Label-free optical microscopy has matured as a noninvasive tool for biological imaging; yet, it is criticized for its lack of specificity, slow acquisition and processing times, and weak and noisy optical signals that lead to inaccuracies in quantification. We introduce FOCALS (Fast Optical Coherence, Autofluorescence Lifetime imaging, and Second harmonic generation) microscopy capable of generating NAD(P)H fluorescence lifetime, second harmonic generation (SHG), and polarization-sensitive optical coherence microscopy (OCM) images simultaneously. Multimodal imaging generates quantitative metabolic and morphological profiles of biological samples in vitro, ex vivo, and in vivo. Fast analog detection of fluorescence lifetime and real-time processing on a graphical processing unit enables longitudinal imaging of biological dynamics. We detail the effect of optical aberrations on the accuracy of FLIM beyond the context of undistorting image features. To compensate for the sample-induced aberrations, we implemented a closed-loop single-shot sensorless adaptive optics solution, which uses computational adaptive optics of OCM for wavefront estimation within 2 s and improves the quality of quantitative fluorescence imaging in thick tissues. Multimodal imaging with complementary contrasts improves the specificity and enables multidimensional quantification of the optical signatures in vitro, ex vivo, and in vivo, fast acquisition and real-time processing improve imaging speed by 4–40 × while maintaining enough signal for quantitative nonlinear microscopy, and adaptive optics improves the overall versatility, which enable FOCALS microscopy to overcome the limits of traditional label-free imaging techniques.
Optical coherence microscopy (OCM) uses interferometric detection to capture the complex optical field with high sensitivity, which enables computational wavefront retrieval using back-scattered light from the sample. Compared to a conventional wavefront sensor, aberration sensing with OCM via computational adaptive optics (CAO) leverages coherence and confocal gating to obtain signals from the focus with less cross-talk from other depths or transverse locations within the field-of-view. Here, we present an investigation of the performance of CAO-based aberration sensing in simulation, bead phantoms, and
- Award ID(s):
- 1752405
- NSF-PAR ID:
- 10276179
- Publisher / Repository:
- Optical Society of America
- Date Published:
- Journal Name:
- Biomedical Optics Express
- Volume:
- 12
- Issue:
- 8
- ISSN:
- 2156-7085
- Page Range / eLocation ID:
- Article No. 4934
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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