Metabolic imaging with the use of fluorescence lifetime imaging microscopy (FLIM) accurately detects mitochondrial
Tim Sanchez1, Tianren Wang2, Marta Venturas Pedro1
1Departments of Applied Physics and Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts.
Objective:
To determine whether metabolic imaging with the use of fluorescence lifetime imaging microscopy (FLIM) identifies metabolic differences between normal oocytes and those with metabolic dysfunction.
Design:
Experimental study.
Setting:
Academic research laboratories.
Patient(S):
None.
Intervention(S):
Oocytes from mice with global knockout of Clpp (caseinolytic peptidase P; n = 52) were compared with wild-type (WT) oocytes (n = 55) as a model of severe oocyte dysfunction. Oocytes from old mice (1 year old; n = 29) were compared with oocytes from young mice (12 weeks old; n = 35) as a model of mild oocyte dysfunction.
Main Outcome Measure(S):
FLIM was used to measure the naturally occurring nicotinamide adenine dinucleotide dehydrogenase (NADH) and flavin adenine dinucleotide (FAD) autofluorescence in individual oocytes. Eight metabolic parameters were obtained from each measurement (4 per fluorophore): short (τ1) and long (τ2) fluorescence lifetime, fluorescence intensity (I), and fraction of the molecule engaged with enzyme (F). Reactive oxygen species (ROS) levels and blastocyst development rates were measured to assess illumination safety.
Result(S):
In Clpp-knockout oocytes compared with WT, FAD τ1 and τ2 were longer and I was higher, NADH τ2 was longer, and F was lower. In old oocytes compared with young ones, FAD τ1 was longer and I was lower, NADH τ1 and τ2 were shorter, and I and F were lower. FLIM did not affect ROS levels or blastocyst development rates.
Conclusion(S):
FLIM parameters exhibit strong differentiation between Clpp-knockout versus WT, and old versus young oocytes. FLIM could potentially be used as a noninvasive tool to assess mitochondrial function in oocytes.
Insights
Fluorescence lifetime imaging microscopy (FLIM) effectively distinguishes metabolic differences in oocytes, offering a potential non-invasive method for assessing mitochondrial function and oocyte health.
Area of Science:
- Reproductive biology and developmental science.
- Cellular metabolism and mitochondrial function.
- Advanced microscopy techniques.
Background:
- Oocyte quality is crucial for successful reproduction and is influenced by mitochondrial function.
- Metabolic dysfunction in oocytes can lead to infertility and poor developmental outcomes.
- Current methods for assessing oocyte metabolic status are limited.
Purpose of the Study:
- To evaluate the utility of fluorescence lifetime imaging microscopy (FLIM) in identifying metabolic differences between normal and dysfunctional oocytes.
- To establish FLIM as a non-invasive tool for assessing oocyte mitochondrial health.
Main Methods:
- Experimental study comparing oocytes from Clpp-knockout mice (severe dysfunction) and old mice (mild dysfunction) with wild-type and young oocytes, respectively.
- Utilized FLIM to measure autofluorescence of nicotinamide adenine dinucleotide dehydrogenase (NADH) and flavin adenine dinucleotide (FAD) in individual oocytes.
- Quantified metabolic parameters including fluorescence lifetime (τ1, τ2), intensity (I), and fraction (F), while assessing safety via reactive oxygen species (ROS) and blastocyst development rates.
Main Results:
- FLIM parameters significantly differentiated Clpp-knockout oocytes from wild-type, and old oocytes from young ones.
- Specific alterations in FAD and NADH lifetimes and intensities were observed in dysfunctional oocytes.
- FLIM measurements did not adversely affect oocyte viability or developmental potential.
Conclusions:
- FLIM successfully identifies distinct metabolic profiles associated with oocyte dysfunction.
- This technique shows promise as a non-invasive method for evaluating mitochondrial function in oocytes.
- FLIM could aid in understanding oocyte aging and infertility.
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