Go Beyond Traditional Microscopy: How To Use Label-Free Techniques for Live Cell Imaging

05 Jun, 2019 | Guides & Resources
Go Beyond Traditional Microscopy: How To Use Label-Free Techniques for Live Cell Imaging

Live Cell Imaging is a major area of growth in the microscopy world as researchers search for techniques to elucidate further mysteries of the human body. Traditional microscopy systems often fail to automatically identify individual cells due to lack of contrast and while labelled techniques can produce high contrast images, they ultimately perturb the cells and can be phototoxic. This can ultimately limit the type of cell that can be used and the duration that they can be imaged before measurement-induced cell behaviour changes emerge.

Given these challenges, the search for a panacea can be futile as an ever-increasing number of technologies refine previous versions, all of which have a valid use and solve a question.

The process of defining the desired outcome of an experiment can be clouded by the latest and greatest shiny new toy that is a ‘must have’ for any self-respecting imaging facility. Is it 2D or 3D? Can we do spheroids? What about angiogenesis, Matrigel, bespoke slides, polylysine, fibronectin, or even hydrogel matrices? Do I need super-res, and what about a new confocal? Do we all still fluoresce – how many Photons?

Clearly, it’s a minefield of instrumentation – but what about applications of your science?

We’re in a position to discuss some interesting solutions to application needs.

Let’s put the quest to dive deep into cellular activities to one side, and let’s consider how cells interact with each other in a natural state. Have you ever thought about how cells react to fluorescent probes? Do they alter how the cells interact with each other? What about intense light? High light intensity does not bode well for the humble cell, are you perturbing the cells?
Population studies are normally the realm of cytometry, not the microscopist’s bread and butter.
Perhaps a system that allows both population and individual analysis would be of value, helping to understand kinetic data about your cells such as speed, displacement, Euclidean distance, meandering index and directionality associated with your treatments. Do you wish to track them? Understand their lineage? You could then re-culture the cells without any reservation or take them for testing elsewhere. Phasefocus in Sheffield, UK, have developed the Livecyte that employs a different modality in imaging called Ptychography – this method suits Live Cell Imaging with its 650nm <1mW laser that delivers incredibly low light toxicity. The instrument creates a diffraction pattern and stacks these patterns to run through the ptychographic algorithm, resulting in images that emulate the resolution of fluorescently tagged cells, ultimately allowing for excellent segmentation and tracking. They also have thought of the needs of scientists grappling with all this data. Whilst it is open for external analysis programs such as Image J, Cell Profiler and Metlab, the Livecyte has some incredible dashboards built into the software designed for popular applications such as Mitotic Time, Wound Healing, Angiogenesis, Proliferation and Morphology.

Ok, shall we dive into these cells now? Consider you have viewed the subtle interactions in your cells, identified some interesting behaviours on the Livecyte and now you would like to put these cells through a confocal microscope for depth selectivity, optical sectioning or to create 3D images. Damn – it is tied up for the next month! Maybe we should consider a benchtop confocal! Let’s make it simple and Laser free! Now, this is interesting if you are averse to long term costs of ownership – have you ever added up what you spend on all the confocal microscope maintenance? Aurox in Oxford, UK, has added a new system to their already successful “Bolt-On” Clarity range of confocal Microscopes with the soon to launch Unity. This super small, sit on the lab bench system has the hallmarks of a winning combination of ease of use (yes finally a confocal without the need of a flight licence to operate it) and powerful imagery. The spinning disk method they have patented is elegant and fundamentally simple. Normally the pinhole arrangement of a spinning disk allows for around 1% of light to be utilised, hence the need for high power lasers. Aurox has developed a novel optical arrangement that uses over 50% of the Cool LED light. Images are produced at a very rapid rate – say 150 images in 11 seconds – without the photobleaching evident in high intensity lasered systems and no need for laser safety restrictions. Often, it is difficult to obtain both high resolution and widefield images, let alone the ultra-large field of view – full overview images of the sample/mount can be detected with the sCMOS detector in the Aurox.

In keeping with the need to make it easy, the instrument is operated with a large format tablet over a secure wireless network with no more messy cables. They have a software called Visionary – a truly simple to use, easy to access web browser with a network connection that enables z -stack, time-lapse and multichannel imaging with OME-TIFF formats.

As instrument manufacturers increase the production of technology, generally they create efficiencies in manufacturing and the economies of scale kick in. As a result, one usually experiences a price drop in subsequent iterations of the model, and additional R&D costs are absorbed by higher sales volumes. This might be a common occurrence, but is not always the case…

I have noted a rise in the cost of a relatively simple technology employing basic fluorescence as its method of choice to perform live cell imaging inside an incubator. I doubt this rise is a result of additional functionality due to component pricing – not all systems need to be action packed with all capabilities to suit a broad range of users. People have also communicated to me their abhorrence that such an expensive system be locked away into individual researchers’ incubator. Clearly, there are always exceptions to the rule – and at times the application may warrant it. I think we have found a solid alternative at a fraction of the price with an impressive imaging system to boot.

NanoEnTek in Seoul, Korea, have developed the JuLI Stage – an individualised Real-Time Cell History Recorder. It is a nice little system, being fully automated, multi-channel, and having Multi-Well and Multi-position analysis all inside your incubator. The tech is more than sufficient for the applications it is used for, and the images are exceptional. Spheroid application is possible along with the bank of usual suspects such as wound healing, apoptosis, proliferation, neurite growth and many more.
What I like the most about the JuLI Stage is the quality of the images, particularly for this price. I guess the proof is in the pudding with this one – I suggest a demo.

It is worth noting that there are super low-cost versions of the JuLI Stage, namely the JuLI BR and JuLI FL, cut down to brightfield and fluorescence only respectively.

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