1. 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.
2. 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)
3. 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.