This document discusses a study on a Single-Photon Avalanche Camera (SPC3) for fluorescence lifetime imaging using time-gated technique. It provides theoretical background on photodiodes, fluorescence, fluorescence lifetime imaging (FLIM), and the SPC3 system. It describes the student's internship building a demonstration system for the company Micro Photon Devices and the measures taken. In conclusion, the student gained knowledge in single-photon detection and its applications in medical research fluorescence analysis.

1. UNIVERSITY OF TRENTO
DEPARTMENT OF INFORMATION ENGINEERING AND COMPUTER SCIENCE
UNDERGRADUATE COURSE IN ELECTRONICS AND TELECOMMUNICATIONS ENGINEERING
Single-Photon Avalanche Camera for Time-Gated
Fluorescence Lifetime Imaging
Supervisor Graduand
Ph.D. Lucio Pancheri Giorgio Marchina
ACADEMIC YEAR 2014/2015

3. I am thankful to professor Lucio Pancheri, who assisted me during the writing of this work.
I also wish to thank Dr. Andrea Giudice, Dr. Simone Tisa and the Micro Photon Devices S.R.L. staff,
for being so helpful and friendly over the period of my internship.
Finally, I am grateful to my parents for all the support they have given me over the years.
1

4. Table of contents
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1. THEORETICAL BACKGROUND
1.1. The photodiode . . . . . . . . . . . . . . . . . . . . . . . 4
1.1.1. APD . . . . . . . . . . . . . . . . . . . . . . . . 6
1.1.2. SPAD . . . . . . . . . . . . . . . . . . . . . . . . 8
1.2. Fluorescence . . . . . . . . . . . . . . . . . . . . . . . . 14
1.3. FLIM . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
1.3.1. TCSPC . . . . . . . . . . . . . . . . . . . . . . . 16
1.3.2. Time-Gated . . . . . . . . . . . . . . . . . . . . . 18
1.4. SPC​3 ​
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
1.4.1. Acquisition phase . . . . . . . . . . . . . . . . . 21
1.4.2. Quenching circuit . . . . . . . . . . . . . . . . . 22
1.4.3. Technical advantages . . . . . . . . . . . . . . . 23
2. INTERNSHIP REPORT​ . . . . . . . . . . . . . . . . . . . . . 24
2.1. System description . . . . . . . . . . . . . . . . . . . . . 25
2.2. Measures description . . . . . . . . . . . . . . . . . . . . 28
CONCLUSIONS​ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
REFERENCES ​ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
2

5. INTRODUCTION
I hereby present a study about the Single-Photon Avalanche Camera (SPC​3​
) for
Fluorescence Lifetime Imaging Microscopy (FLIM) using the Time-Gated
technique.
In the field of medical research a wide range of methodologies are commonly
used for biological and chemical analysis of organic substances. Many of these
research projects are based on spectroscopy which provide detailed information
about cellular structure and natural processes for energy exchange.
The simplest imaging systems based on light intensity provide a partial view of
the molecular structure, while the introduction of FLIM analysis broadens the
observation through the records of fluorescence lifetime.
Some substances are able to absorb partially the incident radiation and emit a
small lapse of fluorescence radiation with a higher wavelength.
The lifetime is an additional information which is not linked to the intensity and
the wavelength. Since the lifetime is very short, ranged in nanoseconds,
nowadays it is not possible to measure it in only one acquisition. It is necessary to
repeat the observation on the same sample with Time-Correlated methods. One
way to do this is through the Time-Gated technique where the time is divided in
different acquisition gates. The intensity measure of each gate is correlated to the
time reference.
In this work I mean to explain the results achieved and the knowledge acquired in
this field during my internship by the Micro Photon Devices company of
Bolzano, Italy. The goal was to build a demonstrative layout for marketing and
customer information purposes. In order to clarify the final internship report a
theoretical explanation is given. Following a logical order, the document starts
referring to the physical phenomena, secondly it treats the photodetectors and
how they are used and finally it offers a description of the entire system.
3

6. Chapter 1
THEORETICAL BACKGROUND
1.1. The Photodiode
Photodiodes are photosensors which can detect some radiation of the
electromagnetic spectrum and generate an electrical signal through the
photovoltaic effect.
Transduction happens by means of absorption, which describes the photon’s
behaviour in the crystalline grid. The photon which impacts on the PN junction,
can interact with a Si atom, thus being annihilated and yielding its energy to the
mentioned atom. If the energy of the incident photon on the PN junction is
greater than the band gap energy of the Si photodiode then an electron-hole pair
is generated inside the molecular structure. The photons that are converted in
electron-hole pairs are separated and accelerated by the electric field toward P and
N layers (Figure 1).
The particles collected throughout the layers are called carriers and increase the
potential difference between the junction poles. However, not all the incident
photons generate an electron-hole pair. The probability of it happening is called
quantum efficiency​.
Figure 1. Schematics of Si photodiode cross section during the photovoltaic effect
4

7. Figure 2. shows the circuital behavior of the photodiode. In absence of light the
characteristic function is like any normal diode. When an incident light occurs,
the function shifts downwards.
Figure 2. Current vs voltage characteristic
In order to get a linear light-current response, some circuital configurations are
used. Since some of the electron-hole pairs are generated by thermal agitation,
not all the current is given by the light. The extra carriers are called dark current
and are a thermal noise component. Each material used for the PN junction has a
different spectral response which means a different sensibility-wavelength
function.
5

8. 1.1.1. APD
Normally the current-light conversion follows a linear function with a light slope.
Therefore, in order to have a better output, next generation photodiodes are
developed. Based on the amplification of the current, this type of devices are
called avalanche photodiodes, because an avalanche multiplication inside the PN
junction generates more carriers per photon.
Increasing the inverse voltage, the high electric field throughout the depletion
layer accelerates the carriers and increases the probability of crashing against the
Si atoms. In this case a new carrier is generated composing the avalanche current.
This chain process is called ionization and results in a current gain.
Figure 3. Schematics of cross section during avalanche multiplication
6

9. The amplification of the output signal depends on the inverse voltage and the
junction temperature as shown in Figure 4. In addition, the wavelength of the
incident radiation changes the avalanche gain of the device [1].
Figure 4. Temperature characteristic of avalanche gain
7

10. 1.1.2. SPAD
The state-of-the-art photodetector is the Single-Photon Avalanche Diode
(SPAD), which benefits from the avalanche multiplication in order to detect
every single light photon.
This type of sensor is particularly used to reveal extremely fast optical impulses
and very weak light signals. It is very similar to the APD but the main difference
is the digital light acquisition. Indeed the light intensity level is obtained counting
the single incident photons. A light quantum absorbed inside the depletion layer
produces an avalanche multiplication that saturates the active zone giving a
strong output current. This device provides a digital light acquisition, so
analogical features are not included.
Initially, the voltage supply is higher than the APD configuration, even more
than the breakdown threshold. Therefore, when a photon is absorbed producing
an electron-hole pair, the carriers are strongly accelerated and the extremely high
probability of crash generates an avalanche by ionizing.
In this case the chain reaction is a positive gain process where the current follows
an exponential increase until the balance point between the level of the electric
field and the amount of carriers. The avalanche is now stable if the supply is over
the breakdown threshold.
The most important feature is the speed of the current rise which results from the
photon absorption. The faster, the better, because it means less delay between
photon arrival and output signal.
After the avalanche generation, all the incident photons are not detected because
they do not produce enough carriers that stand out with remarkable output
alteration. A cyclical “blind-state” of the sensor is expected, hence a reset process
is regularly needed. In order to stop the avalanche the photodiode is switched off
by a quenching process where the voltage supply goes under the breakdown
threshold. In the end, the high voltage is restored and the device is ready to detect
another photon.
8

11. Figure 5. Quenching
The ​quenching circuit is responsible for managing the photodetector. Figure 5
shows the steps chronologically:
1. Waiting for carrier generation
2. Avalanche multiplication is triggered by detecting carrier
3. Voltage between terminals is reduced and multiplication stopped
4. Bias voltage is restored for next carrier
The time between the first absorption and the voltage reset is called dead-time,
over this period the photodetector is “blind” and the incident photons are lost.
Since the dead-time is related with the photon counting rate and affects the
performance of the sensor, it is of the utmost importance to shorten the duration
of this blind state. This feature has a huge effect in many applications of this
technology.
The reason why many of the SPAD literature contains lot of quenching circuit
descriptions is because they have a direct impact on the dead-time duration.
There are two different types of quenching circuit: Passive Quenching Circuit
(PQC) and Active Quenching Circuit (AQC).
9

12. The SPAD photodetection process is described in many stages which involve the
phenomenon of absorption and avalanche. Specifically, the avalanche dynamics
inside the active volume of the photodiode presents several propagation levels.
After the absorption, all around the spot of photon absorption, an exponential
carriers multiplication occurs. Subsequently the electric field decreases until the
breakdown level. At this point, the multiplication process is self-sustained, and
the next stage begins: the diffusion phenomenon. It consists in the carriers
starting the avalanche throughout the depletion layer. Consequently, the active
volume is going to be saturated. The avalanche propagation, which is caused by
diffusion and multiplication as we mentioned, has a different duration whether
the photon is absorbed in the center or on the edge of the layer. If it arrives on
the edge, the time between absorption and saturation is going to be longer. This
is relevant because it implies a margin of error in the measurement. Namely, the
moment of the impulse’s arrival can be recorded differently based on the point of
arrival of the photon in the layer.
Another important SPAD characteristic is the ​photon detection efficiency that
gives the ratio between incident photons and number of output impulses. This
efficiency is given by the quantum efficiency and the avalanche trigger efficiency.
Figure 6. Dependence of the photon detection efficiency of SPAD’s on excess bias voltage V​E
Figure 6 shows how an increase of excess bias voltage implies an increase of
efficiency where a higher electric field increases the probability of avalanche
triggering.
10

13. Temporal resolution is a basic feature that strongly affects the performance of
the device. It is also called statistical delay distribution between the photon arrival
and the output impulse.
Figure 7. Dependence of the FWHM resolution in photon timing on excess bias voltage V​E​ , thin-junction
SPAD at room temperature (filled circles) and cooled to -65 °C (filled squares)
Main delay causes:
1. Spot of photon absorption: the activation of the volume is different
throughout the depletion layer.
2. Type of electric field: the more narrow the field is, the better time
resolution results. The value of breakdown voltage is also significant.
3. Statistical delay of the carrier through the high electric field zone. Figure
7 shows the relationship between time resolution and excess bias voltage.
A higher electric field increases the probability of avalanche triggering
and accelerates the carriers. However, practical experience reveals a very
low impact of this phenomenon.
11

14. SPAD operates by discrete acquisition because the analog to digital conversion is
completely absent. Therefore every type of analog noise does not affect its
functioning. Admittedly, SPAD technology presents some undesirable effects. In
fact, it could happen that some counts do not come from photon absorption.
These are called ​dark counts​ and are contemplated as noise.
The main cause of dark counts is the thermal carriers generation. The rate of
electron-hole pairs that spontaneously occur throughout the depletion layer is a
consequence of temperature and excess bias voltage rising. Output impulses
follow a Poisson distribution, which is the internal noise source. For application
purposes, it is desirable to keep the temperature as low as possible. Setting the
right excess bias voltage implies finding the best compromise between dark
counts, photon detection efficiency and temporal resolution. As a result of this,
the photodiode design must tread carefully about the thermal generation: namely,
the photon wait period should be counted in milliseconds.
Another cause of dark counts comes from the afterpulsing phenomenon. During
the avalanche multiplication some carriers are trapped in deep levels of the
depletion layer. Subsequently the carriers which were left with delay become free
carriers inside the high electric field. The relevance of this is that they could
generate other avalanches and consequently other output impulses not related
with photon absorption. Since the number of trapped carriers is proportional to
the current, the excess bias voltage must be carefully adapted. In order to decrease
the afterpulsing the photodiode is suddenly switched off after the output impulse
detection. In addition a hold-off time is implemented to guarantee the
afterpulsing depletion. However, this creates a slightly worse performance of
photon counting rates, because during this time the photodiode is switched off
increasing the dead-time.
12

15. Figure 8 portrays two different dark count rises as a consequence of different
hold-off times.
Figure 8. Dependence of the dark-count rate on excess bias voltage V​E ​, thin SPAD at room temperature, the
parameter quoted is the hold-off time after each avalanche pulse
As a general rule a great device design is focused on silicon pureness and glitch
production while the circuital solutions are secondary.
In conclusion the SPAD is a type of APD which operates in “Geiger Mode”
configuration because it behaves similarly than a Geiger counter where photons
are counted using the avalanche multiplication. This sensor is able to appreciate
the discrete light nature and provides detailed information without analog to
digital conversion.
13

16. 1.2. Fluorescence
Fluorescence is a light radiation emitted by some substances after an excitement.
The process involves the absorption of a limited range of electromagnetic
spectrum radiation. The incident photons’s energy is partially converted in a new
type of radiation composed by photons with longer wavelength.
An incident photon against the atom is able to promote ​an electron to a higher
energetic level. This is possible only if the photon’s energy is equal to the
difference between two energetic levels. Since the excited state is unstable the
atom tends to return to the natural balance. Figure 9 shows the process from the
excited state to the ground state called relaxation in which the energy is dissipated
by heat and light radiation.
Figure 9. Jablonski diagram of the fluorescence process [8]
The Planck law explains this physical phenomenon, based on the wave-particle
duality: the photon’s energy ​E has an inverse proportion to the radiation
wavelength, where ​h​ is the Planck constant and ​c​ the light speed.
E = λ
hc
Therefore the quantum jump does not depend on the light intensity or photon
speed. Since the photons of the decay process have less energy, the resulting light
has a longer wavelength.
14

17. 1.3. FLIM
Analyses based on fluorescence give more information about the molecular
structure. For instance the Wood lamp is able to stimulate the fluorescence
substances that stand out inside the inspected area. In this way it is possible to
classify the substances according to their different light intensity and wavelength.
Deeper analyses consist in stimulation with impulses where fluorescent materials
reveal different decay periods. Indeed the relaxation process follows an
exponential decrease called fluorescence lifetime where the function type and the
decay period give additional information about the molecular structure.
FLIM is the analysis based on the classification of the different fluorescence
lifetime of each substance. Biologists in the field of biomedical research are the
main users of this technique for spectroscopy, for instance in the Confocal Laser
Scanning Microscopy (CLSM).
A complete research commonly includes also observation about light intensity
and wavelength in order to broaden the knowledge about molecular nature,
disease detection, DNA sequencing, FRET analysis.
Since lifetime is very fast, normal analog photodetectors are unable to appreciate
the fluorescence. As a consequence new devices and systems are developed in
order to achieve a precise acquisition of the decay behavior [4][5][6][7].
15

18. 1.3.1. TCSPC
The Time-Correlated Single Photon Counting (TCSPC) is a system that works
over multiple cycles of single photon acquisition. Basically the system collects the
arrival time of each photon in a temporal histogram. After a sufficient number of
cycles it is possible to build the decay curve. A photodetector that counts single
photons is a necessary part of the system.
Figure 10. Measurement of start-stop times in time-resolved fluorescence measurement with TCSPC
Figure 10 shows the function of the implemented electronics ​that acquires the
period of time between the LASER signal and the photon arrival. An impulse
LASER generator is employed to stimulate the substance. The histogram in
Figure 11 shows how each photon is counted and saved in a “time container”
called time bin, in relation to the time of arrival.
16

19. Figure 11. Histogram of start-stop times in time-resolved fluorescence measurement with TCSPC
Initially, counts are collected in the histogram time bins following a statistical
distribution. Subsequently the fitting process is able to give the same decay curve
of a single acquisition. Since single photon detectors have a dead-time bigger than
the fluorescence lifetime, it is possible to acquire just one photon per cycle. Other
photons are lost and this causes a huge amount of errors. Repeated acquisitions
are based on statistical distribution. Therefore, in order to reduce errors caused
by lost photons, arrival photons probability must be lower than one per cycle.
The practical solution to this problem is to lower the incident light until it
amounts only to one photon. In most practical applications the probability of one
photon arrival is set on 5%.
TCSPC works with single photon detectors such as Photomultiplier Tube (PMT),
Micro Channel Plate (MCP) and SPAD. It can be argued that TCSPC is the most
precise and reliable system. On the other hand, it is very expensive and bulky. For
these reasons it is usually used by large research centres [2][4][5][7].
17

20. 1.3.2. Time-gated
Alternatively to the TCSPC for FLIM analysis the Time-Gated counting is
implemented in cheaper and simpler systems. Even if this type of technique
provides a worse quality of data, the acquisition speed is faster, which is more
suitable for some applications. The reason why FLIM literature involves
Time-Gated counting is because of its Time-Correlated capability that can
provide repeated acquisition cycles.
Basically the photons are acquired in a limited temporal window called gate while
integration period is not considered at all. Originally Time-Gated technique was
implemented in analog systems with CCD and CMOS sensor in order to reduce
the amount of incident light against the sensor. In single photon application this
means a reduction of counts in a specific window of time.
The LASER and the gate are synchronized so that the intensity level of each gate
is linked to a time reference. In order to have a full correlation between time
reference and intensity level the gate is shifted through the entire integration
period. This operation is called exposure time division where each window of
time is correlated to a precise impulse arrival time. To sum up, it can be said that
the final histogram is almost the same as the TCSPC histogram.
18

21. Figure 12. Time-gated detection scheme with two and with eight time gates
Figure 12 shows an example of the time division in two gates, also known as two
channel time-gating, where a mono-exponential decay of fluorescence quenching
is given by:
​ = ΔT/ln (I​2 ​/I​1 ​)
is the offset time between the start of the windows of time and I​1​, I​2 theTΔ
fluorescence intensity levels respectively.
Multi-exponential decays are treated with more than two gates [5].
State-of-the-art techniques use Time-gating ​with single photon devices for FLIM
applications. SPAD is the most suitable technology for this purpose because it is
compact, versatile and with high performance [6][7].
19

22. 1.4. SPC​3
The information given so far is useful in order to explain the core subject of this
work, which is the Single-Photon Counting Camera (SPC​3​
). It is a device made by
a 64x32 matrix of SPAD and it is able to acquire FLIM data with Time-Gating.
Originally it was designed for intensity-based acquisition with high
photodetection efficiency for visible spectrum and near-UV, high noise immunity
and low dark counts. FLIM function is available and implemented in a FPGA
module which manages the photon counting and the LASER synch signal. As a
consequence of the implemented Time-Gated technique, SPC​3
is an extremely
compact and reasonably cheap device, due to the fact that it does not contain
photon timing but only counting. Each pixel has an AQC, a comparator and a 9
bit counter.
20

23. 1.4.1. Acquisition phase
Firstly LASER generator and SPC​3
must be synchronized. A suitable gate width
and steps number is set. Usually for factual analyses there is an overlapping of
gates that improve the accuracy. Since as mentioned dead-time is longer than
fluorescence lifetime, light signal is lowered in order to avoid photon loss.
Statistical distribution must be respected, therefore photon arrival probability is
very low and it occurs just one photon per gate.
Figure 13. Optical waveform reconstruction using the Gated-FLIM approach with the SPC​3​
camera [9]
Figure 13 shows the acquisition process with the control signals. The final FLIM
image is the composition of the FLIM step frames, which are obtained shifting
the gate over the entire fluorescence lifetime. Each FLIM step frame is the
repeated acquisition of a limited decay period in which only the photons inside
the gate are considered. Actually the gating operation is implemented inside the
counter. In fact the comparator provides the impulse of all the absorbed photons
but only the photons inside the gate are counted. At the end of each FLIM step
frame acquisition the number of counted photons is saved in a register, the
counter is reset and the gate is shifted by a precise delay step. Usually the process
takes seconds but it depends on the set parameters such as gate width, FLIM steps
and integration time. These parameters are adapted to the scene conditions such
as brightness and lifetime.
21

24. 1.4.2. Quenching circuit
The signal conditioning stage of each diode involves the quenching circuit
designed in order to maximize SPAD performance.
Figure 14. Quenching circuit
The circuit shown in Figure 14 is a simplified ACQ similar to the PQC.
The initial situation appears as follows: T1 and T2 are open, there is no current
passing through R1 and D1 and difference of potential between R​1 is zero instead
between D​1 is V​D1​=Va + |-Vc| which is higher than breakdown voltage (V​B​). In
this phase the photodiode is waiting for a photon. After the first absorption the
avalanche produce current through D1, the parasite diode capacity is discharging
with a time constant =RC​D1 and V​D1 decreases. When the comparator detects
the avalanche, the system closes T2 so V​D1 goes under breakdown voltage. When
the avalanche has expired T2 is reopened and T1 is closed in order to recharge
C​D1 (reset phase). Finally T1 is reopened and the system is ready for a new
photon.
22

25. 1.4.3. Technical advantages
The SPC​3
hardware has a C-Mount adapter for most of the multifocal and
confocal microscope systems used by biomedical and biological researchers. The
main feature is the acquisition speed, which is faster than the other systems.
Indeed analyses which include strong light stimulation face several sample
problems such as photobleaching, phototoxicity or photoirritation. Some organic
substances are easy to be damaged by LASER exposure and this provide
unreliable measurements. The camera provides a high frame rate, which is also
useful for Förster Resonance Energy Transfer (FRET) analyses. High speed
performance is given by the absence of analog ​noise and limited only by dark
counts. In addition, optomechanics scanner systems have shorter acquisition time
thanks to the 64x32 SPAD matrix [6].
23

26. Chapter 2
INTERNSHIP REPORT
The present report covers all the SPC3 related information, acquired during my
internship by Micro Photon Devices S.R.L. Bolzano, Italy (MPD). This company
designs and produces single photon devices for a wide range of applications such
as biological analysis and quantum cryptography. MPD provides single photon
counting cameras for intensity-based image or FLIM.
The target of the internship was the production of a demonstrative FLIM setup
for customers and hi-tech fairs. As a trainee I joined the team and together we
developed an SPC​3
presentation of FLIM acquisition. Firstly I had to learn about
single photon counting in order to get acquainted with the topic. Secondly as beta
tester I provided a review on the SPC​3
manual focusing on possible errors and
misunderstandings. Finally I looked after the optical layout for FLIM with
fluorescent samples.
24

27. 2.1. System description
The measurements are based on fluorescent light emitted by substances, which
are stimulated by LASER impluses. The goal of the layout was to classify the
samples based on their fluorescence. Since SPC​3
does not involve electronics for
photon timing the only way to get FLIM images with photon counting is through
the time-gating technique. Photons are counted only in limited windows called
gates, which are shifted over the entire observation time.
Figure 15 shows the elements of the main layout. From the SPC​3
software is
possible to control the camera connected to the PC with an USB connection, as
with data acquisition. FLIM mode requires synchronization between camera and
LASER generator. Therefore, a coaxial cable connects SYNC OUT with
TRIGGER IN. Usually coaxial cables introduce a delay for sync signal so the
system must be calibrated beforehand.
Figure 15. Layout description
An image of the actual layer is shown in Figure 16. There is an aluminium plate
which provides a stable sustain for LASER, SPC3 and samples to be fixed on. The
final arrangement allows to acquire several samples in the same scene in order to
highlight their differences.
25

28. Figure 16. Actual layout
LASER power must be set on an appropriate value because it is important to keep
samples visible but at the same time avoid any kind of reflection that could affect
the measures.
The LASER beam stimulates the samples and if some of these contain fluorescent
substances a fluorescent light is emitted and detected by the sensor. An
intensity-based image obtained by means of photon counting is displayed on the
PC screen. Further information is given after clicking on a pixel of the image in
which the number of counts is displayed. In the case of fluorescent emission a
second window shows the time-intensity correlation of the photons captured by
the selected pixel. It is also possible to see a graph illustrating the fluorescence’s
lifetime decay.
In order to avoid the detection of the LASER beam, an optical filter is connected
to the camera’s objective lens.
26

29. The software provided with the SPC​3​
implements several acquisition modes:
● Live Acquisition: It does not acquire any kind of data. Instead, it displays
the view of the camera on the screen. This mode is useful for calibration
or samples arrangement.
● Snap Mode: It provides one or more accurate images with maximum
frame rate. This mode is used for FLIM and scientific analysis.
● Continuous Acquisition: This mode acquires images continuously with
high frame rate and it sends them to the PC until the stop signal. It
provides high accuracy movies to avoid data lost, provided that the PC
hardware is fast enough.
Through the acquisition options it is possible to adapt the camera to different
scene conditions. This could improve the signal-noise ratio and as a result images
would appear clearer and with more detail. Snap Mode also provides movies by
incrementing the number of acquired images.
27

30. 2.2. Measures description
SPC​3
contains a SPAD matrix, therefore acquired images are formed by 64x32
pixels. The monochromatic intensity-based image represents the number of
counted pixels over the exposition period. Fluorescence’s lifetime decay is
available per single pixel only as shown in Figures 17 and 18.
Figure 17. Time correlated counts with a slow decay.
Figure 18. Time correlated counts with a fast decay.
It must be acknowledged that SPC​3
software does not provide final FLIM images
based on false colours. It provides only the counts-time correlation. In fact, the
full FLIM process involves the post-processing phase where a fitting of acquired
data is performed in order to extract time constants of the exponential decays.
28

31. Based on the experience with the customers, SPC​3
designers prefer to build the
software without the post-processing phase and provide only the raw data
because each researcher uses a different fitting operation and time constant
extraction. This allows customers to classify the fluorescence lifetime freely
therefore they can produce FLIM images which highlight the subject of their
specific research.
Initially the FLIM final image was not the subject of the internship. But since I
was curious to get more information about the post-processing phase, the
colleagues suggested me a tool for the purpose. FLIMfit (developed by Imperial
College London) is able to elaborate the OME-TIFF files which are provided by
the camera as raw data. Even though the FLIMfit manual is limited and the time
constant extraction is not easy, I was able to obtain some images in which
different lifetimes were displayed. I have my colleagues to thank for that.
Figure 19. Real photo of the scene Figure 20. Fluorescence’s lifetime classification with false colours
Figure 20 shows the various lifetimes with different colours. The samples are a
coloured piece of plexiglas and a candy. If stimulated by LASER UV impulses
(405 nm wavelength) the samples emit visible light fluorescence. Shorter lifetimes
are classified with colder colours (blue), while longer lifetimes with warmer
colours (red). The candy on the right of the scene has a short fluorescence
lifetime while in the case of the plexiglas it is longer. It has to be kept in mind that
the colours used for the classification are not related to the real colours of the
samples.
29

32. Figura 21. Two pieces of plexiglas
Figure 21 shows two pieces of plexiglas which have different lifetimes. The right
one has a shorter lifetime. In the middle there is a fluorescent zone as a
consequence of the LASER beam. In order to improve the signal-to-noise ratio
and get a better decay representation, a black cover is used over the scene.
30

33. Figure 21. Complete scene representation of the decay colour classification
Figure 22 shows the more complex of my FLIM acquisitions, in which the scene
involves three samples with different lifetimes. On the right side the green
plexiglas has a long lifetime which is classified as green colour (the connection
with the real colour is coincidental). The candy in the middle has the shortest
lifetime in all the scene (classified as blue colour). Finally, the blue plexiglas on
the left side has a lifetime which is shorter than the green one’s but longer than
the candy’s. Fluorescent areas are not well defined because of a not very precise
fitting process. Red shades represent the background noise.
An inefficient sample arrangement could produce unwanted reflections that
could alter the measures. For instance, pieces of plexiglas emit stronger
fluorescence which could be reflected by non fluorescent materials.
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34. CONCLUSION
Biological fluorescence lifetime is usually only a few nanoseconds long.
Unfortunately, a photodetector fast enough to measure it does not yet exist.
Therefore, systems which use FLIM are based on time-correlated techniques in
which measures are taken with repeated acquisition of the same sample. This
function is implemented mainly by systems as TCSPC or any system which
operates with the Time-Gated technique.
TCSPC uses only single photon sensors and timing electronics synchronized with
the LASER generator. Typically, a photodetector contains a one pixel sensor. An
optomechanical device moves the sensor and scans the interested area.
Instead, a system based on Time-Gated techniques is able to use also CCD and
CMOS. Synchronization is always necessary in order to obtain FLIM data.
The topic of this work is to recognise that the SPC​3
offers several advantages as
far as FLIM analyses is concerned. In fact, employing Time-Gated technique with
a single photon detector is an innovative solution because it avoids the timing
electronics and provides less noise than the analog photodetectors. SPAD
technology acquires full digital data with high signal-to-noise ratio thanks to the
low dark-counting and afterpulsing. Due to its simple hardware the production
costs are not so high. And, last but not least, it is faster than TCSPC systems,
which makes it suitable for analysis that have issues of photobleaching.
During the internship I acquired a large amount of knowledge about FLIM
theory and applications. Many universities and companies are conducting
research in this field, therefore I had the opportunity to come across a
state-of-the-art technology such as SPAD. Although my university lessons did
not cover this specific technology, I was prepared enough to understand such a
sophisticated method. Finally, this experience improved my skills in the field of
electronics and gave me a useful first approach to the professional world which
constitute the very first step of my career.
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35. REFERENCES
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https://www.hamamatsu-news.de/hamamatsu_optosemiconductor_handbook
In this link the cited document provides hints of Si photodiodes and Si APD.
[2] Michael Wahl, “Time-Correlated Single Photon Counting”, PicoQuant GmbH.
http://www.picoquant.com/images/uploads/page/files/7253/technote_tcspc.pdf
[3] S. Cova, M. Ghioni, A. Lacaita, C. Samori, and F. Zappa, “Avalanche photodiodes and quenching
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[7] David Stoppa et al., “Single-Photon Avalanche Diode CMOS Sensor for Time-Resolved
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