Optical projection tomography (OPT) is a powerful imaging modality for attaining high resolution absorption and fluorescence imaging in tissue samples and embryos with a diameter of roughly 1 mm. Moving past this 1 mm limit, scattered light becomes the dominant fraction detected, adding significant “blur” to OPT. Time-domain OPT has been used to select out early-arriving photons that have taken a more direct route through the tissue to reduce detection of scattered photons in these larger samples, which are the cause of image domain blur1. In addition, it was recently demonstrated by our group that detection of scattered photons could be further depressed by running in a “deadtime” regime where laser repetition rates are selected such that the deadtime incurred by early-arriving photons acts as a shutter to later-arriving scattered photons2. By running in this deadtime regime, far greater early photon count rates are achievable than with standard early photon OPT.
In this work, another advantage of this enhanced early photon collection approach is demonstrated: specifically, a significant improvement in signal-to-noise ratio. In single photon counting detectors, the main source of noise is “afterpulsing,” which is essentially leftover charge from a detected photon that spuriously results in a second photon count. When the arrival of the photons are time-stamped by the time correlated single photon counting (TCSPC) module , the rate constant governing afterpusling is slow compared to the time-scale of the light pulse detected so it is observed as a background signal with very little time-correlation. This signal is present in all time-gates and so adds noise to the detection of early photons. However, since the afterpusling signal is proportional to the total rate of photon detection, our enhanced early photon approach is uniquely able to have increased early photon counts with no appreciable increase in the afterpulsing since overall count-rate does not change. This is because as the rate of early photon detection goes up, the rate of late-photon detection reduces commensurately, yielding no net change in the overall rate of photons detected.
This hypothesis was tested on a 4 mm diameter tissue-mimicking phantom (μa = 0.02 mm-1, μs’ = 1 mm-1) by ranging the power of a 10 MHz pulse 780-nm laser with pulse spread of < 100 fs (Calmar, USA) and an avalanche photodiode (MPD, Picoquant, Germany) and TCSPC module (HydraHarp, Picoquant, Germany) for light detection. Details of the results are in Fig. 1a, but of note is that we observed more than a 60-times improvement in SNR compared to conventional early photon detection that would have taken 1000-times longer to achieve the same early photon count. A demonstration of the type of resolution possible is in Fig 1b with an image of a 4-mm-thick human breast cancer biopsy where tumor spiculations of less than 100 μm diameter are observable.
1Fieramonti, L. et al. PloS one (2012).
2Sinha, L., et al. Optics letters (2016).
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Time-magnified photon counting with 550-fs resolution
Time-correlated single-photon counting (TCSPC) is an enabling technology for applications such as low-light fluorescence lifetime microscopy and photon counting time-of-flight (ToF) 3D imaging. However, state-of-the-art TCSPC single-photon timing resolution (SPTR) is limited to 3–100 ps by single-photon detectors. Here, we experimentally demonstrate a time-magnified TCSPC (TM-TCSPC) that achieves an ultrashort SPTR of 550 fs with an off-the-shelf single-photon detector. The TM-TCSPC can resolve ultrashort pulses with a 130-fs pulse width difference at a 22-fs accuracy. When applied to photon counting ToF 3D imaging, the TM-TCSPC greatly suppresses the range walk error that limits all photon counting ToF 3D imaging systems by 99.2% and thus provides high depth accuracy and precision of 26 µm and 3 µm, respectively.
more » « less- Award ID(s):
- 2016244
- NSF-PAR ID:
- 10287584
- Publisher / Repository:
- Optical Society of America
- Date Published:
- Journal Name:
- Optica
- Volume:
- 8
- Issue:
- 8
- ISSN:
- 2334-2536
- Page Range / eLocation ID:
- Article No. 1109
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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In time-correlated single-photon counting (TCSPC), photons that arrive during the detector and timing electronics dead times are missed, causing distortion of the detection time distribution. Conventional wisdom holds that TCSPC should be performed with detections in fewer than 5% of illumination cycles to avoid substantial distortion. This requires attenuation and leads to longer acquisition times if the incident flux is too high. Through the example of ranging with a single-photon lidar system, this work demonstrates that accurately modeling the sequence of detection times as a Markov chain allows for measurements at much higher incident flux without attenuation. Our probabilistic model is validated by the close match between the limiting distribution of the Markov chain and both simulated and experimental data, so long as issues of calibration and afterpulsing are minimal. We propose an algorithm that corrects for the distortion in detection histograms caused by dead times without assumptions on the form of the transient light intensity. Our histogram correction yields substantially improved depth imaging performance, and modest additional improvement is achieved with a parametric model assuming a single depth per pixel. We show results for depth and flux estimation with up to 5 photoelectrons per illumination cycle on average, facilitating an increase in time efficiency of more than two orders of magnitude. The use of identical TCSPC equipment in other fields suggests that our modeling and histogram correction could likewise enable high-flux acquisitions in fluorescence lifetime microscopy or quantum optics applications.
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Multiple ultrafast spectroscopic techniques and quantum chemical simulations (QCS) were used to investigate the excited state dynamics of BCC-TPTA. This organic chromophore is believed to possess excited state dynamics governed by a thermally activated delayed fluorescence (TADF) mechanism with a reported internal quantum efficiency ( η IQE ) of 84%. In addition, a significant enhancement in its quantum yield ( Φ ) in solution after purging oxygen has been reported. This Φ enhancement has been widely accepted as due to a delayed fluorescence process occurring on the μs time-scale. The spectroscopic measurements were carried out both in solution and blended films, and from fs to μs time-scales. The excited state dynamics of Rhodamine B and Ir(BT) 2 (acac) were also probed for comparison. Investigations in the absence of oxygen were also carried out. Our time-correlated single photon counting (TCSPC) measurements revealed a lack of a long-lived emissive lifetime for BCC-TPTA in any of the media tested. Our ns transient absorption spectroscopy (ns TAS) experiments revealed that BCC-TPTA does not possess triplet transient states that could be linked to a delayed fluorescence process. Instead, the evidence obtained from our spectroscopic tools suggests that BCC-TPTA has the excited state dynamics of a typical fluorescence chromophore and that just comparing the Φ difference before and after purging oxygen from the solution is not an accurate method to claim excited state dynamics governed by a delayed fluorescence mechanism. Consequently, we believe that previous studies, in which the photo-physics of organic chromophores with TADF characteristics are reported, may have overlooked the influence of the host materials on the obtained optical properties in blended films.more » « less
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Ultrafast Laser Microlaryngeal Surgery for In Vivo Subepithelial Void Creation in Canine Vocal Foldsmore » « less
Background/Objectives Tightly‐focused ultrafast laser pulses (pulse widths of 100 fs–10 ps) provide high peak intensities to produce a spatially confined tissue ablation effect. The creation of sub‐epithelial voids within scarred vocal folds (VFs) via ultrafast laser ablation may help to localize injectable biomaterials to treat VF scarring. Here, we demonstrate the feasibility of this technique in an animal model using a custom‐designed endolaryngeal laser surgery probe.
Methods Unilateral VF mucosal injuries were created in two canines. Four months later, ultrashort laser pulses (5 ps pulses at 500 kHz) were delivered via the custom laser probe to create sub‐epithelial voids of ~3 × 3‐mm2in both healthy and scarred VFs. PEG‐rhodamine was injected into these voids. Ex vivo optical imaging and histology were used to assess void morphology and biomaterial localization.
Results Large sub‐epithelial voids were observed in both healthy and scarred VFs immediately following in vivo laser treatment. Two‐photon imaging and histology confirmed ~3‐mm wide subsurface voids in healthy and scarred VFs of canine #2. Biomaterial localization within a void created in the scarred VF of canine #2 was confirmed with fluorescence imaging but was not visualized during follow‐up two‐photon imaging. As an alternative, the biomaterial was injected into the excised VF and could be observed to localize within the void.
Conclusions We demonstrated sub‐epithelial void formation and the ability to inject biomaterials into voids in a chronic VF scarring model. This proof‐of‐concept study provides preliminary evidence towards the clinical feasibility of such an approach to treating VF scarring using injectable biomaterials.
Level of Evidences N/A
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