US 11,022,538 B2

9
lens 234 and filter 236 in light collector 214 to receive
scattered light 232 from confluence tube 250; disposing light
collector 214 in optical communication with beam splitters
216 and photon detectors 218 via optical path 238; and

electrically connecting photon detectors 218 with analyzer 5

258.

In an embodiment, the process for making quantum flow
cytometer 200 also includes omission of light collector 214.

The process for making quantum flow cytometer 200 also
can include the above steps, except that beam splitter 216
and single photon detectors 218.1, 218.2 . . . can be replaced
by a single photon-counting detector 218 in optical com-
munication with optical output 232 via optical path 238; and
electrically connecting single photon-counting detector 218
with analyzer 258,

Quantum flow cytometer 200 has numerous advantageous
and unexpected benefits and uses. In an embodiment, a
process for detecting analyte 210 with photon-number sta-
tistics with the quantum flow cytometer 200 includes:
receiving, by flow cytometer 212, pump light 220 in first
direction 222; receiving, by flow cytometer 212, analyte
flow 228 including analyte 210 in second direction 224;
producing, by analyte 210, scattered light 232 from scatter-
ing pump light 220: receiving, by a photon number resolving
detector 218, scattered light 232; producing, by photon
number resolving detector 218, detection signal 254 in
response to detecting scattered light beam 232; and subject-
ing detection signal 254 to an analyzer 258 to detect analyte
210 by analyzing the number of detected photons and their

photon number statistics, wherein first direction 222, second 3

direction 224, and third direction 226 are arranged at oblique
angles.

Quantum flow cytometer 200 has numerous advantageous
and unexpected benefits and uses. In an embodiment, a
process for detecting analyte 210 with photon-number sta-
tistics with the quantum flow cytometer 200 includes:
receiving, by flow cytometer 212, pump light 220 in first
direction 222: receiving, by flow cytometer 212, analyte
flow 228 including analyte 210 in second direction 224;
producing, by analyte 210, scattered light 232 from scatter-
ing pump light 220; receiving, by beam splitter 216, scat-
tered light 232 in third direction 226 from flow cytometer
212; splitting, by beam splitter 216, scattered light 232 into
first scattered light beam 244.1 and second scattered light
beam 244.2; detecting, by first single photon detector 218.1,
first scattered light beam 244.1 from beam splitter 216;
producing, by first single photon detector 218.1, first detec-
tion signal 254.1 in response to detecting first scattered light
beam 244.1; detecting, by second single photon detector
218.2, second scattered light beam 244.2 from beam splitter
216; producing, by second single photon detector 218.2,
second detection signal 254.2 in response to detecting sec-
ond scattered light beam 244.2; and subjecting first detection
signal 254.1 and second detection signal 254.2 to an ana-
lyzer 258 to detect analyte 210 by analyzing the number of
detected photons and their photon number statistics, wherein
first direction 222, second direction 224, and third direction
226 are arranged at oblique angles.

In the process for detecting analyte 210 with the quantum
flow cytometer 200, receiving, by flow cytometer 212, pump
light 220 in first direction 222 includes generating pulsed or
continuous wave light with a light source for a selected
wavelength and pulse duration and focusing pump light 220
on a flow cytometer 212 such that the focus coincides with
the geometrical center of the analyte flow 210.

In the process for detecting analyte 210 with the quantum
flow cytometer 200, receiving, by flow cytometer 212,

S

2

40

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10

analyte flow 228 including analyte 210 in second direction
224 includes determining the flow rate for the analyte pump
and for the sheath pump to establish stable laminar flow of
selected analyte cross-section.

In the process for detecting analyte 210 with the quantum
flow cytometer 200, producing, by analyte 210, scattered
light 232 from scattering pump light 220 includes collecting
light from the region of analyte flow 228 at a focal point of
pump light 220 with a selected fraction of solid angle and
optically connecting scattered light with a single photon
number resolving detector or a detector tree with lenses,
filters, fiber and beam splitters.

In the process for detecting analyte 210 with the quantum
flow cytometer 200, receiving, by beam splitter 216, scat-

$s tered light 232 in third direction 226 from flow cytometer

212 includes aligning pump beam 220, analyte flow 228 and,
optionally light collector 214 and detectors 218 to detect
signal.

In the process for detecting analyte 210 with the quantum
flow cytometer 200, splitting, by beam splitter 216, scattered
light 232 into first scattered light beam 244.1 and second
scattered light beam 244.2 includes selecting a beam-splitter
such that the splitting ratio is nearly constant for the wave-
length range of interest.

In the process for detecting analyte 210 with the quantum
flow cytometer 200, detecting, by photon number resolving
detector 218, scattered light beam 232 from beam splitter
216 includes mutual alignment of light beam 232 and
detector 218

In the process for detecting analyte 210 with quantum
flow cytometer 200, producing, by photon number resolving
detector 218, detection signal 254 in response to detecting
scattered light beam 232 includes establishing normal oper-
ating conditions for detector 218 and amplifying its output
signal with a comparator or analog amplifier so that detec-
tion signal 254 can be processed by analyzer 258.

Alternatively, in the process for detecting analyte 210
with quantum flow cytometer 200, detecting, by first single
photon detector 218.1, first scattered light beam 244.1 from
beam splitter 216 includes mutual alignment of light beam
244.1 and detector 218.1

Alternatively, in the process for detecting analyte 210
with the quantum flow cytometer 200 producing, by first
single photon detector 218.1, first detection signal 254.1 in

5 response to detecting first scattered light beam 244.1

includes establishing operating conditions for detector 218.1
and amplifying an output signal with a comparator or analog
amplifier so that detection signal 254.1 can be processed by
analyzer 258.

Alternatively, detecting, by second single photon detector
218.2, second scattered light beam 244.2 from beam splitter
216 includes mutual alignment of light beam 244.2 and
detector 218.2.

Alternatively, producing, by second single photon detec-

5 tor 218.2, second detection signal 254.2 in response to

detecting second scattered light beam 244.2 includes estab-
lishing operating conditions for detector 218.2 and ampli-
fying the output signal with a comparator or analog amplifier
so that detection signal 254.2 can be processed by analyzer
258.

In the process for detecting analyte 210 with quantum
flow cytometer 200, subjecting detection signal 254 or,
alternatively, first detection signal 254.1 and second detec-
tion signal 254.2 to an analyzer 258 to detect analyte 210
includes calculating time-resolved cross-correlations con-
sidering temporal location of detection signals 254.1 and
254.2 with respect to the closest optical pump 220 pulse.


US 11,022,538 B2

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producing, by the analyte, scattered light from scattering
the pump light;

receiving, by the beam splitter, the scattered light in the
third direction from the flow cytometer;

splitting, by the beam splitter, the scattered light into the
first scattered light beam and the second scattered light
beam, such that the beam splitter splits light based on
intensity of the scattered light according to a selected
splitting ratio of the beam splitter and so that the first
scattered light beam and a second scattered light beam
are subjected to intensity interferometry by the inten-
sity interferometer;

detecting, by the first single photon detector, the first
scattered light beam from the beam splitter;

producing, by the first single photon detector, a first
detection signal in response to detecting the first scat-
tered light beam;

detecting, by the second single photon detector, the sec-
ond scattered light beam from the beam splitter;

producing, by the second single photon detector, a second
detection signal in response to detecting the second
scattered light beam; and

subjecting the first detection signal and the second detec-
tion signal to an analyzer to detect the analyte by the
photon number statistics,

wherein the first direction, the second direction, and the
third direction are arranged at oblique angles.

7. The process of claim 6 for detecting the analyte with

photon-number statistics, further comprising:

performing. by the quantum flow cytometer, self-calibra- 3

tion,

8. The process of claim 7 for detecting the analyte with
photon-number statistics, wherein performing self-calibra-
tion comprises:

sequentially and individually providing a plurality of 3

samples as the analyte flow in the quantum flow
cytometer, wherein the samples comprise:
a first composition comprising a first concentration of
the analyte;
a second composition comprising a second concentra-
tion of the analyte, such that the first concentration is
a known multiple of the second concentration.
9. The process of claim 8 for detecting the analyte with
photon-number statistics, further comprising:
determining a noise, a concentration of the analyte, or a
photon flux detected from a single fluorophore, or a
combination comprising at least one of the foregoing
parameters.
10. The process of claim 6 for detecting the analyte with
photon-number statistics, further comprising:
setting a temporal area of interest for detection by the
single photon detector based on a minimum in corre-
lation obtained from the analyzer.

10

3

40

50

18

11. The process of claim 6 for detecting the analyte with
photon-number statistics, further comprising:

flowing, through a sheath flow tube of the flow cytometer,

the sheath flow; and

flowing, through an inner flow tube disposed in the sheath

flow tube, the analyte flow.

12. The process of claim 6 for detecting the analyte with
photon-number statistics, further comprising:

receiving, by a lens, the scattered light from the flow

cytometer;

receiving, by a filter, the scattered light from the flow

cytometer,

reducing, by the filter, an amount of the pump light or

Raman scattering background light prior to communi-
cation of the scattered light to the single photon detec-
tors; and

receiving, by an optical fiber, the scattered light from the

lens and the filter; and

communicating, by the optical fiber, the scattered light to

the beam splitter,

13. The process of claim 6 for detecting the analyte with
photon-number statistics, further comprising:

flowing, through a sheath flow tube of the flow cytometer,

the sheath flow;

flowing, through an inner flow tube disposed in the sheath

flow tube, the analyte flow;

receiving, by a confluence tube, the sheath flow and the

analyte flow:

confluence tube:

hydrodynamically focusing; by the confluence tube, the

analyte flow;

constraining, in the confluence tube, analyte flow ina flow

column within the sheath flow;

receiving, by the analyte flow in the confluence tube, the

pump light;

producing, by the analyte in the confluence tube, the

scattered light; and

communicating the scattered light from the confluence

tube to the intensity interferometer in the third direc-
tion.

14. The process of claim 6 for detecting the analyte with
photon-number statistics, wherein a fluorophore is attached
to the analyte, and

producing, by the analyte, scattered light from scattering

the pump light comprises emitting fluorescence from
the fluorophore.

15. The process of claim 6 for detecting the analyte with
photon-number statistics, herein the single photon detector
comprises a single photon avalanche diode.

16. The process of claim 6 for detecting the analyte with
photon-number statistics, wherein the analyte detected by
the intensity interferometer is one molecule.

+ ke ee


US 11,022,538 B2

1
QUANTUM FLOW CYTOMETER

CROSS REFERENCE TO RELATED
APPLICATIONS

The application claims priority to U.S. Provisional Patent
Application Ser. No. 62/671,907 filed May 15, 2018, the
disclosure of which is incorporated herein by reference in its
entirety.

STATEMENT REGARDING FEDERALLY
SPONSORED RESEARCH

This invention was made with United States Government
support from the National Institute of Standards and Tech-
nology (NIST), an agency of the United States Department
of Commerce and under Cooperative Agreement No.
7ONANB16H168 awarded by United States Department of
Commerce. The Government has certain rights in the inven-
tion. Licensing inquiries may be directed to the Technology
Partnerships Office, NIST, Gaithersburg, Md., 20899; voice
(301) 301-975-2573; email tpo@nist.gov; reference NIST
Docket Number 18-031US1.

BRIEF DESCRIPTION

Disclosed is a quantum flow cytometer for detecting an
analyte with photon-number statistics, the quantum flow
cytometer comprising: a flow cytometer that: receives a

pump light in a first direction; receives an analyte flow 3

comprising the analyte in a second direction; and produces
scattered light from scattering the pump light by the analyte;
a photon-number resolving detector or an intensity interfer-
ometer in optical communication with the flow cytometer

and that comprises: a beam splitter in optical communication 3

with the flow cytometer that: receives, in a third direction,
the scattered light from the flow cytometer; splits the scat-
tered light into a first scattered light beam and a second
scattered light beam; and a first single photon detector in
optical communication with the beam splitter and that
receives the first scattered light beam from the beam splitter;
and a second single photon detector in optical communica-
tion with the beam splitter and that receives the second
scattered light beam from the beam splitter, such that the
beam splitter and the single photon detectors provide a
Hanbury Brown-Twiss (HBT) effect that detects the analyte,
wherein the first direction, the second direction, and the third
direction are arranged at oblique angles.

Disclosed is a process for detecting an analyte with
photon-number statistics with the quantum flow cytometer,
the process comprising: receiving, by the flow cytometer, the
pump light in the first direction; receiving, by the flow
cytometer, the analyte flow comprising the analyte in the
second direction; producing, by the analyte, scattered light
from scattering the pump light; detecting, by the photon-
number resolving detector, the scattered light, producing, by
the photon-number resolving detector of the detection signal
comprising of detection times and detected photon numbers
in response to detecting of the optical signal from the
detector and subjecting the detection signal to second, third
or higher order auto-correlation to detect the analyte or
splitting, by the beam splitter, the scattered light into the first
scattered light beam and the second scattered light beam;
detecting, by the first single photon detectors, the first
scattered light beam from the beam splitter; producing, by
the first single photon detectors, a first detection signal in
response to detecting the first scattered light beam: detect-

40

&

2

ing, by the second single photon detectors, the second
scattered light beam from the beam splitter; producing, by
the second single photon detectors, a second detection signal
in response to detecting the second scattered light beam;
wherein a beam splitter can be replaced with a plurality of
beam splitters arranged in a binary tree wherein an input to
each subsequent beam splitter is in optical communication
with an output from a previous beam splitter; and subjecting
the first detection signal and the second detection signal to
a second, third, or higher order cross-correlation to detect
the analyte by the Hanbury Brown-Twiss effect. wherein the
first direction, the second direction, and the third direction
are arranged at oblique angles.

BRIEF DESCRIPTION OF THE DRAWINGS.

The following description should not be considered lim-
iting in any way. With reference to the accompanying
drawings, like elements are numbered alike.

FIG. 1 shows a quantum flow cytometer with a photon-
number resolving detector;

FIG. 2 shows a quantum flow cytometer with a plurality
of single-photon detectors in a tree configuration;

FIG. 3 shows a perspective view of the quantum flow
cytometer shown in FIG. 2;

FIG. 4 shows a quantum flow cytometer;

FIG. 5 shows a graph of cross-correlation versus time
delay;

FIG. 6 shows a graph of cross-correlation versus esti-
mated number of quantum dots in a focal volume; and

FIG. 7 shows a graph of photon correlation and average
photon number per cell versus the number of fluorescent
molecules per cell.

DETAILED DESCRIPTION

A detailed description of one or more embodiments is
presented herein by way of exemplification and not limita-
tion.

Flow cytometry is an optical method to aid diagnosis of
health disorders and disease monitoring. It has been discov-
ered that a quantum flow cytometer herein provides optical
flow cytometry with photon number statistics determination.
Advantageously, the quantum flow cytometer provides an
absolute measurement of biomarker concentration and has
sensitivity for a single biomarker. Moreover, the quantum
flow cytometer provides cell-by-cell discrimination based
on a number of biomarkers, e.g., a fluorophore, present in
each cell. Unexpectedly, the quantum flow cytometer pro-
vides single-biomarker sensitive flow-cytometry and can
include cell sorting.

Moreover, the quantum flow cytometer can self-calibrate
via an absolute self-calibration and has sensitivity for quan-
tification and enumeration of a single or small number of

5 biological markers per a biological entity such as a cell,

peptide, or antigen. The quantum flow cytometer uses a
photon-number resolving detector or detector arrangement
that resolves photon numbers such as a Hanbury Brown-
Twiss (HBT) arrangement that accumulates times-of-arrival
and photon number statistics and uses first principles of
quantum optics that provide a number of participating
emitters. Beneficially, the quantum flow cytometer deter-
mines photon number information, detection timestamps,
and absolute calibration. At a selected concentration of

5 fluorophores per cell, the quantum flow cytometer accumu-

lates an optical signal from exposures, e.g., from cells, and
statistically determines second-, third-, or higher-order cor-


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a2 United States Patent
Polyakov et al.

US011022538B2

(10) Patent No.: US 11,022,538 B2
(45) Date of Patent: Jun. 1, 2021

(54)
(71)

(72)

(73)

(*)

2)
(22)
(65)

(60)

(51)

(52)

QUANTUM FLOW CYTOMETER

Applicant: Government of the United States of
America, as represented by the
Secretary of Commerce, Gaithersburg,

MD (US)

Inventors: Sergey V. Polyakov, Gaithersburg, MD
(US); Ivan A. Burenkoy, Germantown,
MD (US); Yu-Hsiang Cheng, North
Brunswick, NJ (US)

Assignee: GOVERNMENT OF THE UNITED

STATES OF AMERICA, AS.

REPRESENTED BY THE
SECRETARY OF COMMERCE,
Gaithersburg, MD (US)

Notice: Subject to any disclaimer, the term of this
patent is extended or adjusted under 35
U.S.C. 154(b) by 0 days.

Appl. No.: 16/413,078

Filed: May 15, 2019

US 2019/0353578 Al

Prior Publication Data

Nov. 21, 2019

Related U.S. Application Data
Provisional application No. 62/671,907, filed on May

15, 2018.

Int. Cl.
GOIN 15/14
US. Cl.

(2006.01)

CPC . GOIN 15/1434 (2013.01); GOIN 2015/1454

220

230 296

(2013.01)

(58) Field of Classification Search
CPC ... GOIN 2015/1006; GOIN 2015/1454; GOIN
15/1459; GOIN 15/1434: GOIN 15/1436:
GOIN 15/1411: GOIN 15/14; GOIN
15/1404; GOIN 15/1409
See application file for complete search history.

(56) References Cited
U.S. PATENT DOCUMENTS

6,133,995 A * 10/2000 Kubota GOIN 15/1459
356/337
6,139,800 A * 10/2000 Chandler GOIN 21/645
356/72

(Continued)

Primary Examiner — Sang H Nguyen
(74) Attorney, Agent, or Firm — Otlice of Chief Counsel
for National Institute of Standards and Technology

(67) ABSTRACT

A quantum flow cytometer detects an analyte with photon-
number statistics and includes: a flow cytometer that
receives a pump light, an analyte and produces scattered
light from the analyte; an intensity interferometer that
includes a beam splitter and receives the scattered light;
splits the scattered light; and single photon detectors that
receive the scattered light, such that the beam splitter and the
single photon detectors provide photon number statistics that
detects the analyte. A process for detecting the analyte with
photon-number statistics includes: receiving the pump light;
producing, by the analyte, scattered light from scattering the
pump light; splitting the scattered light into first scattered
light and second scattered light; detecting, by single photon
detectors, the first scattered light and second scattered light;
producing, by the single photon detectors, detection signals;
and subjecting the detection signals to an analyzer to detect
the analyte by photon number statistics.

16 Claims, 7 Drawing Sheets

oar)



US 11,022,538 B2

5

solution or organic solvent. Analyte flow 228 can be pro-
duced from a syringe with a syringe pump. A flow rate of
analyte flow 228 can be from 0 uL/hour to 1000 uL/hour,
specifically from 0.001 uL/hour to 100 uL/hour, and more
specifically from 0.1 to 10 uL/hour. Moreover, a ratio
between analyte flow rate and sheath flow rate can be
maintained at a selected value for laminar flow and selected
dimensions of hydrodynamic focus.

‘Analyte 210 is disposed in analyte flow 228. Analyte 210
can include biological cells, peptides, or antigens that
include appropriate fluorescent labels, e.g., one per biologi-
cal entity of interest to produce optical signal upon optical
excitation and can be organic dye-labeled organic molecules
or quantum-dot-labeled organic molecules mixed with
water, saline solution or organic solvent. Exemplary analytes
210 include colloidal quantum dots dissolved in an inorganic
solvent, e.g., in water, or organic solvent, e.g., decane.
Moreover, to achieve enumeration, each biological entity
can be labeled by a number of fluorescent labels, e.g., by one
such label. In an embodiment, analyte 210 includes quantum
dots with a biotin-binding protein such as streptavidin that
can be covalently attached to a fluorescent label for attach-
meant to biological entities.

In confluence tube 250 analyte 210 is subjected to pump
light 220 and produces scattered light 232. Scattered light
232 can include photons due to fluorescence, photons due to
Raman and elastic scattering, or noise photons that can be
collected for subsequent detection and analysis. A temporal
pulse width of scattered light 232 can be from 0 s to 1 s,

specifically from 1 fs to 100 us, and more specifically from 3

100 ps to 100 ns. A wavelength of scattered light 232 can be
from 200 nm to 4000 nm, specifically from 350 nm to 2000
nm, and more specifically from 350 nm to 1000 nm. An
average power of scattered light 232 can be from 0 mW to

100 mW, specifically from 0 W to 2*10°-10 W, and more 3

specifically from 0 W to 5*10°-12 W. In an embodiment,
scattered light 232 includes photons at the range of wave-
lengths from 350 nm to 1000 nm that originate from
different processes such as fluorescence, Raman scattering,
elastic and inelastic scattering and has energy per pulse of 0
J to 5*10°-17 J. The temporal length of the pulse depends on
the fluorophores and conjugation methods use, and pump
temporal length and can be from 0 s to 100 ns.

Scattered light 232 can include Raman scattering back-
ground light 240 that can have a wavelength from 200 nm
to 4000 nm, specifically from 350 nm to 2000 nm, and more
specifically from 350 nm to 1000 nm. An average power of
Raman scattering background light 240 can be from 0 mW
to 100 mW, specifically from 0 W to 2*10°-10 W, and more
specifically from 0 W to 5*10°-17 W. In an embodiment,
Raman scattering background light 240 includes photons at
the range of wavelengths from 350 nm to 1000 nm and has
energy per pulse of 0 J to 5*10°-17 J. The temporal length
of the pulse of scattered light 232 depends on pump temporal
length is co-temporal with the pump pulse and can be from
500 fs to 2 ns.

Scattered light 232 is received by light collector 214 from
confluence tube 250. Light collector 214 can include focus-
ing lenses, microscope objectives, optical filters, single
mode and multi-mode optical fiber to collect, filter and route
the scattered light 232 to a light detector and can be a
confocal microscope. Exemplary light collectors 214
include high numerical aperture lenses, multimode optical
fiber and optical filters based on wavelength selection.
Moreover, the light collector is assembled with minimal
light losses due to possible surface reflection by using broad
optical coating surfaces that best transmit the light of

0

40

0

6

expected wavelength. In an embodiment, light collector 214
includes a 0.5 numerical aperture aspheric lens, a set of
band-pass filters that only transmit light of wavelengths
expected from the fluorophore used, e.g., a 50 nm-wide
bandpass centered around 655 nm, and a coupling aspheric
lens with a numerical aperture matched to that of the fiber.

Lens 234 can reduce aberrations and anti-reflection coated
to transmit light at 655 nm wavelength and can be a
microscope objective or evanescently coupled waveguide or
shaped tip of the waveguide or fiber. In an embodiment, lens
234 includes aspheric lens coated for 600-1050 nm. A
number of lenses can be selected for collimation and the
like.

Filter 236 can include dichroic mirrors, low-pass optical
filters, high-pas optical filters, notch filters with one or many
pass-bands and a combination thereof to filter by wavelength
selection an optical signal including optical pump residual
and Raman scatter and can be thin-film deposited optical
wavelength filters or volume Bragg gratings or colored
glass. Exemplary filters 236 include thin-film deposited
optical wavelength filters. Moreover, peak absorption or
reflection bands of the filters should be spectrally overlapped
with the wavelengths of peak scattering activity, such as
Raman resonances of water and solvents, pump wavelength
and its second harmonic. In an embodiment, filter 236
includes a set of thin-film filters with combined optical
density of ~8 to >12 to block the unwanted light and
combined transmittance of ~90% in the wavelength region
of interest.

Optical path 238 receives scattered light 232 from light
collector 214. Optical path 238 can include fiber coupling
optics, a single or multimode optical fiber or a free-space
link to the detector to route collected light to a detector 218
or beam splitter 216. Exemplary optical path 238 include
connectorized fiber patch cords. Moreover, length of fiber or
a free space link must be sufficiently short to avoid propa-
gation losses. In an embodiment, optical path 238 includes
a connectorized multimode patch cord 2 meters in length.

Photon-number resolved detector 218 can include a super-
conducting transition-edge sensor detector or an interleaved
superconducting nanowires that generate electrical signal
upon photon arrival and absorption in such a way that the
analysis of that electrical signal can discriminate between
none, one, two, and a few photons with enumeration. The
arrival times could be derived from the temporal dependence
of the output electrical signal as leading edges. The photon
number can be obtained from peak amplitude or integral
amplitude of the electrical pulses or as digital information.
In an embodiment, the detector 218 can be a superconduct-
ing transition edge sensor or a visible-light photon counter,
or an interleaved superconductor nanowire single photon
detector.

The analyzer 258 electrically connected to photon-num-
ber resolved detector can include an amplitude to digital

5 coder, and a processing unit such as a field programmable

gate array (FPGA) or a microprocessor. The processing unit
should be arranged to extract number of photons from the
signal 254 produced by detector 218 and calculate second,
third or higher order correlation of photon arrivals.

In another embodiment, beam splitter 216 receives scat-
tered light 232 from optical path 238. Beam splitter 216 can
include fiber beam splitters, fiber pigtailed beam splitters, or
free space beam splitters to split the collected scattered light
232 into secondary scattered light beams 244. Moreover,

5 several beam splitters can be used one after another to

further split the scattered light in multiple paths 244 for
subsequent detection and correlation analysis. In an embodi-


US 11,022,538 B2

3

relation functions and their dependence on various time
delays. The quantum flow cytometer measures average
photon flux as a function of time and time delays and can
include measurement for different concentrations. The cor-
relation functions relate to concentrations via characteristics
of quantum optics in an absence of prior knowledge of
transmittance loss or detection inefficiency in an unknown
optical background. An optical flux to moles calibration is
obtained from first principles, and dependence of second-,
third-, fourth-, or higher-order correlation functions on time
provides advantageous signal processing in a sorting mode
and enhances signal to noise.

Quantum flow cytometer 200 detects analyte 210 with
photon-number statistics. In an embodiment, with reference
to FIG. 1, quantum flow cytometer 200 includes flow
cytometer 212 that receives pump light 220 in first direction
222; receives analyte flow 228 including analyte 210 in
second direction 224; and produces scattered light 232 from
scattering pump light 220 by analyte 210. Quantum flow
cytometer 200 also includes a photon number resolving
detector 218 in optical communication with flow cytometer
212 and receives the scattered light beam 232.

In another embodiment, with reference to FIG. 2 and FIG.
3, quantum flow cytometer 200 includes flow cytometer 212
that receives pump light 220 in first direction 222; receives
analyte flow 228 including analyte 210 in second direction
224; and produces scattered light 232 from scattering pump
light 220 by analyte 210. Quantum flow cytometer 200 also
includes intensity interferometer 242 in optical communi-

cation with flow cytometer 212. Intensity interferometer 242 3

includes one or more beam splitters 216 in optical commu-
nication with flow cytometer 212. Beam splitter 216
receives, in third direction 226, scattered light 232 from flow
cytometer 212; and splits scattered light 232 into first

scattered light beam 244.1 and second scattered light beam 3

244.2. If more than one beam splitter is used, the input of
each subsequent beam splitter is optically connected to the
output of a previous beam splitter 244, and the scattered
light 232 is further subdivide into scattered light beams 244.
In intensity interferometer 242, first single photon detector
218.1, which can be either a photon number resolving
detector or non-photon number resolving detector, is in
optical communication with beam splitter 216 and receives
first scattered light beam 244.1 from beam splitter 216, and
second single photon detector 218.2, which can be either a
photon number resolving detector or non photon number
resolving detector, is in optical communication with beam
splitter 216 and receives second scattered light beam 244.2
from beam splitter 216. Here, beam splitter 216 and single
photon detectors 218 provide a Hanbury Brown-Twiss
(HBT) effect for detection of analyte 210. It is contemplated
that first direction 222, second direction 224, and third
direction 226 are arranged at oblique angles to one another.

Light source 256 can provide pump light 220. Light
source 256 can be a laser, a frequency doubler, an optical
amplifier, a parametric light converter, light-emitting diode,
a lamp including a gain medium, heating elements or other
sources of energy to produce coherent or incoherent, pulsed
or continuous-wave light. Such includes quantum sources of
light with exotic photon number statistics such as a photon
pair source, squeezed vacuum, and squeezed coherent state
sources for multiphoton absorption in analyte 210 with
lower pump power and to enhance a signal-to-noise ratio.
Exemplary light sources 256 include diode lasers, optically
pumped Ti: Sapphire lasers, Argon ion lasers, parametric
frequency doublers, and optical parametric oscillators.
Moreover, incoherent sources of pulsed or continuous radia-

0

40

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4

tion can be used. In an embodiment, light source 256
includes a pulsed femtosecond Ti: Sapphire laser at 900 nm
whose light is frequency doubled to provide input light 220
at 450 nm.

Pump light 220 can include incoherent or coherent radia-
tion and can be either pulsed or continuous-wave to optically
excite analytes 210. A wavelength of pump light 220 can be
from 4000 nm to 300 nm, specifically from 800 nm to 300
nm to produce single-photon excitations, from 3500 nm to
550 nm to produce multiphoton excitations, and more spe-
cifically from 550 nm to 400 nm to use with fluorophores
excite fluorescence with an optical single photon excitation.
‘A temporal pulse width of pump light 220 can be from 5
attoseconds (as) to continuous wave, specifically from 5 fs
to 10 ns, and more specifically from 500 fs to 5 ps. An
average power of pump light 220 can be from 1 fW to 100
W, specifically from 1 microwatt (uW) to 1 W, and more
specifically from 1 mW to 100 mW. A repetition rate of
pump light 220 can be from 0.001 mHz to 100 GHz or
continuous wave, specifically from 1 Hz to 1 GHz and more
specifically from 1 MHz to 100 MHz. As used herein,
“average power” refers to averaged instantaneous power of
the laser over many (e.g., 100 or more) pulses or time-
averaged power of a continuous wave radiation over 1 ms or
longer. In an embodiment, pump light 220 includes a pulsed
laser with the wavelength of 450 nm, pulses of approxi-
mately 1 ps, repetition rate of 76 MHz and average power of
5 mW produced from a Ti: Sapphire laser with the wave-
length of 900 nm, average power of 1.5 W, pulses of
approximately 2 ps, and repetition rate of 76 MHz.

Flow cytometer 212 receives analyte flow 228 and sheath
flow 230 and hydrodynamically focuses analyte flow 228 in
flow column 252 surroundingly sheathed by sheath flow 230
in confluence tube 250. Flow cytometer 212 can include a
flow cell or a microfluidic cell to channel the analyte flow
228 and sheath flow 230 and establish the optical access for
the excitation beam 220 and the scattered light 232 and can
be a rectangular parallelepiped glass with polished sides and
a rectangular polished through hole with tapered dimensions
inside. Exemplary flow cytometers 212 include a rectangular
parallelepiped flow cell with approximate dimensions of 4
mmx5 mmx15 mm, a rectangular hole with dimensions of
250 umx250 um through its longest dimension. Moreover,
the hole can be tapered from one end with a largest dimen-
sion at the surface of 0.5 mmx0.5 mm to focus analyte flow
228 in sheath flow 230. In an embodiment, flow cytometer
212 includes two independent pumps, one to push the
syringe with the analyte flow 228 and the other to push
sheath flow 230.

Sheath flow 230 flows in sheath flow tube 248 and merges
with the analyte flow at confluence tube 250. Sheath flow
230 can include water, saline fluid, organic solvent that
provides an external flow to the analyte flow for hydrody-
namic focusing. Sheath flow 230 can be provided by a

5. syringe controlled by a syringe pump. A flow rate of sheath

flow 230 can be from 0 microliter (uL)/min to 100000
uL/min, specifically from 0.01 uL/min to 1000 uL/min, and
more specifically from 0.1 uL/min to 100 uL/min. The flow
rate can be controlled to remain in laminar flow in flow
cytometer 212.

Analyte flow 228 flows in inner flow tube 246 and merges
with sheath flow 230 at confluence tube 250. Analyte flow
228 can include an analyte that includes biological cells,
peptides, or antigens that include appropriate fluorescent

5 labels, at least one per biological entity of interest to produce

optical signal upon optical excitation and can be quantum-
dot-labeled organic molecules mixed with water, saline


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The process for detecting analyte 210 with quantum flow
cytometer 200 also can include measuring single photon
arrival times and wavelengths within regions of interest that
are established with the help of the correlation measurement
ona diluted analyte when approximately the average num-
ber of fluorophores within the volume from which the
scattered light 232 is collected is around one, and selecting
those regions where correlation function g(2) is minimal. In
this way the uncorrelated background is reduced

The process for detecting analyte 210 with the quantum
flow cytometer 200 can also include absolute self-calibration
to relate photon flux of the collected scattered light 232 to
the number of fluorophores under measurement. To perform
self-calibration, a given initial concentration of analyte in
the sample flow 228 is achieved through dilution of the
analyte 210 in an organic or inorganic solvent. This con-
centration can be unknown. Several concentrations of ana-
lyte 210 are prepared from the initial concentration of
analyte 210 by further dilution in a solvent, where this
further relative dilution factors need to be known. The
quantum cytometer signal 260 is obtained for several analyte
flows 228 with different dilutions of analyte 210 using the
constant parameters of the pump light 220, and the same
position of the optical path 238 relative to scattered light
232, light collector 214 (if present), and keeping same
alignment and operating conditions of the detectors 218,
sheath flow 230, and analyte flow 228. Instead of using a
plurality of dilutions for the analyte flow 228, only one
initial dilution may be used, and a plurality of measurements
may be taken with different flow rates of analyte flow 228
and sheath flow 230. A model that predicts the energy per
pulse of scattered light 232 with changing analyte concen-
trations or flow parameters can be derived. An example of
such a model under assumption that an optical scattered light

232 consists of light from few fluorophores and uncorrelated 3

background is presented below in the Example
Quantum flow cytometer 200 and processes disclosed
herein have numerous beneficial uses, including first-prin-
ciple based noise elimination, absolute self-calibration, and
increased sensitivity. Advantageously, quantum flow cytom-
eter 200 overcomes limitations of technical deficiencies of
conventional articles such as low specificity of observed
signal and poor correlation between the number of fluoro-
phores and detected optical signal when only a few (~10 or
fewer) fluorophores are used per biological entity.
Quantum flow cytometer 200 provides sensitivity and
specificity that exceeds that of conventional flow cytometer
technology, wherein saturation is reached and after which
further increase in sensitivity does not result in improved
specificity. Quantum flow cytometer 200 enumerates bio-
logical targets, such as antibodies, micro RNA, exosomes
and the like at low concentrations and provides an absolute
scale for such enumeration in situ with inter-comparability
of among samples. Accordingly, quantum flow cytometer
200 overcomes conventional problems of calibration of flow
cytometers for intercomparing and quantification. More-
over, use of calibration information from quantum flow
cytometer 200 optimizes sensitivity in real-time cell-to-cell
measurement at a level of a single fluorophore. Light pro-
duced by a system of few optically active biomarkers
(fluorescent molecules, quantum dots, color centers in
nanocrystals and the like) exhibits quantum behavior, and
quantum flow cytometer 200 detects such via quantum
measurement. Here, photon number statistics unambigu-
ously resolves the number of emitters and is resilient to loss.
This unambiguous measurement provides self-calibration of
flow cytometer 212 in quantum flow cytometer 200. A

30

40

45

12

sample of an unknown concentration of fluorophores diluted
by a selected factor provides a range of concentrations with
an average of fluorophores from approximately 0.1 (or
lower) to approximately 10 per interrogation volume. Quan-
tum flow cytometer 200 acquires photon number statistics
for each of diluted samples. A continuous wave laser or
pulsed laser can be used to optically excite the fluorophores.
In addition to absolute calibration, time-resolving statistical
analysis identifies temporal properties of signal (nonclassi-
cal) and noise (classical). Although conventional instru-
ments collect signal from a biological entity for a short time
microseconds to a millisecond with a number of collected
photons from trace concentrations of biomarkers being,
limited, quantum flow cytometer 200 overcomes such limi-
tations and separates signal from background due to accurate
time of single photon detection being collected with tem-
poral properties of signal and noise independently identified,
wherein separation of signal from background can be made
sub-shot noise.

Moreover, quantum flow cytometer 200 and processes
herein have numerous advantageous properties. In an aspect,
quantum signal 260 can be used together with conventional
classical signals from classical detectors providing for mea-
surement of several different types of analyte simultaneously
or to improve overall specificity. Examples of such use
include triggering the collection of the quantum cytometer
signal upon detecting a certain signal within scattered light
232, correlation of classical signal with quantum cytometer
signal and the like. The classical signal can be obtained with
the same or different pump light beam 220. Further, the
classical signal can be obtained with the arbitrary wave-
length and temporal region of interest. Further, the classical
signal can be obtained in the same or different direction than
directions 222, 224, and 226. Classical signal can originate
from any physically allowed interaction of pump light 220
with the same or different or combined analyte 210, for
instance from scattering, fluorescence and Raman scattering.
Classical signal can be detected by any light detector, such
as a photodiode, an avalanche photodiode, a photomultiplier
tube and a single-photon detector. A difference between
classical signal and quantum signal is that with classical
signal no quantum correlation is expected or measured.

Moreover, quantum flow cytometer 200 can be combined
with another quantum flow cytometer that may be using the
same or different pump light 220, measuring same or dif-
ferent scattered light 232, and measure that light in the same
or different wavelength or temporal region of interest, pro-
viding for measurement of several different types of analyte
simultaneously or to improve overall specificity. The quan-
tum cytometer outputs 260 can be correlated or the presence
of a particular feature in one output can trigger the mea-
surement of the other.

Quantum flow cytometer 200 and processes herein unex-
pectedly provides the means for absolute calibration and
enumeration of the number of fluorophores per biological
entity by observing the photon detections. Moreover, quan-
tum flow cytometer 200 provides signal-to-noise optimiza-
tion from first principles.

The articles and processes herein are illustrated further by
the following Example, which is non-limiting.

EXAMPLE

Absolute measurement of biomarker concentration with
sensitivity for detecting a single biomarker is accomplished
by a quantum flow cytometer. As a mimic for a biological
analyte, a colloidal quantum dot is used. It should be


US 11,022,538 B2

15

nucleic acids, and other molecules and can be used in flow
cytometry so that a functionalized biomarker can be used to
count biological entities by the numbers in a quantum-
enabled flow cytometer.

While one or more embodiments have been shown and
described, modifications and substitutions may be made
thereto without departing from the spirit and scope of the
invention. Accordingly, it is to be understood that the present
invention has been described by way of illustrations and not
limitation. Embodiments herein can be used independently
or can be combined.

All ranges disclosed herein are inclusive of the endpoints,
and the endpoints are independently combinable with each
other. The ranges are continuous and thus contain every
value and subset thereof in the range. Unless otherwise
stated or contextually inapplicable, all percentages, when
expressing a quantity, are weight percentages. The suflix
“(s)” as used herein is intended to include both the singular
and the plural of the term that it modifies, thereby including
at least one of that term (¢.g., the colorant(s) includes at least
one colorants). “Optional” or “optionally” means that the
subsequently described event or circumstance can or cannot
occur, and that the description includes instances where the
event occurs and instances where it does not. As used herein,
“combination” is inclusive of blends, mixtures, alloys, reac-
tion products, and the like.

As used herein, an “oblique angle” is any angle including,
but not limited to right angle, zero angle, straight angle,
acute angle and obtuse angle in a two-dimensional or
three-dimensional space.

As used herein, “a combination thereof” refers to a
combination comprising at least one of the named constitu-
ents, components, compounds, or elements, optionally
together with one or more of the same class of constituents,
components, compounds, or elements.

All references are incorporated herein by reference.

The use of the terms “a” and “an” and “the” and similar
referents in the context of describing the invention (espe-
cially in the context of the following claims) are to be
construed to cover both the singular and the plural, unless
otherwise indicated herein or clearly contradicted by con-
text. “Or” means “and/or.” It should further be noted that the
terms “first,” “second,” “primary.” “secondary,” and the like
herein do not denote any order, quantity, or importance, but
rather are used to distinguish one element from another. The
modifier “about” used in connection with a quantity is
inclusive of the stated value and has the meaning dictated by
the context (e.g., it includes the degree of error associated
with measurement of the particular quantity). The conjunc-
tion “or” is used to link objects of a list or alternatives and
is not disjunctive; rather the elements can be used separately
or can be combined together under appropriate circum-
stances.

What is claimed is:

1. Aquantum flow cytometer for detecting an analyte with
photon-number statistics, the quantum flow cytometer com-
prising:

a flow cytometer that:

receives a pump light in a first direction;

receives an analyte flow comprising the analyte in a
second direction; and

produces scattered light from scattering the pump light
by the analyte;

an intensity interferometer in optical communication with

the flow cytometer and that comprises:
a beam splitter in optical communication with the flow
cytometer that:

S

25

40

16

receives, in a third direction, the scattered light from
the flow cytometer;

splits the scattered light into a first scattered light
beam and a second scattered light beam; and

a first single photon detector in optical communication
with the beam splitter and that receives the first
scattered light beam from the beam splitter; and

a second single photon detector in optical communica-
tion with the beam splitter and that receives the
second scattered light beam from the beam splitter,
such that the beam splitter and the single photon
detectors provide a photon number statistics mea-
surement that detects the analyte,

wherein the first direction, the second direction, and the
third direction are arranged at oblique angles, and the
beam splitter splits light based on intensity of the
scattered light according to a selected splitting ratio
of the beam splitter so that the first scattered light
beam and a second scattered light beam are subject
to intensity interferometry by the intensity interfer-
ometer.

2. The quantum flow cytometer of claim 1, further com-
prising a light collector in optical communication with the
flow cytometer and the intensity interferometer such that the
light collector:

is optically interposed between the flow cytometer and the
beam splitter; and

comprises:

a lens that receives the scattered light from the flow
cytometer;

a filter that receives the scattered light from the flow
cytometer and that reduces the pump light or Raman
scattering background light from communication to
the single photon detectors: and

an optical fiber that receives the scattered light from the
lens and the filter and communicates the scattered
light to the beam splitter.

3. The quantum flow cytometer of claim 1, wherein the
flow cytometer comprises:

a sheath flow tube through which the sheath flow flows:

an inner flow tube disposed in the sheath flow tube
through which the analyte flow flows:

a confluence tube in fluid communication with the sheath
flow tube and the inner flow tube and in optical
communication with the intensity interferometer such
that the confluence tube:
receives the sheath flow and the analyte flow;
hydrodynamically focuses the analyte;
constrains the flow of the analyte flow ina flow column

within sheath flow in confluence tube;

receives the pump light; and

communicates the scattered light to the intensity inter-
ferometer.

4. The quantum flow cytometer of claim 1, wherein the
single photon detector comprises a single photon avalanche
diode.

5. The quantum flow cytometer of claim 1; wherein the
analyte detected by the intensity interferometer is one mol-
ecule.

6. A process for detecting an analyte with photon-number
statistics with the quantum flow cytometer of claim 1, the
process comprising:

receiving, by the flow cytometer, the pump light in the
first direction;

receiving, by the flow cytometer, the analyte flow com-
prising the analyte in the second direction;


US 11,022,538 B2

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US 11,022,538 B2

Page 2
(56) References Cited
U.S. PATENT DOCUMENTS
652 BL* 5/2001 Rodriguez GOIN 15/14
356/335
6,507,391 B2* 1/2003 Riley GOIN 15/1012
356/28
7,630,063 B2* 12/2009 Padmanabhan GOIN 15/1484
356/246
2002/0028434 Al* 3/2002 Goix GOIN 15/1404
435/4
2009/0323061 Al* 12/2009 Novotny GOIN 15/1456
356/336
2012/0122084 Al* 5/2012 Wagner GOIN 15/147
435/61
2012/0129190 Al* 5/2012 Chiu GOIN 33/5302
435/7.23
2013/0126755 Al* 5/2013. Kemnitz GOIN 21/6456
250/459.1
2015/0049333 Al* 2/2015 Gourley GOIN 21/6428
356/338
2016/0178518 Al* 6/2016 Schwabe GOIN 15/1459
422/82.08
2017/0248508 Al* 8/2017 Ward GOIN 15/1404

* cited by examiner


US 11,022,538 B2

7

ment, beam splitter 216 includes a fiber beam splitter with
a splitting ratio close to 50%/50%.

Single photon detectors 218.1 and 218.2 receive scattered
light beams 244.1 and 244.2 correspondingly from beam
splitter 216. Single photon detectors 218.1 and 218.2 can
include avalanche photodiode in a photon detection mode,
superconducting nanowire single photon detector, single-
photon sensitive camera, or a photomultiplier tube. Each of
detectors 218.1 and 218.2 should provide an electrical pulse
upon detection of a photon with a rising edge that contains
information about arrival time of the photon at the detector.
In an embodiment, single photon detectors 218.1 and 218.2
include a thick avalanche photodiode.

Scattered light beams 244.1 and 244.2 can include optical
photons emitted in the flow cytometer 212 by optical pro-
cesses such as Raman scattering and fluorescence. A tem-
poral pulse width and wavelengths of scattered light beams
244.1 and 244.2 can be the same as that of scattered light
232. An intensity of scattered light beams 244.1 and 244.2
can be proportional to that of scattered light 232, albeit the
coefficient of proportionality is lower than unity. In an
embodiment, scattered light beams 244.1 and 244.2 have the
same optical characteristics as scattered light 232, except for
energy of optical pulses. The energy of optical pulses is
proportional to and approximately 50% of the energy of
scattered light pulses.

In response to detecting scattered light beams 244.1 and
244.2, single photon detector 218.1 and 218.2 produce
detection signals 254.1 and 254.2. Detection signals 254.1
and 254.2 can include digital electronic pulses whose lead-
ing edge reflects the information about arrival time of
corresponding optical photons from the light beams 244.1
and 244.2. A temporal pulse width of detection signals 254.1
and 254.2 can be from 0 s to 1 s, specifically from 1 ps to
1 ms, and more specifically from 1 ns to 100 ns. An
amplitude of detection signal 254 can be from OV to 10000
specifically from 0.01 uV to 100V, and more specifically
from 1 uV to 10V, and can have rectangular shape. If
detection signal has amplitude that an analyzer logic cannot
directly detect signal, the voltage can be increased by an
analog amplifier or digital comparator. In an embodiment,
detection signal 254 is a rectangular pulse that is approxi-
mately 2V in amplitude and 25 ns in duration.

Detection signal 254 is received by analyzer 258 from
single photon detectors 218 after optional amplification or a
digital comparator. Analyzer 258 can include a micropro-
cessor or an FPGA to digitally process the temporal char-
acteristics of the signal and compute second, third, or higher
order correlation function of the detection signal 254. Alter-
natively, temporal records of leading edge pulse arrivals
from the detection signal 254 can be measured and stored on
a computer for further processing using the same or other
personal computer. In an embodiment, analyzer 258 includes
an electronic time tagger that records times of arrival of the
electronic pulses, stores them on a hard disc drive and
computes correlation functions at a later time.

Analyzer 258 produces quantum cytometer signal 260
from detection signals 254. Quantum cytometer signal 260
can include photon number resolved rates and correlations
as a function of time from the beginning of the optical
excitation via pump light 220 and time intervals between
detections, known as number of counts at a given time
interval and time-dependent correlation functions. In most
general case with pulsed optical excitation 220 the pulse
number p can be used as an enumerator. Moreover, for each
detected photon, detection time of a photon on one of the
detectors 218 labeled i, occurs at time of p, pulse with the

0

0

40

45

0

8

temporal offset t, from the beginning of the pulse p,. Then,
average n-order correlations can be computed as C(n)(t,, t,,

> ty is Po-Pis PsP» > P,P). To obtain C(n),
continuous times t, break into temporal bins of duration dt.
Then t, are relabeled such that t,,,,,,-floor(t/dt), floor(x)
denotes finding a nearest integer that is less than or equal to
the argument x. From this point on, only t,,,,,, are used and
index “new” is dropped. Of the highest interest is C(n)(0)
defined as C(n)(0)=C(a) (t,. ty... t,,. dt, 0, 0,... 0), which
describes correlations within the same pump pulse. C(n) can
be normalized with either computed probabilities of random
coincidences between photon arrival times or by using
averaged values of C(n) with sufficiently large p.-p,, p3-P).
. «+ P,P such that no correlations between arrival times of
photons occurs in practice. Finally, normalized g(n)(tyjars dt,
P2P;; PsP; - - - P,-P:) is computed such that all detections
whose detection delays at all detectors t, fall into the interval
[tcares tsearetdt] are added and then normalized by the number
of expected uncorrelated coincidence detections that would
occur within the time interval [tyne tesarrtdt]. A series of
observed cross correlation functions are calculated to find
instances where the functions deviate from unity. A temporal
quantum cytometer signal 260 can be a one-dimensional
function of average rates of photon detection versus elapsed
time from the beginning of the excitation pulse 220.

Further, process for making quantum flow cytometer 200
includes analysis of quantum cytometer signal 260. The
measured time-resolved correlation functions are screened
for values statistically different from unity. Because uncor-
related noise is statistically different from correlated signal
from individual fluorophores within analyte 210, quantum
cytometer signal 260 is evaluated for regular temporal
regions where correlation functions significantly deviate
from unity. Such temporal regions repeat regularly after each
excitation pulse. Particularly, quantum signal from few
fluorophores is detected when correlation function of any
order gets significantly below unity. That signal may contain
residual uncorrelated background, but its influence can be
avoided by measuring the part of correlations that are
specifically due to uncorrelated background. To do so, one
either analyzes higher-order correlations or observes quan-
tum signal 260 from analyte flow 228 with a range of analyte
210 concentrations. Specifically, for uncorrelated optical
and electronic dark count noise correlated signal from the
analyte can be determined. The signal, collection efficiency,
and concentration can be determined in situ to relate photon
flux per unit time to the number of fluorophores.

To identify the number of fluorophores in the absolute
way, photon number statistics can be used. Photon number
statistics can be measured using the quantum cytometer
signal 260. For instance, the second-order photon number
statistics with no background present gives the number of
fluorophores in a given analyte entity directly, g(2)=1-1/N,
where N is the number of fluorophores in a given analyte

5 entity, illustrated in FIG. 7. In case when background is

present, the background should be characterized via a cali-
brating procedure described later in the example.
Quantum flow cytometer 200 can be made in various
ways. In an embodiment, a process for making quantum
flow cytometer 200 includes connecting inner flow tube 246
of flow cytometer 212 to a source of analyte flow 228;
connecting sheath flow tube 248 of flow cytometer 212 to a
source of sheath flow 230; disposing confluence tube 250 of
flow cytometer 212 in optical communication with light

5 source 256 for receipt of pump light 220 by analyte 210 in

confluence tube 250; disposing confluence tube 250 in
optical communication with light collector 214; providing


US 11,022,538 B2

13

appreciated that flow cytometry is a powerfull optical method
used in diagnosis of health disorders and disease monitoring
The quantum flow cytometer receives light that optically
excites single biological entities such as cells having
attached a fluorescent biomarker, wherein the light can be
from a laser. Emitted light is assessed with photon-number
statistics.

Discrimination of a cell containing a single desired bio-
marker is detected with the quantum flow cytometer. Low
concentrations of biomarkers and faint levels of fluorescent
light are handled by quantum measurement. Light produced
from an analyte in a hydrodynamically focused analyte flow
is subjected to pump light, wherein few optically active
emitters including a fluorescent molecule, quantum dot
(QD), and the like exhibits quantum behavior. The quantum
flow cytometer unambiguously resolves the number of emit-
ters in the analyte flow by a cross-correlation function of
emitted photons in a photon-number resolving detection
arrangement that is resilient to loss. By virtue of this
unambiguous measurement, the quantum flow cytometer
provides self-calibration of the flow cytometer through
quantum statistics of detected photons as scattered light so
that the quantum flow cytometer delivers absolute quantifi-
cation and a verification that the sensitivity limit of one
biomarker is achieved. Third and higher-order correlation
functions exhibit their own unique dependence on analyte
concentration and can be used to help measure or to verify
absolute quantification in the presence of any optical noise.

The experimental setup is shown in FIG. 4, wherein

hydrodynamic focusing confine a diluted biological material 3

in a channel that has a 10-100 um diameter and surrounded
by a sheath flow of distilled water. Single semiconductor
nanocrystal quantum dots were used as analytes with an
emission maximum at 655 nm. Pump light was provided as

pulsed excitation by a second harmonic of Ti:Sapphire laser 3

at 450 nm with a repetition rate of 76 MHz and approximate
pulse duration of 1 ps. Scattered light is collected in a
direction perpendicular to the pump laser beam with 0.5 NA
aspheric lens and coupled to a multimode fiber. A low-pass
filter at 488 nm rejects pump photons, and a 40 nm band-
pass filter at 655 nm minimizes contamination of a signal
with Raman scattering background. A primitive photon-
number resolving photon detector (a Hanbury-Brown-Twiss
setup) that includes a 50:50 beam splitter with a single
photon avalanche diode in each of its output ports receives
the scattered light.

In contrast to a continuous-wave pump laser source in a
conventional flow cytometer, the quantum flow cytometer
included a pulsed pump source. Ultrashort laser pulses lead
to time dependence in re-emitted photons, providing an
advantage in time-resolved light measurements. Particularly,
Raman scattering from water molecules and fluorescence
from the sample have different emission times and are
distinguished by the detector.

The biomarker is chosen so that its emission wavelength
is detuned from the water Raman peak around 525 nm.
Raman background is still present near the fluorescence
peak. The pulsed pump source and different time depen-
dence of Raman and fluorescent light is leveraged. Raman-
scattered light decays faster than QD fluorescence so data
may be rejected from early photon detection times, relative
to pump pulse arrival. Single photon detectors have a
non-negligible timing resolution also referred to as jitter.
The three time-dependent processes are combined in the
data. Temporal separation of fluorescence and Raman pro-
cesses in the presence of jitter is seen in a time-dependent
cross-correlation measurement because fluorescence from a

S

2

40

14

few particles is anti-bunched, whereas multimode Raman
light has Poisson statistics. A function, g(2) (t,dt,0), defined
in [0037] is used, wherein t is a delay between the arrival of
a pump pulse and the beginning of the region of interest, and
dt=2 ns is the duration of the region of interest. This function
is plotted against tin FIG. 5 and shows three intervals. At
early times, detection is dominated by Poisson statistics due
to Raman scattering. At medium times, emission from
quantum dots takes dominance, and anti-bunching occurs.
At long times, intensity of light emission decreases by more
than an order of magnitude, and residual background domi-
nates. The interval with the pronounced non-classical light
statistics is used for further concentration analysis. In FIG.
5, a temporal profile of detected signal shows separation of
Raman scattering and QD fluorescence based on measured
time-resolved photon number statistics that is shown as
circles with error bars.

QD concentrations are tested with the quantum flow
cytometer such that the number of dots in the focal volume
of the pump light is low. Non-classical behavior was
observed in all these experiments. The nonclassical signal
was used to assess the concentration by fitting the data to a
model. The model assumes an unknown concentration of
perfect single emitters supplemented with an unknown
intensity of both Raman scattering and fluorescence. FIG. 6
shows a graph of cross-correlation versus estimated number
of quantum dots in a focal volume for the best matching fit.
The number of quantum dots in the focal volume is esti-
mated using the model. Assumptions of the model can be
replaced with an independent measurement for absolute
calibration. Raman noise can be reduced or eliminated with
a faster detector, more efficient filter, or two-photon excita-
tion by the pump light. In FIG. 6, measured cross-correla-
tions g(2)(t.dt.0) with t=5 ns and dt=4 ns (circles) were fit to
a model (dashed line). The model can be that for second
order correlations

1
wrei-zh)
CN) Pa + PoP

Pe
+N) Pad + 5]

so that an unknown concentration <N> which measures the
average number of fluorophores in the volume from which
scattered light 232 is collected, probability to get fluores-
cence photon p, from a single emitter and probability to get
noise photon from the entire collection area p,,, are extracted
from the fit. Importantly, probability to get a fluorescence
photon from a given number of fluorophores in the collec-
tion area scales with <N> as shown in the model. The
average number of quantum dots in focal volume. x-axis in
FIG. 6 comes from the model, and error bars in FIG. 6
correspond to one standard deviation. In a series of calibrat-
ing experiments with different concentrations <N,>, only
ratios <N,>/<N> need to be known, but not the absolute
concentrations. Therefore, starting from an unknown con-
centration <N> a series of <N,> can be obtained by dilution,
and dilution factors need to be retained for analysis.

The quantum flow cytometer provides a nonclassical
photonics measurement. The detection of single biomarkers
in flow cytometry occurs with the quantum measurement
that unambiguously verifies that the signal observed comes
from a small number of optically active particles. Addition-
ally, an absolute calibration for flow cytometers based on
quantum properties of single emitters is provided. The QD
biomarker binds with a variety of proteins, protein motifs,