A multiplane two-photon microscope with non-descanned detection provides imaging speeds up to 200 MP/s with four spectral channels (800 MSpectra/s) with area imaging speeds of up to 30 mm2/s. This enables high volumetric throughput deep-tissue imaging at speeds approaching light-sheet imaging without sacrificing resilience to scattering and high axial resolution. Multiplane excitation is achieved by multifocal scanner-synchronous strip-scanning with tilted translation, while spherical lens arrays with custom SiPM array boards provide non-descanned detection that is both inexpensive and easy to spectrally multiplex.
Most two photon and confocal microscopes are limited by the sequential detection of pixels. I present recent work using low-cost silicon photomultiplier (SiPM) technology to build arrays of single-photon sensitive detectors. I present both spatially multiplexed imaging in highly scattering tissue and hyperspectral detection using 16 parallel SiPMs in two detector configurations. These enable 200 MP/s imaging through 4 spectral channels or hyperspectral read out at 50 MP/s and 20 nm spectral resolution at very low cost.
In this study, we developed an improved piston-based specimen holder to provide even pressure distribution across an irregular tissue surface. A series of support fixtures are also developed to facilitate the pressure distribution from the piston to image specimens with small contact area relative to thickness such as bisected shave skin biopsies. Using this capability, we demonstrate imaging of tall and narrow biopsy specimens with precise coregistration to conventional histology as well as rapid imaging of Mohs margins during surgery.
We developed a rapid tissue clearing technique combined with two-photon fluorescence microscopy (cFTPM) to image melanoma in situ biopsy volumetrically. cFTPM can provide rapid volumetric melanoma in situ biopsy images with the potential to provide same-day melanoma biopsy turnaround time with full volumetric images. The overall time span of our process, including tissue clearing and imaging, is faster than conventional histology processing.
Diffuse optical spectroscopy is a widely used method for the non-invasive recovery of important biological factors such as tissue oxygenation and total hemoglobin concentration. Frequency domain-diffuse optical spectroscopy improves the accuracy of parameter recovery over continuous wave-diffuse optical spectroscopy by enabling the decoupling of tissue absorption from tissue scattering. However, this comes at the price of increased instrumentation cost and complexity. Here we detail an easy to build, low-cost, and robust frequency domain-diffuse optical spectroscopy system to increase accessibility to this technology along with testing of the system’s stability and accuracy to ensure its applicability for biological measurements.
An integrated clinical two photon fluorescence microscope system allows for real-time assessment of freshly excised non-melanoma skin cancer skin biopsies with 2 minutes of preparation and enables imaging of multi-centimeter lesions in under 5 minutes. This system simulates the conventional workflow of a brightfield microscope to minimize pathologist retraining. A blinded study comparing two photon images and coregistered H&E paraffin section images is performed to show degree of concordance between the two modalities.
We perform micron-level co-registration of two-photon fluorescence microscopy (TPFM) images with en face frozen section analysis (FSA) histology. We demonstrate that TPFM has excellent sensitivity and specificity for evaluating squamous cell carcinoma (SCC) on surgical margins.
Significance: Two-photon and confocal microscopy can obtain high frame rates; however, mosaic imaging of large tissue specimens remains time-consuming and inefficient, with higher imaging rates leading to a larger fraction of time wasted translating between imaging locations. Strip scanning obtains faster mosaic imaging rates by translating a specimen at constant velocity through a line scanner at the expense of more complex stitching and geometric distortion due to the difficulty of translating at completely constant velocity.
Aim: We aim to develop an approach to mosaic imaging that can obtain higher accuracy and faster imaging rates while reducing computational complexity.
Approach: We introduce an approach based on scanner-synchronous position sampling that enables subwavelength accurate imaging of specimens moving at a nonuniform velocity, eliminating distortion.
Results: We demonstrate that this approach increases mosaic imaging rates while reducing computational complexity, retaining high SNR, and retaining geometric accuracy.
Conclusions: Scanner synchronous strip scanning enables accurate, high-speed mosaic imaging of large specimens by reducing acquisition and processing time.
Revascularization is required to deliver the factors necessary for bone injury healing to the injury site. Therefore, vascularization is usually monitored to assess the bone healing outcome in preclinical settings. Previously, blood flow changes measured by diffuse correlation tomography have shown the potential to predict the healing outcome of the mouse femoral graft in vivo. To obtain more comprehensive hemodynamic information in addition to blood flow, we adapted spatial frequency domain imaging (SFDI) method to quantify the total hemoglobin concentration and oxygen saturation in the mouse bone graft model.
An in-house SFDI system was built based on a Texas Instrument digital micromirror device (DMD) and a near-infrared camera. The system was tested using a simplified tissue phantom mimicking the mouse hindlimb with a femoral allograft (avascular) implanted. A single time-point measurement for mouse hindlimbs with and without allograft was performed. The SFDI results were compared with traditional contrast agent-mediated micro-CT for validation. Longitudinal measurements are being performed before and weekly after the allograft surgery. The SFDI-derived properties will be related to the biomechanical outcomes of the healed bones.
Preliminary results of tissue phantom experiments showed the capability of SFDI for mapping the absorption and scattering properties of the graft mimicking tube at a 2 mm depth. Since the mouse femur is usually ~1-2 mm under the skin surface, the SFDI technique has the potential for monitoring the vascularization in healing bone grafts.
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