SubscribeLiving cells communicate at lightning speed using electrochemical transport mechanisms.
For example, neuronal switches in the brain co-ordinate our emotions, senses, movement, memories, learning – in fact, almost everything we do, consciously and unconsciously, every second we are alive. A primary driver for our pursuit of knowledge about these processes is to establish what is normal so that we can contrast and compare between the healthy and disease states. However, the study of these quick processes can be tricky both in terms of technological limitations and biological models.
It is not possible to use human models for most studies, so we make do with the next best thing - animal models or cells in culture. Living model organisms, like the hair worm Caenorhabditis elegans, or the fruit fly Drosophila melanogaster, are preferred subjects for investigation because of their short life cycle but the developmental processes of such organisms are correspondingly fast.
Together with cell culture studies, they have provided a wealth of knowledge about what is ‘normal’ in these key intracellular signalling pathways and leverage in monitoring the effect of potential drug candidates. And, this allows us to investigate how treatments may work. For instance, what channels are being used in cells to get to the target and correct the problem? Once again, these are fast, detailed events that need to be observed in a way that gives us information in a form that can be clearly investigated.
Going “Live”
The rapid growth of live cell imaging in recent years goes hand in hand with developments in fluorescence microscopy and the emergence of Green Fluorescent Protein (GFP) and its multicoloured derivatives such as BFP (blue), CFP (cyan) and YFP (yellow). By fusing the GFP sequence to a gene of interest, researchers can follow expression or movement of the protein they are trying to study. This has allowed scientists to observe living cells and unravel the complex processes that control every aspect of cellular function, right down to the single molecule level.
Using today’s fluorescence microscopes we can substantiate the interactions of protein partners, observe the movement of individual molecules and measure the dynamics of the cytoskeleton during cell adhesion, cell motility. This has been satisfactory when observing slower cellular events, as spatial resolution can be maintained and clear images acquired.
However, when it comes to fast events such as calcium sparks and nerve impulses, technological limitations mean that the researcher always has to choose between speed and resolution. Image fast enough to capture the highly transient events under investigation and spatial information is sacrificed. Maintain resolution and you miss the event altogether. Even worse, try to go faster by increasing the light intensity and you risk damage and destruction of the sample itself.
Figure 1
Speed, Resolution and Sensitivity
It’s a dilemma that may not have to be faced in future. The introduction of a dedicated live cell imaging system from Carl Zeiss radically alters the balance that can be struck between speed, resolution and sensitivity. Capable of collecting up to 1,010 images per second, the LSM 5 LIVE (Figure 1) delivers faster, brighter, more evenly illuminated and better contrasted images than ever before and extends biological imaging to new levels.
Unlike traditional point-by-point laser scanning microscopes, the LSM 5 LIVE uses an ultra-fast CCD line detector to pick up specially shaped laser light 512 pixels at a time. It can attain 1,010 frames per second at a format of 512 x 50 pixels and will capture up to 120 full-frame images per second at a resolution of 512 x 512 pixels, 20 times faster than any other confocal system.
This high speed X-Y scanning system is combined with a new design of fast Z drive that allows 3D image stacks to be acquired every second, a feature that will be especially useful for developmental biologists. And, because the precisely tuned zoom optics and sophisticated scanning concept permits the size and position of the scan field to be varied precisely without changing the major optical parameters of the system, the ultra-fast imaging doesn’t compromise spatial resolution or sample viability (Figure 2).
Making use of the speed advantage
Using GFP fusion proteins, researchers can follow expression or movement of a protein and examine whether the expressed protein is inhibited, stimulated or unaffected by a drug. With the development of GFP fusion proteins that report cell cycle position of individual cells, one can study, for example, the effect of a drug on cell cycle promotion or inhibition. GFP fusion proteins can also be used to report the effects of a drug on cell cycle progression, a crucial indicator of cancerous states. Alternatively, if the drug under test is labelled, then live cell imaging studies could be used to track the uptake of the drug into the cells or the cellular compartments.
Meanwhile, changes in mitochondrial transmembrane potential, calcium homeostasis, nuclear morphology and plasma membrane permeability in living cells can be used to monitor cell death by apoptosis or necrosis. And, if we determine the effect of drugs or test compounds on these parameters it may help elucidate the sequence of events leading to cell death, which might lead to effective treatments for disorders including cancer, stroke, osteoporosis and autoimmune disorders
Work like this has been reported. For instance, Graham Dunn and his colleagues in London have developed a new method for tracking molecules within living cells following photobleaching (1). In their FLAP (fluorescence localisation after photobleaching) method, the molecule to be tracked carries two fluorophores. One is photobleached, just as in FRAP and FLIP, and the other acts as a reference label enabling the distribution of the molecules to be easily located merely by image differencing.
The group has used FLAP to study actin dynamics during protrusion in cancer cells and has shown that actin is delivered to the leading edge of cells at a speed exceeding 5 micrometers per second, a rate much faster than can be accounted for by diffusion. This is a very significant finding, suggesting that cancer cells can rapidly propel materials to the places where they need them and may point the way to preventing cancer cells from growing and spreading if a way can be found of stopping the active transport mechanism. Using the technique, they can track both the fast relocation of monomeric (globular) actin and the much slower dynamics of filamentous F-actin simultaneously in living cells (Figure 2).
Figure 2
The results were achieved using a Zeiss LSM 510 operating at 514nm (YFP) and 458nm (CFP), at that time the only microscope capable of switching between wavelengths and scanning fast enough to achieve a result. However, when pushed to its limits, the LSM 510 produced results that showed a new phenomenon – active transport rather than passive diffusion – that might point the way to treatment options. Even then, what is being observed is a 3D object but the speed limitations of the instrument means that the results are only being obtained in 2D. The LSM 5 LIVEopens up at least two possible new areas of investigation.
Firstly, is the speed of the mechanism constant across the entire front of the wave or are there some microsites where the movement is much faster compared to the rest of the wave? Finding out whether this is so demands a much faster scan rate than current scanning microscopes, and the 5 LIVE is as much as 100x faster.
Secondly, what is happening in 3D over time. If we were able to see through the lateral wave, would we observe differences in the mechanism or speed of the movement. This demands the ability to collect very fast 3D sections and, once again, the LSM 5LIVE has the crucial fast Z-drive technology to begin to address this area.
Calcium and the need for speed
Cells use calcium changes, sometimes incredibly minute, through calcium channels to mediate important functional processes. This includes several key cellular processes - apoptosis, differentiation, proliferation and cytoprotection – all of which are relevant to drug discovery. The abnormal release of calcium can lead to heart failure, arrhythmia and coronary heart disease. Electrophysiology and calcium imaging can enable us to study the effects of test compounds on calcium channels and how this affects cell physiology in vivo and in vitro. The cell also uses calcium changes as a beacon to call for immune back-up in case of infection. As well as studying infections, it is important to study these processes to correct situations when back up is called in against the self, in autoimmune disorders such as arthritis, multiple sclerosis, and uveitis.
The nervous system also uses calcium. Studies of this kind require very fast sampling speeds and fast switching of excitation wavelengths when studying emission ratios. However, voltage-sensitive dyes to study membrane potential dynamics are reported to be slow, lack spatial resolution, and exhibit poor voltage discrimination.
Several fluorescent calcium indicators are available to help in the study of calcium sparks and transients in response to stimulation by a drug or external stimuli (e.g. an electric current). The challenge for researchers has been to obtain speed and resolution and, once again, technology constraints have led to a reduction in the number of dimensions that can be studied, in this case one.
Until now, the choice was singular as well – the Nipkow disk real-time scanning confocal microscope. The principle behind this system goes back to the early days of television as a way of transmitting pictures. German engineering student, Paul Nipkow took a disc and cut into it a spiral series of holes positioned so that they could scan every part of an image in turn as the disk spun around. This raster scanning concept was later applied to microscopes. Unlike conventional laser scanning systems, the laser does not need to be moved and this leads to a fast frame rate (30+ fps). However, it suffers from a very low illumination efficiency of usually less than a few percent, which compromises spatial resolution. In calcium waves, this trade-off has been acceptable although it has limited its usefulness for very fast events, such as following electrical nerve impulses.
The LSM 5 LIVE combines fast frame rate technology with high illumination efficiency and high resolution. It opens up the possibility of fast imaging for fluorescent markers, allowing the study of the mechanics of calcium waves and the determination of the steps in the molecular cascade activated in the immune response.
Drug effects on cells
It is generally accepted that for a tumour drug to work it has to target and inactivate cell surface receptors. A critical step in this pathway is internalisation of the receptor to make it immune to tumour-causing agents. Therefore, antibody-based drugs and the study of their capability of inducing receptor internalisation are important.
In the live cell, subcellular compartments including cell surface, endosomes, lysosomes and Golgi, can be labelled to study where these internalised receptors are going. This, in turn enables the study of protein trafficking in these antibody induced live cells.
The movements and changes of molecules in or between specific intracellular regions are frequent and important in cellular functioning. Simultaneous analysis of multiple macromolecules in different cellular compartments helps localise molecular targets within a cell.
The vertebrate model for the human system being currently developed for rapid in vivo drug screening is Zebrafish. These embryos and larvae are useful in drug screening applications because they are transparent and produced in large numbers.
Once again, fluorescent technologies have been developed for disease-relevant assays, including a Zebrafish with fluorescent blood vessels to monitor angiogenesis in a living organism. Given the availability of this model, the affect of various drugs on angiogenesis can be studied.
Another area for research is fluorescent lipid absorption (e.g. cholesterol) and drugs to inhibit this mechanism. And, certain drugs can induce cytoskeletal rearrangements that may affect the whole cell and its morphology. Quantitative fluorescence imaging to automatically measure these changes over time and space could prove crucial in studying the affect of various pharmacological targets.
Not for the first time, the common themes here are fluorescence and speed. Given that the LSM 5 LIVE is approximately twenty times faster than any other confocal microscope, he possibilities are obvious.
Figure 3
5 LIVE – today and tomorrow
Clearly the LSM 5 LIVE’s capacity to image events in excess of 1,000 frames per second extends our imaging capabilities significantly. In doing so, it opens a Pandora’s Box of possibilities.
We have talked already about slow and fast events but what is our evidence? Is a slow event real or is it a combination of countless faster events that we just haven’t been able to see yet?
We have seen fast events, such as the work of Dunn’s group. They pushed the limits of confocal to 10fps to study FLAP in 3 dimensions; X,Y and time. Using the 5 LIVE, they could now exceed these speeds easily. Crucially, they could explore four dimensions of information by investigating changes that might be occurring in the Z axis.
In 2003, Bullock et.al. (2), studied the kinetics of mRNA along microtubules in Drosophila embryos. In order to visualise these processes, fluorescently labelled mRNA was injected into Drosophila blastoderm embryos. They studied the kinetics of mRNA localisation using time-lapse microscopy but this was limited to the lateral axis in order to obtain temporal information. With the LSM 5LIVE, they could extend these studies to include both temporal and spatial information. Using the fast 3D imaging, they could study any motion of mRNA along these microtubules in the Z-axis.
The unique technology behind the LSM 5 LIVE is capable of allowing us exclusive insights into cellular motion and interaction and offers a new dimension to all biological applications. In demonstrating just a few areas where it might impact the drug discovery process, we hope to spark new research ideas and possibilities. The combination of speed, image quality and sensitivity has never before been within reach of scientists.
Figures
Figure 1 - LSM 5 LIVE: the new confocal live cell imaging system from Carl Zeiss shown on the new Axio Imager microscope
Figure 2 - Motion of erythroblasts during one heartbeat cycle in 8-day old mouse embryo. GFP expression, colour-coded projection over time, recorded at 88 frames per second. Specimen: Dr. Mary Dickinson, Biological Imaging Centre, Caltech Pasadena, USA.
Figure 3 - FLAP ratio image of the leading lamella of a T15 cell immediately after a strip (red band) was photobleached for 4s. Three regions of strongly labelled transported actin can be seen at the top right leading edge. Zicha et. Al., 2003
References
1) Dunn GA, Dobbie IM, Monypenny J, Holt MR, Zicha D. Fluorescence localization after photobleaching (FLAP): a new method for studying protein dynamics in living cells.
J Microsc. 2002 Jan; 205(Pt 1): 109-12.
2) Bullock SL, Zicha D, Ish-Horowicz D. The Drosophila hairy RNA localization signal modulates the kinetics of cytoplasmic mRNA transport. EMBO J. 2003 May 15; 22(10): 2484-94.