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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.