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.
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.
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.
Microscopy with ultraviolet surface excitation (MUSE) is investigated as a means to enhance curricula and education in the life sciences based on simplicity of use, the incorporation of inexpensive hardware, and the simplest methods of tissue preparation. Ultraviolet excitation in effect replaces tissue sectioning because it penetrates only a few micrometers below the tissue surface at the single cell level, preventing the generation of out-of-focus light. Although tissue autofluorescence may be used, image quality and content can be enhanced by a brief immersion in a solution of nontoxic fluorescent dyes that selectively highlight different cellular compartments. Safe mixed-dye powder combinations have been developed to provide students who have minimal lab proficiencies with a one-step tissue staining process for rapid tissue preparation.
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