Transcript Focal Modulation microscopy

Sowmya Vasa, Umar Alqasemi, Aditya Bhargava
Objectives
 This paper aims in bringing out a novel light
microscopy method called Focal Modulation
Microscopy (FMM).
 To achieve high resolution molecular imaging of the
thick biological tissues embedded in turbid medium.
 Single photon excitation fluorescence microscopy with
effective in up to 600 microns.
Literature Review
Comparison of the
FMM
with
the
following microscopy
techniques:
1. Confocal Microscopy (CM)
2. Multi-Photon Microscopy
(MPM)
3. Optical Coherence
Tomography (OCT)
4. Photo-Acoustic Tomography
(PAT) and others
Confocal Single-Photon and MultiPhoton Microscopy
Confocal Single-Photon and MultiPhoton Microscopy
MPM Concept
 first observed in 1962 in cesium vapor using laser
excitation by Isaac Abella
 Two photons with lower energy can excite a
fluorophore in one quantum event, Each photon
carries approximately half the energy necessary
to excite the molecule.
 Simultaneous absorption of three or more
photons is also possible, allowing for threephoton or multi-photon excitation microscopy.
MPM Concept
 The probability of the near-simultaneous
absorption of two photons is extremely low,
increases quadratically with the intensity.
 Therefore a high flux of excitation photons is
typically required, usually 100 femtosecond laser,
PRR of 80MHz.
 For 1PE 400–500 nm range, 2PE is in the ~700–
1000 nm (infrared) range
Advantages of
MPM over CM
 Light scatter much less for longer wavelengths
 Ballistic photons have higher probability to
excite
 The nonlinear absorption rate decays rapidly out
of focus giving higher resolution images.
 This selective excitation method is effective
when the imaging depth is less than 1 mm.
Disadvantages of
MPM
 very expensive technique that uses laser sources
of ultra-short pulses
 single photon excitation is preferred over multiphoton excitation in some situations in which
nonlinear photo-damage and availability of
fluorescence probes are of concern
Optical Coherence
Tomography (OCT)
Michelson Interferometer
Optical Coherence
Tomography (OCT)
cross-sectional scanning
rate over 30 frames per
second is readily
achievable with an
imaging depth up to 3
mm.
Unfortunately OCT is not
compatible with
fluorescence.
Also, its molecular
imaging capability is
rather limited
Other Modalities
 Photo-Acoustic Tomography (PAT): resolution is
limited by ultrasound wavelength which is minimum
of 10microns even with highest frequency transducer.
 Diffused Optical Tomography (DOT): resolution is
limited a few millimeters
PMM
 Advantages of Single Photon Excitation
 Much cheaper than PMM, no high cost laser
 Optical Diffraction-Limit resolution
 Inspired by some of the ideas in OCT technique.
METHOD
I = A [Sin(2πft) + Sin(2πft + Φ)]
I = A Sin(2πft) + A Sin(2πft) Cos(Φ) + A Cos(2πft) Sin(Φ)
I = A [(1+ Cos(Φ)) Sin(2πft) + Sin(Φ) Cos(2πft)]
I = A√[1 + Sin2(Φ) + Cos2(Φ) + 2 Cos(Φ)] Cos(2πft)
I = A√[2(1 + Cos(Φ))] Cos(2πft)
E = K1 + K2 Sin(2π×5000t)
E = CM + FMM
Effect of increasing pinhole size
The pinhole
radius a is
normalized as ν =
2π NA a /λ ,
where λ is
emission
wavelength and
NA is the
numerical
aperture of the
achromat.
ac part
v=3
The SM fiber we are using has a mode diameter around 4.3 microns and the
measured modulation depth is roughly 70%, agreeing with the numerical
simulation result.
Experiment and results
 Used chicken cartilage as a sample tissue to evaluate
the performance of FMM
 FMM images from 500 and 600 microns in Depths are
obtained with 640nm excitation wavelength. It is
evident that resolution is still high enough to visualize
cellular structures.
 Beyond 600 microns the shot noise associated with the
background started to overwhelm the FMM signal
CM and FMM @ 280µm depth
CM and FMM @ 280µm depth
FMM images at 500 and 600µm
Conclusion
 Developed and experimentally demonstrated a novel
microscopy method for molecular imaging of thick
biological tissues with one photon excited
fluorescence.
 FMM will find numerous applications in basic
biological research and clinical diagnoses with further
improvement in imaging speed using higher frequency
(in MHz) (achieved is 0.2ms per pixel vs. 10µs in CM)
and compatibility with more excitation wavelengths
and fluorescence dyes.
References

Focal Modulation Microscopy; Nanguang Chen, Chee-Howe Wong, and Colin J. R. Sheppard; OSA; 10
November 2008 / Vol. 16, No. 23 / OPTICS EXPRESS 18764.

Spatially Localized Ballistic Two-Photon Excitation in Scattering Media; HENRYK SZMACINSKI,
IGNACY GRYCZYNSKI, JOSEPH R. LAKOWICZ; Center for Fluorescence Spectroscopy, Department
of Biochemistry and Molecular Biology, University of Maryland, School of Medicine, 725 West
Lombard Street, Baltimore, Maryland 21201; Received 17 November.

http://en.wikipedia.org/wiki/Optical_coherence_tomography; visited 11/16/2010.

http://en.wikipedia.org/wiki/Michelson_interferometer; visited 11/16/2010.