Abstract
The invention and advancement of biological microscopy depends critically on an ability to accurately simulate imaging of complex biological structures embedded within complex scattering media. Unfortunately no technique exists for rigorous simulation of the complete imaging process, including the source, instrument, sample and detector. Monte-Carlo modelling is the gold standard for the modelling of light propagation in tissue, but is somewhat laborious to implement and does not incorporate the rejection of scattered light by the microscope. On the other hand microscopes may be rigorously and rapidly modelled using commercial ray-tracing software, but excluding the interaction with the biological sample. We report a hybrid Monte-Carlo optical ray-tracing technique for modelling of complete imaging systems of arbitrary complexity. We make the software available to enable user-friendly and rigorous virtual prototyping of biological microscopy of arbitrary complexity involving light scattering, fluorescence, polarised light propagation, diffraction and coherence. Examples are presented for the modelling and optimisation of representative imaging of neural cells using light-sheet and micro-endoscopic fluorescence microscopy and imaging of retinal vasculature using confocal and non-confocal scanning-laser ophthalmoscopes.
Highlights
The invention and advancement of biological microscopy depends critically on an ability to accurately simulate imaging of complex biological structures embedded within complex scattering media
We report the first rigorous and holistic modelling of the complete imaging process in scattering media: that is light propagation is modelled in the imaging regime, where polarisation and non-diffuse light propagation is important in addition to interaction of all light with the optical instrument
We present three illustrative holistic Monte-Carlo optical modelling (HMCOM) simulations of imaging of biological structures: fluorescence imaging of a neuron, labelled with green-fluorescent protein (GFP), embedded in a scattering medium using a graded-index microendoscope; volumetric imaging and reconstruction of the same neuron using a diffraction-limited light-sheet fluorescence microscope; and imaging of retinal vasculature using a scanning laser ophthalmoscope (SLO)
Summary
The invention and advancement of biological microscopy depends critically on an ability to accurately simulate imaging of complex biological structures embedded within complex scattering media. Holistic modelling incorporates the coherent propagation of polarised light through the complete image-formation process: from source, through the instrument illumination optics, through the biological sample, through the imaging instrument, to the detector.
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