Adam Straub

Frequency Multiplexed In Vivo Multiphoton Phosphorescence Lifetime Microscopy

Multiphoton microscopy (MPM) is widely used for optical sectioning deep in scattering tissue, in vivo [1–2]. Phosphorescence lifetime imaging microscopy (PLIM) [3] is a powerful technique for obtaining biologically relevant chemical information through Förster resonance energy transfer and phosphorescence quenching [4–5]. Point-measurement PLIM [6] of phosphorescence quenching probes has recently provided oxygen partial pressure measurements in small rodent brain vasculature identified by high-resolution MPM [7, 8]. However, the maximum fluorescence generation rate, which is inversely proportional to the phosphorescence lifetime, fundamentally limits PLIM pixel rates. Here we experimentally demonstrate a parallel-excitation/parallel collection MPM-PLIM system that increases pixel rate by a factor of 100 compared with conventional configurations while simultaneously acquiring lifetime and intensity images at depth in vivo. Full-frame three-dimensional in vivo PLIM imaging of phosphorescent quenching dye is presented for the first time and defines a new platform for biological and medical imaging.

High speed multiphoton axial scanning through an optical fiber in a remotely scanned temporal focusing setup

Simultaneous spatial and temporal focusing is used to acquire high speed (200Hz), chemically specific axial scans of mouse skin through a single-mode fiber. The temporal focus is remotely scanned by modulating the group delay dispersion (GDD) at the proximal end of the fiber. No moving parts or electronics are required at the distal end. A novel GDD modulation technique is implemented using a piezo bimorph mirror in a folded grating pair to achieve a large GDD tuning range at high speed.

High sensitivity third-order autocorrelation measurement by intensity modulation and third harmonic detection

We demonstrate a novel (to our knowledge) method for high sensitivity measurement of pulsed light at the telecommunication wavelength of 1558 nm using a GaAsP photomultiplier tube. By modulating the input power and analyzing the harmonic components, high sensitivity third-order autocorrelation is obtained in the wavelength and power regimes where a mixture of two-photon absorption and three-photon absorption in the photomultiplier tube is present.

Enhanced axial confinement of sum-frequency generation in a temporal focusing setup.

We demonstrate enhanced axial confinement in a temporal focusing setup with a shaped spectrum and a narrow emission filter, achieving a reduction of 1 order of magnitude of the out-of-focus background when compared with conventional point-scanning two-photon microscopy. By rejecting the background in the optical domain, our technique circumvents the noise problems common in other background subtraction techniques.

Multiphoton modulation microscopy for high-speed deep biological imaging

We demonstrate a novel line scanning multiphoton microscope with a single element detector, potentially allowing fast imaging deep into scattering tissue. Multiphoton biological imaging of ex vivo rat tendon using this technique is presented.

Pulse measurement based on simultaneous two- and three-photon autocorrelation in a GaAsP photomultiplier tube

We demonstrate a novel method for high sensitivity measurement of pulsed light using a GaAsP photomultiplier tube. 2nd- and 3rd-order autocorrelations are obtained simultaneously by modulating the input power and analyzing the RF harmonic components.

Enhanced axial confinement of sum frequency generation in a temporal focusing setup

We demonstrate enhanced axial confinement in a temporal focusing setup using a shaped spectrum and a narrow emission filter, achieving 10× reduction of out-of-focus background when compared to conventional two-photon microscopy.