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

• What is Multi-photon microscopy ?
• Fluorophores
• Uses in Acute Kidney Injury
Multiphoton Microscopy
• The MFM uses pulsed long-wavelength light to
excite fluorophores within the specimen being
observ...
A typical system is comprised
of an
1. excitation laser,
2. scanning and imaging
optics,
3. sensitive and optical filters
...
The advantages offered by multiphoton imaging
systems

• True three-dimensional imaging.
• The ability to image deep insid...
FLUOROPHORE
• A fluorophore is a fluorescent chemical compound
that can re-emit light upon light excitation.

• Different ...
Uses in AKI
• Multi-photon microscopy allows for utilization of multiple
fluorescent probes simultaneously, enabling label...
• Intracellular uptake, compartmentation and metabolism can
be studied and quantified once the fluorescent probe has
enter...
• Intracellular organelles such as mitochondria and lysosomes
can be studiedin acute injury states by specific labeling of...
Experiments
CONCLUSION ???
Multiphoton imaging techniques in AKI
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Multiphoton imaging techniques in AKI

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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.

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Multiphoton imaging techniques in AKI

  1. 1. Multiphoton Imaging Techniques in Acute Kidney Injury Dr. Sandeep G. Huilgol MBBS., DNB (Int.Med)., MMedSci (Nephro)
  2. 2. OUTLINE OF DISCUSSION • What is Multi-photon microscopy ? • Fluorophores • Uses in Acute Kidney Injury
  3. 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. 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. 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
  6. 6. FLUOROPHORE • A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation. • Different types of probes are available.
  7. 7. 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.
  8. 8. • 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.
  9. 9. • 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
  10. 10. Experiments
  11. 11. CONCLUSION ???

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