As life scientists, the study of cells and live-cell imaging are intrinsic to your research. Cellular biology encompasses a vast field, from the unicellular to complete living systems. Imaging of inter- and intracellular events range from the nanometre scale of molecular and ultrastructural investigations to the millimetre level required for developmental biology. Depending on your research interests, cells may derive from an immortalized cell line, primary cells, stem cells or ex vivo tumour cells for cancer cell imaging.
Live-cell imaging aims to capture as much information as possible while maintaining cellular health. This information can take the form of images, fluorescent signals, cell location, motility and interaction. The imaging environment can be detrimental to living cells, as they may experience changes to their natural environment in terms of pH, humidity, temperature and gases. As well as homeostatic controls, living cells require gentle and fast imaging to enable you to collect high-quality data, while limiting phototoxicity. It is therefore imperative that you choose the correct imaging system to meet the requirements of your biological specimen.
Maintaining a healthy and stable cell culture requires daily microscopic examination, which should be quick and easy in order to prevent environmental stress and minimise the risk of contamination, as well as to keep your work load low. Cell culture imaging can provide information on growth, contamination, cell differentiation and status, and transfection rates. Good cell culture practice and characterisation are prerequisites to ensure reproducible results.
The microscope you use for cell culture should ideally fit inside your culture hood, or be compact enough to place on an adjacent bench. As cultured cells can appear transparent under brightfield illumination, phase contrast enables the visualisation of cellular morphology and imaging of organelles. Using a light-emitting diode (LED) source can be beneficial as LEDs can be instantly switched on and off, produce minimal amounts of heat or unwanted ultraviolet (UV) light, and thereby reduce the phototoxic effects compared to sources such as mercury arc lamps.
Visualisation of the finest structures in your cells requires a perfectly fitting contrasting technique, such as differential interference contrast (DIC), PlasDIC and improved Hoffman modulation contrast (iHMC). DIC improves the contrast of unstained specimens and produces high-resolution images. In PlasDIC, the specimen is illuminated with non-polarized light, making this an ideal technique when using plastic culture containers. HMC uses a modulator with three zones, each of which allows a specific transmission of light. With a combined slit aperture providing oblique specimen illumination, light is either refracted or passes through the modulator in accordance to the specimen structure. Improved HMC ensures sharp contrast and pseudo-3D images of the finest cellular structures.
Temporal and time-lapse fluorescence imaging of live cells offers insights into intracellular and molecular dynamics, cell proliferation, cell motility and many other aspects. In the case of cancer cells this can, for example, lead to a better understanding of drug resistance to develop novel therapies. However, light can react with intracellular molecules or fluorophores to produce free radicals, and long-term imaging of cells may result in phototoxicity.
Fast, gentle imaging captures dynamic processes, but also reduces light exposure. For long-term live-cell imaging, over hours, days, or even weeks, it is imperative that the imaging platform is enabled to capture information while maintaining cellular homeostasis and system stability.
Life exists in three dimensions. Therefore, live-cell imaging often requires the multi-dimensional capabilities of confocal laser scanning microscopy (CLSM). Acquiring optical sections with CLSM ensures that only light from the focal plane of interest is captured. As CLSM rejects out-of-focus light, low noise images are obtained, and optical sections from thick samples can be combined into a 3D Z-stack, with an additional temporal dimension for live cells. CLSM has proved useful in cell biology and particularly useful in studying cancer cells, with this technique being used to reveal the structure of telomeres and the underlying mechanism to how they shorten with age.
CLSM additionally offers the ability to perform photomanipulation experiments such as fluorescence recovery after photobleaching (FRAP) and associated methods. Fluorescently-labelled intracellular targets can be bleached within a region of interest, followed by imaging in the area to determine kinetic and dynamic properties of proteins and structures in live cells. In addition, CLSM can be used for raster image correlation spectroscopy (RICS) experiments. Within the small, diffraction-limited confocal volume, the molecular dynamics of single molecules in live cells can be examined. RICS can provide information on molecular concentration, reaction rates and diffusion coefficients.
The solutions to your live-cell imaging problems may lie beyond the approximate 200 nm resolution limit of light microscopy. Techniques for imaging your cells beyond this limit include superresolution (SR) and electron microscopy (EM), enabling the detailed visualisation of structures such as actin filaments and the nuclear pore complex.
Photoactivated localization microscopy (PALM) is one of a number of SR techniques known as single molecule localization microscopy (SMLM). In PALM, one photo-switchable fluorophore is activated in a single point spread function (PSF), recorded and then deactivated. This cycle continues until all fluorophore localizations are captured to form a SR image. PALM produces images with 20 to 30 nm lateral resolution, and 50 to 80 nm axial resolution. Structured illumination microscopy (SIM) is another SR technique that uses an illuminating grid pattern to obtain high frequency information, producing images with up to twice the resolution of diffraction-limited microscopy.
Using SR-SIM light microscopy, the resolution of fluorescently labelled cellular components can be improved compared to conventional widefield or confocal imaging. When combined with the ultrastructural topography obtained using SEM, precise fluorescent signals can be assigned to sub-cellular structures, which greatly increases the information gain you can pull out of your sample.
With focused ion beam SEM (FIB-SEM), you can image and reconstruct large volume 3D tomography for your biological samples. FIB-SEM combines an ion beam to mill the surface of a sample, with the power of SEM. This sequential, automated milling and imaging in FIB-SEM allows precise reconstruction of 3D volumes for subcellular high-resolution results.
ZEISS Primovert is a complete solution for your cell culture laboratory and is perfect for your cancer research requirements. Primovert has a compact design allowing it to be placed directly inside your culture hood. With a universal phase slider for all objectives, you can quickly and easily assess the condition of your cells without needing to adjust phase position when changing magnification. Primovert also comes with mounting frames for different culture vessels, and by simply removing the condenser, you can increase the working distance for culture flasks. Primovert iLED with integrated fluorescence allows quick and efficient contrast imaging of green fluorescent protein (GFP)-labelled cells by switching the contrast technique on the stand. Primovert HDcam, with integrated five-megapixel camera, lets you capture images, videos and produce reports. In combination with your iPad and the free imaging App, Labscope, you can share and discuss observations with colleagues.
ZEISS Axio Vert.A1
ZEISS Axio Vert.A1 is the only system in its class to provide all the standard contrast techniques, yet is compact enough to sit beside your incubator. With the new IVF contrast system, you can switch freely between DIC, PlasDIC and iHMC to investigate your samples. Axio Vert.A1 offers LED excitation with no UV component, enabling you to image fluorescently labelled cells or specify transfection rates without a negative impact on cell survival rates. With the ergonomic design of Axio Vert.A1, including tilting eyepieces, your routine tasks can be performed quicker and easier.
ZEISS Cell Observer
All your live-cell imaging requirements are met with the ZEISS Cell Observer, a fully integrated research platform, based on ZEISS Axio Observer 7, with convenient workflow for the most complex applications. Optional light sources include Colibri, the innovative, high-performance LED source for bright, fast and gentle imaging to greatly reduce phototoxicity. Without the reliance on mechanical shutters, required wavelengths can be switched in microseconds. Your application may require image capture at maximum speed, maximum resolution, or both. A range of monochrome cameras can be integrated in Cell Observer to meet the highest possible sensitivity and resolution requirements. To collect every single photon from a weakly fluorescent sample, Cell Observer supports the full range of ZEISS Axiocams and is also compatible with sCMOS and EM-CCD cameras from third-party manufacturers.
An environment that is as close to the living organism as possible ensures you produce physiologically relevant results. Incubator XL encases all beam path components ensuring environmental stability of your cells, and of Cell Observer. In addition, Definite Focus 2 keeps your cells in focus, hours and days after starting your experiment.
For optical sectioning and Z-stacks, the Apotome slider module transforms Cell Observer into a 3D workstation. Based on structured illumination microscopy (SIM), Apotome generates extremely high-resolution optical sections of your live cells.
ZEISS Celldiscoverer 7
For multiple live-cell imaging experiments with increased throughput, ZEISS Celldiscoverer 7 is your fully automated imaging platform, incorporating an inverted microscope, darkroom, and various incubation and detection options. For your most demanding long-term live-cell experiments with rapid time-lapse imaging, you can choose fast, sensitive sCMOS or EM-CCD cameras. With Celldiscoverer 7, you can undertake long-term observation of the physiological and morphological parameters of living tissue sections or spheroids during growth, motility and interaction. Celldiscoverer 7 excitation unit combines up to seven LED’s providing broad spectrum excitation, gentle illumination, low phototoxicity and fast switching times. Integrated incubation options ensure environmental stability for your cells, and with automatic recognition and adaptation, you are free to use any number of cell carriers. With Auto-immersion, hardware-based focus and Autocorr objectives, you can produce images of a quality unseen before. You can further enhance these images with the computational deconvolution option, which greatly increases signal-to-noise ratio (SNR) and produces crystal clear 3D images. For confocal imaging of 3D samples, simply add the LSM 900 with Airyscan 2 and profit from a system that seamlessly combines camera and confocal imaging like never before.
ZEISS LSM 900 with Airyscan 2
For observing dynamic processes in live cells, ZEISS LSM 900 with Airyscan 2 provides the highest sensitivity and frame rates for gentle imaging and is tailored precisely to your live-cell applications. The LSM 900 with Airyscan 2 offers a range of imaging solutions including fluorescence resonance energy transfer (FRET) and FRAP, as well as parallel acquisition and linear unmixing in samples with multiple overlapping fluorophores. The LSM 900 with Airyscan 2 has a genuinely small footprint and reduced complexity, saving valuable lab space and minimizing training time. With ZEN imaging software, setup and use is simple, enabling reproducible results in the shortest possible time to increase your productivity. You can analyse bleach events within a time series using the FRAP Efficiency Analysis Module for ZEN.
ZEISS LSM 980 with Airyscan 2
The ZEISS LSM 9 family of confocal microscopes with Airyscan 2 includes the LSM 900 and the LSM 980. In conventional confocal systems, the pinhole blocks out-of-focus light from reaching the detector. Although this achieves higher resolution, photons are lost for the final image. With the Airyscan 2 detector simply more photons are collected. Replacing the conventional pinhole/detector arrangement, the Airyscan 2 detection element array extracts more information from the fluorescence signal while simultaneously increasing resolution and SNR with improved optical sectioning. The new Multiplex mode for Airyscan 2 offers more flexibility for your live-cell experiments enabling you to rapidly image whole sample volumes and large fields of view in high resolution. In a single sweep, Airyscan 2 in Multiplex mode can acquire up to four high SNR superresolution image lines.
ZEISS Elyra 7
ZEISS Elyra 7 with Lattice SIM is the flexible platform for fast and gentle 3D SR microscopy that brings SIM to a new level. Whether you work with living cells, organoids or tissue, Elyra 7 with Lattice SIM offers a range of SMLM techniques including PALM. The Lattice SIM spot pattern has a sampling efficiency twice as high as classic SIM, an imaging speed of up to 255 fps, and a lower laser dose ensuring reduced phototoxicity. This fast, gentle and light-efficient imaging ensures you can reveal mechanistic and dynamic details in live cells for the first time. Applications such as vesicle trafficking and cytoskeleton and membrane reorganization are examples in cell biology where you can never have enough temporal and spatial resolution to discover new pathways and mechanisms. The Apotome mode of Elyra 7 gives you fast optical sectioning with high lateral and axial resolution for crisp contrast, minus out-of-focus and background signal. With a wealth of contrast and SR techniques, combined with optical sectioning, Elyra 7 is tailored precisely to your live-cell imaging applications. From molecular-level imaging such as focal adhesion dynamics of cancer cells, to whole-cell capture in a single image, Elyra 7 is an outstanding SR platform with the flexibility to perfectly match your applications.
The ZEISS Sigma field emission SEM (FE-SEM) family comprises the Sigma 300 and Sigma 500. The new generation of secondary electron detectors in the Sigma family produces images with 50% more signal and 85% greater contrast with the novel C2D (Cascade Current Detector) and VPSE (Variable Pressure SE) detectors in variable pressure mode. With a semi-automated, high-speed 4-step workflow, you can image across multiple regions of interest benefiting from fast time-to-image, as well as saving time on training, especially in a multi-user environment. The C2D is able to deliver sharp, ultrastructural images of cryo-fixed biological samples, and non-conductive biomaterials (such as teeth and bone) can be studied without coating using the Advanced VP mode or low voltage approaches.
The ZEISS Gemini FE-SEM family includes the GeminiSEM 300, GeminiSEM 450 and GeminiSEM 500. The GeminiSEM 500 offers you more signal and more detail, with minimal sample damage. Perfect image quality and a resolution of 1.0 nm are obtained at low voltages without requiring beam deceleration. Applying beam deceleration with the Tandem decel option resolves to 0.8 nm at 1 kV. The imaging capabilities of the GeminiSEM 500, combined with the annular scanning transmission electron microscope (aSTEM) detector, provide ultrastructural details in biological specimens such as brain cell lipid bilayers.
Your specialist instrument for speed and surface sensitivity in imaging and analytics is the GeminiSEM 450. The optical design ensures there are no time-consuming realignments as you work. With the GeminiSEM 450, you can seamlessly switch between analytical modes at high beam currents and high-resolution imaging at low beam currents. Using the high beam current, you can capture large areas of high-resolution cellular ultrastructure.
For imaging flexibility, the GeminiSEM 300 provides high resolution and high contrast, even on extremely large fields of view. Even with non-conductive samples, the novel high gun resolution mode achieves excellent resolution and distortion-free images. When combined with the variable pressure mode, sputter coating is not required to analyse the topology of biological samples.
Based on the novel Gemini optics and the low voltage capabilities of Ion-sculptor FIB column, ZEISS Crossbeam 350 and Crossbeam 550 provide true sample information from high-resolution images, minimal sample damage and ultra-thin samples. For your most demanding characterisations, Crossbeam 550 features an optional large chamber with 22 configurable ports. The imaging capabilities of the Crossbeam family can be applied to FIB-tomography of biological samples. This allows you to investigate different cell compartments in single cells, or understand the 3D morphology of complete model organisms with the highest resolution and reliability.
ZEISS ZEN Connect
For greater insights into your cell biology research, you can utilize a connected microscopy approach with ZEISS ZEN Connect, which lets you combine and correlate images and data from different imaging modalities. For example, you can overlay a large area widefield scan of your specimen with detailed regions acquired with a confocal or superresolution microscope, or you can combine and correlate light and electron microscopic images with each other. Correlating information from different resolution and size scales will allow you to identify new cellular functions and mechanisms more precisely and faster than before.
ZEISS Shuttle & Find
Bridging the gap between structure and function, ZEISS Zen Shuttle & Find for Life Sciences offers the first easy to use, highly productive workflow for correlative light with electron microscopy (CLEM). Fully integrated into the ZEN imaging software, Shuttle & Find is a correlative interface between your light and electron microscopy results, controlling the required functions of both systems. On your light microscope, you can use the Shuttle & Find holder, or any sample holder, and ZEN performs a fast calibration. After capturing light microscopy images, transfer your holder to a SEM. Easy relocation of the corresponding positions is performed with the click of a button, saving time searching for your cells or region of interest. After SEM imaging, the built-in functions of Shuttle & Find generate precise correlative images, superimposing the imaging data from your light and electron microscopes.