Rapid Examination of Nonprocessed Renal Cell Carcinoma Using Nonlinear Microscopy.

CONTEXT.­: Histology, the traditional method of examining surgical tissue under a microscope, is a time-consuming process involving the fixation of tissue in formalin, dehydration, embedding in paraffin, and cutting into thin sections for hematoxylin-eosin (H&E) staining. Frozen section analysis is a faster alternative used in surgery to quickly evaluate tissue, but it has limitations, such as the size of the specimens that can be analyzed and difficulties with fatty and bony tissues. OBJECTIVE.­: To rapidly examine nonprocessed kidney tumors using nonlinear microscopy (NLM), a fluorescence microscopy technique that can rapidly visualize fresh or fixed, rapidly stained, nonprocessed tissue resembling H&E histology. This technology eliminates the need for fixation, embedding, microtome sectioning, or slide preparation. DESIGN.­: In this study, a total of 190 tissue specimens were collected from 46 patients who underwent partial or radical nephrectomy. RESULTS.­: Two genitourinary pathologists confirmed that diagnostically important features present in the H&E images could also be identified in the NLM images. CONCLUSIONS.­: The results of this study demonstrated that NLM had a high degree of correspondence with H&E staining for the classical variants of renal cell carcinoma. NLM offers several clinical benefits, such as facilitating rapid renal cell carcinoma diagnosis, assessment of targeted kidney biopsies for both tumor and medical kidney diseases, and collection of fresh renal cell carcinoma tissue for molecular studies.

Rapid examination of lung tissues by nonlinear microscopy.

OBJECTIVES: Traditional histopathology is a time-intensive and labor-intensive process involving tissue formalin fixation, paraffin embedding, and microtoming into thin sections for H&E staining. Frozen section analysis is a modality used during surgery to quickly evaluate tissue, but it has limitations, such as the size and number of the specimens that can be analyzed as well as difficulties with fatty and bony tissues. Our objective was to investigate the performance of nonlinear microscopy, a fluorescence microscopy technique, for the rapid examination of resected lung tumors. METHODS: In this proof-of-principle study, nonlinear microscopy imaging of resected lung tissue was performed on a total of 73 tissue specimens collected from 13 patients who underwent lobectomy, segmentectomy, or wedge resection for pulmonary nodules. RESULTS: Two pathologists reviewed the digital nonlinear microscopy images in comparison to the corresponding histopathologic H&E slides from a variety of pulmonary pathologies. CONCLUSIONS: This study demonstrated that nonlinear microscopy readily replicates traditional H&E staining for both lung tumors and nonneoplastic pulmonary structures. Nonlinear microscopy provides many advantages over frozen section analysis and is an optical imaging platform that has the potential to augment rapid pathologic evaluation of resected tissues in the age of digital pathology.

Data retrieval from archival renal biopsies using nonlinear microscopy.

Thorough examination of renal biopsies may improve understanding of renal disease. Imaging of renal biopsies with fluorescence nonlinear microscopy (NLM) and optical clearing enables three-dimensional (3D) visualization of pathology without microtome sectioning. Archival renal paraffin blocks from 12 patients were deparaffinized and stained with Hoechst and Eosin for fluorescent nuclear and cytoplasmic/stromal contrast, then optically cleared using benzyl alcohol benzyl benzoate (BABB). NLM images of entire biopsy fragments (thickness range 88-660 µm) were acquired using NLM with fluorescent signals mapped to an H&E color scale. Cysts, glomeruli, exudative lesions, and Kimmelstiel-Wilson nodules were segmented in 3D and their volumes, diameters, and percent composition could be obtained. The glomerular count on 3D NLM volumes was high indicating that archival blocks could be a vast tissue resource to enable larger-scale retrospective studies. Rapid optical clearing and NLM imaging enables more thorough biopsy examination and is a promising technique for analysis of archival paraffin blocks.

High-speed multiplane confocal microscopy for voltage imaging in densely labeled neuronal populations.

Genetically encoded voltage indicators (GEVIs) hold immense potential for monitoring neuronal population activity. To date, best-in-class GEVIs rely on one-photon excitation. However, GEVI imaging of dense neuronal populations remains difficult because out-of-focus background fluorescence produces low contrast and excess noise when paired with conventional one-photon widefield imaging methods. To address this challenge, we developed an imaging system capable of efficient, high-contrast GEVI imaging at near-kHz rates and demonstrate it for in vivo and ex vivo imaging applications in the mouse neocortex. Our approach uses simultaneous multiplane imaging to monitor activity within contiguous tissue volumes with no penalty in speed or requirement for high excitation power. This approach, multi-Z imaging with confocal detection (MuZIC), permits high signal-to-noise ratio voltage imaging in densely labeled neuronal populations and is compatible with imaging through micro-optics. Moreover, it minimizes artifacts associated with concurrent imaging and optogenetic photostimulation for all-optical electrophysiology.

Sensitivity optimization of a rhodopsin-based fluorescent voltage indicator.

The ability to optically image cellular transmembrane voltages at millisecond-timescale resolutions can offer unprecedented insight into the function of living brains in behaving animals. Here, we present a point mutation that increases the sensitivity of Ace2 opsin-based voltage indicators. We use the mutation to develop Voltron2, an improved chemigeneic voltage indicator that has a 65% higher sensitivity to single APs and 3-fold higher sensitivity to subthreshold potentials than Voltron. Voltron2 retained the sub-millisecond kinetics and photostability of its predecessor, although with lower baseline fluorescence. In multiple in vitro and in vivo comparisons with its predecessor across multiple species, we found Voltron2 to be more sensitive to APs and subthreshold fluctuations. Finally, we used Voltron2 to study and evaluate the possible mechanisms of interneuron synchronization in the mouse hippocampus. Overall, we have discovered a generalizable mutation that significantly increases the sensitivity of Ace2 rhodopsin-based sensors, improving their voltage reporting capability.

Ultrasound differential phase contrast using backscattering and the memory effect.

We describe a simple and fast technique to perform ultrasound differential phase contrast (DPC) imaging in arbitrarily thick scattering media. Although configured in a reflection geometry, DPC is based on transmission imaging and is a direct analog of optical differential interference contrast. DPC exploits the memory effect and works in combination with standard pulse-echo imaging, with no additional hardware or data requirements, enabling complementary phase contrast (in the transverse direction) without any need for intensive numerical computation. We experimentally demonstrate the principle of DPC using tissue phantoms with calibrated speed-of-sound inclusions.

Fast, multiplane line-scan confocal microscopy using axially distributed slits.

The inherent constraints on resolution, speed and field of view have hindered the development of high-speed, three-dimensional microscopy techniques over large scales. Here, we present a multiplane line-scan imaging strategy, which uses a series of axially distributed reflecting slits to probe different depths within a sample volume. Our technique enables the simultaneous imaging of an optically sectioned image stack with a single camera at frame rates of hundreds of hertz, without the need for axial scanning. We demonstrate the applicability of our system to monitor fast dynamics in biological samples by performing calcium imaging of neuronal activity in mouse brains and voltage imaging of cardiomyocytes in cardiac samples.

In vivo corneal and lenticular microscopy with asymmetric fundus retroillumination.

We describe a new technique for non-contact in vivo corneal and lenticular microscopy. It is based on fundus retro-reflection and back-illumination of the crystalline lens and cornea. To enhance phase-gradient contrast, we apply asymmetric illumination by illuminating one side of the fundus. The technique produces micron-scale lateral resolution images across a 1 mm diagonal field of view in the central cornea. We show representative images of the epithelium, the subbasal nerve plexus, large stromal nerves, dendritic immune cells, endothelial nuclei, and the anterior crystalline lens, demonstrating the potential of this instrument for clinical applications.

Non-mydriatic chorioretinal imaging in a transmission geometry and application to retinal oximetry.

The human retina is typically imaged in a reflection geometry, where light is delivered through the pupil and images are formed from the light reflected back from the retina. In this configuration, artifacts caused by retinal surface reflex are often encountered, which complicate quantitative interpretation of the reflection images. We present an alternative illumination method, which avoids these artifacts. The method uses deeply penetrating near-infrared (NIR) light delivered transcranially from the side of the head, and exploits multiple scattering to redirect a portion of the light towards the posterior eye. This unique transmission geometry simplifies absorption measurements and enables flash-free, non-mydriatic imaging as deep as the choroid. Images taken with this new transillumination approach are applied to retinal oximetry.

Neuronal imaging with ultrahigh dynamic range multiphoton microscopy.

Multiphoton microscopes are hampered by limited dynamic range, preventing weak sample features from being detected in the presence of strong features, or preventing the capture of unpredictable bursts in sample strength. We present a digital electronic add-on technique that vastly improves the dynamic range of a multiphoton microscope while limiting potential photodamage. The add-on provides real-time negative feedback to regulate the laser power delivered to the sample, and a log representation of the sample strength to accommodate ultrahigh dynamic range without loss of information. No microscope hardware modifications are required, making the technique readily compatible with commercial instruments. Benefits are shown in both structural and in-vivo functional mouse brain imaging applications.