DCS2022 Orland Oral Abstract
Deadtime-less Single Photon Detection and potential applications
Masanobu Yamamoto1,2, John Jaiber González Murillo3, Keegan Hernandez1,Valery Patsekin1,2, J. Paul Robinson1,2,4
1Miftek Corporation, West Lafayette, IN, United States. 2Basic Medical Sciences, Purdue University, West Lafayette, IN United States. 3Dept. of Electronics and Biomedical Engineering, Universitat de Barcelona, Barcelona, Spain. 4Weldon School of Biomedical Engineering,Purdue University, West Lafayette, IN, United States.
Single photon counting is the most sensitive optical measurement method available. The counting range is limited by photoelectron (PE) pulse width and dark count operating in Geiger-mode typical in SPAD and SiPM sensors. The PE width is determined by the recharge process after typical picoseconds avalanche and the sensor time constant by its capacitance. We achieved sub-ns PE pulses using pF range capacitance coupled with each arrayed pixel and GHz electronics. Dark count was reduced by thermoelectric cooling. Current photon counting performance shows 580ps average PE width, saturation count 500Mcps and dark count <100cps/mm2. Counting electronics can perform up to 1Gcps with ECL logic after 200ps resolution comparator.
Time correlated single photon counting (TCSPC) is an important single photon application. Due to sensor deadtime issues, START-STOP period per excitation impulse becomes the determining factor for a measurement. Deadtime-less photon detection enables the counting of multiple photons within an excitation pulse, enabling simultaneous measure of fluorescence intensity and fluorescence lifetime. The PE pulse stream is captured by a digital oscilloscope and analyzed by MATLAB script, avoiding pulse pair resolution limitations using peak detection and statistical analysis. Time resolution is decided by the sampling rate even in overlapped PE signals. Experiments were performed using a commercial oscilloscope with 8M sampling/2ms at 4Gs=250ps, showing that higher bandwidth and sampling rate instruments improve the measurements. This approach is termed Time Correlated Multi-Photon Counting (TCMPC). When combined with a wide dynamic range photon counting sensor, it is a powerful tool for fluorescence analysis, laser induced photon spectroscopy (LIPS), photon flow cytometry and potentially photon communications in deep and free-space or even underwater.
ISAC Cytometry A Abstract
Quantum approach for nanoparticle fluorescence by sub-ns photon detection
Masanobu Yamamoto1,2 | J. Paul Robinson1,2,3
Nanoparticle optical detection is advantageous with relation to extracellular vesicles or viruses for potential disease diagnostics. This is quite challenging when using conventional flow cytometry optical methods. The reason is that the particle is smaller than the diffraction limit making it difficult to detect. An alternative approach is using fluorescence detection via conjugated fluorochrome on nanoparticles, however, the challenge in this case is the photodetection of a limited number of emitted photons often compared with high background photons. Emitted fluorescence is described by the well-known equation kf = σa I Q. This equation describes the emitted fluorescence rate(kf) [photons/s] as the multiplication of molecular absorption cross section(σa), excitation intensity(I) and quantum yield(Q). In addition, the excitation rate is described by kf=1/t which is the inverse of the lifetime of several ns representing typical conjugated fluorescent molecules used in flow cytometry(2). We recently developed a sub-ns photon sensor that is faster than most fluorescence lifetimes – sub-ns speed is a critically important parameter for the separation of individual emitted photons. Based on observation of fluorescence and background levels on typical commercial flow cytometers. A significant component of the background is induced by water-molecular vibrations. Understanding what constitutes all of the components that contribute to the signals we measure in flow cytometry would help in defining what we currently call “background signals”. To define a theoretical model to try to unravel these issues, a reflective-dry-surface was tested as an experimental set-up. Using this model, it is possible to minimize background and enhance signal allowing us to confirm a single 50nm diameter particle fluorescence signal on the surfaces with minimum background. Our results suggest this quantum approach closely follows established photon base theory and may be predictive for practical nanoparticle fluorescence analysis while enhancing our knowledge of the contribution of background properties.
CYTO2019 Vancouver Poster Abstract
Photon Statistics for Particle Detection
Masanobu Yamamoto1,2 , Keegan Hernandez1 , J. Paul Robinson1,2,3
1Miftek Corporation, West Lafayette, IN, USA, 2Basic Medical Sciences, Purdue University, West Lafayette, IN, USA, 3Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN USA
Introduction & Background: The important function of flow cytometry is particle population analysis by statistics. Statistics is also important to understand particle signal created by single photon in photon stream and photon burst. Photon Statistics is new concept in flow cytometry, but it has been popular topic in quantum mechanics since 1920s. Quantum theory mentions photon has three kind of correlation called as Poissonian with random and independent event, super-Poissonian with bunching and sub-Poissonian with anti-bunching. Development of nanosecond pulse pair resolution photon sensor is now possible to analyze many photoelectron (PE) pulses from photon burst. How about photon statistics on flow signal of excitation laser, Raman, scatter and fluorescence – this is the key question for measured signal computation and quantitative existence probability. In order to evaluate photon statistics, we applied particle signal simulator presenting on separate poster.
Method & Results: Particle signal simulator consists of light source, modulator, intensity control, optical filter and photon sensor. Simulating for typical flow signal, pulse or gate width is 10us at 20kHz and 10k events are captured. Detected PE pulse number is from 0 to 7,000 in 10us. Max. PE is determined by PE pulse width. Measured PE number M is calibrated to true number N with equation M/N= 1- Mt (t: pulse pair resolution). Defining Poissonian Factor PF=σ/√n, it indicates correlation status. Under measurement condition, laser and scatter shows typical Poissonian characteristics as expected. Tungsten halogen thermal light indicates super-Poissonian with peak around 3k PE and close to Poissonian at small PE number. In order to evaluate fluorescence property, dyed microsphere is sandwiched with cover glass and illuminated by focused 405nm laser pulse. Laser photon is eliminated with optical filter and FL PE pulse is captured. This method is also possible to evaluate FL life time and photobleaching at stationary condition. Water Raman, flow check bead and several FL materials indicates random characteristics. Some FL material shows bunching. In addition, it is observed pulse excitation keeps constant emission, but photobleaching by continuous exposure. FL photon looks to depend on molecular energy transfer scheme including recovery process. It is necessary and interesting to study various material FL characteristics from the view point of photon statistics, photobleaching and emission/excitation photon ratio.
Conclusion: In principle, single photon indicates quantum information of molecules on surface or inside cellular particle. The sub-ns single photon sensor opens new analytical approach for photon counting statistics – especially important for nanoparticle measurement. We are developing 100ps resolution time addressing electronics. As next step, it may be possible to analyze sub-ns time correlation among single photons as new tool in flow cytometry.
Photonics West 2017 BiOS Proceeding Paper
Masanobu Yamamoto, Keegan Hernandez, J. Paul Robinson, “Photon spectroscopy by picoseconds differential Geiger-mode Si photomultiplier,”
Proc. SPIE 10500, Single Molecule Spectroscopy and Superresolution Imaging XI, 1050002 (20 February 2018); doi: 10.1117/12.2286743
The pixel array silicon photomultiplier (SiPM) is known as an excellent photon sensor with picoseconds avalanche process with the capacity for millions amplification of photoelectrons. In addition, a higher quantum efficiency(QE), small size, low bias voltage, light durability are attractive features for biological applications. The primary disadvantage is the limited dynamic range due to the 50ns recharge process and a high dark count which is an additional hurdle. We have developed a wide dynamic Si photon detection system applying ultra-fast differentiation signal processing, temperature control by thermoelectric device and Giga photon counter with 9 decimal digits dynamic range. The tested performance is six orders of magnitude with 600ps pulse width and sub-fW sensitivity. Combined with 405nm laser illumination and motored monochromator, Laser Induced Fluorescence Photon Spectrometry (LIPS) has been developed with a scan range from 200~900nm at maximum of 500nm/sec and 1nm FWHM. Based on the Planck equation E=hν, this photon counting spectrum provides a fundamental advance in spectral analysis by digital processing. Advantages include its ultimate sensitivity, theoretical linearity, as well as quantitative and logarithmic analysis without use of arbitrary units. Laser excitation is also useful for evaluation of photobleaching or oxidation in materials by higher energy illumination. Traditional typical photocurrent detection limit is about 1pW which includes millions of photons, however using our system it is possible to evaluate the photon spectrum and determine background noise and auto fluorescence(AFL) in optics in any cytometry or imaging system component. In addition, the photon-stream digital signal opens up a new approach for picosecond time-domain analysis. Photon spectroscopy is a powerful method for analysis of fluorescence and optical properties in biology.
Keywords: Single Photon, Silicon Photomultiplier, Differential Geiger-mode, Motored Monochromator, Laser Induced Photon Spectroscopy(LIPS), Auto-fluorescence, Raman, Photon Stream Digital (PSD)
Book Chapter “Single Cell Analysis” 2017
Photon Detection: Current Status. M. Yamamoto( P227-242) in Single Cell Analysis, J. P. Robinson & A. Cossarizza, Springer Series in Bioengineering, 2017
Fluorescence analysis at low-level light intensity is important and inevitable for flow cytometry and cell biology. The photomultiplier (PMT) has been used as a photon-detection device for many years because of its high sensitivity; it can amplify a single photoelectron to millions of electrons by a cascade of dynodes in a vacuum. In addition, the photocathode in the PMT has the advantage of a wide detection area and wide dynamic range through analog photocurrent detection.
Recently, microelectromechanical system (MEMS)-based PMTs and many solid-state sensors such as Si photodiodes (PDs), avalanche photodiodes (APDs), and Si photomultipliers (SiPMs) have been developed and improved in UV to near-IR wavelengths. Advancements in photosensors especially for photon detection and potential applications are described in this chapter.