- Last Updated on Wednesday, 21 October 2015 05:27
Modern image sensors and lasers give cell biologists a new revolutionary tool to observe and quantify cellular dynamics.
Holography is based on that light waves create interference patterns just like water waves. A hologram is created by dividing the illuminating laser light in two beams. One beam illuminates the sample, the sample beam. The other beam bypasses the sample, the reference beam.
By either reflection or transmission, the sample will make an imprint on the illuminating sample beam. To be able to record the imprint, the sample beam is rejoined with the reference beam. The resulting interference pattern is the hologram.
Traditionally holograms are 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 3-dimensional sample will visually 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 differs from a traditional microscope in that the illuminating light is split into a sample beam and a reference beam by a beam splitter (above). After the sample beam has illuminated the sample, it is rejoined with the reference beam by a beam combiner to create the hologram.
Another distinction from a traditional microscope is that a holographic microscope records the information needed to create 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 allow images to be refocused after they have been recorded. In a holographic microscope, re-focusing to compensate for focus drift is entirely done in software. This is achieved by creating images at 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 may be manually set to focus on a plane of interest.
Quantitative phase imaging
The recorded hologram contains both intensity and phase information. A holographic microscope therefore create two separate images, an ordinary bright field image and a phase shift image.