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Light Microscopy Techniques for Live Cell Imaging David J. Stephens, et al. Science 300, 82 (2003); DOI: 10.


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once a sample is being imaged. School of Medical Sciences. UK. In time-lapse experiments. many other potential probes that can be introduced into cells. but often overlooked. it is better to use probes with distinct excitation and emission spectra that are separable at the point of image acquisition. and there are many ways of trying to limit light-induced damage. results in a “speckled” appearance. Commercial systems are now available for “spectral unmixing” of data. Stephens1 and Victoria J. University of Bristol. snapshot view of cells.sciencemag. biologists have been fascinated by observing cells using light microscopy. but. and the usefulness of such probes is also continually driving the technology for studying intracellular dynamics. However. In addition. particularly in the presence of fluorophores (which generate free radicals upon photobleaching). allowing specific detection only after a pulse of illumination (14–16 ). The spectral variants of GFP and the unrelated red fluorescent protein (4 ) make it feasible to perform multicolor imaging of living cells. These speckles can then be imaged over time and tracked within the cell to provide accurate quantitative analysis of polymer dynamics. Environmental considerations.sciencemag. There are. as determined from biochemical and genetic experiments spanning diverse approaches from in vitro reconstitution of cellular processes to atomic resolution structure determination.allan@man. and CO2. Perhaps the commonest approach for studying dynamic cellular events is live cell fluorescence microscopy. of course. with some mention of their pros and cons. Specific fluorescent lipid molecules and organelle-specific dyes are often cell permeable and can simply be added to the culture medium (12). The simultaneous study of multiple fluorophores or ratiometric analysis of a single probe requires spectral separation of both the excitation and emission light. a number of probes can be activated by light. 2008 routines are available. .BIOLOGICAL IMAGING SPECIAL SECTION REVIEW Light Microscopy Techniques for Live Cell Imaging David J. A prime example of this is the development of fluorescence speckle microscopy (13). and the viability of the specimen. speed of acquisition. We have an incredibly detailed view of how proteins and lipids interact inside cells to govern the generation. and function of cellular organization. Light microscopy of living versus fixed samples is essentially a trade-off between acquiring images with a high signal-to-noise ratio and damaging the sample under observation. transmitted light techniques also have an important part to play (1) and not just as an adjunct to fluorescence imaging. such as a microtubule or actin filament. There are a number of solutions to this problem. It is also vital to keep the cellular environment constant. cause of focus drift is air conditioning units. these techniques only provide a static. one should consider three things: sensitivity of detection. which can compensate for focus drift to some extent. One important. including the control of temperature. Environmental control ranges from simple heating jackets to Perspex boxes that fully encase a system. Fluorescently labeled proteins can be introduced by microinjection. Finally. and we will discuss this in some detail. which can cause cyclic changes in focus as they turn on and off. Regardless of the imaging technique to be used. the cysteine residues must be in the reduced state for labeling to occur. where possible. allowing organelle-specific analysis of calcium dynamics. it is crucial to consider the cells’ health on the microscope stage. For anyone new to this area. Manchester. this is a particularly critical issue in live cell Downloaded from www. humidity. It is mediated by engineering a tetracysteine motif into the target protein and then incubating cells with a nonfluorescent biarsenical compound that becomes strongly fluorescent upon binding to this tetracysteine motif. 2School of Biological Sciences. introduction of a limited amount of fluorescent protein to a polymeric structure. The relative importance of each parameter will vary between samples. it can be daunting to decide which techniques or equipment to try. Is it thick or thin? Is the process to be observed fast or slow? Do you need to image for 82 4 APRIL 2003 VOL 300 SCIENCE www. Cells are sensitive to photodamage. Autofocus 1 Fluorescence Imaging Whilst it is sometimes possible to image endogenous cellular molecules such as NAD(P)H (2) by their inherent fluorescence. the focal plane must remain stable. minutes. The advent of fluorescent labeling technologies plus the plethora of sophisticated light microscope techniques now available make studying dynamic processes in living cells almost commonplace. maintenance. BS8 1TD. Oxford Road. Being able to observe processes as they happen within the cell by light microscopy adds a vital extra dimension to our understanding of cell function. Other important questions center on the sample you want to image. or days. GFP-tagging is also being applied to high-throughput analyses to provide further functional annotation of genome sequences (8). it is far more common to introduce exogenous fluorescent molecules. on July 4. but the overriding concern for all three is stability. M13 9PT. and this allows the use of closely related fluorophores. Email: viki. FlAsH (fluorescent arsenical helix binder) labeling provides another means for fluorescent labeling of genetically encoded probes Live Cell Imaging When selecting which system to use for imaging living cells. (9). GFP-based biosensors are opening many fields to optical techniques. University of Manchester. *To whom correspondence should be addressed. The field of calcium imaging also makes use of GFP-based probes (7 ). The advent of green fluorescent protein (GFP) technology has revolutionized live cell imaging because an autofluorescent molecule can be genetically encoded as a fusion with the cDNA of interest (3). but they require additional illumination of the sample. however. Allan2* Since the earliest examination of cellular structures. and at how many different wavelengths does the image need to be sampled? How bright is your signal? You also need to consider several further ques- Department of Biochemistry. notably the spatio-temporal analysis of signaling events following the development of probes for diverse processes including heterotrimeric G protein activity (5) and phosphoinositide signaling using GFP-tagged pleckstrin homology (PH) domain constructs (6 ). Unfortunately FlAsH compounds can also label endogenous proteins containing similar tetracysteine motifs (11). Bristol. we aim to give a brief overview of the main approaches to live cell imaging. University Walk. and antidotes must be added simultaneously with labeling to avoid toxicity problems. Here. Here. hours. A recent development enables multicolor labeling and photoconversion of diaminobenzidine for correlative electron microscopy (10).

A good understanding of the pros and cons of different microscopes is needed. omitting phenol red and serum from the medium (again. and if so. The basic features of three types of fluorescence microscope systems are illustrated in Fig. and it is also helpful to understand the resolution of the light microscope (17 ). A minimum requirement is the ability to shut off illumination light when it is not needed. scanning confocal. Scanning speed in confocal microscopy can also be improved with the use of multifocal imaging (20). Because illumination of fluorophores causes photobleaching and therefore cell damage. the excitation light Fig. the lower the illumination intensity needed. www. Care should also be taken to remove unwanted wavelengths of light and not to rely simply on the excitation on July 4. one may obtain faster scanning by using resonant galvanometers (which are optional on many commercial systems).sciencemag. if using a confocal) will be vital (18). Here. although this process reduces image resolution. providing the cells tolerate oxygen withdrawal. so high numerical aperture objectives should be used. Comparison of widefield. sensitive back-illuminated charge-coupled device (CCD) cameras with thinned chips are available. Alternatively. no single microscope system will be best. 1). emission beams. filters. Monochromator-based systems have the advantage of rapid switching between excitation wavelengths (typically Ͻ3 ms) but suffer from reduced illumination intensity. if your cells will stand it) will help reduce background fluorescence. 2008 . and there should be as few optical elements in the light path as possible. Typical system configurations are shown. and spinning disk confocal systems. with schematics of each. The system must also make best possible use of the light. 1. Using an intensified camera is one way of increasing sensitivity. at the expense of increasing noise in the image.sciencemag. Filter wheel configurations usually have higher light throughput but are often slower in switching. particularly when multiple fluorophores are imaged simultaneously or when a single probe is analyzed ratiometrically. of what quality? In many cases. whereas CCD cameras acquire a whole field of view at once. All systems are capable of being equipped for 3D and 4D data acquisition. because the more sensitive the detector. or output from a monochromator will slow data acquisition (Fig. Data acquisition rates of conventional scanning confocal microscopes are fast enough for rapid imaging if only small regions are sampled. principally due to fiber optic coupling to the microscope.BIOLOGICAL IMAGING SPECIAL SECTION tions: Will you want to use a specialized technique such as photobleaching? Are transmitted light images required. and compromises will have to be made. Limiting cell damage. Oxygen can be removed from the medium as long as the cells are in a sealed chamber (13).org SCIENCE VOL 300 4 APRIL 2003 83 Downloaded from www. Switching between laser lines. Excitation beams are shown in green. 1. The differences between these systems mean that no single system is suited for every experiment. Reducing the level of oxygen can help reduce photobleaching and free radical production. The sensitivity of the camera (or photomultiplier tube. A new type of camera that amplifies the CCD readout signal on the chip (19) offers further possible advantages. this is inherent in confocal systems and can easily be achieved for widefield systems that use electronic shutters controlled by computer (which will usually control the image acquisition as well). A key consideration is speed of data acquisition. To image very fast processes such as neuronal network activity. Speed of acquisition. Finally. Another simple way of increasing camera sensitivity is to combine signals from multiple pixels (called binning). and user modification and options allow great flexibility. in blue. everything possible should be done to limit the duration and intensity of illumination. Another important consideration is that scanning systems acquire data pixel by pixel.

(A) A mitotic spin. and (F). which is not encountered often [(C). The nucleus (N) and centrosomal area (C) are marked in (A). However. imaged as described (61). in reality (25). and (E).org Downloaded from www. further developments are needed to address this issue. and (E).and four-dimensional (3. and correct matching of optics and tissues greatly benefits from this technique. This is absolutely vital when studying processes such as apoptosis and mitosis.sciencemag. Perhaps the most important consideration is the on July 4. The contribution of the analysis of gene expression (31) in living light from an infinitely small point source to a animals. (D). also called Normarski) microscopy are the most commonly used. (C). lishing]. Scale bar. from movie S2]. Deconvolution must be applied with great care and accuracy. with permission from Eaton Pub. 2. and motility. Imaging living cells by video-enhanced differential interference contrast (A and B) and phase contrast (C and D) microscopy reveals a wealth of information on organelles including mitochondria (arrows) and the endoplasmic reticulum (ER) (arrowheads). to avoid the generation of artefacts (21). Multiphoton confocal systems are now (Fig. position. and it can fluorescent reporters and of monitoring can- cer progression and treatment. Deconvolution of large 4D data series can now be achieved in minutes to hours with the use of dual processor personal computers. from movie S1] and a Xenopus tissue culture ( XTC) cell [(C) and (D). immany experiments. Examples of 3D images obtained by confocal and tidimensional live cell imaging also rewidefield deconvolution microscopy.BIOLOGICAL IMAGING SPECIAL SECTION be particularly advantageous in imaging very weakly fluorescent structures such as microtubules (Fig. (B and C) A mitotic spindle in a Xenopus XLK2 cell imaged by 3D widefield microscopy ticle tracking of objects moving inside [adapted from (21). 1). volution of the data series. so to get a complete picture we need to image cells in four dimensions. Researchers are often attracted to convelopment (28) and the pathophysiology of focal systems because high-resolution 3D imAlzheimer’s (29) by replacing a small part of ages (Fig. Most cellular processes occur in three dimensions over time. depend on the quality length excitation. However. Similarly. 2 ␮m . and (F) are enlargements of the boxed areas in (A). 1 ␮m. Piezoelectric objective drives have the edge here. confocal imaging has recently been coupled with endoscopy (32) with diagnostic potential. No into samples and reduces photobleaching. (B). Such systems Three. This enables the use of longer wavewidefield microscopes. Phase contrast and differential interference contrast (DIC. and reproducibility of the z position change. 2) can be acquired simply. Other Imaging Modes Bright-field imaging. other (conventional) systems with subsequent decontechniques. Alternatively. Determination extending the capabilities of existing magnetof the PSF of a system enables mathematical ic resonance imaging and positron emission reassignment of the out-of-focus light back to tomography technologies. The twoavailable commercially through a number of photon effect excites a chromophore not by a suppliers and can achieve speeds of 360 single photon but from two photons being frames per second. The ER is more obvious in the associated movie clips of a Vero cell [(A) and (B). 2) (22). single camera will perform optimally for all Notably. This likely to be of great benefit to the application approach has been used with great success in of gene therapy in combination with nontoxic both cell and developmental biology. 2008 . To switch between transmitted Fig. they collect it all. A single plane of a z series without additional Most reconstruction approaches assume processing [(B).cells such as transport vesicles (24 ). particularly those using live aging of neuronal processes through thinned cells. These approaches are likely to be plane of focus some distance away from that developed toward medically applicable syspoint source is described by the point spread tems for diagnosis and treatment of patients. enabling high-speed acquisition of stacks at multiple wavelengths over time. the analysis of intact organisms or tasks. (D).quires tools for data analysis (23). like (26 ). MulFig. restored] are shown. Tools dle in a DLD1 cell imaged by single photon confocal are continually being developed for parmicroscopy (61). using single photon excitation. electronics is essential for best performance. accuracy. Most confocal systems and epi-illumination microscopes are either provided with or can be simply adapted to include a means of acquiring data series in four dimensions. beam is split into multiple foci from which Multiphoton approaches to in vivo imagdata are collected simultaneously with a CCD ing. Sensitivity and data acabsorbed within a femtosecond time scale quisition rates of Nipkow disk systems. in (B). 2 ␮m. which penetrates deeper and the readout time of the detector CCD. however. Nipkow disk confocal microscopy is available from several companies. (C). 3. function (PSF) of the objective. Video-enhanced transmitted light microscopy. where cells undergo drastic shape changes. the skull with a coverslip. Optical imaging is its point source by deconvolution (21).and 4D) have been used for imaging both tumor deimaging. original data] and the same data set a uniform refractive index through the after restoration by constrained iterative deconvolution sample. Imaging living cells with transmitted light is often used along with fluorescence microscopy in order to provide information on cell shape. allowing imaging in situ (27 ). 84 4 APRIL 2003 VOL 300 SCIENCE www. Scale bars in (A). An immunofluorescence image of the ER in an XTC cell (61) is shown for comparison (E and F). may be better performed using widefield skulls is also possible (27 ).sciencemag. Widefield microhave also been applied with great success to scopes do not exclude light from any plane of the imaging of protein interactions (30) and focus.

this alrequire specialist equipment and lows measurement of protein-proknowledge. membrane traffic and nuclear architecture Cellular processes can also be imaged using a which have a motorized analyzer that can be and function (46 ). not those lying after caspase cleavage of a linker vides a means of direct imaging of further away (green). 3 and associated another. However. Excellent examples of this tal internal reflection fluorescence coverslip. depends on the inverse sixth powHowever. the coupling of these technologies path in addition to a fluorescence on July 4. there is huge potenmains of cells and organelles. the role of actin and dynamin in endocytosis technique is prone to imaging artefacts such Fluorescence lifetime imaging (FLIM). such as reflection contrast ping fluorophores are very close microscopy (34 ) and DRIMAPS together and in an orientation [Digitally Recorded Interference such that dipole-dipole coupling Microscopy with Automatic results in a transfer of energy Phase Shifting. microscopy. The most reliable method of choice.sciencemag. Excitation FRET to biosensors measuring at a critical angle generates an evaintracellular processes such as nescent field of excitation light that decays rap. Kaede (14 ).direct proximity to the plasma membrane (shown in red). there are other spewith the use of FRET. This illumination mode can be coupled with between donor and acceptor molprocesses within very close proxim. all of these advanced er of the distance between the transmitted light techniques do donor and acceptor (47 ). An alternative approach is to Photobleaching and photoactivation apmolecules in living cells (39). require skilled use and intensity of the image reaching the camera. 44 ).conventional widefield microscopy to allow combined imaging of events ecules (49) and the application of close to and away from the coverslip. only illuminates ϳ100 nm of the sample closest to the (48). system will be enough for many allow selective labeling of subdoexperiments. 4. that provide from one probe to another (47 ). this can be achieved In addition. tial for obtaining detailed insight Fluorescence resonance enerinto cell function by pushing gy transfer (FRET). This field decays rapidly and. 2008 and fluorescence imaging under computer contion. (35)]. and if this is left in place during GFP-tagged proteins in cells. fluorophore. ity to the coverslip (36 ). a key cally achieved by counting the number of used to quantitatively measure local concalcium flux (7 ).therefore. This provides a means (40) and viral infection (41). A bleaching (FRAP) and fluorescence loss in phowith GFP-based expression strategies is sure to complication with DIC is that it needs a polartobleaching (FLIP) (43. An evanescent field occurs when incident light passes from a FRET occur when the donor and cellular processes occur in specifi. Probes that can be lightvery small area of illumination by fluoresmoved out of the light path. In addition. which works only at certain wavelengths of light. To. Both are now lead to further developments in the near future. Fluorescence correlation spectroscopy. Many Fig. incident light exceeds a critical angle ␣. and reproducible examples of Total internal reflection. Because scanning confocal microsthe use of FLIM (51). Due to its high sensitivity. researchers that FRET is extremely ineffiwill often only be interested in a cient.copy has control over the region of illuminafor detecting multiple fluorophores in live www. Intermolecutransmitted systems to the limits lar interactions form the basis of by using the best possible optics all processes in live cells and can and specialized image-processing be monitored by measuring the equipment (33). different types of information Because the efficiency of FRET about cell structure and SCIENCE VOL 300 4 APRIL 2003 . 4) pro. FRET occialized light microscopy techcurs when two spectrally overlapniques.medium of high refractive index (glass) to one of low refractive index acceptor fluorophores are co(water or a cell). the plicability of FRET to live cell studies. both of which show greatly enWhilst a simple image caphanced fluorescence emission foltured by the microscope camera lowing activation at ϳ400 nm. they available but. izing filter (the analyzer) between the objective widely used to measure diffusional mobility of Systems for TIRFM are now commercially and the camera. or an analyzer that activated such as photoactivatable-GFP and cence correlation spectroscopy (FCS). limiting centration and diffusion of particles through a FRET pairs and improvements in imaging the depth of excitation to a distance of ϳ100 very small volume and can now be applied to methods (50) will doubtless enhance the apnm. There are some systems available to avoid this. In the context of light movies S1 and S2 [see also (1)].sciencemag. The detection of fluorescent probes is typiniques such as photobleaching (38) or widefield including photobleaching. plains their rather limited use at Limitations of this approach are present. Further developments of idly with distance from the coverslip. valently linked to one another such as the plasma membrane. and many hypothetical single organelle or structure. This enables specific visualization of only those fluorophores in include the elimination of FRET microscopy (TIRFM) (Fig.advantage of FCS is that it is also applicable photons emitted by the excited state of a opments has been the ability to image single to single molecule studies. which probably extein interactions in live cells.BIOLOGICAL IMAGING SPECIAL SECTION 85 Downloaded from www. and examples of measure the lifetime of this excited state with this include growth factor receptor signaling proaches. it is ideal for photobleaching techniques cally demanding and requiring state-of-the-art trol requires a shutter on the transmitted light such as fluorescence recovery after intramolecular changes in fluorescence. TIRFM of live cells has given insight into live cells (42). Increasingly. Although techni. One of the most exciting recent devel. FRET pairs do not produce FRET which will make fluorescence the in live cells. as with most of the recent develfluorescence image capture it will reduce the are combined with kinetic modeling of cellular opments in microscopy. (37 ) and can also be combined with other tech. processes (45) to study topics as diverse as careful interpretation of data. Total internal reflection occurs when the angle of cally restricted areas of the cell. This is clearly proximity of one component to illustrated in Fig.

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