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):
- NSF-PAR ID:
- Publisher / Repository:
- Optical Society of America
- Date Published:
- Journal Name:
- Biomedical Optics Express
- Page Range / eLocation ID:
- Article No. 4934
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
Fourier ptychographic microscopy is a computational imaging technique that provides quantitative phase information and high resolution over a large field-of-view. Although the technique presents numerous advantages over conventional microscopy, model mismatch due to unknown optical aberrations can significantly limit reconstruction quality. A practical way of correcting for aberrations without additional data capture is through algorithmic self-calibration, in which a pupil recovery step is embedded into the reconstruction algorithm. However, software-only aberration correction is limited in accuracy. Here, we evaluate the merits of implementing a simple, dedicated calibration procedure for applications requiring high accuracy. In simulations, we find that for a target sample reconstruction error, we can image without any aberration corrections only up to a maximum aberration magnitude of
λ/40. When we use algorithmic self-calibration, we can tolerate an aberration magnitude up to λ/10 and with our proposed diffuser calibration technique, this working range is extended further to λ/3. Hence, one can trade off complexity for accuracy by using a separate calibration process, which is particularly useful for larger aberrations.
Multi-parametric photoacoustic microscopy (PAM) is uniquely capable of simultaneous high-resolution mapping of blood oxygenation and flow
in vivo. However, its speed has been limited by the dense sampling required for blood flow quantification. To overcome this limitation, we have developed a high-speed multi-parametric PAM system, which enables simultaneous acquisition of ∼500 densely sampled B-scans by superposing the rapid optical scanning across the line-shaped focus of a cylindrically focused ultrasonic transducer over the conventional mechanical scan of the optical-acoustic dual foci. A novel, to the best of our knowledge, optical-acoustic combiner (OAC) is designed and implemented to accommodate the short working distance of the transducer, enabling convenient confocal alignment of the dual foci in reflection mode. A resonant galvanometer (GM) provides stabilized high-speed large-angle scanning. This new system can continuously monitor microvascular blood oxygenation (sO2) and flow over a 4.5 × 3 mm2area in the awake mouse brain with high spatial and temporal resolutions (6.9 µm and 0.3 Hz, respectively).
In this Letter, we report a low-cost, portable, two-photon excitation fluorescence microscopy imager that uses a fiber-based approach for both femtosecond supercontinuum (SC) generation and light delivery to the optical head. The SC generation is based on a tapered polarization-maintaining photonic crystal fiber that uses pre-chirped femtosecond narrowband pulses to generate a coherent SC spectrum with a bandwidth of approximately 300 nm. Using this approach, high-power, near-transform-limited, wavelength-selectable SC pulses are generated and directly delivered to the imaging optical head. Preliminary testing of this imager on brain slices is presented, demonstrating a high signal-to-noise ratio and sub-cellular imaging capabilities to a depth of approximately 200 µm. These results demonstrate the suitability of the technology for
ex vivoand potentially in vivocellular-level biomedical imaging applications.
Optical coherence tomography (OCT) has seen widespread success as an
in vivoclinical diagnostic 3D imaging modality, impacting areas including ophthalmology, cardiology, and gastroenterology. Despite its many advantages, such as high sensitivity, speed, and depth penetration, OCT suffers from several shortcomings that ultimately limit its utility as a 3D microscopy tool, such as its pervasive coherent speckle noise and poor lateral resolution required to maintain millimeter-scale imaging depths. Here, we present 3D optical coherence refraction tomography (OCRT), a computational extension of OCT that synthesizes an incoherent contrast mechanism by combining multiple OCT volumes, acquired across two rotation axes, to form a resolution-enhanced, speckle-reduced, refraction-corrected 3D reconstruction. Our label-free computational 3D microscope features a novel optical design incorporating a parabolic mirror to enable the capture of 5D plenoptic datasets, consisting of millimetric 3D fields of view over up to without moving the sample. We demonstrate that 3D OCRT reveals 3D features unobserved by conventional OCT in fruit fly, zebrafish, and mouse samples.