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Advances in fluorescence microscopy techniques to study kidney function

Abstract

Fluorescence microscopy, in particular immunofluorescence microscopy, has been used extensively for the assessment of kidney function and pathology for both research and diagnostic purposes. The development of confocal microscopy in the 1950s enabled imaging of live cells and intravital imaging of the kidney; however, confocal microscopy is limited by its maximal spatial resolution and depth. More recent advances in fluorescence microscopy techniques have enabled increasingly detailed assessment of kidney structure and provided extraordinary insights into kidney function. For example, nanoscale precise imaging by rapid beam oscillation (nSPIRO) is a super-resolution microscopy technique that was originally developed for functional imaging of kidney microvilli and enables detection of dynamic physiological events in the kidney. A variety of techniques such as fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS) and Förster resonance energy transfer (FRET) enable assessment of interaction between proteins. The emergence of other super-resolution techniques, including super-resolution stimulated emission depletion (STED), photoactivated localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM) and structured illumination microscopy (SIM), has enabled functional imaging of cellular and subcellular organelles at ≤50 nm resolution. The deep imaging via emission recovery (DIVER) detector allows deep, label-free and high-sensitivity imaging of second harmonics, enabling assessment of processes such as fibrosis, whereas fluorescence lifetime imaging microscopy (FLIM) enables assessment of metabolic processes.

Key points

  • Microscopic imaging has revolutionized our understanding of kidney structure and physiology.

  • Kidney physiology is determined by dynamic processes and, although understanding of kidney structures at super-resolution is needed to understand kidney function, knowledge of kidney structure per se cannot explain kidney function.

  • Only a few super-resolution microscopy techniques, including super-resolution stimulated emission depletion (STED) microscopy and nanoscale precise imaging by rapid beam oscillation (nSPIRO), can simultaneously provide high-resolution structural information and insights into kidney dynamics.

  • Imaging of individual cells in tissues requires methods that are capable of imaging deep into the tissue, such as two-photon excitation and autofluorescence methods, imaged using methodologies such as the deep imaging via emission recovery (DIVER) detector; single photon excitation using dyes and immunofluorescence approaches cannot penetrate deep into live tissue.

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Fig. 1: Microscopy techniques to assess mobility and interactions in live cells.
Fig. 2: nSPIRO microscopy to track protein dynamics.
Fig. 3: The principles of phasor-FLIM analysis.
Fig. 4: Autofluorescence FLIM for assessment of metabolism.
Fig. 5: Label-free quantification kidney fibrosis by SHG and FLIM imaging DIVER.
Fig. 6: Super-resolution techniques used in imaging.
Fig. 7: Examples of super-resolution imaging techniques in kidney.

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Acknowledgements

L.L. was supported by Associazione Italiana per la Ricerca sul Cancro (AIRC) under MFAG 2018-ID. 21931 project. E.G. and S.R. were supported by National Institutes of Health (NIH) P50 GM076516 and NIH P41GM103540 to E.G. A.E.L. and M.L. were supported by NIH 5R01DK116567 (NIDDK), and National Institute on Ageing (NIA) 5R01AG049493 to M.L.

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Glossary

Two-photon excitation microscopy

A microscopy technique in which fluorophores can be excited at wavelengths that are double the characteristic wavelengths of excitation, as a consequence of the simultaneous absorption of two photons.

Deep imaging via emission recovery

(DIVER). A detector assembly that improves imaging in deep tissues by maximizing the amount of fluorescence emission collected by a large area (6.45 cm2) photomultiplier tube in the forward direction of the excitation propagation.

Fluorescence recovery after photobleaching

(FRAP). A microscopy method whereby a small area of the sample is photobleached and as the molecules repopulate that area, intensity is recovered. This increase in intensity enables calculation of diffusion properties.

Fluorescence correlation spectroscopy

(FCS). A technique that analyses the intensity fluctuations resulting from the movement of fluorescent molecules in and out of an optically restricted volume (~1 µl), which enables calculations of the diffusion parameters, concentrations and photophysical properties of the molecules.

Förster resonance energy transfer

(FRET). An event that results from the transfer of excitation energy from one fluorescent molecule (the donor) to another (the acceptor) when they are in close proximity (<10 nm). FRET decreases the donor intensity and fluorescence lifetime and increases the acceptor fluorescence.

Nanoscale precise imaging by rapid beam oscillation

(nSPIRO). A microscopy technique based on the scanning of a laser beam around the surface of apical microvilli or other cellular protrusions. The detected signal and the position of the scanner are used to perform high-resolution imaging of the protrusions.

Confocal microscopy

An optical imaging technique that provides rejection of out-of-focus signal by combining laser scanning with a pinhole in the detection path. The typical resolution of confocal fluorescence microscopy is ~200 nm in the focal plane and ~600 nm along the axial direction owing to its efficient rejection of out-of-focus signals.

Laurdan

An environment-sensitive fluorophore used to perform biophysical studies on membranes. The spectral properties of Laurdan are sensitive to the penetration of water into membranes.

Generalized polarization domains

Portions of a membrane characterized by the same value of generalized polarization, a parameter that quantifies the spectral shift of Laurdan in different lipid environments.

Giant unilamellar vesicles

(GUVs). Simplified model membrane systems, 1–100 μm in size, in which components of the cell membrane, including lipids and transmembrane proteins, are combined with cell components of the membrane to create a lipid bilayer.

Total internal reflection microscopy

(TIRF). A microscopy approach in which total internal reflection of the excitation light limits fluorescence detection to a sub-diffraction, ~100 nm region above the glass, called the evanescent field. Only the fluorophores that are in this small area are excited and give light.

Fluorescence lifetime imaging microscopy

(FLIM). A microscopy approach that measures the lifetime (average time the fluorophore stays in the excited state) in each of the pixels of an image. FLIM can be used to distinguish signals from different species even if they have similar emission spectra.

Diffusion coefficients

A parameter that describes how fast a molecule undergoes Brownian motion in a given medium.

Single-particle tracking

(SPT). A class of microscopy methods for tracking the movement of molecules or cell components. It involves continuous updating of the centre of a scanned orbit based on feedback provided by the recorded fluorescence intensity, which keeps the component of interest at the orbital centre with nanometre precision.

Fluorescence cross-correlation spectroscopy

An extension of fluorescence correlation spectroscopy (FCS) that allows analysis of intensity fluctuations in two different colours from two different molecules. Correlated fluctuations in both channels results from two interacting molecules diffusing together whereas a lack of correlated intensity fluctuations shows the absence of interaction.

Scanning FCS

An extension of fluorescence correlation spectroscopy (FCS) that allows analysis of intensity fluctuations at multiple locations of a sample as a result of fast laser scanning.

Raster image correlation spectroscopy

(RICS). A spatiotemporal fluorescence correlation technique that measures the diffusion of a molecule of interest in images and determines the diffusion coefficients by fitting the resulting correlation function to a diffusion model.

Number and brightness

(N&B). A fluorescence correlation technique that measures the temporal fluctuations of intensity at each pixel of an image and provides maps of the concentration and the degree of oligomerization of a molecule or proteins.

Pair correlation functions

A fluorescence correlation technique that analyses the fluctuations detected at two separate locations of a sample. It can be used to determine the presence of barriers to diffusion.

Orbital tracking

A single-particle tracking technique that involves continuous updating of the centre of a scanned orbit based on feedback provided by the recorded fluorescence intensity, which keeps the component of interest at the orbital centre with nanometre precision.

Second harmonic generation

(SHG). A non-linear optical process where two photons of the same wavelengths are combined to create a photon of half of the initial wavelength and double the initial photon energy. In biological systems, collagen I and II and myelin fibres lack a centre of symmetry and generate an SHG signal.

Third harmonic generation

(THG). A non-linear optical process where three photons of the same wavelengths are combined to create a photon of one-third of the initial wavelength and three times the initial photon energy. This is generated because of changes in the refractive index and is especially useful for identifying tissue boundaries and lipid droplets.

Photomultiplier tube

A detection device that converts the incident light into an electrical signal.

FLIMBox

A digital device for detection of fluorescence lifetime imaging microscopy (FLIM) data in the frequency domain. The device measures the demodulation and phase shift of the fluorescence intensity with respect to a modulated excitation source.

Phasor plot

A method of describing fluorescence lifetime imaging microscopy (FLIM) data by converting the intensity decay (time domain) and modulation and phase shifts (frequency domain) to the polar plot to separate pixels of FLIM images into different phasor points for a fast fit-free analysis of lifetime data.

π–π interactions

Non-covalent interactions that can occur between the aromatic groups of different molecules.

Phasor-FLIM

A graphical fit-free way of analysing the fluorescence lifetime data from an image. Reciprocity between the phasor plot and the fluorescence lifetime imaging microscopy (FLIM) image means that selection of a particular area on an image can show the lifetime signature of those areas whereas selection of an area on the phasor plot can show areas of the image that have similar lifetime signatures.

Optical elements

Components of an optical setup (for example, a lens, a mirror, a filter).

Optical clearing

The treatment of biological tissues with exogenous agents capable of reducing scattering and increasing imaging penetration depth.

Expansion microscopy

A technique whereby the tissue is put in a polyelectrolyte hydrogel that can isotropically expand the biological specimen.

Moiré patterns

Interference patterns generated when two identical periodic patterns are rotated one with respect to the other.

Bessel beam excitation

The excitation of fluorescence by a special type of laser beam whose shape is described by a Bessel function.

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Ranjit, S., Lanzanò, L., Libby, A.E. et al. Advances in fluorescence microscopy techniques to study kidney function. Nat Rev Nephrol 17, 128–144 (2021). https://doi.org/10.1038/s41581-020-00337-8

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