monoexponential lifetimes of the QD sample can be indicative of a high fraction of the probes being primarily in on states during excitation. Fisher et al. For application of QDs in biological experiments, the particles synthesized in organic solvents must be transferred to an aqueous phase. Decreases in quantum yield are typically observed upon ligand exchange in the aqueous solubilization of QDs; however, fluorescence efficiencies can be maintained if the shell potential barrier is sufficient to keep exciton carriers insulated within the core.

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The QDs coated with three different hydrophilic ligands were also compared in blinking behavior. Remarkably, the blinking suppression was invariant to the ligands in both the frequency and duration of the off times see Figure 4. The distributions of ensemble on time percentages were similar for QDs cast from hexane solution To our knowledge, this is the first QD synthesis method that is able to preserve the optical properties of quantum yield and blinking suppression after aqueous transfer using a variety of ligands.