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Modern image sensors and lasers have given cell biologists a new revolutionary tool for observing and quantifying cellular dynamics.
Holography is based on the fact that light waves create interference patterns just as water waves do. A hologram is created by dividing the illuminating laser light into two beams: one beam, the sample beam, illuminates the sample; the other beam, the reference beam, bypasses the sample.
By either reflection or transmission, the sample will make an imprint on the illuminating sample beam. To record the imprint, the sample beam is rejoined with the reference beam, and the resulting interference pattern is the hologram.
Holograms have traditionally been recorded on a photographic plate. After development, the photographic plate is again illuminated with the reference beam. Amazingly, the imprinted sample beam reappears. As the recreated sample beam is a perfect copy of the original, the three-dimensional sample will appear as if it is physically present.
Modern image sensors allow holograms to be digitally recorded. Instead of physically recreating the imprinted sample beam and the final image, the image-creation process is simulated by a computer.
A holographic microscope, like the HoloMonitor time-lapse cytometer, differs from a traditional microscope in that the illuminating light is split into a sample beam and a reference beam using a beam splitter. After the sample beam has illuminated the sample, it is rejoined with the reference beam using a beam combiner to create the hologram.
The recorded hologram is an interference pattern, created by joining the sample beam and the reference beam.
Fine focusing is done entirely in software, after recording. The digitally recorded hologram is computationally processed to create holographic images over a range of focal distances.
Another difference from a traditional microscope is that a holographic microscope records the information needed to create the image, not the image itself. The traditional image-creating lens is replaced by a computer algorithm – a digital lens.
The flexibility of a digital lens allows images to be refocused after they have been recorded. In a holographic microscope, refocusing to compensate for focus drift is done entirely in software. This is achieved by creating images on several focal planes. From this temporary stack of images, the best-in-focus image is automatically selected to produce the final holographic image. Alternatively, the focal distance can be manually set to focus on a plane of interest.
The recorded hologram contains both intensity and phase information. A holographic microscope therefore creates two separate images, an ordinary bright-field image and a phase-shift image.
Time-lapse video microscopy, based on image sequences acquired at regular intervals, was until recently complicated to set up and generated limited data. Nevertheless, understanding a living system entails understanding how the fundamental building blocks of life interact over time to form the system in its present state.
Semiconductor technology has advanced to a point at which it enables affordable time-lapse microscopes for routine use, without the issues commonly associated with conventional time-lapse microscopy.
State-of-the-art digital image sensors are extremely light sensitive, reducing the amount of required light and consequently reducing the risk of phototoxicity.
Semiconductor light sources dissipate very small amounts of heat, allowing time-lapse microscopes to be placed inside standard cell incubators.
Phase-shift imaging techniques allow unstained cell populations to be imaged, tracked, and automatically quantified for extended periods.
Microfluidic devices, adapted for microscopic observation, are rapidly becoming available to enhance the in vitro cell environment and push the limit of how long living cells can be kept for observation under the microscope.
The average computer hard disk has the capacity to store millions of images. This is sufficient storage capacity to record years of cellular dynamics with time-lapse microscopy.
The availability of light-sensitive, high-resolution digital image sensors, light-emitting diodes (LEDs), and high-speed computers means that microscopes can now be adapted to an environment suitable for cells (Liu & Nolan 2012, Piston 2009) – rather than cells having to be adapted to an environment suitable for microscopes.