Effect of Fixation and Mounting on Fluorescence Lifetime of Cellular Autofluorescence
01 Jan 2019-IEEE Journal of Selected Topics in Quantum Electronics (Institute of Electrical and Electronics Engineers (IEEE))-Vol. 25, Iss: 1, pp 1-6
TL;DR: It was found that the bound-FAD had two different groups, which was related to the cell division cycle, indicating glycerol has a negative impact on the fluorescence lifetime compared with neutral balsam.
Abstract: Fluorescence lifetime measurements are often performed on live as well as fixed cells and tissues. Fixation and mounting processes are routinely used in cellular research or clinical diagnosis. In this paper, the effects of fixation and mounting on the fluorescence lifetime of cellular autofluorescence were studied by fluorescence lifetime imaging microscopy over time. Two endogenous fluorescent fluorophores, reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), showed different results between live cells and fixed cells. The average lifetime of NADH in live HeLa cells was about 1.02 ns, while maintained about 1.57 ns during the fixation periods of 14 days. The average lifetimes of FAD in live and fixed HeLa cells within 11 days were similar around 1.75 ns but increased to 2.10 ns after 12 days. The free and bound states of the two kinds of fluorophores were further analyzed. It was found that the bound-FAD had two different groups, which was related to the cell division cycle. The effect of mounting medium on fluorescence lifetimes was also studied, indicating glycerol has a negative impact on the fluorescence lifetime compared with neutral balsam.
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TL;DR: It is shown that the lifetime based metabolic contrast in a sample is preserved after chemical fixation and the capability to draw metabolic interpretations in fixed tissues even after long periods of storage is demonstrated.
Abstract: Autofluorescence based fluorescence lifetime imaging microscopy (AF-FLIM) techniques have come a long way from early studies on cancer characterization and have now been widely employed in several cellular and animal studies covering a wide range of diseases. The majority of research in autofluorescence imaging (AFI) study metabolic fluxes in live biological samples. However, tissues from clinical or scientific studies are often chemically fixed for preservation and stabilization of tissue morphology. Fixation is particularly crucial for enzymatic, functional, or histopathology studies. Interpretations of metabolic imaging such as optical redox intensity imaging and AF-FLIM, have often been viewed as potentially unreliable in a fixed sample due to lack of studies in this field. In this study, we carefully evaluate the possibility of extracting microenvironment information in fixed tissues using reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) endogenous fluorescence. The ability to distinguish changes such as metabolism and pH using intrinsic fluorescence in fixed tissues has great pathological value. In this work, we show that the lifetime based metabolic contrast in a sample is preserved after chemical fixation. The fluorescence lifetime of a sample increases with an additive fixative like formaldehyde; however, the fixed tissues retain metabolic signatures even after fixation. This study presents an opportunity to successfully image archived unstained histopathology tissues, and generate useful AF-FLIM signatures. We demonstrate the capability to draw metabolic interpretations in fixed tissues even after long periods of storage.
18 citations
TL;DR: It is demonstrated that fast-developing embryos present a 'quiet' metabolic pattern on Day 2 and Day 4 of development, compared to on-time embryos, the first collective use of confocal and hyperspectral imaging in cleavage-stage bovine embryos in the absence of fluorescent tags.
16 citations
TL;DR: In this paper, two polyarginine conjugates of the complex Os(II) [bis-(4'-(4-carboxyphenyl)-2,2':6',2″-terpyridine)] [Os-(Rn)2]x+ (n = 4 and 8; x = 10 and 18) are reported, to explore whether the R8 peptide sequence that promotes cell uptake requires a contiguous amino acid sequence for membrane permeation or if this can be accomplished in a linearly bridged structure with the additive effect of shorter peptide
Abstract: The preparation of two polyarginine conjugates of the complex Os(II) [bis-(4'-(4-carboxyphenyl)-2,2':6',2″-terpyridine)] [Os-(Rn)2]x+ (n = 4 and 8; x = 10 and 18) is reported, to explore whether the R8 peptide sequence that promotes cell uptake requires a contiguous amino acid sequence for membrane permeation or if this can be accomplished in a linearly bridged structure with the additive effect of shorter peptide sequences. The conjugates exhibit NIR emission centered at 754 nm and essentially oxygen-insensitive emission with a lifetime of 89 ns in phosphate-buffered saline. The uptake, distribution, and cytotoxicity of the parent complex and peptide derivatives were compared in 2D cell monolayers and a three-dimensional (3D) multicellular tumor spheroid (MCTS) model. Whereas, the bis-octaarginine sequences were impermeable to cells and spheroids, and the bis-tetraarginine conjugate showed excellent cellular uptake and accumulation in two 2D monolayer cell lines and remarkable in-depth penetration of 3D MCTSs of pancreatic cancer cells. Overall, the data indicates that cell permeability can be promoted via non-contiguous sequences of arginine residues bridged across the metal centre.
8 citations
TL;DR: In this paper, the metabolic status of rat diabetic cardiomyopathy (DCM) models is assessed by status changes of reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) in both myocardial tissues and blood.
Abstract: This study assesses the metabolic status of rat diabetic cardiomyopathy (DCM) models. Echocardiography is used to detect the diastolic dysfunction in type 2 diabetic rats, and a lower threshold for inducible atrial fibrillation is found in type 2 diabetic rats with diastolic dysfunction compared to the control. Metabolic abnormalities are detected by status changes of reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H), which is an essential coenzyme in cells or tissues. Fluorescence lifetime imaging microscopy (FLIM) is used to monitor changes in NAD(P)H in both myocardial tissues and blood. FLIM reveals that the protein-bound proportion of NAD(P)H in rat myocardium in the DCM group is smaller than the control group, which indicates the oxidative phosphorylation rate of the DCM group decreased. Similar results are found for blood plasma of DCM rats by the FLIM study. FLIM exhibits high potential for screening DCM as a label-free, sensitive, and noninvasive method.
5 citations
TL;DR: It was found that the benign uterine tumors can be detected by measuring the fluorescence lifetime of NAD(P)H and FAD in adjacent healthy cervical tissues, which opened a novel strategy for afflicted women to undergo the cervical biopsies instead of hysterectomies for detecting tumors, which can preserve the fertility of patients.
Abstract: The endogenous fluorophores such as reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD) in cells and tissues can be imaged by fluorescence lifetime...
5 citations
References
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TL;DR: It is argued that immunostaining experiments in dead, permeabilized cells should be complemented with live-cell imaging when scrutinizing protein localization, and pitfalls of immunofluorescence labeling that often receive little attention are discussed.
Abstract: Fluorescent fusion proteins have revolutionized examination of proteins in living cells. Still, studies using these proteins are met with criticism because proteins are modified and ectopically expressed, in contrast to immunofluorescence studies. However, introducing immunoreagents inside cells can cause protein extraction or relocalization, not reflecting the in vivo situation. Here we discuss pitfalls of immunofluorescence labeling that often receive little attention and argue that immunostaining experiments in dead, permeabilized cells should be complemented with live-cell imaging when scrutinizing protein localization.
444 citations
"Effect of Fixation and Mounting on ..." refers background in this paper
...could lead to the change of fluorescence lifetime [16], [17], [21], [22] as well as fluorescence intensity [18], [23], [24]....
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TL;DR: Recent studies using fluorescence lifetime imaging microscopy (FLIM) which offer the potential to discriminate between the two separate pools of NADH and NADPH are discussed, offering biochemical insights into the changes in time-resolved NAD(P)H fluorescence signals observed in diseased tissues.
260 citations
"Effect of Fixation and Mounting on ..." refers background in this paper
...They observed enhanced fluorescence intensity in fixed samples than live or fresh samples [10], [23], [25]....
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TL;DR: Time-resolved fluorescence measurements of the enhanced forms of ECFP and EYFP in water–glycerol mixtures were performed to quantify the effects of the refractive index and viscosity on the fluorescence lifetimes of these proteins.
Abstract: The fluorescence lifetime strongly depends on the immediate environment of the fluorophore Time-resolved fluorescence measurements of the enhanced forms of ECFP and EYFP in water-glycerol mixtures were performed to quantify the effects of the refractive index and viscosity on the fluorescence lifetimes of these proteins The experimental data show for ECFP and EYFP two fluorescence lifetime components: one short lifetime of about 1 ns and a longer lifetime of about 37 ns of ECFP and for EYFP 34 The fluorescence of ECFP is very heterogeneous, which can be explained by the presence of two populations: a conformation (67% present) where the fluorophore is less quenched than in the other conformation (33% present) The fluorescence decay of EYFP is much more homogeneous and the amplitude of the short fluorescence lifetime is about 5% The fluorescence anisotropy decays show that the rotational correlation time of both proteins scales with increasing viscosity of the solvent similarly as shown earlier for GFP The rotational correlation times are identical for ECFP and EYFP, which can be expected since both proteins have the same shape and size The only difference observed is the slightly lower initial anisotropy for ECFP as compared to the one of EYFP
160 citations
"Effect of Fixation and Mounting on ..." refers background in this paper
...could lead to the change of fluorescence lifetime [16], [17], [21], [22] as well as fluorescence intensity [18], [23], [24]....
[...]
TL;DR: This work identifies an autofluorescent, endogenous species with a characteristic fluorescent lifetime distribution as a probe for oxidative stress and correlates the spectroscopic signals arising from lipid droplets by combining FLIM with THG and CARS microscopy which are established techniques for selective lipid body imaging.
Abstract: Presence of reactive oxygen species (ROS) in excess of normal physiological level results in oxidative stress. This can lead to a range of pathological conditions including inflammation, diabetes mellitus, cancer, cardiovascular and neurodegenerative disease. Biomarkers of oxidative stress play an important role in understanding the pathogenesis and treatment of these diseases. A number of fluorescent biomarkers exist. However, a non-invasive and label-free identification technique would be advantageous for in vivo measurements. In this work we establish a spectroscopic method to identify oxidative stress in cells and tissues by fluorescence lifetime imaging (FLIM). We identified an autofluorescent, endogenous species with a characteristic fluorescent lifetime distribution as a probe for oxidative stress. To corroborate our hypothesis that these species are products of lipid oxidation by ROS, we correlate the spectroscopic signals arising from lipid droplets by combining FLIM with THG and CARS microscopy which are established techniques for selective lipid body imaging. Further, we performed spontaneous Raman spectral analysis at single points of the sample which provided molecular vibration information characteristics of lipid droplets.
103 citations
"Effect of Fixation and Mounting on ..." refers background or methods in this paper
...used to evaluate oncological processes in tumor cells [5], [6], stem cell differentiation [7]–[9], oxidative stress [10], differential diagnosis of tissues on breast cancer [11], lung cancer...
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...They observed enhanced fluorescence intensity in fixed samples than live or fresh samples [10], [23], [25]....
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...These fluorescence measurements were often performed on fixed cells [5], [6], [10] as well as fixed tissues [11]–[13] since the easier type of samples we...
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TL;DR: Current understanding of the interactions between FAO and OXPHOS proteins is reviewed and how defects in these two metabolic pathways contribute to mitochondrial disease pathogenesis is reviewed.
Abstract: Mitochondria provide the main source of energy to eukaryotic cells, oxidizing fats and sugars to generate ATP. Mitochondrial fatty acid β-oxidation (FAO) and oxidative phosphorylation (OXPHOS) are two metabolic pathways which are central to this process. Defects in these pathways can result in diseases of the brain, skeletal muscle, heart and liver, affecting approximately 1 in 5000 live births. There are no effective therapies for these disorders, with quality of life severely reduced for most patients. The pathology underlying many aspects of these diseases is not well understood; for example, it is not clear why some patients with primary FAO deficiencies exhibit secondary OXPHOS defects. However, recent findings suggest that physical interactions exist between FAO and OXPHOS proteins, and that these interactions are critical for both FAO and OXPHOS function. Here, we review our current understanding of the interactions between FAO and OXPHOS proteins and how defects in these two metabolic pathways contribute to mitochondrial disease pathogenesis.
97 citations
"Effect of Fixation and Mounting on ..." refers background in this paper
...on the binding-protein and vary in a range of several hundred picoseconds [4]....
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