How to measure your microscope's HPF. A critical guide for residents.

Counting stuff under the microscope is part of the duties of a surgical pathologist. Many textbooks and articles still report the surface area as the number of high-power fields (HPFs) counted. This is bad, since the area displayed by an HPF varies between two microscopes. It is therefore necessary to express the surface as mm2. This is a how to guide written for the resident who has to measure the HPF of the microscope for the first time. The Resident can either calibrate the microscope with a stage micrometer slide (a small ruler on a glass slide) or compute the surface area of the HPF using the numbers on the eyepiece and the magnification objective. for "10X/22" eyepiece and a "40X" objective, the diameter of the HPF is 22/40 = 0.55 (if no other magnification is present), and the surface is 0.238 mm2. The young resident might then ask: "How far off-target was I when I counted the number of HPFs that the chief resident declared to be correct?" Probably not that much: although legitimate in principle and correct in math, the size of the problem is often overstated since microscopes are not that different after all and because pathology is not just about counting.

2022 Golden Goose Award honors serendipitous science.

Ceremony recognizes unexpected breakthroughs on pain relievers, microscopes, and laser surgery.

High-throughput digital pathology via a handheld, multiplexed, and AI-powered ptychographic whole slide scanner.

The recent advent of whole slide imaging (WSI) systems has moved digital pathology closer to diagnostic applications and clinical practices. Integrating WSI with machine learning promises the growth of this field in upcoming years. Here we report the design and implementation of a handheld, colour-multiplexed, and AI-powered ptychographic whole slide scanner for digital pathology applications. This handheld scanner is built using low-cost and off-the-shelf components, including red, green, and blue laser diodes for sample illumination, a modified stage for programmable sample positioning, and a synchronized image sensor pair for data acquisition. We smear a monolayer of goat blood cells on the main sensor for high-resolution lensless coded ptychographic imaging. The synchronized secondary sensor acts as a non-contact encoder for precisely tracking the absolute object position for ptychographic reconstruction. For WSI, we introduce a new phase-contrast-based focus metric for post-acquisition autofocusing of both stained and unstained specimens. We show that the scanner can resolve the 388-nm linewidth on the resolution target and acquire gigapixel images with a 14 mm × 11 mm area in ∼70 seconds. The imaging performance is validated with regular stained pathology slides, unstained thyroid smears, and malaria-infected blood smears. The deep neural network developed in this study further enables high-throughput cytometric analysis using the recovered complex amplitude. The reported do-it-yourself scanner offers a portable solution to transform the high-end WSI system into one that can be made widely available at a low cost. The capability of high-throughput quantitative phase imaging may also find applications in rapid on-site evaluations.

Visualizing cellular and tissue ultrastructure using Ten-fold Robust Expansion Microscopy (TREx).

Expansion microscopy (ExM) is a powerful technique to overcome the diffraction limit of light microscopy that can be applied in both tissues and cells. In ExM, samples are embedded in a swellable polymer gel to physically expand the sample and isotropically increase resolution in x, y, and z. The maximum resolution increase is limited by the expansion factor of the gel, which is four-fold for the original ExM protocol. Variations on the original ExM method have been reported that allow for greater expansion factors but at the cost of ease of adoption or versatility. Here, we systematically explore the ExM recipe space and present a novel method termed Ten-fold Robust Expansion Microscopy (TREx) that, like the original ExM method, requires no specialized equipment or procedures. We demonstrate that TREx gels expand 10-fold, can be handled easily, and can be applied to both thick mouse brain tissue sections and cultured human cells enabling high-resolution subcellular imaging with a single expansion step. Furthermore, we show that TREx can provide ultrastructural context to subcellular protein localization by combining antibody-stained samples with off-the-shelf small-molecule stains for both total protein and membranes.

Functional network topography of the medial entorhinal cortex.

The medial entorhinal cortex (MEC) creates a map of local space, based on the firing patterns of grid, head-direction (HD), border, and object-vector (OV) cells. How these cell types are organized anatomically is debated. In-depth analysis of this question requires collection of precise anatomical and activity data across large populations of neurons during unrestrained behavior, which neither electrophysiological nor previous imaging methods fully afford. Here, we examined the topographic arrangement of spatially modulated neurons in the superficial layers of MEC and adjacent parasubiculum using miniaturized, portable two-photon microscopes, which allow mice to roam freely in open fields. Grid cells exhibited low levels of co-occurrence with OV cells and clustered anatomically, while border, HD, and OV cells tended to intermingle. These data suggest that grid cell networks might be largely distinct from those of border, HD, and OV cells and that grid cells exhibit strong coupling among themselves but weaker links to other cell types.

Handheld and Cost-Effective Fourier Lightfield Microscope.

In this work, the design, building, and testing of the most portable, easy-to-build, robust, handheld, and cost-effective Fourier Lightfield Microscope (FLMic) to date is reported. The FLMic is built by means of a surveillance camera lens and additional off-the-shelf optical elements, resulting in a cost-effective FLMic exhibiting all the regular sought features in lightfield microscopy, such as refocusing and gathering 3D information of samples by means of a single-shot approach. The proposed FLMic features reduced dimensions and light weight, which, combined with its low cost, turn the presented FLMic into a strong candidate for in-field application where 3D imaging capabilities are pursued. The use of cost-effective optical elements has a relatively low impact on the optical performance, regarding the figures dictated by the theory, while its price can be at least 100 times lower than that of a regular FLMic. The system operability is tested in both bright-field and fluorescent modes by imaging a resolution target, a honeybee wing, and a knot of dyed cotton fibers.

Binocular stereo-microscopy for deforming intact amoeba.

A powerful and convenient method for measuring three-dimensional (3D) deformation of moving amoeboid cells will assist the progress of environmental and cytological studies as protists amoebae play a role in the fundamental environmental ecosystem. Here we develop an inexpensive and useful method for measuring 3D deformation of single protists amoeba through binocular microscopy and a newly proposed algorithm of stereo-scopy. From the movies taken from the left and right optical tubes of the binocular microscope, we detect the 3D positions of many intrinsic intracellular vesicles and reconstruct cellular surfaces of amoeboid cells in 3D space. Some observations of sampled behaviors are shown in a single-celled organism of Amoeba proteus. The resultant surface time series is then analyzed to obtain surface velocity, curvature and volume increasing rates of pseudo-pods for characterizing the movements of amoeboid cells. The limitations and errors of this method are also discussed.

Low-cost 3D-printed inverted microscope to detect Mycobacterium tuberculosis in a MODS culture.

MODS, an assay for diagnosis of tuberculosis and drug-susceptibility, is based in the microscopic observation of the characteristic cords of Mycobacterium tuberculosis colonies grown in liquid media. An inverted optical microscope (100× magnification) is required to observe and interpret MODS cultures. Unfortunately, the cost of commercial inverted microscopes is not affordable in low resource settings. To perform a diagnosis of tuberculosis using the MODS assay, images with modest quality are enough for proper interpretation. Therefore, the use of a high cost commercial inverted optical microscope is not indispensable. In this study, we designed a prototype of an optical inverted microscope created by 3D-printing and based on a smartphone. The system was evaluated with 226 MODS TB positive and 207 MODS TB negative digital images. These images were obtained from 10 sputum samples MODS positive and 10 sputum samples MODS negative. The quality of all images was assessed by a qualified technician, in terms of adequacy to interpret and classify them as positive or negative for tuberculosis. The quality of the images was considered appropriate for MODS interpretation. All the 20 samples were correctly classified (as TB positive/negative) by reading with the prototype 3D-printed inverted microscope.

The utility of the exoscope compared to the operating microscope in simulated temporal bone surgery.

OBJECTIVE: To objectively assess the utility of an exoscope during simulated otological surgery. DESIGN: Cohort study. SETTING: Tertiary referral otolaryngology centre. PARTICIPANTS: Seven experienced otologists undertook simulated temporal bone surgery on plastic temporal bones using the Zeiss Kinevo microscope with both a microscope and exoscope facility. OUTCOME MEASURES: The utility of microscope and exoscope was compared using a Likert scale from 1 to 10 with and without PPE. Attributes assessed included image quality, depth perception, adequacy of view, exoscope positioning, surgeon comfort, surgeon safety and adequacy of image and protection for assistants and observers. RESULTS: The exoscope in 3D mode performed as well as or better than the microscope for image quality, field of view and manoeuvrability. It outperformed the microscope for compatibility with PPE, surgeon comfort and assistant/observer experience. It scored almost as highly as the microscope for depth perception. CONCLUSION: There is likely to be a learning curve but this initial assessment of the exoscope shows significant potential as an alternative to the operating microscope in otological surgery but with the advantage of allowing the use of appropriate PPE and better ergonomics for both surgeon and assistant/observer.

Cord factor producer Mycobacterium abscessus subsp. bolletii in asymptomatic immunocompetent host sputa samples

Abstract We report a case of Mycobacterium abscessus subsp. bolletii colonization in upper respiratory tract of an immunocompetent patient, who was misdiagnosed as tuberculosis by Acid Fast Bacilli (AFB) and cord factor formation observed directly from the sputa culture in liquid medium. This fact reflected a significant impact on the individual case's life and showed the importance to identify the mycobacteria isolated from clinical sample at species level, and to determine the true implication of nontuberculous mycobacteria (NTM) detected in clinical samples.

OME-NGFF: a next-generation file format for expanding bioimaging data-access strategies.

The rapid pace of innovation in biological imaging and the diversity of its applications have prevented the establishment of a community-agreed standardized data format. We propose that complementing established open formats such as OME-TIFF and HDF5 with a next-generation file format such as Zarr will satisfy the majority of use cases in bioimaging. Critically, a common metadata format used in all these vessels can deliver truly findable, accessible, interoperable and reusable bioimaging data.

Towards non-blind optical tweezing by finding 3D refractive index changes through off-focus interferometric tracking.

In modern 3D microscopy, holding and orienting arbitrary biological objects with optical forces instead of using coverslips and gel cylinders is still a vision. Although optical trapping forces are strong enough and related photodamage is acceptable, the precise (re-) orientation of large specimen with multiple optical traps is difficult, since they grab blindly at the object and often slip off. Here, we present an approach to localize and track regions with increased refractive index using several holographic optical traps with a single camera in an off-focus position. We estimate the 3D grabbing positions around several trapping foci in parallel through analysis of the beam deformations, which are continuously measured by defocused camera images of cellular structures inside cell clusters. Although non-blind optical trapping is still a vision, this is an important step towards fully computer-controlled orientation and feature-optimized laser scanning of sub-mm sized biological specimen for future 3D light microscopy.

Multimodal on-chip nanoscopy and quantitative phase imaging reveals the nanoscale morphology of liver sinusoidal endothelial cells.

Visualization of three-dimensional (3D) morphological changes in the subcellular structures of a biological specimen is a major challenge in life science. Here, we present an integrated chip-based optical nanoscopy combined with quantitative phase microscopy (QPM) to obtain 3D morphology of liver sinusoidal endothelial cells (LSEC). LSEC have unique morphology with small nanopores (50-300 nm in diameter) in the plasma membrane, called fenestrations. The fenestrations are grouped in discrete clusters, which are around 100 to 200 nm thick. Thus, imaging and quantification of fenestrations and sieve plate thickness require resolution and sensitivity of sub-100 nm along both the lateral and the axial directions, respectively. In chip-based nanoscopy, the optical waveguides are used both for hosting and illuminating the sample. The fluorescence signal is captured by an upright microscope, which is converted into a Linnik-type interferometer to sequentially acquire both superresolved images and phase information of the sample. The multimodal microscope provided an estimate of the fenestration diameter of 119 ± 53 nm and average thickness of the sieve plates of 136.6 ± 42.4 nm, assuming the constant refractive index of cell membrane to be 1.38. Further, LSEC were treated with cytochalasin B to demonstrate the possibility of precise detection in the cell height. The mean phase value of the fenestrated area in normal and treated cells was found to be 161 ± 50 mrad and 109 ± 49 mrad, respectively. The proposed multimodal technique offers nanoscale visualization of both the lateral size and the thickness map, which would be of broader interest in the fields of cell biology and bioimaging.

Deep learning-enabled whole slide imaging (DeepWSI): oil-immersion quality using dry objectives, longer depth of field, higher system throughput, and better functionality.

Whole slide imaging (WSI) has moved the traditional manual slide inspection process to the era of digital pathology. A typical WSI system translates the sample to different positions and captures images using a high numerical aperture (NA) objective lens. Performing oil-immersion microscopy is a major obstacle for WSI as it requires careful liquid handling during the scanning process. Switching between dry objective and oil-immersion lens is often impossible as it disrupts the acquisition process. For a high-NA objective lens, the sub-micron depth of field also poses a challenge to acquiring in-focus images of samples with uneven topography. Additionally, it implies a small field of view for each tile, thus limiting the system throughput and resulting in a long acquisition time. Here we report a deep learning-enabled WSI platform, termed DeepWSI, to substantially improve the system performance and imaging throughput. With this platform, we show that images captured with a regular dry objective lens can be transformed into images comparable to that of a 1.4-NA oil immersion lens. Blurred images with defocus distance from -5 µm to +5 µm can be virtually refocused to the in-focus plane post measurement. We demonstrate an equivalent data throughput of >2 gigapixels per second, the highest among existing WSI systems. Using the same deep neural network, we also report a high-resolution virtual staining strategy and demonstrate it for Fourier ptychographic WSI. The DeepWSI platform may provide a turnkey solution for developing high-performance diagnostic tools for digital pathology.