Optical clearing-assisted optical sectioning microscopy for point-of-care tissue imaging applications
Medical imaging techniques are crucial in providing medical professionals insight into human health, specifically for pathologists making diagnostic decisions from human biopsy samples. The standard technique for digital pathology imaging first requires generation of tissue sections on glass slides using extensive and slow tissue preparation techniques. However, an emerging method for imaging tissues near the patient and space and in time is a form of ex vivo microscopy called structured illumination microscopy (SIM). SIM is an optical sectioning technique that preferentially extracts the imaging focal plane from within a thick sample. Samples stained with fluorescent dyes and illuminated with the proper wavelength of light generate fluorescence in all directions, enabling images to be obtained in a back-reflection geometry rather than trans-illumination as required by traditional histopathology. However, the efficacy of SIM for thick tissue imaging is affected by the amount of light scattering in the tissue. As light passes through tissue, photons encounter different refractive indices as a result of inhomogeneous tissue, causing them to bend, changing their direction and impeding the modulation depth of the structured illumination, which in turn reduces the signal-to-noise ratio (SNR) of the resulting images. As photons moves deeper into a sample, they encounter more and more changes in refractive indices and eventually the illumination patterns become completely demodulated, and the optical section benefit of the SIM is lost. This study aims to reduce light scattering in SIM using rapid optical clearing. Optical clearing is a method of tissue preparation that makes tissues more homogenous, reducing the amount of differing refractive index, therefore, reducing light scattering. Using simple, non-toxic clearing agents in aqueous solution and a user-designed optical clearing chamber, image clarity and the depth at which structures can be viewed have been studied in response to rapid clearing protocols in tissue phantoms and human prostate biopsy samples.