This study aimed to describe the morphologic characteristics of the SARS-CoV-2 present in human nasopharyngeal specimens using high-resolution microscopy.
Clinical specimens. Two nasopharyngeal swabs tested by Real Time-PCR to SARS CoV-2 were studied. The first one, with a negative Real-Time -PCR result, was collected from a contact of a confirmed COVID-19 patient (with a second negative PCR result after 21 days). While the other sample belongs to a confirmed COVID-19 patient and resulted in a positive Real -Time-PCR. These samples were received and tested at the National Reference Laboratory of Viral Respiratory Infections of the Institute of Tropical Medicine for virologic diagnostic. Real Time-PCR was performed as previously described(15).
Inactivation of clinical specimens. 200 µL of the clinical specimens were inactivated for 12 hours in a solution of 25% formaldehyde and 5% glutaraldehyde before microscopy study. Inactivated samples were processed at the Center for Advanced Studies of Cuba by SEM, CM, and AFM.
Scanning Electron Microscopy. Ten microliters of the inactivated clinical specimen were placed in a glass-coverslip and dry-in air oven overnight. Then, the coverslips were fixed with 5% glutaraldehyde and dehydrated through a series of increasing concentrations (25%–100%) of ethanol. Coverslips were further subjected to critical point drying for 1.5 h and left in a 37°C oven overnight. Subsequently, the coverslips were sputter-coated with gold (thickness of 10 nm) and viewed under the MIRA3-TESCAN Scanning Electron Microscope (TESCAN, Czech Republic) at 10 kV.
Atomic Force Microscopy. Inactivated clinical samples were processed similarly. Normally, samples for the Atomic Force Microscopy should be subjected to minimal processing to maintain its original condition. However, because of the biohazard of SARS-CoV-2, fixed, and gold-coated samples were used for this technique. The di-Innova Scanning Probe Microscope (Veeco Instruments, Santa Barbara, California) was used in tapping mode. Golden silicon probes NSG30-A, supplied by NT-MDT (Zelenograd, Russia), with a curvature radius of 10 nm and a resonant frequency of 240-440 kHz were used.
Confocal Microscopy. For the immunofluorescence staining, inactivated clinical samples were hydrated for 10 min in PBS and incubated with PBS-Tween (PBS-T) for 20 min. To block non-specific antibody reaction, the best results were obtained by incubating the sections with 0.2% bovine serum albumin (free of IgG) (Sigma Chemical Co. St. Louis, Mo. USA), for 20 min. After two washes in PBS-T, samples were incubated for 1h at 4 ºC with the primary antibody (hyper-immune serum of the COVID-19 -convalescent Cuban patient, dilutions 1:40 in PBS-T). Incubations were followed by washes with PBS-T. The second incubation was accomplished with FITC-conjugated Anti-human Polyvalent Immunoglobulins (IgA- IgG-IgM (dilutions 1:40 in PBS-T, Sigma Co. St. Louis, Mo.USA) for 1 h. After three washes with PBS-T, the sections from all samples were counterstained with propidium iodide (dilution 1:1000, Vector laboratories, Inc. Burlingame CA., USA), followed by extensive washing in PBS-T. Immunostained samples were coverslipped in Vectashield mounting medium (Vector Laboratories, Inc. Burlingame, CA., USA), Fluorescent images were observed on a Confocal laser scanning microscope OLYMPUS FV1000 IX81. Background correction of the images was performed in both, control and samples images using the Olympus Flowview FV-ASW. Software version 3.1 (Olympus, Japan).
Improvement and segmentation of coronavirus images by mathematics algorithm (16) . In this section, we will slightly expose some details of the mathematical algorithms used in the enhancement of microscopic images of the novel coronavirus. A second paper will deeply explain all related to these algorithms.
The Gaussian filter was used to diminishing the noise in the original images. In this case, the best performance was obtained using σ = 1.5. The used window size was 3x3. A larger dimensional window caused a loss of information in the microphotographs (I).
Definition 1: (Grayscale reconstruction): The grayscale reconstruction of I from J obtained by iterating grayscale dilations of J “under” I until stability is reached (II), that is,
Definition 2: The h-dome image D h (I) of the h-domes of a greyscale image I given by
In expressions (I) and (II), the letters J and I are two grayscale images, while the symbol “V” means the pointwise maximum and is an elementary geodesic dilation. For more details on these expressions.
The h-dome transformation extracts light structures without involving any size or shape criterion. The only parameter (h) related to the height of these structures. In the case of coronavirus S-spikes enhancement, this parameter was of very importance, which will be analyzed deeply in a next publication.