Multiphoton imaging helps analyzing cellular pathophysiology of acute kidney injury (AKI) and the effects of potential treatments in the context of an intact functioning organ.

1. Multiphoton Imaging
Techniques in
Acute Kidney Injury
Dr. Sandeep G. Huilgol
MBBS., DNB (Int.Med)., MMedSci (Nephro)

2. OUTLINE OF DISCUSSION
• What is Multi-photon microscopy ?
• Fluorophores
• Uses in Acute Kidney Injury

3. Multiphoton Microscopy
• The MFM uses pulsed long-wavelength light to
excite fluorophores within the specimen being
observed.
• The fluorophore absorbs the energy from two longwavelength photons which must arrive
simultaneously in order to excite an electron into a
higher energy state, from which it can decay,
emitting a fluorescence signal.

4. A typical system is comprised
of an
1. excitation laser,
2. scanning and imaging
optics,
3. sensitive and optical filters
for separating the
fluorescence from the laser
(dichroic beamsplitter) and
blocking the laser light from
reaching the detector
(emission filter).

5. The advantages offered by multiphoton imaging
systems
• True three-dimensional imaging.
• The ability to image deep inside of live tissue.
• Independent of the amount of fluorescent probes
injected, the excitation power and the depth of field
being imaged

7. FLUOROPHORE
• A fluorophore is a fluorescent chemical compound
that can re-emit light upon light excitation.
• Different types of probes are available.

8. Uses in AKI
• Multi-photon microscopy allows for utilization of multiple
fluorescent probes simultaneously, enabling labeling of
different physiological compartments.
• Helps studying cells within their natural living environment,
rather than in isolated ex-vivo controlled settings.
• Analyzing cellular pathophysiology of acute kidney injury (AKI)
and the effects of potential treatments in the context of an
intact functioning organ.
• Additional diagnostic tools for diagnosis, stratification and
prognostic purposes.

9. • Intracellular uptake, compartmentation and metabolism can
be studied and quantified once the fluorescent probe has
entered the cell.
• It is now possible to observe and quantify endocytosis
occurring across the apical membrane of the proximal tubule
cells.
• It is possible to follow the intracellular accumulation and
subcellular distribution over time.
• To undertake repeated observations in the same animal at
varying intervals over days to weeks.
• Understanding drug delivery for AKI states.

10. • Intracellular organelles such as mitochondria and lysosomes
can be studiedin acute injury states by specific labeling of
these organelles and quantifying individual number and
fluorescence potential of respective organelles.
• DNA fluorescent markers can help identify specific cell types
based on their nuclear morphology (e.g. nuclei of podocytes
are characteristically bean-shaped, while endothelial cells
have characteristic flattened elongated morphology).
• It also permits evaluation of intranuclear uptake of other
fluorescent compounds in disease and therapeutic states.
• Analysis of necrosis and apoptosis

11. Experiments

12. CONCLUSION ???