School on Optical Biosensors 27 May 2014
Time-gated luminescence sensing
for biomedical diagnostics
Prof Jim Piper [email protected]
Acknowledgements: Duncan Veal, Graham Vesey, Mark Gauci,
Belinda Ferrari, Russell Connally, Jin Dayong, Ewa Goldys, Lu Yiqing,
Yuan Jingli, Paul Robinson, Robert Lief, Tom Lawson, John Iredell,
Subra Vermulpad, Zhang Run, Zhao Jianbo, Lu Jie, Zhang Lixin
School on Optical Biosensors 27 May 2014
Outline of presentation
• Beginnings-the Great Sydney Cryptosporidium crisis
• Concept of Time-Gated Luminescence sensing
• TGL Microscopy
• TGL Flow Cytometry
• TGL Scanning Microscopy
• New developments of long-lifetime luminescent
probes for TGL sensing
• Developments of spectrally and temporally coded
luminescent probes-upconversion nanoparticles
• Latest developments in temporally coded microscopy
School on Optical Biosensors 27 May 2014
The Great Sydney Cryptosporidium Crisis
Ten Cryptosporidium parvum
oocysts in one litre of drinking water
is a health hazard
Conventional microbiological
methods are slow and do not apply
to un-culturable organisms, such as
Cryptosporidium
In the early 1990s Australian Water
Technologies funded the
Fluorescence Applications group
from MQ Biological Sciences and
Physics to develop fast detection
techniques
Immuno-fluorescence labelling of individual target organisms
enables direct observation but the extremely high level of
autofluorescent from detritis and other organisms in the sample
makes unambigous identification impossible without using special
techniques to enhance signal-to-background
School on Optical Biosensors 27 May 2014
Protocol for detection of Cryptosporidium*
* Veal et al J. Immun. Methods 243 (2000) 191-210
Immuno-Magnetic
Separation (IMS)
IMS concentrated pellet
(1 oocyst/ 103-5) Biosolid pellet
(1 oocyst/109-11)
Fluorescence-Activated Cell
Sorting (1 oocyst/ 101-3)
Fluorescence
microscopy
Sample incubated with mAb-
CRY104 paramagnetic beads
and after IMS stained with
fluorescent mAb CRY104-FITC
School on Optical Biosensors 27 May 2014
Can we do this better?
Time Gated Luminescence detection
TGL techniques exploit significant
difference in emission lifetime ()
between the luminescent bioprobe
(>100S) and auto-fluorophors (1-10nS)
Available luminescent lanthanide
chelates have peak excitation at 335nm
and sharp emission at 617nm with
lifetime ~350s
O
OS
OO
OO C
3F
7F7C
3
Cl
Eu3+
BHHCT
G203 Giardia
lamblia monoclonal
IgG antibody - anti-
mouse polyvalent
IgG antibody -
BHHCT
School on Optical Biosensors 27 May 2014
First-generation TGL Microscope (Connally, Veal and Piper)
Flash Lamp
CCD camera
Image intensifier
Epifluorescence
microscope
50W
Mercury
vapour lamp
Micro-controller
Flash lamp
controller
Operation at 50Hz
School on Optical Biosensors 27 May 2014
TGL microscopy for direct detection of
Giardia lamblia*
*Connally et al FEMS Microbiology Ecology 41 (2002) 239-245
School on Optical Biosensors 27 May 2014
Second-generation fully-electronic
TGL microscope*
*Connally et al Cytometry
69A (2006) 1020-1027
Annals New York Acad Sc
1130 (2008) 106-116,
School on Optical Biosensors 27 May 2014
Self-synchronised module for low-cost TGL
microscopy
Russell Connally’s GALD module fits in the filter
slot of a standard fluorescence microscope and
converts it to a TGL microscope
With Olympus Australia and Westmead hospital
we are developing new rapid-testing for
Stapphylococcus aureas (including MRSA)
Immunoflourescence FISH (RNA) probe
School on Optical Biosensors 27 May 2014
Low-cost retrofit for practical TGL
microscopy*
*Jin & Piper Analytical Chemistry 83 (2011) 2294-2300
School on Optical Biosensors 27 May 2014
Immuno-luminescence detection of Cryptosporidium
True-colour time-gated luminescence observation of a BHHCT
europium complex labelled Cryptosporidium parvum oocyst
School on Optical Biosensors 27 May 2014
Future of TGL Microscopy
• TGL Microscopy remains a relatively cheap practical
option in respect of equipment but expensive in
terms of trained personnel
• Automated image processing is an option subject to
technical requirements on the camera and data
processing
• Extensions of TGL Microscopy rely on availability of
appropriate sources and new luminescent probes
• We are currently exploring UV-excitation options
using new-generation high-pulse-rate Xenon lamps
School on Optical Biosensors 27 May 2014
TGL Flow Cytometry
Sample flow
(~10 m/s)
Single element gated
detector
Temporal
delay
Previous approaches to TRFC involve fast
modulation of laser source and phase-
sensitive detection (complex and
expensive)
TGL offers major advantages in detection
of target microoganisms in highly
autofluorescent backgrounds
(environmental & biomedical diagnostics)
New long-lifetime metal chelate
fluorochromes (BHHCT, BHHST), UV LED,
LD or new solid-state laser (336nm)
sources, and cheap single-element gated
detectors, offer potential for low-cost
instrumentation
Careful attention must be given to
synchronising sample flow-rates, pulsed
excitation, gate delay times and detection
intervals to ensure 100% sampling
Pulsed excitation
School on Optical Biosensors 27 May 2014
TGL flow cytometry*
*Jin et al Cytometry Part A 71A (2007) 783-796, 797-808
A prototype TGLFC constructed by Jin
Dayong has detected single, BHHCT-labelled
Giardia cells in dirty samples using mW
pulsed UV LED illumination at TGL cycle
rates up to 6 kHz
School on Optical Biosensors 27 May 2014
TGL Flow Cytometry model
The TGL sequence (pulsed excitation, gate delay & time-gated detection) at fixed repetition rate (shown in (B)) is
applied continuously to the flow sample. The continuous flowing stream can be conceived as adjacent continuous flow
sections, and in proper design of sizes and positions of the excitation and detection spots in consideration of the sample
flow velocity, one pulsed excitation and detection cycle will be responsible for screening of TGL event in one
corresponding section, so that theoretically all parts of flow will be analyzed sequentially.
School on Optical Biosensors 27 May 2014
TGL Scanning Cytometry
TGL detection can be applied in
high-speed scanning configurations
for automated diagnostics of 2-D
sample arraysTGL
Scanning Cytometry represents a
compromise between wide-field
microscopy and flow cytometry
Stable long-lifetime metal chelate
fluorochromes (BHHST), UV Laser
Diodes or solid-state lasers (eg
355nm), cheap single-element gated
detectors and high-speed translation
stages give promise to low-cost
instrumentation
high-prf pulsed
UV laser
scanning dichroic
mirror
single-element
gated detector
sample substrate
translation
capture
optics
beamscan
School on Optical Biosensors 27 May 2014
Schematics show two-step scanning
strategy to discover and locate targets-of-
interest, allowing cytometric data
collection simultaneously. (a) The sample
is first examined in a serpentine pattern,
of which the continuous movement is
along X-axis, to obtain precise X
coordinates as well as rough Y
coordinates for each targets. (b) Then, the
targets are scanned sequentially at
respective X coordinates along Y-axis to
obtain precise Y coordinates, where their
luminescent intensities are acquired of
cytometric accuracy.
Diagrams illustrate the temporal
waveform of time-gated luminescence
(TGL) signal when a target is spatially
scanned across. (a) depicts the detailed
pulse trains when the detection time
window TW is relatively-long, with one
cycle enlarged to facilitate the
explanation of TGL detection.. Pulsed
excitation (blue) illuminates the
interrogation field while a gating signal
(grey) turns the detector off, leaving a
delay period for residual excitation and
autofluorescence to diminish. The long-
lifetime TGL signal is recorded during
the detection time window .The profile
(pink) indicates the tendency in average
intensity of luminescence decay. (b)
represents the interrogation field
scanning with velocity v(t) across a
long-lifetime target, which appears to
travel a distance of L from P1 to P2. (c)
draws the real signal when the TGL
cycle was compressed to 0.2 ms, along
with its profile of average intensity.
High-speed scanning TGL cytometry * *Lu et al Scientific Reports 2 (2012) Art. 837
School on Optical Biosensors 27 May 2014
High-speed scanning cytometry for rare-event detection
The TGL scanning system was used to analyse BHHCT-Eu chelate labeled Giardia cysts spiked onto normal glass slides.
(a) sums up from seven slide samples in a form of histogram the distribution of luminescence intensity from a total number
of 920 labeled Giardia cysts. The great contrast between the events and the threshold is an evidence that the system is free
of false-negatives. (b) and (c) are mapping result and further imaging confirmation of one sample containing 24 Giardia
cyst (marked A to R; scale = 100 μm; CCD camera exposure time of 150 ms for luminescence imaging, 8 ms for bright-
field imaging), proving no false-positive errors.
School on Optical Biosensors 27 May 2014
New luminescent molecular probe technologies
• BHHCT (Prof Yuan Jingli, Dalian University of Technology) is the
foundation for a variety of long-lifetime luminescent (lanthanide) molecular
probes-variants such as BHBCB (Yuan), BHHST and BHTEGS (Connally,
Macquarie University) have improved stability in conjugation
• Silica-encapsulated lanthanide-doped nanospheres have been
demonstrated (Wu et al Chemical Commun. 3 (2008) 365-367)
• Co-doping microspheres with lanthanide complex donor and acceptor dye
offers the prospect of tuning the Eu3+ lifetime
• Extensive studies have been conducted of conjugation of Eu3+-BHHCT and
derivatives to specific antibodies as a basis for detection via Immuno-
luminescence assays (Giardia, Cryptosporidium, PSA etc)
• First demonstration of TGL-DNA probe using Eu3+-BHTEGS in
Luminescence-In-Situ-Hybridisation protocol for detection of
Staphylococcus aureus
• Incorporation of lanthanide complexes with co-dopants in microspheres
allows the luminescence lifetime to be engineered, offering the prospect of
lifetime coding
School on Optical Biosensors 27 May 2014
First demonstration of TGL detection of S.
aureus using DNA probe (LISH)
(a) BHTEGS (b) Alexa Fluor
Tom Lawson PhD thesis Macquarie University 2012, Figure 5.3: SA separated from
whole-blood labeled with (a) BHTEGS and (b) Alexa Fluor R 488. The BHTEGS
signal is time-resolved and the Alexa Fluor signal is not.
School on Optical Biosensors 27 May 2014
Tom Lawson PhD thesis Macquarie University 2012, Figure 5.2: Staphylococci
separated from whole-blood and SA labeled with KT68 and BHTEGS and visualized
with (a) bright-field and (b) LISH and TGLM. S. epidermidis labeled with KT68 and
visualized with (c) bright-field and (d) LISH and TGLM. Bar = 5 microns.
School on Optical Biosensors 27 May 2014
Using coded lifetimes: Time-Resolved
Luminescence detection*
Schematic diagram illustrates the concept of time-resolved orthogonal scanning automated microscopy
(TR-OSAM), which can identify micron-sized targets randomly distributed on a slide and distinguish them
by individuals’ luminescent lifetimes. (a) It typically takes 3 minutes for the TR-OSAM to map these
targets in background-free condition via UV LED pulsed excitation and time-gated luminescence
detection in anti-phase. The signal trains of luminescence intensity recorded from the detection field-of-
view during the transit of the targets are used to obtain their precise locations along the continuous
scanning direction. (b) The positional coordinates guide sequential orthogonal scans for spot-by-spot
inspection of targets at the centre of the field-of-view, and the luminescence lifetime identity of each target
can be decoded in real-time.
*Method of Successive Integration
Lu et al Nature Communications (2014)
School on Optical Biosensors 27 May 2014
Lifetime-coded co-doped polymer microspheres* Lu et al Nature Communications (2014)
Lifetime measurement results from individual Eu-containing microspheres engineered by LRET. Different solutions
containing identical amount of Eu complexes as donor but incremental amount of acceptor dyes were encapsulated
into individual groups of polymer microspheres, following by the TR-OSAM analysis. (a) Luminescence lifetime
measured at Eu3+ red emission band shortens as the acceptor concentration in the original dye solution increases,
as a result of stronger LRET effect. The inset curves are the luminescence decay signals measured from single Eu-
LRET microspheres. (b) Among all samples, 5 types of microspheres give completely separate lifetime histograms,
so that they are capable of definite discrimination by the TR-OSAM. The numerals at the left to each histogram are
the mean lifetime ± the lifetime CV for its Gaussian fitting.
School on Optical Biosensors 27 May 2014
TR-scanning cytometry: lifetime coding
Results illustrate the multiplexing detection process for the lifetime-encoded Eu-LRET microspheres using the TR-OSAM. (a) A mapping result (locations on a microscopic slide) of a mixture of 5 selected types of Eu-LRET microspheres is shown (refer to Fig. 4b). The color tones (hues) bar represents the lifetime values. (b) Individual types of microspheres in the mixed sample are recognized based on the separation of lifetime populations using definite boundaries in between. (c) The initial mapping result for the mixed sample is thus decomposed into 5 planes of different lifetime regions for individual types of Eu-LRET microspheres carrying lifetime identities.
School on Optical Biosensors 27 May 2014
Size-dependent lifetime of NaYF4:Yb:Er upconversion
nanocrystals*
*Zhao et al, Nanoscale
5 (2013) 944-952
School on Optical Biosensors 27 May 2014
NaYF4:Yb:Er upconversion luminescent
nanocrystals
School on Optical Biosensors 27 May 2014
Concentration-dependent lifetime of NaYF4:Yb:Tm
upconversion nanocrystals (-Dots)*
Lifetime tuning scheme and time-resolved confocal images for NaYF4:Yb,Tm
upconversion nanocrystals. The colour tone (hue) for each pixel represents its lifetime
value decoded from the decay curve. The nanocrystals in the images from left to right
have Tm doping concentrations of 4, 2, 1, 0.5 and 0.2 mol%, respectively.
*Lu et al Nature Photonics (2014)
School on Optical Biosensors 27 May 2014
-Dot encoded microspheres
Results for -Dots-encoded populations of microspheres as the multiplexing suspension
arrays carrying the unique lifetime codes: (a) The synthesized monodispersed Tm
upconversion nanocrystals (top TEM image) can be embedded onto the microsphere
shell (bottom SEM image). (b) The mechanism of upconversion energy transfer, by
adjusting the co-dopant concentration of sensitizer-activator, can generate 8 lifetime
populations of microspheres at Tm blue-emission band. (c) The 2-D (intensity vs.
lifetime) scattered plots show all lifetime populations independent of intensitiy .
School on Optical Biosensors 27 May 2014
Super-multiplexing arrays: a new library of
optical codes
School on Optical Biosensors 27 May 2014
Superdots! (Jin Dayong)
(Jin Dayong
School on Optical Biosensors 27 May 2014
Summary • TGL detection has come a long way from very simple beginnings
• Practical instrumentation has been demonstrated for a variety of
different optical, sample-handling and detection platforms
• Initial aims of achieving high background signal suppression have
been overtaken by the opportunities implicit in lifetime coding
• There has been concurrent development of long-lifetime luminescent
molecular probes and demonstration of practical application,
particularly for environmental and clinical pathogen detection, but
including cancer diagnostics
• Recent developments of high-brightness nanoparticle probes with
potential for large-scale temporal and spectral multiplexing are
extremely promising, provided the challenge of interfacing with the
biology can be effectively met
• Time-Resolved Luminescence detection offers enormous scope for
innovative research and development, and the potential to make a
real difference in human disease diagnostics
Thank you!