Insights into Metabolic Activity and Structure of the Retina through Multiphoton Fluorescence Lifetime Imaging Microscopy in Mice

Abstract

Fluorescence lifetime imaging microscopy (FLIM) evaluates the metabolic state of tissue based on reduced nicotinamide adenine dinucleotide (NAD(P)H) and flavin adenine dinucleotide (FAD). Fluorescence lifetime imaging ophthalmoscopy (FLIO) can image the fundus of the eyes, but cannot detect NAD(P)H. We used multiphoton FLIM to study the metabolic state of the retina in fixed eyes of wild-type mice C57BL6/J. We sectioned the eye using a polyacrylamide gel-embedding technique and estimated the percentage of bound NAD(P)H. We found that oxidative phosphorylation was the predominant metabolic state, particularly in the inner retina, when a fixed retina was used. We also demonstrated the feasibility of FAD imaging of the retina. In addition, we demonstrated that autofluorescence and various FLIM channels, such as hemoglobin, melanin and collagen, can be used to evaluate the structure of the retina and other parts of the eye without any special staining.

Comparison of Retinal Metabolic Activity and Structural Development between rd10 Mice and Normal Mice Using Multiphoton Fluorescence Lifetime Imaging Microscopy

“…In total, 121 eyes were included in this study. Tissue preparation was performed as previously described [14]. Cautery was used to mark the superior aspect of each cornea before enucleation to preserve eyeball orientation.…”

“…The complex wavelet filter was applied to the phasor data [20]. The excitation and emission spectra were chosen based on our previous protocol and that of others [10,14,[21][22][23].…”

“…Free NAD(P)H has a shorter lifetime than bound NAD(P)H, with the former signaling glycolysis, the latter oxidative phosphorylation (OXPHOS), and the relative shifts between them indicating the exchanging of electrons [6,8]. Together, their relative fluorescence lifetimes can be studied in different conditions and used to show cellular metabolic states in various tissues, including in human organoids, mouse oocytes, kidneys, and eyes [6,[8][9][10][11][12][13][14][15]. These metabolic shifts can also be helpful when assessing the degree of oxidative stress [11].…”

“…To date, FLIM eye studies have mostly been performed on healthy, functional eyes [12,14]. Of the ocular pathologies investigated so far, FLIM has been performed on eyes with age-related macular degeneration (AMD) to investigate retinal pigment epithelium (RPE) and sub-RPE deposits [16].…”

FLUTE: A Python GUI for interactive phasor analysis of FLIM data

“…( 15 , 16 , 21 , 22 ) Phasor analysis of FLIM data is increasingly used in several fields of biomedical research ( 23 – 40 ) and is particularly useful for metabolic imaging with the endogenous biomarkers NAD(P)H and FAD to map their complex autofluorescence distributions in live tissues in the context of different physiopathological processes. ( 5 , 16 , 41 – 63 ) .…”