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Improving Diagnosis of Cervical Pre-Cancer: Combination of PCA and SVM Applied on Fluorescence Lifetime Images
摘要： We report a signi?cant improvement in the diagnosis of cervical cancer through a combined application of principal component analysis (PCA) and support vector machine (SVM) on the average ?uorescence decay pro?le of Fluorescence Lifetime Images (FLI) of epithelial hyperplasia (EH) and CIN-I cervical tissue samples, obtained ex-vivo. The fast and slow components of double exponential ?tted ?uorescence lifetimes were found to be higher for EH compared to the lifetimes of CIN-I samples. Application of PCA to the average time-resolved ?uorescence decay pro?les showed that the 2nd PC, in combination with 1st PC, enhanced the discrimination between EH and CIN-I tissues. Fluorescence lifetime and PC scores were then classi?ed separately by using SVM support vector machine to identify the two. On applying SVM to a combination of ?uorescence lifetime and PC scores, diagnostic capability improved signi?cantly.
关键词： PCA,?uorescence lifetime,SVM,PC scores
更新于2025-09-23 15:22:29
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Two Orders of Magnitude Variation of Diffusion-Enhanced F?rster Resonance Energy Transfer in Polypeptide Chains
摘要： A ?exible peptide chain displays structural and dynamic properties that correspond to its folding and biological activity. These properties are mirrored in intrachain site-to-site distances and diffusion coef?cients of mutual site-to-site motion. Both distance distribution and diffusion determine the extent of F?rster resonance energy transfer (FRET) between two sites labeled with a FRET donor and acceptor. The relatively large F?rster radii of traditional FRET methods (R0 > 20 ?) lead to a fairly low contribution of diffusion. We introduced short-distance FRET (sdFRET) where Dbo, an asparagine residue conjugated to 2,3-diazabicyclo[2.2.2]octane, acts as acceptor paired with donors, such as naphtylalanine (NAla), tryptophan, 5-L-?uorotryptophan, or tyrosine. The F?rster radii are always close to 10 ?, which makes sdFRET highly sensitive to diffusional motion. We recently found indications that the FRET enhancement caused by diffusion depends symmetrically on the product of the radiative ?uorescence lifetime of the donor and the diffusion coef?cient. In this study, we varied this product by two orders of magnitude, using both donors of different lifetime, NAla and FTrp, as well as a varying viscogen concentration, to corroborate this statement. We demonstrate the consequences of this relationship in evaluating the impact of viscogenic coadditives on peptide dimensions.
关键词： chain dynamics,viscosity,radiative ?uorescence lifetime,diffusion,short-distance FRET,peptide structure
更新于2025-09-04 15:30:14
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Label‐Free Metabolic Classification of Single Cells in Droplets Using the Phasor Approach to Fluorescence Lifetime Imaging Microscopy
摘要： Characterization of single cell metabolism is imperative for understanding subcellular functional and biochemical changes associated with healthy tissue development and the progression of numerous diseases. However, single-cell analysis often requires the use of ?uorescent tags and cell lysis followed by genomic pro?ling to identify the cellular heterogeneity. Identifying individual cells in a noninvasive and label-free manner is crucial for the detection of energy metabolism which will discriminate cell types and most importantly critical for maintaining cell viability for further analysis. Here, we have developed a robust assay using the droplet micro?uidic technology together with the phasor approach to ?uorescence lifetime imaging microscopy to study cell heterogeneity within and among the leukemia cell lines (K-562 and Jurkat). We have extended these techniques to characterize metabolic differences between proliferating and quiescent cells—a critical step toward label-free single cancer cell dormancy research. The result suggests a droplet-based noninvasive and label-free method to distinguish individual cells based on their metabolic states, which could be used as an upstream phenotypic platform to correlate with genomic statistics.
关键词： single cell analysis,metabolism,circulating tumor cells,droplet micro?uidics,?uorescence lifetime imaging microscopy,phasor analysis,quiescent stage
更新于2025-09-04 15:30:14