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 Multi- Photon 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 three- photon or multi-photon excitation microscopy.

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 MPM Concept

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. Advantages of MPM over CM

very expensive technique that uses laser sources of ultra-short pulses single photon excitation is preferred over multi- photon excitation in some situations in which nonlinear photo-damage and availability of fluorescence probes are of concern Disadvantages of MPM

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 METHOD E = K 1 + K 2 Sin(2π×5000t) E = CM + FMM 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 + Sin 2 (Φ) + Cos 2 (Φ) + 2 Cos(Φ)] Cos(2πft) I = A√[2(1 + Cos(Φ))] Cos(2πft)

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 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. v = 3

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 280µm depth

FMM images at 500 and 600µm

Conclusion 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 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. visited 11/16/ visited 11/16/