In order to get high-resolution glomerulus image with large field of view (FOV), stitching multiple small FOV images with high-resolution is necessary. Directly stitching images without properly correction is not acceptable and cannot afford any significant assistance in pathological diagnosis for intensity inhomogeneity and geometric distortion. Therefore we proposed a method of distortion correction and intensity inhomogeneity correction of glomerulus transmission electron microscope (TEM) image. In this paper, we firstly describe how these two distortions degrade images. Secondly, based on the TEM imaging system, image acquisition model and distortion correction model were proposed. Then according to these two models, distortions were greatly degraded and stitching results were improved by respectively applying two corrections, intensity inhomogeneity correction and geometric distortion correction. With the method proposed here, the result was improved significantly and stripes, fuzzy and artifacts were decreased dramatically. Our method has been proved to be valid to solve the problems of TEM glomerulus image distortion and at the same time to improve the result of multiple TEM glomerulus image stitching.
ObjectiveTo design a method for observing pulmonary microcirculation in vivo in a native tissue environment, due to the high incidence of lung diseases and the advantages of animal experiments in vivo.MethodsTen BALB/c male mice were randomly divided into group A and group B, with five mice in each group. A self-made apparatus was used to control the movement towards local lung tissues in order to get a stabilized observation plane, and then a 5-minute video was shot with laser confocal scanning microscope. During the filming, the mice in group A were injected with fluorescein isothiocyanate-dextran via the tail vein, and the mice in group B were injected with green fluorescent protein-platelets (extracted from the blood of tie2-cre&rosa26-tomato-EGFP transgenic black C57 male mice). The data of group A was used to observe the lungs perfusion and the damage to tissue by this method, and the data of group B was used to observe the movement of platelets.ResultsImage of lung structure obtained by this method was clear and stable. Mean areas of alveolus in an instant and at the 30th, 60th, 120th, 180th, and 300th second were (1 603±181), (1 588±183), (1 528±363), (1 506±353), (1 437±369), (1 549±307) μm2, respectively, and there were no significant differences between each time point (P>0.05). The video was smooth, the rapid movement of platelets was recorded and the particles were clear and without tailing; after the observation, hematoxylin-eosin staining showed no obvious damage to the lung tissue.ConclusionThe method can be used for the observation and research of the lung microcirculatory system in the living state of the mouse, and provides a methodological basis for studies of other lung diseases in vivo.
【Abstract】Objective To observe the bacteria in cholesterol stones by electronic microscope and to explore the role of bacteria in the stone formation.Methods Twelve patients with cholelithiasis underwent operations (male 6, female 6, average age 54.6 years） with cholecystolithiasis 5, extrahepatic and intrahepatic bile duct stone 1, common bile duct stone combining with gallstone 6. The cholesterol stones were observed by electronic microscope.Results There were bacterial structures in the cholesterol stones and cholesterol crystals.Conclusion There are bacteria in the core and peripheral of cholesterol stones, which suggests that bacteria may play an initial role in the formation of cholesterol stones.
Objective To compare the mid-term effectiveness of unilateral biportal endoscopy (UBE)-transforaminal lumbar interbody fusion (TLIF) and minimally invasive surgery-transforaminal lumbar interbody fusion (MIS-TLIF) assisted with three-dimensional microscope in the treatment of single-level lumbar spondylolisthesis. Methods A total of 41 single level lumbar spondylolisthesis patients who met the selection criteria were retrospectively collected between June 2018 and September 2019. Twenty-three patients were treated with UBE-TLIF (study group) and 18 with MIS-TLIF assisted with three-dimensional microscope (control group). There was no significant difference in gender, age, Meyerding degree of slippage, type of spondylolisthesis, lesion segment, course of disease, and preoperative hemoglobin (Hb) level, visual analogue scale (VAS) score, Oswestry disability index (ODI), lumbar lordosis (LL), and disc height (DH) between the two groups (P>0.05). The operation time, hospitalization time, intraoperative blood loss, Hb level between preoperative and postoperative at 1 day, and complications were compared between the two groups. The recovery of clinical sign and symptom was evaluated by VAS score and ODI before operation, and at 1 month, 3 months, 1 year, and 3 years after operation. The LL and DH were measured by radiography before operation and at last follow-up, and the fusion rate was calculated according to Suk grade at last follow-up. ResultsAll the operations were successfully completed. There was no significant difference in operation time between the two groups (P>0.05); the hospitalization time, intraoperative blood loss, and Hb difference between pre- and post-operation in the study group were significantly less than those in the control group (P<0.05). Both groups were followed up 36-48 months, with an average of 39.2 months. In the study group, 1 case of dural tear and 2 cases of Cage subsidence occurred, without postoperative infection and epidural hematoma; in the control group, infection occurred in 1 case, dural tear in 2 cases, Cage subsidence in 1 case, and no epidural hematoma occurred; there was no significant difference in the incidence of complications between the two groups (13.04% vs. 22.22%) (χ2=0.601, P=0.438). The VAS score and ODI at each time point after operation in both groups significantly improved when compared with those before operation, and further improved with time (P<0.05). There was no significant difference in VAS scores between the two groups at each time point after operation (P>0.05); the ODI of the study group was significantly lower than that of the control group at 1 and 3 months after operation (P<0.05), and there was no significant difference between the two groups at other time points (P>0.05). The imaging test showed that the intervertebral fusion rates were 95.7% in the study group and 94.4% in the control group at last follow-up, with no significant difference (χ2=0.032, P=0.859). At last follow-up, LL and DH in the two groups significantly improved when compared with those before operation (P<0.05), and the difference between before and after operation showed no significant difference between the two groups (P>0.05). ConclusionBoth UBE-TLIF and MIS-TLIF assisted with three-dimensional microscope have the advantages of clear intraoperative field and high surgical efficiency in treating lumbar spondylolisthesis, and can obtain satisfactory mid-term effectiveness. Compared with MIS-TLIF assisted with three-dimensional microscope, UBE-TLIF has the advantages of less bleeding and faster recovery.
Objective The combined appl ication of green fluorescent protein (GFP) and confocal laser scanning microscope three-dimensional reconstruction (CLSM-3DR) were used to monitor the construction and in vivo transplantation of tissue engineered bone (TEB), to provide for technology in selection of scaffolds and three-dimensional constructional methods. Methods After bone marrow mesenchymal stem cells (BMSCs) were isolated from a 2-year-old green goat by a combination method of density gradient centrifugation and adherent culture, and the expressions of CD29, CD60L, CD45, and CD44 in BMSCs were detected by flow cytometry. Plasmid of pLEGFP-N1 was ampl ified, digested by enzymes (Hind III, BamH I, Sal I, and Bgl II), and identified. Transfection of pLEGFP-N1 into PT67 cells was performed under the help of l iposome. Positive PT67 cells were picked out with G418, and prol iferated for harvesting virus. Based on the titre of virus, after BMSCs were infected by virus containing pLEGFP-N1, GFP positive BMSCs were collected and prol iferated for seeding cells. TEB was fabricated by GFP positive BMSCs and decalcified bone matrix (DBM) and observed by CLSM-3DR for the evaluation of the distribution and prol iferation of seeding cells. After TEB was transplanted in the defect of goat femur, CLSM was used for observing the survival and distribution of GFP positive cells in the grafts. Results The isolated cells were fibroblast-l ike morphous, with the positive expression of CD29 and CD44, and negative expression of CD60L and CD45. The digested production of pLEGFP-N1 was collected for ionophoresis, whose results showed the correct fragment length (6 900 bp). The virus of pLEGFP-N1 was harvested by transfection of pLEGFP-N1 into PT67 cells and used for further infection to obtain GFP positive BMSCs. The prol iferated GFP positive BMSCs and DBM were used for fabrication of TEB. The distribution, prol iferation, and migration of BMSCs in TEB were observed by CLSM-3DR. GFP positive cells also were observed in images of TEB graft in goat femur 28 days after transplantation. Conclusion The BMSCs labeled by GFP in three-dimensional scaffold in vivo were monitored well by CLSM-3DR. It suggests a wide use potency in monitoring of three-dimensional cultured TEB.
Objective To investigate the effectiveness of microscope assisted anterior lumbar discectomy and fusion (ALDF) and mobile microendoscopic discectomy assisted lumbar interbody fusion (MMED-LIF) for lumbar degenerative diseases. Methods A clinical data of 163 patients with lumbar degenerative diseases who met the criteria between January 2018 and December 2020 was retrospectively analyzed. Fifty-three cases were treated with microscope assisted ALDF (ALDF group) and 110 cases with MMED-LIF (MMED-LIF group). There was no significant difference between the two groups in terms of gender, age, disease type, surgical segments, preoperative visual analogue scale (VAS) scores of low back pain and leg pain, Oswestry disability index (ODI), intervertebral space height, lordosis angle, and spondylolisthesis rate of the patients with lumbar spondylolisthesis (P>0.05). The operation time, intraoperative blood loss, and hospital stay of the two groups were recorded. The effectiveness was evaluated by VAS scores of low back pain and leg pain and ODI. Postoperative lumbar X-ray films were taken to observe the position of Cage and measure the intervertebral space height, lordosis angle, and spondylolisthesis rate of the patients with lumbar spondylolisthesis. Results The operations were successfully completed in both groups. The operation time, intraoperative blood loss, and hospital stay in ALDF group were less than those in MMED-LIF group (P<0.05). The patients in both groups were followed up 12-36 months, with an average of 24 months. The VAS scores of low back pain and leg pain and ODI after operation were lower than those before operation in the two groups, and showed a continuous downward trend, with significant differences between different time points (P<0.05). There were significant differences between two groups in VAS score of low back pain and ODI (P<0.05) and no significant difference in VAS score of leg pain (P>0.05) at each time point. The improvement rates of VAS score of low back pain and ODI in ALDF group were significantly higher than those in MMED-LIF group (t=7.187, P=0.000; t=2.716, P=0.007), but there was no significant difference in the improvement rate of VAS score of leg pain (t=0.556, P=0.579). The postoperative lumbar X-ray films showed the significant recovery of the intervertebral space height, lordosis angle, and spondylolisthesis rate at 2 days after operation when compared with preoperation (P<0.05), and the improvements were maintained until last follow-up (P>0.05). The improvement rates of intervertebral space height and lordosis angle in ALDF group were significantly higher than those in MMED-LIF group (P<0.05). There was no significant difference in the reduction rate of spondylolisthesis between the two groups (t=1.396, P=0.167). During follow-up, there was no loosening or breakage of the implant and no displacement or sinking of the Cage. Conclusion Under appropriate indications, microscope assisted ALDF and MMED-LIF both can achieve good results for lumbar degenerative diseases. Microscope assisted ALDF was superior to MMED-LIF in the improvement of low back pain and function and the recovery of intervertebral space height and lordosis angle.
Objective To investigate the feature and regularity of the collagen change in bone healing during bone lengthening. Methods Bone lengthening model was made in the middle segment of the rabbit tibia. Five days after the model was established, the bone was lengthened 1.5 mm perday for 14 days. The rabbits were put to death after elongation, 7,14,21,30,40,50,60 and 70 days after elongation. The distracted area of the bone was imbedded with paraffin. After being stained by the picric acidsirius red staining, the slice was observed under polarized microscope. Results The features of the collagen change in the distracted bone were as follows: ① In the fibrous tissue of the distracted area during lengthening period and the early stage after lengthening, there was not only collagen Ⅲ but alsomuch collagen Ⅰ. ② Collagen Ⅰ, Ⅱ and Ⅲ were observed in the cartilage. ③ Collagen Ⅰ, Ⅱ and Ⅲ were also observed in the pseudogrowth plate. ④ Collagen Ⅰ took the dominance during lengtheningperiod and the late stage after lengthening. Conclusion New bone formation in bone lengthening is under the distracted force, so the collagen changes have different features compared with that in fracture healing. Collagen Ⅰ, Ⅱ and Ⅲcan be identified by picric-acid-sirius red staining and polarized microscope, so a new method for studying the collagen typing in bone repairing is provided.
Objective To study the influence of the immersed time by hydrogen dioxide on the characteristics of bovine cancellous bone granules in various periods. Methods Ten 24-month-old Qinchuan bovine, male or female, weighing 150-170 g, were selected. Cancellous bone granules from metaphysic of bovine long bone were cut into cubes of 5 mm × 5 mm ×5 mm and immersed by 8.8 mol/L hydrogen dioxide for 0, 12, 24, 36, 48, 60 and 72 hours separately. Determination of ash, scanning electron microscope, X-ray energy spectrum and micro CT were used to investigate the changes of composition, structure and qual ity of bone. Results With the immersed time increasing, the contents of organics in the bone cancellous were reduced gradually, and obviously decreased during the periods of 0 to 24 hours and 60 to 72 hours (P lt; 0.05). The contents of calcium and phosphorus decreased gradually, they could not be detected almost after 60 days (P lt; 0.05). Bone mineral density and bone mineral content were decreased obviously after 60 hours (P lt; 0.05). The bone trabecula became sl immer and trabecular spacing became larger. Conclusion Hydrogen dioxide can be used to remove the antigen in xenogeneic bone; however as the time increasing (more than 60 hours) the composition and structure will be damaged. Thus it is important to control the immersed time for maintaining the biological characteristics of xenogeneic bone substitute as well as el iminating antigen by hydrogen dioxide.
OBJECTIVE: To investigate the expression and distribution of platelet derived growth factor receptor-beta(PDGFR-beta) in normal skin and keloid and to discuss its biological function in keloid formation. METHODS: 1. To detect the expression and distribution of PDGFR-beta in normal skin and keloid tissue by immunohistochemistry; 2. To detect the receptor expression in vitro by Flow cytometry (FCM); 3. To detect the subcellular distribution of receptor by Laser confocal microscope. RESULTS: 1. Immunohistochemistry showed that normal skin and keloid tissue were almost the same in expression but different in distribution of PDGFR-beta; 2. There was more expression of PDGFR-beta in normal fibroblasts than that in keloid fibroblasts in vitro by FCM; 3. Laser confocal microscope revealed that the PDGFR-beta concentrated on the surface of cell membrane in keloid fibroblasts, but in normal skin fibroblasts, the receptors were coagulated on the nuclear membrane and intranucleus. CONCLUSION: Compared with the fibroblasts in vivo, there was a difference of the PDGFR-beta expression in fibroblasts in vitro, more expression of PDGFR-beta in normal fibroblast than that in keloid fibroblast in vitro; and the subcellular distribution of PDGFR-beta was different in normal skin and keloid fibroblasts. The characteristics of the expression and distribution of PDGFR-beta in keloid may contribute to the formation of keloid.
Objective To investigate the microscope-assisted anterior cervical surgery and traditional open surgery for the treatment of cervical myelopathy with ossification of the posterior longitudinal ligament (OPLL). Methods Retrospective selection of patients with OPLL who underwent microscope-assisted and traditional open anterior cervical surgery in West China (Airport) Hospital Sichuan University were selected between January 2016 and August 2020. The patients who underwent traditional open anterior cervical surgery between January 2016 and August 2018 were classified as the conventional group, and the patients who underwent microscope-assisted anterior cervical surgery between September 2018 and August 2020 were classified as the microscope group. The baseline characteristics, operative time, intraoperative blood loss, length of hospital stay, Visual Analogue Scale (VAS) of pain before and after surgery, and surgical complications were collected. Neurological function was assessed using the Japanese Orthopaedic Association (JOA) score. Result A total of 46 patients were included. There were 24 cases in the conventional group and 22 cases in the microscope group. There was no significant difference in baseline characteristics between the two groups (P>0.05). The operation time, intraoperative blood loss and length of hospital stay in the microscope group were lower than those in the conventional group (P<0.001). There was no significant difference in VSA score and JOA score between the two groups before operation (P>0.05). There were statistically significant differences in VAS score and JOA score between the two groups 18 months after operation (P<0.001). The comparison of VAS score and JOA score in the two groups before and after operation showed that there was a statistically significant difference between 18 months after operation and before operation (P<0.05). In the microscope group, the average improvement rate of neurological function [(79.90±16.67)% vs. (58.12±17.47)%, t=4.317, P<0.001], excellent and good rate [95.45% (21/22) vs. 66.67% (16/24), χ2=4.354, P=0.037] were higher than those in the conventional group. The total number of complications in the microscope group was lower than that in the conventional group (P=0.024). Conclusion Compared with the traditional open anterior cervical surgery, the microscope-assisted anterior cervical surgery for OPLL can reduce intraoperative blood loss and length of hospital stay, reduce the incidence of postoperative complications.
