Physiological monitoring of optically trapped cells: assessing the effects of confinement by 1064-nm laser tweezers using microfluorometry.

We report the results of microfluorometric measurements of physiological changes in optically trapped immotile Chinese hamster ovary cells (CHOs) and motile human sperm cells under continuous-wave (CW) and pulsed-mode trapping conditions at 1064 nm. The fluorescence spectra derived from the exogenous fluorescent probes laurdan, acridine orange, propidium iodide, and Snarf are used to assess the effects of optical confinement with respect to temperature, DNA structure, cell viability, and intracellular pH, respectively. In the latter three cases, fluorescence is excited via a two-photon process, using a CW laser trap as the fluorescence excitation source. An average temperature increase of < 0.1 +/- 0.30 degrees C/100 mW is measured for cells when held stationary with CW optical tweezers at powers of up to 400 mW. The same trapping conditions do not appear to alter DNA structure or cellular pH. In contrast, a pulsed 1064-nm laser trap (100-ns pulses at 40 microJ/pulse and average power of 40 mW) produced significant fluorescence spectral alterations in acridine orange, perhaps because of thermally induced DNA structural changes or laser-induced multiphoton processes. The techniques and results presented herein demonstrate the ability to perform in situ monitoring of cellular physiology during CW and pulsed laser trapping, and should prove useful in studying mechanisms by which optical tweezers and microbeams perturb metabolic function and cellular viability.     

Charge-coupled device imaging of rapid calcium transients in cultured arterial smooth muscle cells

Transient changes in the concentration of intracellular free calcium are associated with the transduction of primary signals and the subsequent employment of Ca2+ as a second messenger in a multitude of cell types. These transients, typically monitored with the calcium-sensitive fluorescent dye Fura-2, are known to occur with a time course in the order of seconds. In order to accurately monitor such rapid changes in intracellular free calcium concentration in both single cells and simultaneously in several cells in a single field, we have developed a digital fluorescence imaging system based on a charge-coupled device (CCD) camera. We report here on the detailed kinetics of calcium increases in cultured arterial swine smooth muscle cells in response to the agonist ATP.     

Automatic annular laser trapping: a system for high‐throughput sperm analysis and sorting

An automatic microscope system is designed to study the response of sperm motility to an annular laser trap. A continuous annular laser trap provides a parallel way to analyze and sort sperm based on their motility and to study the effects of laser radiation, optical force and external obstacles. In the described automatic microscope system, the phase contrast images of swimming sperm are digitized to the computer at video rates. The microscope stage is controlled in real‐time to relocate the sperm of interest to the annular trap with a normal or tangential entering angle. The sperm is continuously tracked and the swimming behavior is identified. Using this system, parallel sorting on human and gorilla sperm are achieved and threshold power levels separating the “fast” group and the “slow” group are compared for those two species. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

2D and Trap‐Assisted 2D Langevin Recombination in Polymer:Fullerene Blends

The impact of trapping on the recombination dynamics in polymer:fullerene blends is clarified using the highly ordered bulk heterojunction (BHJ) blend poly[2,5‐bis(3‐tetradecylthiophen‐2‐yl)thieno[3,2‐b]thiophene] (PBTTT) and [6,6]‐phenyl‐C61‐butyric acid methyl ester (PCBM) at different weight ratios as a model system. The recombination dynamics are determined using both transient charge extraction and steady‐state techniques. The results show that both the decay of photogenerated charge and the light ideality factor at a polymer:fullerene weight ratio of 1:4 are fully consistent with 2D Langevin recombination; in the 1:1 case the recombination is seen to be affected by electron trapping. The theory of 2D Langevin recombination is extended to the case with high trap density in agreement with the observations in the 1:1 case. The recombination capture coefficients are derived both for trap‐assisted and band‐to‐band recombination and it can be seen that anisotropic charge transport reduces the capture coefficients in both cases resulting in a reduced overall recombination.     

Avoiding verisimilitude when modelling ecological responses to climate change: the influence of weather conditions on trapping efficiency in European badgers (Meles meles)

The signal for climate change effects can be abstruse; consequently, interpretations of evidence must avoid verisimilitude, or else misattribution of causality could compromise policy decisions. Examining climatic effects on wild animal population dynamics requires ability to trap, observe or photograph and to recapture study individuals consistently. In this regard, we use 19 years of data (1994–2012), detailing the life histories on 1179 individual European badgers over 3288 (re‐) trapping events, to test whether trapping efficiency was associated with season, weather variables (both contemporaneous and time lagged), body‐condition index (BCI) and trapping efficiency (TE). PCA factor loadings demonstrated that TE was affected significantly by temperature and precipitation, as well as time lags in these variables. From multi‐model inference, BCI was the principal driver of TE, where badgers in good condition were less likely to be trapped. Our analyses exposed that this was enacted mechanistically via weather variables driving BCI, affecting TE. Notably, the very conditions that militated for poor trapping success have been associated with actual survival and population abundance benefits in badgers. Using these findings to parameterize simulations, projecting best‐/worst‐case scenario weather conditions and BCI resulted in 8.6% ± 4.9 SD difference in seasonal TE, leading to a potential 55.0% population abundance under‐estimation under the worst‐case scenario; 38.6% over‐estimation under the best case. Interestingly, simulations revealed that while any single trapping session might prove misrepresentative of the true population abundance, due to weather effects, prolonging capture–mark–recapture studies under sub‐optimal conditions decreased the accuracy of population estimates significantly. We also use these projection scenarios to explore how weather could impact government‐led trapping of badgers in the UK, in relation to TB management. We conclude that population monitoring must be calibrated against the likelihood that weather conditions could be altering trap success directly, and therefore biasing model design.     

High-speed digital microscopy

High-speed imaging is an ideal technique to accurately resolve the temporal and spatial characteristics of rapid events at either the molecular or cellular level. In this article, the digital imaging techniques used to simultaneously acquire transillumination phase-contrast images, at 240 images s(-1) (high-speed), to characterize ciliary beat frequency, and fluorescence images, at 30 images s(-1) (fast), to measure intracellular calcium concentration ([Ca2+]i), are described. With this technique, a precise correlation between the changes in ciliary beat frequency with changes in [Ca2+]i can be made. Simultaneous imaging is achieved by using different wavelengths of light to form the phase-contrast and fluorescent images and selectively directing these light wavelengths to different cameras with dichroic mirrors and bandpass filters. High-speed images compatible with standard video recording equipment are obtained by prematurely resetting the raster scan of a CCD camera with additional vertical synchronization pulses. The fast [Ca2+]i images are determined using the ratiometric dye fura-2 and a recording technique that monitors rapid changes in fluorescence at a single wavelength and uses intermittent reference images for calibration.     

Versatile laser‐based cell manipulator

Here we describe a two‐photon microscope and laser ablation setup combined with optical tweezers. We tested the setup on the fission yeast Schizosaccharomyces pombe, a commonly used model organism. We show that long‐term imaging can be achieved without significant photo‐bleaching or damage of the sample. The setup can precisely ablate sub‐micrometer structures, such as microtubules and mitotic spindles, inside living cells, which remain viable after the manipulation. Longer exposure times lead to ablation, while shorter exposures lead to photo‐bleaching of the target structure. We used optical tweezers to trap intracellular particles and to displace the cell nucleus. Two‐photon fluorescence imaging of the manipulated cell can be performed simultaneously with trapping. The combination of techniques described here may help to solve a variety of problems in cell biology, such as positioning of organelles and the forces exerted by the cytoskeleton. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Controlled rotation of biological microscopic objects using optical line tweezers

Controlled, continuous rotation of cells or intracellular objects was achieved using optical tweezers with an elliptic beam profile (line tweezers), which was generated by placing a cylindrical lens in the path of the trapping beam. By rotating the cylindrical lens, rotation of the elliptic trapping beam and hence of the object trapped therein was achieved. Compared to previously reported techniques for rotation of microscopic objects, this approach is much simpler, gives better utilization of available laser power and also allows much easier control of the trap beam profile. We have used this approach for rotation of biological objects varying in size from 2 to 40 m. At 25 mW trapping beam power at the object plane E. coli bacteria could be rotated at speeds approaching 10 Hz and an intracellular object (presumably a calcium oxalate crystal) trapped inside Elodea densa plant cell could be rotated with speeds of up to 4 Hz. To our knowledge, this is the first report for rotation of an intracellular object.     

Real‐time imaging of single cancer‐cell dynamics of lung metastasis

We have developed a new in vivo mouse model to image single cancer‐cell dynamics of metastasis to the lung in real‐time. Regulating airflow volume with a novel endotracheal intubation method enabled controlling lung expansion adequate for imaging of the exposed lung surface. Cancer cells expressing green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm were injected in the tail vein of the mouse. The right chest wall was then opened in order to image metastases on the lung surface directly. After each observation, the chest wall was sutured and the air was suctioned in order to re‐inflate the lung, in order to keep the mice alive. Observations have been carried out for up to 8 h per session and repeated up to six times per mouse thus far. The seeding and arresting of single cancer cells on the lung, accumulation of cancer‐cell emboli, cancer‐cell viability, and metastatic colony formation were imaged in real‐time. This new technology makes it possible to observe real‐time monitoring of cancer‐cell dynamics of metastasis in the lung and to identify potential metastatic stem cells. J. Cell. Biochem. 109: 58–64, 2010. © 2009 Wiley‐Liss, Inc.     

Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images. Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3]. Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons. One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, and maintaining general cell health. Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons. A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde. Our approach to imaging GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events. The imaging approach described here is flexible for a variety of imaging applications and can be adopted by a laboratory for relatively little cost provided a microscope is available.     

Cell calcium of ejaculated rabbit spermatozoa before and following in vitro capacitation

Ejaculated rabbit spermatozoa were loaded with the calcium-selective fluorescent indicator quin-2. Measurements of trypan blue exclusion indicated that cell viability was not affected by quin-2 loading. The concentration of intracellular free calcium of quin-2 loaded sperm was calculated to be 144 +/- 14 nM. Spermatozoa capacitated in vitro either before or following quin-2 loading had intracellular free calcium levels similar to that of non-capacitated sperm. These studies indicate that the concentration of intracellular free calcium of ejaculated rabbit spermatozoa does not change as a result of in vitro capacitation.     

Spatiotemporal relationships among early events of fertilization in sea urchin eggs revealed by multiview microscopy.

Four early events of egg fertilization, changes in intracellular calcium concentration and intracellular pH, reorientation of the surface membrane, and the elevation of the fertilization envelope, were imaged in real time and in pairs in single sea urchin eggs. The paired imaging allowed the correlation of the four events spatially and temporally. Three of them propagated as waves starting at the sperm entry site. The earliest was the calcium wave, visualized with fluorescent indicator dyes. After a delay of 10 s there followed a large decrease in the fluorescence polarization of membrane-bound dyes, which we interpret as arising from membrane reorientation as a result of cortical granule exocytosis and microvillar elongation. With a further delay of 15 s the fertilization envelope was seen to rise in transmitted light. All three waves propagated with similar velocities of approximately 10 microns/s, supporting the view that calcium triggers the latter two events. The fluorescence polarization changed in two steps with a clear pause of 10-20 s in between. The second step, which also propagated as wave, reflects either further elongation of microvilli or straightening of irregular microvilli. This second step was abolished by cytochalasin B and was coincident with an increase in cytoplasmic pH, suggesting that pH-induced actin reorganization may play a role. The cytoplasmic alkalinization, imaged with a fluorescent probe, was quite different from the other events in that it took place homogeneously throughout the egg and slowly (over 100 s). Apparently, the alkalinization is not on a direct downstream pathway of calcium origin. An opposing possibility, that the alkalinization may in fact be triggered by the traveling calcium wave, is also discussed.     

Detection of cooling-induced membrane changes in the response of boar sperm to capacitating conditions

There is a need for methods of rapid and sensitive sperm function assessment. As spermatozoa are not able to fertilize an oocyte before having undergone a series of complex physiological changes collectively called capacitation, it is logical to assess sperm function under fertilizing conditions in vitro. In this study, the responsiveness of sperm to capacitating conditions in vitro was monitored by changes in sperm response to ionophore and by changes in the amount of intracellular calcium ions in stored boar semen. Boar semen was diluted at 32 and 20 degrees C and stored for 24 and 72 h at 16 and 10 degrees C. Ionophore-induced changes and increased intracellular calcium ion content in boar spermatozoa were recorded by flow cytometry and found to progress as a function of time during incubation under capacitating conditions. All responsiveness parameters (increases in proportions of membrane-defective spermatozoa, acrosome-reacted spermatozoa, and cells with high intracellular calcium levels) were shown to be sensitive to subtle physiological changes occurring at low storage temperatures. The initial levels of sperm with a high calcium content were higher in semen stored at 10 degrees C, but the accumulation of internal calcium was lower than in semen stored at 16 degrees C. The loss of membrane integrity and increase in the proportion of acrosome-reacted cells were higher in semen stored at 10 degrees C. Dilution at 20 degrees C had no negative effect on membrane integrity or responsiveness to capacitating conditions. There was no significant difference between semen stored for 24 and 72 h in terms of membrane integrity, acrosome reaction, and intracellular calcium after capacitation treatment. However, dynamics of cell death and acrosome reaction in response to capacitating conditions were somewhat accelerated after 72 h storage, especially in semen stored at 10 degrees C. It can be concluded that the simultaneous use of the sperm membrane responsiveness and kinetic parameters is a sensitive tool for the detection of storage-related membrane changes in boar semen.     

Calcium, mitochondria and apoptosis studied by fluorescence measurements

Among the many unsolved problems of calcium signalling, the role of calcium elevations in apoptotic and necrotic cell death has been a focus of research in recent years. Evidence has been presented that calcium oscillations can effectively trigger apoptosis under certain conditions and that dysregulation of calcium signalling is a common cause of cell death. These effects are regularly mediated through calcium signal propagation to the mitochondria and the ensuing mitochondrial membrane permeabilization and release of pro-apoptotic factors from mitochondria to the cytoplasm. The progress in this area depended on the development of (1) fluorescent/luminescent probes, including fluorescent proteins that can be genetically targeted to different intracellular locations and (2) the digital imaging technology, fluorescence-activated cell sorting and fluorescent high throughput approaches, which allowed dynamic measurements of both [Ca2+] in the intracellular compartments of interest and the downstream processes. Fluorescence single cell imaging has been the only possible approach to resolve the cell-to-cell heterogeneity and the complex subcellular spatiotemporal organization of the cytoplasmic and mitochondrial calcium signals and downstream events. We outline here fluorometric and fluorescence imaging protocols that we set up for the study of calcium in the context of apoptosis.     

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis¹. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol². It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology³. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)⁴ or aequorin⁵ targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of ''ratiometric-pericam'' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin⁴. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 μM⁴. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.     

Dynamic fluorescence anisotropy imaging microscopy in the frequency domain (rFLIM)

We describe a novel variant of fluorescence lifetime imaging microscopy (FLIM), denoted anisotropy-FLIM or rFLIM, which enables the wide-field measurement of the anisotropy decay of fluorophores on a pixel-by-pixel basis. We adapted existing frequency-domain FLIM technology for rFLIM by introducing linear polarizers in the excitation and emission paths. The phase delay and intensity ratios (AC and DC) between the polarized components of the fluorescence signal are recorded, leading to estimations of rotational correlation times and limiting anisotropies. Theory is developed that allows all the parameters of the hindered rotator model to be extracted from measurements carried out at a single modulation frequency. Two-dimensional image detection with a sensitive CCD camera provides wide-field imaging of dynamic depolarization with parallel interrogation of different compartments of a complex biological structure such as a cell. The concepts and technique of rFLIM are illustrated with a fluorophore-solvent (fluorescein-glycerol) system as a model for isotropic rotational dynamics and with bacteria expressing enhanced green fluorescent protein (EGFP) exhibiting depolarization due to homotransfer of electronic excitation energy (emFRET). The frequency-domain formalism was extended to cover the phenomenon of emFRET and yielded data consistent with a concentration depolarization mechanism resulting from the high intracellular concentration of EGFP. These investigations establish rFLIM as a powerful tool for cellular imaging based on rotational dynamics and molecular proximity.     

Real-time multi-wavelength fluorescence imaging of living cells

We describe a new real-time fluorescence video microscope design for capturing intensified images of cells containing dual wavelength "ratio" dyes or multiple dyes. The microscope will perform real-time capture of two separate fluorescence emission images simultaneously, improving the time resolution of spatial distribution of fluorescence to video frame rates (30 frames/s or faster). Each emission wavelength is imaged simultaneously by one of two cameras, then digitized, background corrected and appropriately combined at standard video frame rates to be stored at high resolution on tape or digital video disk for further off-line analysis. Use of low noise, high sensitivity image intensifiers, coupled to CCD cameras produce stable, high contrast images using ultra low light levels with little persistence or bloom. The design has no moving parts in its optical train, which overcomes a number of technical difficulties encountered in the present filter wheel designs for multiple imaging. Coupled to compatible image processing software utilizing PC-AT computers, the new design can be built for a significantly lower cost than many presently available commercial machines. The system is ideal for ratio imaging applications; the software can calculate the ratio of fluorescence intensities pixel by pixel and provide the information to generate false-color images of the intensity data as well as other calculations based on the two images. Thus, it provides a powerful, inexpensive tool for studying the real-time kinetics of changes in living cells. Examples are presented for the kinetics of rapidly changing intracellular calcium detected by the calcium indicator probe indo-1 and the redistribution kinetics of multiple vital dyes placed in cells undergoing cell fusion.     

Calcium mobilization and catecholamine secretion in adrenal chromaffin cells. A Quin-2 fluorescence study

To better understand the relation between cell calcium and exocytotic secretion, a quantitative dependence of adrenal catecholamine secretion on cytosolic free calcium has been determined for isolated, intact, bovine chromaffin cells, using the fluorescent probe Quin-2. The cells required a threshold of 250-300 nM cytosolic calcium to be reached before detectable secretion occurred and half-maximal secretion occurred near 2 microM cytosolic calcium. Nicotinic receptors mediated an increase of cytosolic calcium from resting levels near 100 nM to levels in the 1-10 microM range within seconds followed by a decay back to resting levels over several minutes. Muscarinic receptors mediated a smaller rise in cytosolic free calcium from 100 to about 200 nM, within seconds. The nicotinic response required extracellular calcium, while the muscarinic response was largely independent of extracellular calcium, suggesting the latter mobilizes intracellular calcium. The acetylcholine-evoked rise in cytosolic calcium decayed by at least two kinetically distinct processes with half-time constants: t1 = 0.6 min and t2 = 3.2 min. Extracellular Na+ deprivation caused a more prolonged elevation of the acetylcholine-evoked calcium transient, suggesting a possible role of Na+/Ca2+ exchange and/or other Na+ -dependent processes in lowering cytosolic calcium following stimulation. The possible perturbing effects of Quin-2 on resting and stimulated cytosolic calcium levels and on secretion were examined and a novel use of Quin-2 to measure membrane calcium flux was demonstrated.     

A new multiparameter flow cytometer: optical and electrical cell analysis in combination with video microscopy in flow

BACKGROUND: Flow cytometers, which are commercially available, do not necessarily meet all demands of actual biomedical research. This is the case for the investigation of mechanisms involved in cell volume regulation, which requires electrical volume measurement and ratiometric multichannel fluorescence analysis for the simultaneous assessment of different physiologic parameters (intracellular pH and the intracellular concentration of calcium ions, etc). METHODS AND RESULTS: We describe the construction of a new nonsorting flow cytometer designed for the simultaneous acquisition of seven parameters including fluorescence signals, forward and perpendicular light scatter, cell volume according to the electrical Coulter principle, and flow cytometric imaging. The instrument is equipped with three different light sources. A tunable argon-ion laser generates efficient excitation of the most standard fluorescent probes in the visible spectral range, and an arc lamp provides the means for ultraviolet excitation at low cost. Because of the spatial filtering by the excitation and detection optics, two independent sets of dual fluorescence measurements can be performed, a prerequisite for flexible ratiometric fluorescence analysis. A flow video microscope integrated into the optical system optionally generates either brightfield or phase images of selected flowing particles. Only particles whose individual datasets meet predefined gating conditions are imaged in real time. To avoid smear effects, the motion of the object to be imaged (speed approximately 8 m/s) is frozen on the target of a CCD camera by flash illumination. For this purpose, a high radiance gas discharge lamp with 25-mJ electric pulse energy provides an illumination time of 18 ns (full width half maximum). Test results obtained from latex spheres and cells are shown. CONCLUSIONS: Test results indicate that our instrument can perform Coulter measurements in combination with flexible optical analysis. Moreover, integration of an adapted video microscope into a flow cytometer is an approach to overcome the gap between flow and image cytometry.     

Imaging of single molecules in live cells

Progress in optical microscopy, combined to the emergence of new fluorescent probes and advanced instrumentation, now permits the imaging of single molecules in fixed and live cells. This extreme detection sensitivity has opened new modalities in cellular imaging. On the one hand, optical images with an unprecedented resolution in the 10-50 nm range, well below the diffraction limit of light, can be recorded. These super-resolution images give new insights into the properties of cellular structures. On the other hand, proteins, either in the membrane or intracellular, can be tracked in live cells and in physiological conditions. Their individual trajectories provide invaluable information on the molecular interactions that control their dynamics and their spatial organization. Single molecule imaging is rapidly becoming a unique tool to understand the biochemical and biophysical processes that determine the properties of molecular assemblies in a cellular context.