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.
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.
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.
Photomultiplier tubes are the standard for high sensitivity detection of fluorescence signals, but are costly, fragile, have limited QE and worse sensitivity at far-red wavelengths. Silicon photomultipliers (SIPMs) combine the higher shot-noise limited sensitivity of silicon sensors with the single photon sensitivity of PMTs. We evaluate the new generation of SIPMs for high speed fluorescent imaging applications and find that for a range of operating conditions, SIPMs offer higher SNR and dynamic range than GaAsP or GaAs PMTs. We show how to design low cost electronics that enable extremely high sensitivity, dynamic range, and bandwidth, discuss how to customize detectors for high speed fluorescent imaging, and show the results of human tissue imaging comparing PMTs and SIPMs.
The silicon photomultiplier (SIPM) is an emerging detector technology that enables both high sensitivity and high dynamic range detection of visible and near-infrared light at a fraction of the cost of conventional vacuum tube photomultiplier tubes (PMTs). A low-cost detection circuit is presented and the performance of a commercial SIPM is evaluated for high-speed laser scanning microscopy applications. For moderate-to-high-speed fluorescent imaging applications, the measurements and imaging results indicate that the SIPM exceeds the sensitivity of GaAsP PMTs, while providing higher dynamic range and better saturation behavior. For low speed or applications requiring large detector areas, the GaAsP PMT retains a sensitivity advantage due to large area and lower dark counts. The calculations presented show that, above a critical detection bandwidth, the SIPM sensitivity exceeds that of a GaAsP PMT.
Rapid histopathological examination of surgical specimen margins using fluorescence microscopy during breast conservation therapy has the potential to reduce the rate of positive margins on postoperative histopathology and the need for repeat surgeries. To assess the suitability of imaging modalities, we perform a direct comparison between confocal fluorescence microscopy and multiphoton microscopy for imaging unfixed tissue and compare to paraffin-embedded histology. An imaging protocol including dual channel detection of two contrast agents to implement virtual hematoxylin and eosin images is introduced that provides high quality imaging under both one and two photon excitation. Corresponding images of unfixed human breast tissue show that both confocal and multiphoton microscopy can reproduce the appearance of conventional histology without the need for physical sectioning. We further compare normal breast tissue and invasive cancer specimens imaged at multiple magnifications, and assess the effects of photobleaching for both modalities using the staining protocol. The results demonstrate that confocal fluorescence microscopy is a promising and cost-effective alternative to multiphoton microscopy for rapid histopathological evaluation of ex vivo breast tissue.
We present a portable multiphoton system designed for evaluating centimeter-scale surgical margins on surgical breast specimens in a clinical setting. The system is designed to produce large field of view images at a high frame rate, while using GPU processing to render low latency, video-rate virtual H&E images for real-time assessment. The imaging system and virtual H&E rendering algorithm are demonstrated by imaging unfixed human breast tissue in a clinical setting.
We developed an ultrahigh speed endoscopic swept source optical coherence tomography (OCT) system for clinical
gastroenterology using a vertical-cavity surface-emitting laser (VCSEL) and micromotor based imaging catheter, which
provided an imaging speed of 600 kHz axial scan rate and 8 μm axial resolution in tissue. The micromotor catheter was
3.2 mm in diameter and could be introduced through the 3.7 mm accessory port of an endoscope. Imaging was
performed at 400 frames per second with an 8 μm spot size using a pullback to generate volumetric data over 16 mm
with a pixel spacing of 5 μm in the longitudinal direction. Three-dimensional OCT (3D-OCT) imaging was performed in
patients with a cross section of pathologies undergoing standard upper and lower endoscopy at the Veterans Affairs
Boston Healthcare System (VABHS). Patients with Barrett’s esophagus, dysplasia, and inflammatory bowel disease
were imaged. The use of distally actuated imaging catheters allowed OCT imaging with more flexibility such as
volumetric imaging in the terminal ileum and the assessment of the hiatal hernia using retroflex imaging. The high
rotational stability of the micromotor enabled 3D volumetric imaging with micron scale volumetric accuracy for both en
face and cross-sectional imaging. The ability to perform 3D OCT imaging in the GI tract with microscopic accuracy
should enable a wide range of studies to investigate the ability of OCT to detect pathology as well as assess treatment
response.
Interferometric based imaging has a number of advantages over direct detection of photons, including
very high time resolution, shot noise limited sensitivity, and reasonable cost. In spite of these advantages,
diffuse optical techniques have almost exclusively used direct detection. In order to explore the feasibility
of interferometrically detected multiply scattered light, we constructed a multiply scattered low coherence
interferometry (MS/LCI) system. Using angle resolved detection and a novel illumination scheme, we
demonstrate direct imaging through nearly 94 mean free scattering paths.
Angle-resolved scattering measurements have shown promise as a method of detecting neoplasia and
analyzing cellular structure. Recently we have developed new systems for interferometric measurement of
two-dimensional, depth resolved scattered fields with excellent depth resolution and polarization sensitivity.
We present inverse analysis of oriented ensembles of micro-spheroidal phantoms and cells showing sub-wavelength
accuracy in size and shape determination, and additionally precise estimates of scatterer
orientation. Finally we show that inverse fits provided are essentially free of multiple solutions over a wide
range of possible scatterer sizes and shapes.
We propose a fiber-optic system for Fourier-domain angle-resolved low coherence interferometry. The system is
based on singlemode fiber couplers and employs a scanning fiber to collect the angular scattering distribution of the
sample. The measured distribution is then fitted to Mie theory to estimate the size of the scatterers. Depth resolution
is achieved with Fourier-domain low coherence Mach-Zehnder interferometry. The sample arm of the interferometer
illuminates the specimen with polarized light and collects back-scattered light from the sample. The probe's optical
performance is evaluated and its depth-resolved sizing capability is demonstrated with subwavelength accuracy
using a two-layer microsphere phantom.
Inverse light scattering methods have been applied by several groups as a means to probe cellular structure in both
clinical and scientific applications with sub-wavelength accuracy. These methods determine the geometric properties
of tissue scatterers based on far field scattering patterns. Generally, structure is determined by measuring scattering
over some range of angles, wavelengths, or polarizations and then fitting the observed data to a database of
simulated scattering selected from a range of probable geometries. We have developed new light scattering software
based on the T-matrix method that creates databases of scattering from spheroidal objects, representing a substantial
improvement over Mie theory, a method limited to simulating scattering from spheres. The computational cost of
the T-matrix method is addressed through a simple but massively parallel program that concurrently simulates
scattering across hundreds of PCs. We are exploring the use of these T-matrix databases in inverting interferometric
measurements of angle-resolved scattering from spheroidal cell nuclei using a technique called angle-resolved low
coherence interferometry (a/LCI). With a/LCI, we have previously distinguished between healthy and dysplastic
tissue in both cell cultures and in ex vivo rat and hamster tissue using Mie theory to measure nuclear diameter. We
now present nuclear volume and spheroidal aspect ratio measurements of unstained, living MCF7 cells using the
improved T-matrix database to analyze a/LCI data. We achieve measurement accuracy equivalent to conventional
image analysis of stained samples. We will further validate the approach by comparing experimental measurements
of scattering from polystyrene microspheroids, and show that the T-matrix is a suitable replacement for Mie theory
in ex vivo tissue samples.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.