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Related Experiment Video

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Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy
12:51

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Published on: December 9, 2013

Two-color, two-photon, and excited-state absorption microscopy.

Dan Fu1, Tong Ye, Thomas E Matthews

  • 1Princeton University, Department of Chemistry, Princeton, New Jersey 08544, USA.

Journal of Biomedical Optics
|November 13, 2007
PubMed
Summary

We developed a novel microscopy technique to image nonfluorescent species by measuring absorption changes. This method allows high-resolution imaging of melanin distribution in cells, aiding melanoma diagnosis.

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Area of Science:

  • Biophysics
  • Optical Microscopy
  • Spectroscopy

Background:

  • Imaging nonfluorescent species remains a challenge in microscopy.
  • Nonlinear absorption phenomena like two-photon absorption (TPA) and excited-state absorption (ESA) offer potential for contrast generation.

Purpose of the Study:

  • To develop a new microscopy approach for imaging nonfluorescent species.
  • To characterize the TPA and ESA properties of sepia melanin.
  • To demonstrate the capability of imaging melanin distribution in cells.

Main Methods:

  • Utilized two-color, two-photon microscopy with synchronized, modulated laser pulse trains.
  • Employed a radio frequency (RF) lock-in amplifier to detect minute absorption changes (10^-6).
  • Performed spectroscopic studies in solution and imaging in B16 cells.

Main Results:

  • Successfully measured TPA and ESA of sepia melanin.
  • Identified two distinct excited states in melanin with lifetimes of 3 ps and hundreds of nanoseconds at 775 nm.
  • Achieved high-resolution imaging of melanin distribution in cells, comparable to two-photon fluorescence microscopy (TPFM).

Conclusions:

  • The developed technique enables sensitive detection and imaging of nonfluorescent species via nonlinear absorption.
  • Melanin's unique TPA/ESA characteristics are suitable for high-resolution cellular imaging.
  • This method holds potential for diagnosing melanoma by visualizing melanin distribution.