PicoMaster

PicoMaster 

Photon Technology International 

TCSPC Fluorescence Lifetime Spectrofluorometer


Fluorescence Lifetime Spectrometer 

PTI has been manufacturing modular fluorescence systems for nearly 

three decades. Fluorescence lifetime instrumentation has always been 

one of the main product lines of our company. In fact, PTI was awarded 

a prestigious R&D 100 Award for developing a new stroboscopic 

lifetime technique back in 1989. Our team of scientists and engineers 

understands that diverse applications require different experimental 

approaches and therefore we develop and offer different techniques 

that can meet these challenges. 

The PicoMaster line represents modular fluorescence lifetime 

systems based on the Time Correlated Single Photon Counting 

(TCSPC) technique. These instruments can meet the strictest 

demands with regard to the lifetime range, from single picoseconds 

Fluorescence Lifetime Measurements 

Applications of time-resolved fluorescence (TRF) have been 

growing very rapidly in recent years. They encompass very diverse 

disciplines, ranging from chemistry and biology to various materials 

science disciplines such as nanotechnology, semiconductors, 

crystals, glasses and ceramics. These applications are based on the 

fluorescence lifetime, which is the average time a molecule spends 

in the excited state before emitting a photon and returning to the 

ground state. 

The fluorescence lifetime provides complementary information to 

the commonly used steady state measurements, as it adds the ability 

to resolve molecular dynamic events and multiple components of 

a system, which are averaged in the steady state experiment. The 

lifetime not only reflects intrinsic properties of the excited molecule, 

but is also affected strongly by properties of the environment and by 

various interactions with surrounding molecules. 

For example, conformational changes in proteins, nucleic acids or 

other macromolecules can be monitored and detected by measuring 

the emission decay of the probe molecule. Distances between two 

chromophore groups of a macromolecule can be determined by 

studying the fluorescence resonance energy transfer (FRET), which 

reduces the lifetime of the donor molecule. Polarity, viscosity of the 

environment, ion transport, and local electric field will be “imprinted” 

in the lifetime of the excited molecule. The presence of impurity or a 

dopant will affect the luminescence decay of a crystal. 

Emission decays often show more than one lifetime and in some cases 

are described by even more complex kinetic behavior. PTI systems 

include a complete software package, which contains analysis 

modules for virtually any time-resolved application. Another 

important technique is time-resolved emission anisotropy, which 

is an emission decay measurement with polarized light. The temporal 

behavior of anisotropy depends on rotational freedom of the emitting 

molecule and thus can be used to determine microviscosity of a 

membrane, the size of a protein, to follow folding and unfolding of a 

protein or to monitor curing processes of polymers. 

to seconds, and provide the highest dynamic range of at least 5 

orders of magnitude. There are many types of light sources that the 

PicoMaster can use, such as pulsed LEDs, laser diodes, and modelocked 

lasers pumping OPO or dye lasers and higher harmonics 

generators. With its Multi-Channel Scaling (MCS) capability, even 

low repetition lasers, such as nitrogen/dye or Q-switched can be 

used for longer fluorescence or phosphorescence lifetimes. A variety 

of detectors are available that can cover different spectral ranges, 

from UV to NIR, making the instruments suitable for very diverse 

applications. And being modular, they can be combined with the 

steady state fluorometer, phosphorescence system or utilized in 

microscopy measurements. 

Protein decay, ex=295 nm, em=320, 340, 400 nm 

Fluorescence decays of bovine serum albumin protein measured with 

PicoMaster 1 (295 nm LED and PMD-2 fast PMT detector). A 3-exponential 

fitting function required to obtain adequate fits (pre-exponential factors in 

parentheses): 

320 nm: 0.3ns (0.43), 2.6ns (0.23) and 6.5ns (0.34) 

340 nm: 0.3ns (0.20), 2.8ns (0.22) and 6.5ns (0.58) 

420 nm: 0.4ns (0.34), 4.6ns (0.38) and 7.9ns (0.28) 

Fluorescence decays shown in the adjacent figure illustrate a complex 

decay of intrinsic tryptophan (Trp) in bovine serum albumin (BSA) 

protein measured across the emission spectrum. The analysis reveals 

3 lifetime components pointing to heterogeneous environment and 

conformational diversity of the protein imprinted on the fluorescence 

decay of polarity sensitive Trp. Such detailed information is not 

revealed in the steady state measurement alone.

The Advantages of TCSPC 

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Signal Statistics - Due to a low probability of photon detection 

in a single excitation event, the standard deviation of the 

measured intensity is governed by Poisson statistics. Therefore, 

the S/N is well-defined and statistical data analysis involving 

numerical reconvolution and fitting of various decay models can 

be performed in a rigorous and reliable way. 

High Dynamic Range - Photons can be counted practically 

indefinitely, so fluorescence decays can be obtained and analyzed 

over many orders of magnitude (typically 4 to 5), which enhances 

the precision of the lifetime determination, especially important 

for complex decays. 

Short Lifetimes - Due to availability of lasers with ultra-short 

pulses (fs-ps), low-jitter multi-channel plate (MCP) detectors and 

single photon avalanche photodiodes (SPAD), lifetimes of a few 

picoseconds can in principle be measured. 

Modern TCSPC Technology 

The TCSPC systems offered by PTI represent the most modern state 

of the art technology. 

Gone are the days when the TCSPC electronics was mounted in a 

huge, floor standing NIM-BIN rack that housed a collection of standalone 

modules like the time-to-amplitude converter (TAC), two 

constant fraction discriminators (CFD), time calibrator, coaxial cablebased 

delay line, a web of interconnecting cables and an assortment 

of knobs and toggle-switches. 

In the modern PTI offering, all these components, including the 

TAC, two CFDs, a fast analog-to-digital converter (ADC) and the 

memory buffer are all integrated in a single PCI board. The board also 

provides two 12 V outputs to power the PMT and an optional trigger 

photodiode. The only connections to the board are the START and 

Max Count Rate: 10 MHz 

Repetition Rates: 0 to 200 MHz 

Frequency Divider: 1-2-4 

Dead Time: 100 ns 

Min Time/Channel: 813 fs 

Max Time Channels/Curve: 4096 

Measurement Times Down to: 0.1 ms 

TAC Range: 50 ns to 5 µs 

Biased TAC Amplifier Gain: 1 to 15 

CFD Threshold (Photon Channel): -20 to -500 mV 

CFD Threshold (Sync Channel): -20 to -500 mV 

Sync Delay: Electronic, automatically calculated and 

applied for the selected TAC time range 

MCS min time/channel: 25 ns 

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Lifetimes in NIR - Due to availability of photon counting PMT 

and SPAD detectors with near-infrared sensitivity, the TCSPC is 

the best choice to measure pico and nanosecond lifetimes in NIR. 

Time Domain Technique - Fluorescence intensity is measured 

directly as a function of time, which makes it more intuitive to 

interpret and easy to follow the kinetic mechanism of a system 

during the measurement. 

Immune to Intensity Fluctuations - Since no more than a 

single photon is counted as a result of a single flash, the intensity 

vs. time histogram is not affected by pulse-to-pulse intensity 

fluctuations of the light source. 

STOP pulses and an optional external TTL trigger. All the board 

functions are conveniently controlled from Felix GX software – a 

comprehensive software platform that operates all PTI instruments 

and data analysis. 

The TCSPC board also has a Multi-Channel Scaling (MCS) function 

incorporated. That makes the TCSPC system really versatile and 

capable of measuring longer fluorescence and phosphorescence 

lifetimes, from a few tens of nanoseconds to seconds. With the MCS 

operation the user can utilize low repetition rate sources, such as 

xenon flash lamps as well as nitrogen and Q-switched lasers. 

PTI TCSPC systems offer femtosecond time resolution, MHz count 

rates and are excellent choices for the most demanding applications. 

Old TCSPC electronics 

Modern TCSPC board


TCSPC Technique 

The TCSPC is the time-domain technique. Like the other PTI lifetime 

technique, the Strobe, it utilizes pulsed light sources (lasers, LEDs and 

old-fashioned ns flash lamps). It also measures the same experimental 

functions as the Strobe, i.e. the fluorescence decay and the IRF. Its 

detection, however, is based on a different principle. 

The block diagram shows the principle of the TCSPC operation. 

The pulse generator triggers the light source and also outputs a sync 

START pulse which triggers the detection electronics. Alternatively, 

for the ultimate in temporal resolution, the START pulse can be 

generated by a fast photodiode in front of the excitation source. 

The START pulse after being fed through a Constant Fraction 

Discriminator (CFD) starts the Time-to-Amplitude Converter (TAC), 

the key element of the technique. After being triggered, the TAC 

starts a voltage ramp, which is linear in time. 

In the mean time, the sample has been excited and has emitted 

fluorescence photons. When the PMT detects the 1st photon, a short 

pulse is created at the output of the PMT. The photoelectron pulses 

from the PMT show considerable pulse-to-pulse amplitude variations 

and are therefore fed through another CFD, which eliminates the 

time jitter caused by the amplitude spread. The signal then enters the 

TAC as a STOP pulse and stops the voltage ramp. 

The voltage value (equivalent to the time difference between the 

start and stop pulses) is read by an analog-to-digital converter (ADC), 

converted to a time channel and the count value in that time channel is 

incremented by 1. The cycle is repeated with each flash and eventually 

after many cycles a histogram of counts vs. time channels is created. 

If the photon detection rate is low enough, so no more than a single 

photon is detected per cycle, the histogram represents undistorted 

fluorescence decay. 

An important feature is that the single photon counting obeys the 

Poisson statistics. Because of that, the standard deviation of each data 

point is well determined, i.e. σ = N 1/2 , where N is the number of 

counts. This makes the data precision very predictable and facilitates 

the analysis process, where the knowledge of standard deviations is 

required. 

As in the Strobe technique, in most cases the analysis requires that the 

decay and the IRF are collected. The model parameters (e.g. lifetimes 

and pre-exponential factors) are recovered from the non-linear least 

squares fitting procedure that involves iterative re-convolution of the 

IRF and model function.

Excitation Sources 

Nanosecond LED and LD Light Sources 

The PicoMaster1 system operates with nanosecond pulsed light 

emitting diodes (LED) and nanosecond laser diodes (LD). LEDs and 

LDs are very stable, versatile, inexpensive and maintenance free light 

sources. They are available from UV to NIR and can cover most of 

LED: 266, 280, 297, 310, 340, 368, 375, 403, 407, 432, 

444, 456, 486, 510, 572 nm 

LDs: 633, 649, 667 nm 

Rep Rate: up to 180 kHz (LED/LD dependent) 

Pulse Width: < 1.5 ns (LED/LD dependent) 

LED and LD Spectra (Normalized) 


fluorescence lifetime applications. The PicoMaster1 comes with an 

LED/LD pulser, which can operate any of the LEDs and LDs listed.


Excitation Sources 

Picosecond LD Light Sources 

The PicoMaster2 utilizes picosecond laser diodes. A number of 

LD heads are available from with wavelengths ranging from UV to 

NIR. The pulse width can be as short as 50 ps, which is sufficient to 

picosecond Laser 

Diodes: 

LDs: 633, 649, 667nm 

375, 405, 445, 473, 488, 635, 650, 660, 670 nm 

(up to 1550 nm available) 

Rep Rate: Variable: 1, 20, 50 MHz (up to 80 MHz 

available) 

Pulse Width: down to 50 ps 

Picosecond/Femtosecond Lasers 

Other optional pulsed light sources will include mode-locked argon 

ion, Nd: YAG or Ti: Sapphire lasers. Argon ion and Nd: YAG provide 

single wavelength outputs and usually need an additional dye laser and 

a frequency doubler. The Ti: sapphire laser is much more versatile 

and stable. It self-mode locks, runs at rep rates around 80 MHz and 

is tunable over ca. 700-1100 nm range. Its output can be frequency 

doubled or tripled with a SHG or THG crystal. In addition, optical 

Nanosecond LED and LD Light Sources Relative Intensities 

measure lifetimes down to about 10 ps. The ps LDs can operate at 

very high rep rates. 

parametric oscillators (OPO) can be used to broaden the excitation 

range of these lasers. These lasers will be at their best when used 

with a fast MCP detector and when combined with the PicoMaster-2 

will provide capability of measuring lifetimes of a few ps. 

These ultra-fast lasers are available on special requests. Please contact 

a sales representative in your area for more details.

Pulse Pile-up Effect, Rep Rate and Speed 

The fundamental requirement of the TCSPC measurement is that the 

photon count rate must be low enough in order to avoid multiple 

photon detection, typically not exceeding 3-5% of the excitation 

repetition rate. At higher count rates, the measured decay curve will 

become distorted. 

This eliminates relatively inexpensive low rep light sources, such as 

nitrogen/dye, Q-switched and excimer lasers as viable excitation 

sources for the TCSPC. On the other hand, these sources are 

excellent choices for PTI’s Strobe Technique and are widely used 

with TM-3 LaserStrobesystem. The lowest practical rep rates for 

TCSPC light sources should start at about 10 kilohertz and higher. 

The ultrafast light sources operating at rep rates of tens of MHz 

seem ideal for TCSPC, as the decay acquisition can be completed 

Lifetime Range 

The PicoMaster systems are excellent choices for measuring all possible 

ranges of fluorescence lifetimes. Depending on the selection of the 

excitation source and the detector, the TCSPC mode of operation 

will cover lifetimes from single picoseconds to microseconds. 

For those users who work in the area of inorganic luminescence or 

phosphorescence where the lifetimes are much longer, the TCSPC 

board has a built-in Multi-Channel Scaling (MCS) capability, which 

Short Lifetimes 

Short lifetimes measured with TD375 ps laser 

diode and PMD-2 fast pmt. Samples: curcumin 

in cyclohexane (43 ps), erythrosine in H2O 

(89 ps), erythrosine in MeOH (465 ps) and 

POPOP in EtOH (1.29 ns). 

Ruthenium Bipyridyl 

Long fluorescence decay of Ru(bpy)3 

measured with TL460 LED and R928 pmt in 

920C cooled pmt housing. 


in seconds or even faster if the sample intensity is reasonable. The 

TCSPC electronics in PTI systems has a dead time of only 100 ns and 

can count photons at up to 10 MHz rates, which is more than enough 

for the fastest lasers available. 

With the high rep rate sources the range of lifetimes that can be 

measured becomes limited. For a laser operating at 80 MHz, the time 

period between flashes is only 12.5 ns, so only the lifetimes shorter 

than about 2 ns can be measured. For longer lifetimes the rep rate has 

to be reduced and the acquisition times will become longer. A pulse 

picker or a cavity dumper to reduce the rep rate is necessary for 

mode-locked ps/fs lasers to ensure that the useful range of lifetimes 

can be measured. The pulsed laser diodes and LEDs from PTI have 

rep rate control built in. 

provides the lifetime range from tens of nanoseconds to seconds, 

depending on the rep rate of the excitation source. Switching from 

TCSPC to MCS operation is done in the software and the TTL trigger 

for the MCS is provided either from the light source pulser or from 

the ASOC-10 system interface. The MCS mode will work well with 

low rep rate sources such as PTI nitrogen/dye laser, Xe flash lamp as 

well as with LEDs and LDs operating at kHz frequencies. 

Tb MCS 

Luminescence decay of Tb +3 ion measured 

with Xe flash lamp excitation and R928 pmt 

in 920C cooled pmt housing using the Multi- 

Channel Scaling (MCS) mode.


Spectral Range 

Excitation 

The PicoMaster1 uses PTI proprietary nanosecond LED and LD 

sources, which cover the excitation range from 260 to 670 nm. There 

are 19 different LED/LD to choose from, which ensures that there is 

a proper excitation source for practically any sample. 

The PicoMaster2 uses picosecond LDs, which are available from 

375 to 1550 nm. Although they lack the deep UV coverage that is 

Emission 

The PicoMaster systems can accommodate a broad range of 

detectors. For UV-VIS there are two fast PMT detectors that cover 

the ranges of 185-650 nm and 185-820 nm, respectively. A number 

of side-on PMTs used with our QuantaMaster systems can also be 

used as TCSPC detectors, especially if ordered with the TE-cooled 

Software Control 

PicoMaster comes with FelixGX software for instrumental control 

and includes new PowerFit-10 analytical software for fluorescence 

lifetime analysis. 

Through the new, powerful ASOC-10 USB interface FelixGX provides 

a full set of data acquisition protocols and controls the hardware for 

all system configurations and operating modes. 

FelixGX controls: 

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Monochromators 

Motorized slits 

Motorized polarizers 

Motorized sample holders 

Temperature control Peltier devices 

Detectors 

External devices such as stopped flow or titrator 

available with ns LEDs, the main benefit here is the short pulse, higher 

energy and higher rep rates. 

Other light sources, like ultra-short mode-locked lasers with higher 

harmonics generators are available on request. These sources, 

depending on the choice, will combine broad excitation wavelength 

coverage with the ability to measure very short lifetimes. 

920C housing. Depending on the choice, these PMTs will cover the 

range of 185-1200 nm. 

There are four PMT detectors that will extend the detection range 

into NIR, either to 1400 nm or 1700 nm. Two of these PMTs will also 

cover UV-VIS range starting from 300 nm. 

FelixGX also fully controls all functions of the TCSPC 

board: 

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Time-to-Amplitude Converter (TAC): 

» 

» 

» 

» 

Range 

Gain 

Offset 

Limit low and limit high 

Sync and Stop channel 

» 

» 

Discriminator threshold 

Zero crossing level 

Time delay of Sync pulse 

» 

» 

Automatically calculated or 

User defined 

Number of channels 

Frequency divider 1, 2 or 4 

Acquisition stop method (stop button, peak channel count or 

time)

Lifetime Analysis 

PowerFit-10 software (incorporated into FelixGX) provides powerful 

analytical package for decay data analysis. All modules include 

reconvolution algorithms, selection of data weighing (Poisson statistics 

or analog signals), shift and offset parameters, statistical goodness-of- 

BSA 420 nm decay 

Protein decay analyzed with a multiexponential model function. At least 

3 lifetimes are required to adequately fit the decay as evidenced by chisquare, 

weighed residuals and autocorrelation’ 


fit parameters (Chi-square, Durbin-Watson, Runs test, residuals and 

autocorrelation), as well as standard deviations of the fit parameters. 

PowerFit-10 contains the following modules: 

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Multi-exponential (1 to 4 lifetimes) decay 

It fits decay to up to 4 lifetimes and corresponding preexponential 

factors, the lifetimes can be floating or individually 

fixed. 

Multi-file multi-exponential 

1 to 4 exponential fitting program operating in a batch mode, will 

analyze up to 100 fluorescence decays. 

Global (1 to 4 lifetimes) analysis 

Analyzes simultaneously several decay data sets assuming that the 

lifetimes are the same for all data sets and only pre-exponential 

factors vary. 

Anisotropy decay 

Free rotor, restricted rotor and other models. 

Stretched exponential 

General fitting function which is applicable to a variety of complex 

kinetics, such as energy transfer in diffusion-controlled systems 

(e.g. Yokota –Tanimoto model), restricted geometries (molecules 

on surfaces, zeolites) etc. 

Micelle quenching kinetics 

Applies to fluorophores in micelles in the presence of external 

quenchers. The decay kinetics follows Infelta-Groetzel-Tachiya 

model, which allows the determination of micelle aggregation 

number and diffusion-controlled quenching rate constant. 

Exponential Series Method (ESM) 

Unconstrained lifetime distribution analysis which uses up to 200 

exponential terms with logarithmically spaced lifetimes. Allows for 

negative pre-exponentials (risetimes). 

Maximum Entropy Method (MEM) 

Unconstrained lifetime distribution analysis (up to 200 exponential 

terms with logarithmically spaced lifetimes) using Shannon-Jaynes 

entropy function – bias free approach to data analysis. Allows for 

negative pre-exponentials (risetimes). 

Time-Resolved Spectra (TRES) 

• 

Decay-Associated Spectra (DAS)


Convolution and Deconvolution 

Because the excitation pulse is not infinitely narrow in time, it is 

necessary to correct for the distorting effect of the instrument response 

function (IRF, which comprises the light source width and detector 

response) on the decay data. This process, called deconvolution, 

requires the light source profile and the fluorescence decay data. For 

fitting a decay model, e.g. one or more exponentials, the method 

of choice world-wide appears to be iterative reconvolution. The 

Lifetime Distribution Analysis Programs 

There is a growing interest in the recovery of distributions of 

fluorescence lifetimes from decay data. PTI offers two programs, which 

quickly accomplish this mathematically complex task. The Exponential 

Series Method (ESM) involves fitting the data to a large sum (up to 

200) of exponentials with fixed, logarithmically spaced lifetimes 

and variable pre-exponential coefficients by minimizing Chi-square. 

The Maximum Entropy Method (MEM) uses the same trial function 

but maximizes the Shannon-Jaynes entropy subject to a constraint 

Fluorescence decay of CdSe quantum dots 

light source profile is convoluted with a trial decay function and the 

parameters varied until the best fit is obtained to the actual data. PTI’s 

software accomplishes this rapidly and efficiently. All PTI decay data 

analysis programs include deconvolution at some stage of the analysis. 

If the decay lifetimes are much longer than the IRF width, the user has 

an option of skipping deconvolution. 

on Chi-square. Both methods are model-free and start with a flat 

distribution function. The MEM is the most rigorous data analysis 

method and offers bias free solution to a problem of fitting complex 

multi-parameter decays. As experimentalists study systems of ever 

increasing complexity (proteins, membranes, micelles, molecules on 

surface, nanoparticles etc.), these distribution analysis programs will 

prove invaluable and in fact necessary. 

Fluorescence decay of CdSe quantum dots in chloroform 

measured with 490 nm pulsed LED excitation and 

monitored at 580 nm. The decay analysis with MEM 

reveals a broad bimodal lifetime distribution reflecting 

polidispersity of QDots.

Specifications 

System 

Detection Technique 

Light Source Range 

Rep Rate 

Pulse Width 

PMT Response 

MCP Response 

Temporal Resolution 

Shortest Lifetime 

Emission Spectral Range 

PicoMaster 1 PicoMaster 2 


Time-Correlated Single Photon Counting (TCSPC) Time-Correlated Single Photon Counting (TCSPC) 

LEDs: 266, 280, 297, 310, 340, 368, 375, 403, 407, 432, 

444, 456, 486, 510, 518, 572 nm 

LDs: 633, 649, 667 nm 

picosecond Laser Diodes: 375, 405, 445, 473, 488, 635, 

650, 660, 670 nm 

(up to 1550 nm available) 

up to 180 kHz (LED dependent) Variable, up to 100 MHz 

< 1.5 ns (LED dependent) Down to 50 ps 

180 ps (UV-VIS), 300 ps (NIR) 180 ps (UV-VIS), 300 ps (NIR) 

25 ps 

813 fs 813 fs 

40 ps < 10 ps 

180–1700 nm (detector dependent) 180–1700 nm (detector dependent) 

PTI has a policy of continuous product development and reserve the right to amend specifications without prior notice (Dec 2010)

Complete Line of Fluorescence Spectroscopy Instruments from PTI 

QuantaMaster Series 

Steady State Fluorescence and Phosphorescence Spectrofluorometers 

TimeMaster Series 

Fluorescence Lifetime Spectrofluorometers 

RatioMaster Series 

Fluorescence Microscopy Spectrofluorometers 

FluoDia 

Fluorescence Microplate Reader 

Photon Technology International 

USA: Photon Technology International, Inc., 300 Birmingham Road, PO Box 272 Birmingham, NJ 08011 

Tel: 609-894-4420, Fax: 609-894-1579, E-mail: marketing@pti-nj.com, www.pti-nj.com 

Canada: Photon Technology International, Inc., 347 Consortium Court, London, Ontario, N6E 2S8 

Tel: 519-668-6920, Fax: 519-668-8437, E-mail: sales@pti-can.com 

UK: Photon Technology International, Inc., Unit M1 Rudford Industrial Estate, Ford Road, Ford, West Sussex BN180BF 

Tel: +44 (0) 1903 719555, Fax: +44 (0) 1903 725722. E-mail: sales@pti-uk.co.uk 

Germany: PhotoMed GmbH, Inninger Str. 1, 82229 Seefeld, Germany, 

Tel: +49 (0) 8152 993090, Fax: +49 (0) 8152 993098, E-mail: sales@photomed.com

PicoMaster

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