Tissue optical characterization with use of immersion and frequency domain measurements

Confocal microscopes have important applications in biology and neural sciences. Right now most of commercial confocal microscopes use mechanical scanning mechanisms to generate confocal images, e.g., using mirrors .to scan a laser beam across the field ofview, or using a rotating disk to scan across the field of view. These confocal systems limit the confocal scanning to only a raster scanning format. They do not offer externally gated and controllable illumination and imaging frames, fields, and subsets. Using the digital micromirror device (DMD) technology, we have developed a confocal microscope system that uses the DMD as a binary spatial light modulator to provide confocal scanning and offers flexible operating modes. For example, users can generate confocal images over any areas of interest, they can easily control the axial resolution, and they can also easily switch between confocal imaging modes and other microscopic imaging modes. The constructed prototype used one DMD for both the illumination aperture and detection aperture. This structure offers a robust system alignment and lowers the synchronization requirements. A reflective imaging system was used to relay the illumination patterns onto a sample and to relay confocal images onto a two-dimensional detector [e.g., charge injection device (CID)]. This design made the whole confocal imaging system less sensitive to chromatic aberration errors. We have built a computer simulation model to associate the performance parameters of a confocal imaging system (such as transversal resolution, axial resolution, and temporal resolution) with the confocal parameters of the DMD (such as confocal pattern period, pixel size, contrast ratio, etc.). Optical experiments were conducted to verify the computer simulation model. The experimental results have confirmed the computer simulation. The experiments showed that good confocal images can be obtained even when the confocal pattern period is as short as five times the aperture size. Right now the consensus for the confocal pattern period is about 10 times the aperture size. Based on our results we can improve the temporal resolution of a confocal imaging system by four times. Because of the nature of programmable confocal patterns, users can trade off among transversal resolution, axial resolution, and temporal resolution by changing the confocal parameters of the DMD. The details of the theoretical analysis, system construction, and experimental results are reported.