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Abstract
Holography was invented by Dennis Gabor in 1948 for which he was awarded the Nobel Prize inPhysics in 1971. A hologram (portmanteau of the Greek words ‘holos’ meaning whole, and ‘gramma’,
meaning record) is a physical recording of an interference pattern which retains the phase information
of a three-dimensional (3D) object, and thus its depth information. The interference pattern is usually
obtained by combining an object wave and a reference wave which are mutually coherent (i.e. the
waves coming from various points of the object are statistically correlated). However, most of the
imaging performed in biological research uses fluorescent light which is inherently incoherent. Creating
holograms with incoherent light relies on the principle of self-interference of light, where beams
originating from the same point source are interfered with each other since they are mutually coherent.
This technique of interfering incoherent light with itself to create holograms is called self-interference
digital holography (SIDH). SIDH has been used for imaging biological samples, however it has been
limited to very bright samples.
In this dissertation we explore the possibility of using SIDH in low-light conditions, particularly
to detect single molecules used in single molecule localization microscopy (SMLM). We demonstrate
the application of SIDH to localize the position of point-like single emitters with high precision over
large axial ranges. We describe the development of a novel 3D imaging system that uses incoherent
digital holography to image single emitters under low-light conditions over a large axial range. We
demonstrate SIDH of particles emitting ~ 50,000 photons over a 20 micron axial range, and show that
particles emitting as few as 10,000 detected photons can be localized. Here we detect 0.2 photons per
pixel, below the quantum level of visibility. To benchmark digital holography as a 3D imaging technique,
we derive the theoretical limit of localization precision and compare the calculated precision
to the 3D single molecule localization precision of different Point Spread Functions.