SignificanceThe emergence of label-free microscopy techniques has significantly improved our ability to precisely characterize biochemical targets, enabling non-invasive visualization of cellular organelles and tissue organization. However, understanding each label-free method with respect to the specific benefits, drawbacks, and varied sensitivities under measurement conditions across different types of specimens remains a challenge.AimWe link all of these disparate label-free optical interactions together and compare the detection sensitivity within the framework of statistical estimation theory.ApproachTo achieve this goal, we introduce a comprehensive unified framework for evaluating the bounds for signal detection with label-free microscopy methods, including second-harmonic generation, third-harmonic generation, coherent anti-Stokes Raman scattering, coherent Stokes Raman scattering, stimulated Raman loss, stimulated Raman gain, stimulated emission, impulsive stimulated Raman scattering, transient absorption, and photothermal effect. A general model for signal generation induced by optical scattering is developed.ResultsBased on this model, the information obtained is quantitatively analyzed using Fisher information, and the fundamental constraints on estimation precision are evaluated through the Cramér–Rao lower bound, offering guidance for optimal experimental design and interpretation.ConclusionsWe provide valuable insights for researchers seeking to leverage label-free techniques for non-invasive imaging applications for biomedical research and clinical practice.
Imaging with third harmonic generation (THG) provides a powerful label-free approach to probing biological systems, particularly looking at structures formed by lipids. We present the first demonstration of THG widefield holographic microscopy. Holographic microscopy preserves the amplitude and phase of the measured field, which enables us to construct a synthetic spatial aperture with expanded field of view for complex valued THG images. Additionally, we implement a matrix approach postprocessing algorithm to reconstruct aberration-free THG holographic images by estimating and correcting for phase aberrations and scattering distortions.
Non-invasive imaging with high resolution deep within biological materials without the use of harmful ionizing radiation is of great interest in the field of medical imaging. Second harmonic generation is an excellent mechanism to circumvent this issue by providing outstanding contrast and optical sectioning. In general, these signals are weak and prone to scattering which introduce great challenges when imaging deep within turbid media. We will discuss recently demonstrated Epi-SHG holography, which can detect very weak backscattered SHG signals and enables the application of recently developed techniques which utilize the phase information to allow diffraction limited imaging within deep tissue.
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