Metabolic trajectory of cellular differentiation in small intestine by Phasor Fluorescence Lifetime Microscopy of NADH

Abstract

There is a lack of fast and high resolution methods to measure metabolic activity of single cells in their native environment. Here we develop a straightforward, non-invasive and sensitive method to measure metabolic phenotype of single cells in a live tissue. By using NADH as optical biomarker and the phasor approach to Fluorescence Lifetime microscopy (FLIM) we identify cellular metabolic fingerprints related to different rates of oxidative phosphorylation and glycolysis. For the first time we measure a three dimensional metabolic gradient in the small intestine (SI) epithelia that appears tightly associated with epithelial cell proliferation, differentiation and the Wnt gradient. The highest free/bound NADH ratios are measured at the base of the crypt within the highly proliferative stem cells, indicating high levels of glycolysis. For the first time mouse small intestinal stem cells in intact live crypts are identified within the tissue by their metabolic fingerprint.

Figure 1. Intrinsic contrast in the small intestine.

(a-b) Fluorescence Intensity images at the base of small intestine crypts excited at 880 nm (a) and 740 nm (b). Images are acquired at the level of the black arrow in fig g. (c–d) Phasor FLIM maps of the tissue excited at 880 nm (c) and 740 nm (d) highlight Lgr5+GFP stem cells (green), collagen (blue), porphyrins within the lamina propria (purple) and NADH within the epithelial cells (cyan). Phasor plot histogram of the FLIM image at 880 nm (e) and 740 nm (f). The color scale (from blue to purple ) corresponds to the 64 levels of the contours that indicate the percent occurrence in the phasor histogram of the pixels of the image. Four clusters corresponding to different tissue components are identified in the phasor distribution with different colors. (g) Epithelium is organized with differentiated villi and crypts that contain the stem cell (in green) while the lamina propria (dotted area) forms the core of each villus and is composed of loose connective tissue containing lymphocytes, myofibroblasts and a network of capillaries and lymphatics.

Figure 2. Label-free identification of stem cells at the base of the small intestine.

Two-photon fluorescence intensity images excited at 740 nm (a) and 880 nm (d) of the small intestine from Lgr5-GFP mice that express eGFP in the crypt base stem cells. Dotted lines indicate the border of the round crypts within the SI tissue. (b) Phasor color maps at 740 nm of porphyrin (blue) and of the relative concentrations of free NADH and bound NADH in the lamina propria. Red-purple color indicates a high free/bound NADH ratio, while violet, cyan and white indicate linearly and progressively decreasing ratios free/bound NADH ratio, as shown in f. (c) Phasor color map at 880 nm highlights GFP (green) stem cells. (e) Phasor histogram of the FLIM image excited at 740 nm. Linear cluster represents all possible relative concentrations of free NADH (purple) and bound NADH (white). (f) Phasor histogram of the FLIM image excited at 880 nm, with a circular cluster that highlights the GFP (green). (g) Scatter plot of the cell phasor of all stem cells (cyan triangles) and Paneth cells (purple squares) excited at 740 nm.

Figure 3. Metabolic gradient in the epithelial cells in the small intestine crypt.

Two-photon fluorescence intensity images (a) and free/bound NADH FLIM maps (b) of the ex-vivo small intestine crypts excited at 740 nm at different depths (z) of the tissue starting from the base of the crypt. (c) Phasor FLIM distribution at different depths (z). The circular blue cluster is used to highlight the porphyrin in the lamina propria. A linear cluster represents relative concentrations of free NADH (purple) and bound NADH (white). Red-purple color indicates a high free/bound NADH ratio, while violet, cyan and white indicate linearly and progressively decreasing ratios of free/bound NADH. (d) Scatter plot of the cell phasor of stem cells and differentiated epithelial cells at different depths from the collagen fibers of the basal membrane (Figure SM3) excited at 740 nm. Cyan diamond for Z = 55 µm, black stars for Z = 44 µm, red triangles for Z = 34 µm, green squares for Z = 24 µm and blue circles for Z = 14 µm). Along the Z axis the cell phasor shifts toward a longer lifetime indicating an increase of bound NADH with respect to free NADH. i.e. a decrease in NADH/NAD+ ratio. (e) Schematic diagram of the small intestine epithelia shows a free/bound NADH gradient along the epithelial cells of the SI crypt.

Figure 4. Metabolic gradient in the epithelial cells in the small intestine villi.

Two-photon fluorescence intensity images (a) and free/bound NADH FLIM maps (b) of ex-vivo small intestine mucosa excited at 740 nm at different depths (z) of the tissue starting from the tip of the villi. (c) Phasor FLIM distribution at different depths z. Circular blue cluster is used to highlight the porphyrin in the lamina propria vessels. A linear cluster represents the relative concentrations of free NADH (purple) and bound NADH (white). Purple color indicates a high free/bound NADH ratio, while violet, cyan and white indicate linearly and progressively decreasing ratios free/bound NADH ratio. (d) Scatter plot of the cell phasor of stem cells and differentiated epithelial cells at different depths from the tip of the villi. Along the Z the cell phasor shifts toward the shorter lifetime indicating an increase of free/bound NADH ratio. (e) Schematic diagram of the small intestine epithelia shows a free/bound NADH gradient along the epithelial cells of the SI villi. (f) Metabolic trajectory (M-trajectory) from free to protein-bound NADH indicated a shift from a glycolytic phenotype to an oxidative phosphorylation phenotype.