Time-gated luminescence sensing for biomedical diagnosticsblog.cnbp.org.au/cnbp/wp/wp-content/uploads/2014/06/june... · 2014. 11. 13. · School on Optical Biosensors 27 May 2014

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

mailto:[email protected]


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


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


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


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


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


TGL microscopy for direct detection of

Giardia lamblia*

*Connally et al FEMS Microbiology Ecology 41 (2002) 239-245


Second-generation fully-electronic

TGL microscope*

*Connally et al Cytometry

69A (2006) 1020-1027

Annals New York Acad Sc

1130 (2008) 106-116,


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


Low-cost retrofit for practical TGL

microscopy*

*Jin & Piper Analytical Chemistry 83 (2011) 2294-2300


Immuno-luminescence detection of Cryptosporidium

True-colour time-gated luminescence observation of a BHHCT

europium complex labelled Cryptosporidium parvum oocyst


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


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


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


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.


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


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


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.


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


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.


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.


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)


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.


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.


Size-dependent lifetime of NaYF4:Yb:Er upconversion

nanocrystals*

*Zhao et al, Nanoscale

5 (2013) 944-952


NaYF4:Yb:Er upconversion luminescent

nanocrystals


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)


-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 .


Super-multiplexing arrays: a new library of

optical codes


Superdots! (Jin Dayong)

(Jin Dayong


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!

Time-gated luminescence sensing for biomedical diagnosticsblog.cnbp.org.au/cnbp/wp/wp-content/uploads/2014/06/june... · 2014. 11. 13. · School on Optical Biosensors 27 May 2014

Documents