Frequency-multiplexed in vivo multiphoton phosphorescence lifetime microscopy

Abstract

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.Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms * Corresponding Authors: Scott Howard < showard@nd.edu>, Chris Xu < cx10@cornell.edu>. AUTHOR CONTRIBUTIONS S.H. coordinated the project, designed and built the microscope, designed and fabricated the linear spatial light modulator, wrote control and image processing software, performed simulations, wrote analysis algorithms, analyzed data, preformed animal preparation and surgery, prepared dye and calibrations, and wrote the paper. A.S. greatly assisted with microscope design and assembly, performed simulations and analysis, wrote analysis algorithms, significantly contributed to the content of the paper, and performed experiments verifying pixel rate increase. N.H. and D.K. assisted in animal preparation and surgery for imaging. C.X. initiated the project, significantly contributed to the design of M4 and experimental design, and greatly contributed to the theoretical and experimental discussions. All authors contributed to manuscript editing. HHS Public Access Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptCurrent technologies for overcoming the fundamental pixel rate limitation of serialacquisition MPM require parallel excitation and imaging a sample onto multi-element detectors (typically CCD) [9][10]. While satisfactory for thin tissue slices or non-scattering samples, thick scattering samples typically encountered in in vivo applications cause crosstalk between excited pixels when imaged onto a detector array, resulting in smeared images [11]. State-of-the-art fast fluorescence lifetime imaging microscopy systems utilize parallel excitation (e.g. LED arrays or pulsed diode excitation) or collection (e.g. gated CCD, single photon avalanche dio...

Nature of relaxation processes revealed by the action signals of intensity-modulated light fields

“…Our analysis of the two-photon PL shows that ratio of the signals modulated at 2φ and φ depends on the excitation intensity. At low excitation intensity, the ratio converges to the previously known values, 7 and at higher excitation intensity the ratio decreases (until it goes up again for extremely high intensities, the regime hardly achievable in experiment). Moreover, when the lifetime of the pholuminescence is short compared to the repetition rate of the laser the ratio becomes 1:4.…”

“…Such modified techniques have been successfully used in multiphoton life-time imaging. 7 Among a wide variety of methods (electro-optic modulation or reflection from vibrating surfaces) that can be used to modulate the intensity of the laser beams, only a few, such as the interference of two phase modulated beams in a Mach-Zehnder interferometer, [8][9][10] have been shown to generate a clean modulation (without undesirable sidebands at the multiples of the modulation frequency) of intensity at a single frequency. Single frequency modulated light-fields have recently been used in phase synchronous detection of different coherent and incoherent nonlinear signals.…”

Encoded multisite two-photon microscopy

“…The principle of multiplexing/demultiplexing with orthogonal functions has been used in many fields such as telecommunications and spectroscopy (40). Initially we considered using frequency multiplexing, an approach that was successfully used in biomedical imaging to encode magnetic resonance images and more recently applied to fluorescence (41) and phosphorescence lifetime (42) imaging with a single detector. With this type of encoding, demultiplexing is easily achieved by analyzing the detector signal in the frequency domain (e.g., with Fourier transform) and extracting the signal at the corresponding frequencies.…”

Multifocal multiphoton microscopy with adaptive optical correction

“…More generally, multifocal generation in the framework of TPM has been successfully developed using optical beams generated with lenslet arrays Buist et al, 1998;Kim et al, 2007), Nipkow--Type microlens array (Bewersdorf et al, 1998), high efficiency beam splitter (Nielsen et al, 2001;Rinnenthal et al, 2013), diffractive optical element (DOE) (Watson et al, 2009) and high--performance electrically addressable SLM (Grier, 2003;Howard et al, 2013;Tyndall et al, 2011).…”