The definite article

Cutting edge live cell imaging experiments can take place over long time scales. This can reveal much but can cause a reduction in optic performance. Here we explore briefly how technological innovation can eliminate thermal drift

LIVE cell imaging is one of life sciences' fastest growing techniques, utilised in biomedical facilities around the globe to unravel the complex processes that control every aspect of cellular function and the molecular mechanisms underlying disease. Modern laser scanning microscopes allow users to capture a 3D image of an entire cell, probe the interactions of the cell's protein partners, map the movement of individual intracellular molecules or measure the dynamics of the cytoskeleton during such processes as cell adhesion, cell motility and cell signalling.

Although the combination of laser scanning microscopes, inexpensive computers, megapixel digital cameras and sophisticated image analysis software is helping to make live cell imaging routine, it does not mean that it is without challenges. For instance, every microscope makes a trade-off between speed and resolution. But, subtle differences in the technology employed mean that some trade-off less, substantially effecting the results that may be achieved, especially when investigating fast, transient events. Multichannel time-lapse experiments, in which several different fluorophores are observed for a longer period of time, are also commonplace and many confront the researcher with thermally unstable conditions. This causes optical drifting in the Z plane as the mechanical expansion and contraction of the microscope components alters their relative positions – a challenge to even the most mechanically and thermally stable designs.

When it comes to dynamic cell events, there is no way around the need to achieve speed with sensitivity. The ZEISS LSM 710 Confocal LSM is a key development, offering a dramatic increase in sensitivity and signal-to-noise ratio to enable even the faintest signals to be clearly observed. Moreover, the LSM 710 includes software tools that automate microscope set up, calibration and image analysis to ensure that results are obtained quickly and easily for even inexperienced users. 

Traditionally, microscope sensitivity is increased by suppressing the amount of laser light reflected from the sample. The light is passed through filters housed in a beamsplitter and then passed through a grating before being directed towards the detector, resulting in a loss of at least 10% of the signal as it hits the grating. To combat this, the new microscope incorporates a spectral recycling loop that decreases this loss by more than 90%, to increase the signal reaching the detector to more than 99%.  Previous designs also made changing the filters in the beamsplitter difficult, a practical limitation on the number of fluorescence markers used experimentally. In the LSM 710, up to 24 filters are divided between two simple filter wheels resulting in the capability to suppress stray light from up to 50 laser lines. This whole assembly is called the TwinGate Beamsplitter and is unique to the LSM 710 (Figure 1).

The PMT detector has also been upgraded to the new QUASAR detector, which

The beampath within the LSM 710.

can operate in 2-, 3- or 34-channel modes, and new electronics incorporating an oversampling strategy increase the sampling time per pixel by 30%. In 34-channel mode, the QUASAR detector enables spectral imaging over the entire wavelength range, a vital asset as researchers look to understand biological systems in more detail and use many different fluorescent dyes to track the location and migration of molecules of interest. With QUASAR in multichannel mode, the brightness distribution and spectral composition of the fluorescent signal is recorded and linear un-mixing of the signals is performed by the systems’ software to resolve the signals from each of the individual fluorophores. Because no part of the signal is discarded, signal intensity is maintained, with a corresponding improvement in sensitivity. The result is a resolution of just 3nm in the recording of lambda stacks. 

This increased sensitivity and resolution enables researchers to discriminate

Definite focus projects a grid onto the bottom of the culture vessel, where it is reflected onto a slanted camera chip. If there is drift, definite focus returns the relative position of the grid projection back to the original state and counteracts drift.

between ten different fluorescent signals within a sample while the high sensitivity allows lower powered lasers to be used to excite the fluorophores in a sample. This is particularly advantageous when conducting time-lapse studies where the viability of cells is crucial to the results, as lasers can cause phototoxic damage to cells at high energy.

The LSM 710’s precise scanner control enables accurate optical manipulation for photoactivation, photoconversion and FRAP (fluorescence recovery after photobleaching) studies where scan and bleach regions of interest can be freely defined. FRAP is particularly useful for studying protein dynamics and the movement of fluorescently tagged particles into, and out of, specific regions. After the dye molecules in the region of interest are bleached using the laser, the movement of non-bleached dye molecules can be followed as they migrate into the bleached area. And, although the LSM will scan a sample pixel by pixel to a greater sensitivity than other systems, this is not always suitable for studying very fast processes, such as blood flows. Therefore, the LSM 710 can be combined with a high-speed line-scanning system to enable a line scanning strategy to be employed.

Long-term, time-lapse studies of physiological processes require some measure

The ZEISS LSM 710 Confocal LSM

of incubation in order to ensure that the cells survive.  However, heating or cooling leads to mechanical expansion of the microscope components and impairs image quality due to drifting in the Z plane.
 
Until now, researchers have had to compensate for this drift through software-based autofocus systems. At the start of the observation period, these systems assess and store image parameters, such as contrast, in the image plane that is being observed. Then, as the experiment progresses and an observation is required, the Z-plane stack is scanned and the individual plane that best cor¬responds to the original evaluation criteria is brought into focus. However, there is no guarantee that the original plane is accurately and consistently re-selected and the entire Z-scanning process introduces significant time delays.

Definite fo¬cus compensates for this drifting by automatically maintaining the distance between the objective and the culture vessel, using infrared light to continuously monitor the gap. Definite Focus overcomes the disadvantages of previous approaches to drift reduction and, for the first time, allows novel experiments such as those requiring rapid temperature changes.

Live Cell Imaging Applications • Dynamic Fluorescence -Fast Imaging of Calcium Signals, Gene expression and localisation with GFP and its derivatives • Cell Motility - Cellular movement and differentiation, morphological response to stress and environment • Neurobiology - Interaction of neurons and neuroglial cells, the growth of axons/dendrites • Dynamic Gene expression with Luminescent reporters • Elucidation of cellular signalling pathways • Protein & vesicle tracking • Cell cycle and development studies • FRET/FLIM analysis of molecular interaction

Via the infrared light of an LED, which is coupled into the nosepiece carrier, a grid is projected onto the bottom of the cul¬ture vessel, which acts as a reflec¬tive surface and to send the image back onto a slanted camera chip. Due to the slanted position of the camera chip, only a small area of the grid is sharply focused (Figure 2) and, in cases involving drifting, the relative position of the grid projection to the camera chip changes. Now, during the experiment, definite focus can control the microscope’s Z drive and correct the position until the original grid area is once again sharply depicted on the camera chip. At this point, the original distance between the objective and the bottom of the culture dish is re-instated and the original plane of examination is automatically brought into sharp focus. Because definite focus runs transparently in the background, users can choose to allow the process to operate continuously without their intervention or to establish precise refocusing at defined intervals.

Definite focus has widespread applicability. It is compatible with all contrast methods and almost all objectives, including LD objectives. Standard fluorescence filter sets can be used as can all the established fluoro¬phores and it works perfectly with plastic dishes. Sensitivity is uncompromised, as the 835nm wavelength of the infrared light source lies well outside the range of excitation and emission spectra. What’s more, if the culture vessel is changed, defi¬nite focus can also be used as a focus finder. In this context, a single activation of the function automatically positions the Z drive and, therefore, the objective in the previous observation plane. Searching for the right Z plane, which can be very time consuming, is not required.

Since definite focus eliminates drifting, waiting for stable temperature conditions is unnecessary. This means that it can be used in nearly all applications involving live cell imaging and overcomes the disadvantages of pre¬vious approaches in many situations. In particular it is suited to long-term experiments - which may run for several hours or more - because drifting be¬comes increasingly noticeable over time, and in applica¬tions with high power objectives, since the image's shallow depth of field makes drifting more rapidly apparent.

Definite focus also opens up additional opportunities for novel experiments which, until now, have not been feasible. For example, studies based on pro¬tein-folding mutants or on heat-shock experiments require that observations are made whilst one or more rapid temperature changes take place.  Naturally, with traditional microscopes, rapid drifting in the Z plane occurs in the process. However, definite focus is able to continuously monitor the dimensional changes involved and re¬act rapidly, and automatically, to keep the initial plane in sharp focus.

It will also allow smaller incubation systems to be developed, concentrating solely on maintaining the condition of the sample rather than attempting to encapsulate the entire microscope system, which can make access to the specimen quite difficult.

Together, the enhanced sensitivity of the LSM 710 and the definite focus system establish firmly the contribution that technological developments can make to live cell imaging, confirming its place as an indispensible tool for studying the various processes involved during cell development, disease onset and progression, and cell death.