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. One, with a negative Real-Time -PCR result, was collected from a contact of a confirmed COVID-19 patient. The other 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 Scanning Electron Microscopy, Confocal Microscopy, and Atomic Force Microscopy.
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 1 h 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.
Improvement and segmentation of coronavirus images by mathematics algorithm. The Gauss filter was used to diminishing the noise in the original images. The best performance was obtained using σ = 3. The used window size was o5 × 5. Smaller dimensional windows produced a lot of noise, mainly due to the change of intensity. A larger dimensional window caused a loss of information in the images. A mathematical approach for the morphological grayscale reconstruction was the following:
Let J and I be two grayscale images defined on the same domain DI, taking their values in the discrete set {0, 1, ........, L-1} and such that J ≤ I (i.e., for each pixel p Є DI, J(p) ≤ I (p)). L is an arbitrary positive integer. In this way, it is useful to introduce the geodesic dilations according to the following definition(16):
The elementary geodesic dilation of grayscale image J ≤ I “under” I (J is called the marker image and I is the mask) is defined as,
where the symbol Λ stands for the pointwise minimum and J ⊕ B is the dilation of J by flat structuring element B. The grayscale geodesic dilation of size n ≥ 0 is obtained by,
The grayscale reconstruction of I from J is obtained by iterating grayscale dilations of J “under” I until stability is reached [1, 2, 3], that is,