Modulated-Alignment Dual-Axis (MAD) Confocal Microscopy Optimized for Speed and Contrast

Abstract

Modulated-alignment dual-axis (MAD) confocal microscopy combines the benefits of dual-axis confocal (DAC) microscopy and focal-modulation microscopy (FMM) for rejecting out-of-focus and multiply scattered light in tissues. The DAC architecture, which utilizes off-axis and separated beam paths for illumination and detection, has previously been shown to be superior to single-axis confocal (SAC) microscopy for the spatial filtering (rejection) of unwanted background light. With the MAD approach, a modulation of the alignment between the illumination and collection beam paths tags ballistic photons emanating from the focal volume with a characteristic radio frequency that can be extracted and separated from background signal using lock-in detection. We report here an optimized form of MAD confocal microscopy where we have fully mitigated tradeoffs in performance in an initial proof-of-concept system in order to recover the imaging speed of DAC microscopy while retaining contrast enhancement of 6 dB (signal-to-background ratio) with a secondary improvement in optical-sectioning and inplane resolution. Validation is demonstrated with light-scattering tissue phantoms and freshly excised tissues.

Bessel‐beam illumination in dual‐axis confocal microscopy mitigates resolution degradation caused by refractive heterogeneities

“…One of the challenges for laser‐scanning microscopy of biological tissues is that refractive heterogeneities due to micro‐architectural structures – such as nuclei, organelles, glands, and vasculature – can cause spatial variations of optical beam foci in terms of shape (aberrations/distortions) and position (beam steering) . In particular, the tissue‐imaging performance of a DAC microscope is highly sensitive to the effects of refractive heterogeneities because of its reliance on two beam paths (illumination and collection) that must intersect at their foci . For example, it has been shown that the in‐focus signal collected by a DAC microscope is significantly decreased when the alignment of the illumination and collection beams is spatially modulated at the micron scale .…”

“…In particular, the tissue‐imaging performance of a DAC microscope is highly sensitive to the effects of refractive heterogeneities because of its reliance on two beam paths (illumination and collection) that must intersect at their foci . For example, it has been shown that the in‐focus signal collected by a DAC microscope is significantly decreased when the alignment of the illumination and collection beams is spatially modulated at the micron scale . In addition, as shown in previous studies with tissue phantoms and through in vivo imaging experiments of human epidermis, refractive heterogeneities in tissues lead to a degradation in spatial resolution for DAC microscopy , with a concomitant loss in sensitivity (signal‐to‐noise ratio, SNR) and contrast (signal‐to‐background ratio, SBR).…”