Confocality & Focal-stack
Confocal imaging is traditionally used for imaging through a scattering medium. Confocal microscopy decreases scattering during illumination and also during imaging with the help of pinhole (or bucket detector) combined with a large aperture lens. In the past, the team demonstrated a scanning confocal endomicroscope (SCEM) using the principles of confocal imaging. The details are as follows:
Endomicroscopy provides clinicians a powerful tool to visualize tissue architecture and cellular morphology for early cancer detection. Due to its minimal invasiveness, probe-based endomicroscopy is widely applicable in the detection and evaluation of neoplasia in many sites, such as the gastrointestinal tract, cervix, pancreas and lung. Since cancer progression is usually associated with increased cell density that impairs image contrast, optical sectioning can be introduced to reject out-of-focus signal and improve the axial resolution. Previously, a differential structured illumination microendoscopy (DSIMe) was developed in our team using a reflective spinning disk to better visualize neoplasia-related alterations. Here, we present the first line-scanning confocal endomicroscope based on a digital light projector (DLP) and a CMOS camera without the need for mechanical scanning. In this novel scanning confocal endomicroscope (SCEM) as shown in the left figure below, the rolling shutter of a CMOS detector is used to achieve versatile slit detection without the need for a physical aperture. On the illumination end, a digital light projector can be synchronized as a spatial light modulator to project matching illumination lines and perform confocal imaging. Our ex vivo and in vivo validations demonstrate that the SCEM improves the visualization of cell architecture with optical sectioning, especially in crowded regions, when compared with a non-confocal endomicroscope (right figure). Also, the quantitative analysis reveals enhancement in parameters such as the nuclear to cytoplasmic ratio, which can potentially facilitate automated objective diagnosis based on cell density and morphology. Built in a compact enclosure at a low cost (<$5,000), the SCEM offers an opportunity to provide real-time histological information with enhanced contrast and can potentially contribute to improved cancer detection in community and low-resource settings.
The micro-vascular structure is located at multiple-depths, self-occluding, and very thin. Hence, using a fixed-focal-length lens or a small-aperture all focus lens creates loss-of-information in imaging micro-vasculatures and we need images captured at different focal length settings.
In the past, the team demonstrated 3D-reconstruction of micro-vessels from a set of images taken with different focal settings.
The team designed a non-invasive camera that allowed for the vein visualization, which operated in real-time, at different body parts and was robust to different types of skins.