Mitochondria play a central role in the regulation of energy metabolism and signal transduction in eukaryotic cells. Although many fluorescent labeling strategies have been developed for mitochondrial studies, the methods that enable labeling efficiency assessment at the single-mitochondrion level are still lacking. By employing the unique advantages of high sensitivity flow cytometry ( HSFCM ) in the sensitive, rapid, and quantitative multiparameter analysis of individual mitochondria, here we examined the performance of several different mitochondrial labeling strategies from the perspectives of brightness, labeling ratio, and stability. Mitochondria isolated from HeLa cells transfected with pAcGFP1-Mito plasmid upon transient or stable transfections, and mitochondria directly labeled with MitoTracker Green or SYTO 62 were analyzed by a laboratory-built three-channel HSFCM. Upon the quantitative measurement of fluorescence brightness, it was found that the fluorescence intensity of green fluorescent protein ( GFP ) in mitochondria isolated from cells with stable transfection was about 17. 7-fold higher than the transient transfection ones, and was approximately two orders of magnitude brighter than mitochondria labeled with MitoTracker Green. On the other hand, the fluorescence signal of SYTO 62 labeling decreased upon washing, indicating its rapid dissociation rate. The strong fluorescence intensity and good labeling stability make stable transfection an efficient method to label mitochondria. The experimental results demonstrates that HSFCM provides a powerful analytical tool to assess the performance of mitochondrial fluorescence labeling via high throughput single mitochondria analysis.

Objective To investigate the radiological features of granulomatous mastitis (GM) in dynamic contrast enhanced MRI (DCE-MRI) and DWI and to differentiate it from the breast cancer in diagnose.Methods Forty five cases of GM and 64 cases of breast cancer confirmed by surgical histopathology or biopsy were retrospectively analyzed in the study.All of the patients were examined preoperatively by DCE-MRI and DWI.Including lesion type,location,enhancement pattern,nipple retraction,supplying arteries,skin thickening and axillary adenopathy in the two groups were evaluated and analyzed by using x2 test.One-way ANOVA was employed to compare the ADC values between the abscess area of GM and non-abscess area,and the difference among the breast cancer lesion area.Dynamic enhancement MR pharmacokinetic parameters were used to measure including early-phase enhancement rate (EER),peak enhancement ratio(Emax),and time to peak ehhancement(Tmax).The statistical differences of EER,Emax and Tmax between the two groups were calculated by using Wilcoxon test.Results In 45 cases of GM,DCE MR images showed nonmass-like lesions (43 patients) and mass-like lesions (2 patients); the nipple involved(16 patients) and segment involved (29 patients);rim-like with heterogeneous enhancement (40 patients) and heterogeneous enhancement (5 patients); nipple retraction (24 patients) supplying arteries dilatation (42 patients),skin thickening (29 patients),and axillary adenopathy (17 patients).Corresponding to the radiological features above,in the 64 breast cancer cases,it showed 54,10,5,59,30,34,16,51,12 and 20,respectively.There were statistical significance between GM and breast cancer in lesion type,location,enhancement pattern,and nipple retraction (x2=67.574,13.075,20.297,20.398 and 23.510,respectively,all P＜0.01).But no differences were existed between 2 groups in supplying arteries and axillary adenopathy(x2=3.928 and 0.502,P＞0.05).EER,Emax and Tmaxin GM were 146.58％,191.13％,195.00 s in GM and 118.13％,159.43％,183.33 s in breast cancer,respectively.Significant statistic differences between GM and breast cancer were found in EER and Emax(Z values were-2.271 and-2.948,P＜0.01).But it did not show significant difference in Tmax (Z =-0.548,P＞0.05).The ADC values of GM on abscess area,non-abscess area,and breast cancer lesion area were (8.0±2.6) × 10-3,(11.3± 1.7) × 10-3 and (8.2± 1.1) × 10-3mm2/s,respectively.There were significant differences in the groups (F=52.167,P＜0.01).Conclusions The characteristic of radiological findings can be found in GM by using advanced MR imaging techniques.DCE-MRI combined with DWI is useful in the differential diagnosis between GM and breast cancer.

Background and purpose: The technique of dynamic contrast-enhanced MRI (DCE-MRI) is widely applied in differential diagnosis between benign and malignant tumor and therapeutic estimation of neoadjuvant chemotherapy in clinic. However, there is no standard quantitative measurement method. This study aimed to assess the variability of different region of interest (ROI) selections for tumor bed of breast cancer using DCE-MRI, and to ascertain the optimal ROI delineation. Methods: We retrospectively analyzed DCE-MRI of 30 patients diagnosed with breast cancer by pathology. The ROIs were delineated by 2 different observers using iCAD software with 4 methods, including whole tumor (Whole), the slice containing the most enhancing voxels (SliceMax), 3 slices centered in SliceMax (Partial) and the 5％ most enhancing contiguous voxels within SliceMax (5Max), to generate the volume transfer constant (Ktrans), the extracellular volume fraction (Ve) and rate constant (Kep). And the reproducibilities of the measurements were assessed using the Bland-Altman method. Results: In the analysis of ROIs delineation, the Ktrans, Ve and Kep reported by different observers were 1.26±0.54 vs 1.25±0.53, 0.75±0.23 vs 0.73±0.22 and 1.93±1.46 vs 1.95±1.51 (P＞0.05) using the method of Whole, and 1.28±0.43 vs 1.26±0.43, 0.74±0.21 vs 0.80±0.27, 1.95±1.53 vs 1.93±1.43 (P＞0.05) using the method of Partial, and 1.30±0.33 vs 1.32±0.33, 0.77±0.20 vs 0.73±0.24, 1.82±1.53 vs 1.87±1.45 (P＞0.05) using the method of SliceMax, and 1.31±0.35 vs 1.35±0.33, 0.77±0.20 vs 0.98±0.25, 1.97±1.36 vs 1.73±1.55 using the method of 5Max (P＜0.05). Using the methods of ROI delineation except 5Max, there was no significant difference between Ktrans, Ve and Kep reported by different observers. The bias vs limits of agreement were 0.002 vs-0.013 to 0.012,-0.003 vs-0.023 to 0.017, 0.006 vs-0.018 to 0.029,-0.035 vs-0.054 to 0.018 measured with Whole method, SliceMax, Partial and 5Max respectively using the Bland-Altman method. Conclusion: It may be reliable to measure functional parameters of primary tumors in breast cancer using DCE-MRI according to the methods of Whole, Partial and SliceMax.