Ultraviolet absorbance Mach–Zehnder interferometric quadrature microscopy

We introduce Absorbance Mach-Zehnder (AMAZE) Interferometric Microscopy, a novel reflection-mode, absorption-contrast imaging technique. AMAZE is a laser-scanning method that employs two co-focused lasers: a continuous-wave interrogation laser and a pulsed excitation laser. A Mach-Zehnder interferometer with optical quadrature detection captures both amplitude and phase modulations of the interrogation beam induced by the excitation pulse. These modulations are proportional to the sample’s optical absorption coefficient at the excitation wavelength and are used to reconstruct absorption images. Unlike its non-interferometric predecessor, photoacoustic remote sensing (PARS) microscopy, which detects intensity modulations of the interrogation beam, AMAZE signals are largely independent of reflectivity or scattering, resulting in much stronger signals in low-reflecting samples, such as biological tissue. Using ultraviolet excitation (UV-AMAZE), we demonstrate label-free imaging of cell nuclei in both fresh unprocessed and formalin-fixed paraffin-embedded (FFPE) tissues. In fresh mouse tissue, UV-AMAZE achieved a 24 dB improvement in signal-to-noise ratio (SNR) over UV-PARS. Additionally, AMAZE phase modulation provides absorption contrast in samples with weak photoelastic responses, such as thin metal films, where PARS fails. This method requires no active stabilization or vibration isolation and avoids sensitivity fluctuations common to interferometric techniques. AMAZE microscopy represents a new paradigm in label-free absorption imaging with promising use cases including slide-free virtual histology and industrial inspection of surface and subsurface defects.