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(12) Hegyi United States Patent
(10 ) Patent No.:
FROM MOVING OBJECTS
(71 ) Applicant: Palo Alto Research Center Incorporated , Palo Alto , CA (US) (72 ) Inventor : Alex Hegyi, San Francisco , CA (US ) ( * ) Notice : Subject to any disclaimer, the term of this patent is extended or adjusted under 35
See application file for complete search history . References Cited (56 ) U .S . PATENT DOCUMENTS
An optical device includes a first polarizer arranged to receive light emanating from an object moving along a trajectory. The first polarizer polarizes the light emanating from the object along a first polarization direction . A wave plate that has an optical retardance that varies as a function of position along the trajectory receives light from the first polarizer. The slow axis of the waveplate is at a first angle
with respect to the first polarization direction . A second polarizer is arranged to receive light from the waveplate . The second polarizer polarizes light along a second polar
ization direction . At least one detector receives light from
the second polarizer and provides an electrical output signal
Kudenov et al., " Compact Snapshot Real- Time Imaging Spectrom eter” , Proc . of SPIE , vol. 8186 , 2011 , 12 pages. Zakrzewski et al., “ Advancements in Hyperspectral and Multi Spectral Imaging” , retrieved from the internet on Oct. 1, 2015 , 11
Aug. 12 , 2010 , p . 781206 /1 . with Increased Field of View ,” Applied Optics, Optical Society of Courtial et al., “ Design of a Static Fourier - Transform Spectrometer
America, Washington , DC , vol. 35 , No. 34, Dec . 1, 1996 , pp.
File History for EP App . No. 15198314 .5 as retrieved from the EP Electronic File System on Aug. 5 , 2016 , 88 pages. File History for U . S . Appl. No . 14 / 944 ,446 , 132 pages. File History for U . S . Appl. No. 14 /944 ,446 , 234 pages.
* cited by examiner
Aug. 14 , 2018
10 101101 - Q 105 ~
US 10 ,048 ,192 B2
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FIG . 1
U . S . Patent
Aug. 14 , 2018
US 10 ,048 ,192 B2
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FIG . 2
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U . S . Patent
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Sheet 5 of 5
US 10 ,048 ,192 B2
LIGHT EMANATES FROM AN OBJECTMOVING ALONGA TRAJECTORY POLARIZE THE LIGHT ALONG A FIRST POLARIZATION DIRECTION CAUSE A VARYING POLARIZATION OF THE LIGHT
CONVERT THE VARYING POLARIZATION OF THE LIGHT TOA VARYING INTENSITY OF LIGHT DETECT THE VARYING INTENSITY OF THE LIGHT AND GENERATE AN ELECTRICAL SIGNAL
EXTRACT INFORMATION ABOUT THE OBJECT FROM THE ELECTRICAL SIGNAL
FIG . 5
US 10 ,048 ,192 B2 mine information about the objects. The processor may
OBTAINING SPECTRAL INFORMATION
FROM MOVING OBJECTS TECHNICAL FIELD
determine the optical spectra of one or more of the objects based on the detector output signal. The processor may
determine object type based on the optical spectra of the
5 objects and / or may determine other object characteristics.
This disclosure relates generally to devices , systems and methods for determining characteristics ofmoving objects.
BACKGROUND Various techniques have been proposed for performing
The processor may use the spatially modulated light to determine velocities of the one or more objects . In some
implementations the portion of the detector output signal
responsive to the spatially modulated light may include a
10 first frequency component that is different from and /or
orthogonal to a second frequency componentof the detector
sample analysis using light emanating from objects. For
output signal that is responsive to light received from the
example , U . S . Pat. No . 7 , 358 ,476 (Kiesel et al.) discusses a fluidic structure with a channel along which is a series of
second polarizer. In some implementations the optical device includes one
sensing components to obtain information about objects 15 or more optical bandpass filters configured to limit a band width of the light received by the detector. The processor objects carried by a fluid . A sensing component includes a may apply various correction factors that correct for prop
traveling within the channel, such as droplets or other set of cells that photosense a range of photon energies that emanate from objects . A processor receives information
erties of the optical components. For example , the processor may correct for optical dispersion in the waveplate and /or
about objects from the sensing components and uses it to 20 the processor may correct for angle of incidence of the
obtain spectral information . Additional techniques are
described , for example, in U . S . Patent Application Publica -
tions 2008 /0181827 (Bassler et al. ), 2008 /0183418 (Bassler
et al.), 2009 /0195773 (Bassler et al.), 2009/0195852
(Bassler et al.), and 2010 /0201988 (Kiesel et al.).
According to some aspects, multiple detectors are used
wherein each detector generates a detector output signal.
The first detector output signalmay be responsive to a light
25 polarized along one direction and the second detector output Also , various flow cytometry techniques have been pro signal may be responsive to light polarized along another posed . direction . The processor combines the output signals of the
multiple detectors and processes the combined output signal to determine information about the one or more objects . 30 According to some configurations , the optical device and Some embodiments described herein are directed to an processor are components of a flow cytometer. In these
optical device that includes a first polarizer arranged to receive light emanating from an object moving along a trajectory of a flow path . The first polarizer polarizes the
configurations the processor may be configured to determine presence and/or amount of one or more of objects and/or analytes based on the detector output signal. The processor
first polarizer, the waveplate having an optical retardance direction and having a slow axis at a first angle , e. g., about
of the flow cytometer. According to some implementations , automatically during operation .
The second polarizer polarizes light received from the
along a first polarization direction. After the light is polar
about parallel or about perpendicular with respect to the first
ing a retardance that varies as a function of position along
detector provides an electrical output signal that varies with time according to intensity of the light received from the
converted to a varying intensity of the light and is detected and a time varying electrical signal is generated in response
light emanating from the object along a first polarization 35 can include multiple electronic (hardware or software ) fil direction . A waveplate is arranged to receive light from the ters , each electronic filter associated with an optical channel
that varies as a function of position along the trajectory
the electronic filters can be modified either manually or
45 degrees , to the first polarization direction . A second 40 Some embodiments are directed to a method . Light ema polarizer is arranged to receive light from the waveplate . nating from an object moving along a trajectory is polarized
waveplate along a second polarization direction that may be
ized along the first polarization direction , a waveplate hav
polarization direction . At least one detector is optically 45 the trajectory direction is used to create a varying polariza coupled to receive light from the second polarizer. The tion of the light. The varying polarization of the light is second polarizer.
The light emanating from the object may be scattered 50 light, reflected light, fluorescence light, phosphorescence light, chemiluminescence light, or bioluminescence light,
for example. The optical device may further include a lens
to the detected light.
BRIEF DESCRIPTION OF THE DRAWINGS FIG . 1 is a block diagram of a system in accordance with
configured to image the light emanating from the object on some embodiments ; the waveplate . Optionally the first polarizer and /or the 55 FIG . 2 illustrates conversion of the detector output to a second polarizer is a polarizing beamsplitter. In some con -
frequency domain signal in accordance with some embodi ments; Optionally, the optical detector may include a spatial FIG . 3 shows an optical device that includes a spatial filter mask , wherein a portion of the light emanating from the in accordance with some embodiments; object, e . g ., about half, is spatially modulated by the spatial 60 FIG . 4 depicts an optical device that includes a polarizing
figurations, the waveplate is a Wollaston prism .
mask and the detector is arranged to receive the spatially
beamsplitter and multiple detectors in accordance with some
modulated light. Optionally , the optical detector may include
embodiments ; and
one or more optical bandpass filters configured to limit a bandwidth of the light received by the detector.
to a some embodiments .
an optical device as discussed above and a processor con -
used in the figures refer to like components . However, it will
figured to process the detector output signal and to deter-
be understood that the use of a number to refer to a
Some embodiments are directed to a system that includes 65
FIG . 5 is a flow diagram illustrating a method according The figures are not necessarily to scale . Like numbers
US 10 ,048 , 192 B2 component in a given figure is not intended to limit the component in another figure labeled with the same number.
direction. Light that has been polarized along the first polarization direction is received by a waveplate 140 , e .g . a multiple (high ) order waveplate, sandwiched between the first polarizer 131 and a second polarizer 132 . The waveplate
DETAILED DESCRIPTION Embodiments described herein involve devices, systems,
5 140 has an optical retardance that varies as a function of
without decreasing the optical throughput or the spectral
propagation through the waveplate when the object is
nating one or more moving objects to an electrical signal,
or more second polarizers . The polarization axes of the first
wherein the electrical signal encodes spectral information
and second polarizers 131, 132 may be arranged in either a
position along the trajectory direction 111 of the object 101 .
and methods for determining characteristics of moving In some configurations, the retardance varies monotonically objects . Many embodiments are particularly applicable to or linearly as a function of position along the trajectory the field of flow cytometry . Approaches involve obtaining a direction 111 of the object 101. In some configurations , the high resolution optical spectrum from a moving object 10 waveplate may be a Wollaston prism or other optical retar emanating light, in some cases , using one single pixel dance device , positioned such that there is about zero optical detector. The optical spectrum is obtained at high resolution , retardance between the two polarizations of light after
bandwidth , by using the object motion to generate an roughly at the center of the field of view of the detector. This interferogram . In the embodiments described below , the 15 ensures that the recorded interferogram is substantially optical device , which may be referred to herein as a “ spec - centered about its zeroth order fringe . trum encoder," converts the optical signal from light emaThe second polarizer 132 shown in FIG . 1 represents one from each of the objects in the frequency domain of the 20 parallel or perpendicular orientation . In some implementa
electrical signal. The electrical signal can be processed by
electrical circuitry, referred to herein as a processor, to extract the spectral information . Extraction of the informa-
tions, the first and second polarizers 131 , 132 are crossed
polarizers , the first polarizer 131 having a polarization axis that is about 90 degrees from the polarization axis of the
tion can involve transformation of the electrical signal from second polarizer 132 . The slow axis of the waveplate 140 time domain to frequency domain such as by performing a 25 makes a first angle , e . g ., about 45 degrees , with respect to the
full Fourier transform of the electrical signal or a partial
polarization axes of the first and second polarizers 131, 132.
predetermined frequencies of interest .
phase delay of the waveplate 140 creates a time- dependent variation in polarization of the emanating light that is
Fourier transform of the electrical signal at one or more FIG . 1 illustrates a system comprising an optical device
The object's movement relative to the position -dependent
103 configured to provide an electrical signal that includes 30 dependent on the optical spectrum of the light. The varying information about characteristics of a moving object. FIG . 1
polarization of light is converted to a varying intensity by the
shows one or more moving objects 101 moving in a trajec tory 111 along a flow path 110 . In some implementations, the flow path may be disposed between channel walls 112 that
second polarizer 132. A detector 150 , e.g ., a single pixel detector, converts the time varying intensity of light to a time varying electrical output signal, e.g ., time varying voltage, at
constrain the object movement to trajectory 111 . Although 35 the output 151 of the detector. For narrow band light, the
not shown in FIG . 1 , in some embodiments , a fluid move-
time variation in the light intensity may be oscillatory , with
ment device , e . g ., comprising one or more pumps and / or one or more valves , may be coupled to the flow channel between
oscillation period dependent on the center wavelength of the band . Graph 160 illustrates an example of the time varying
the channel walls 112 , wherein the fluid movement device is output signal of the detector associated with one object configured to cause the movement of the object 101 along 40 moving along the trajectory 111 while emanating light in a the trajectory 111 . Light 105 emanates from each of the narrow band of wavelengths. The output signal includes
objects 101 . Optionally, the optical device 103 may include a light source 102 configured to provide input light that interacts with the objects 101. In response to the input light,
information about the object 101, such as the spectrum of the light emanating from the object. Optionally , the optical device 103 includes one or more
105 may be or comprise scattered light, reflected light, fluorescence light, phosphorescence light, chemilumines -
optical filter may be arranged anywhere in the light path between the object 101 and the detector 150 , e. g ., between
cence light, bioluminescence light, etc.
the second polarizer 132 and the detector 150 as shown in
parent to the emanating light 105 . The optical device 103 includes one or more first polarizers , represented in this example by first polarizer 131 . The first polarizer 131 is
detector allows full recovery of the spectral information
the object and the first polarizer 131 such that the lens 120
samples per interference fringe ).
the objects emanate light. For example , the emanating light 45 Optical filters 145 . For example , in various embodiments , an
In embodiments that include flow channel walls, at least FIG . 1 . The optical filter 145 can serve to limit the band one of the walls may be optically transparent to the input 50 width of the light incident on the detector 150 to a bandwidth light and at least one of the walls may be optically trans - range of interest which simplifies signal processing . In some
cases, limiting the bandwidth of the light received by the from the optical interferogram generated by the motion of
configured to receive the light 105 emanating from the 55 the object, even if the electrical signal generated by the object 101. Optionally, a lens 120 may be arranged between interferogram is sampled below the Nyquist limit (two
focuses the emanating light 105 , e .g., halfway between the surfaces of the waveplate 140 . Alternatively, the trajectory
The system shown in FIG . 1 includes a processor 180
coupled to receive the electrical output signal from the
111 of the object may bring the object 101 sufficiently close 60 detector 150 . The processor 180 includes circuitry , such as
to the waveplate 140 so that a lens is not needed . In
a processor that executes programmed instructions , config
embodiments that include a lens, the lens can be configured
ured to process the output signal to extract information about
to be chromatically correct so that all frequencies oflight are
the object 101. Note that the size of the object may be small
131 which polarizes the light along a first polarization
by the processor 180 . For example, if N interference fringes
substantially focused halfway between the surfaces of the with respect to the field of view of the detector 150 , so that waveplate 140 . 65 the light interference fringes created by the motion of the The emanating light 105 passes through the first polarizer object 101 and detected by the detector 150 can be resolved
US 10 ,048, 192 B2 are to be recorded as the object traverses the field of view of
light is filtered by optional optical filter 145 which is
the detector , the size of the object 101 along the trajectory
arranged in the light path between the second polarizer 132
direction 111 may be about 1/ 2N of the field of view of the
and the detector 150 .
detector 150 along the trajectory direction 111 , or smaller, to
Optical device 305 additionally includes a spatial mask
5 320 that includes a number, e . g ., 3 , 4 , 5 , or more , mask resolve the interference fringes . The output of the detector can be processed , e .g., such as features that are optically transparent to the emanating light alternating with 3 , 4 , 5, or more optically opaque mask by Fourier transformation or other time to frequency domain features . A portion of the light 105 emanating from the transformation , to determine the optical spectrum of the object may be imaged on the spatial mask 320 . The spatial light emanating from the object. As shown in FIG . 2, the 10 mask 320 with the light emanating 105 from object transformed output signal 170 may include lower frequency 101 and theinteracts interaction a time varying light that is components 170a and higher frequency components 170b . incident on the detector creates 150 . It will be appreciated that the The lower frequency components 170a are associated with movement of object 101 along the trajectory provides a time the envelope of the signal 160 and the higher frequency multiplexed signal at the output 152 of the detector 150 . The components 170b correspond to the optical spectrum of the 15 time emanating light. includes a first time multiplexed portion that corresponds to In one example , the waveplate is a Wollaston prism the varying light intensity from the second polarizer 132 and having a wedge angle a , where wedge angle is defined as the a second time multiplexed portion that corresponds to the angle between the front face of the prism which is normal to time varying light caused by the interaction between the the optical axis, and the interior face where the two halves 20 emanating light 105 and the spatial mask 320 . It will be
of the prism are cemented together. The slow axes of the prism may be oriented at about 45 degrees with respect to
the axes of the polarizers. The wedge direction of the prism
appreciated that the optical device 305 may optionally be arranged so that the first portion of the output signal 152
corresponds to the time varying light from the spatialmask
is defined as the direction , in a plane parallel to the front face 320 and the second portion corresponds to the varying of the prism , along which the thicknesses of the two halves 25 intensity at the output of the second polarizer 132 . The first of the prism trade off the fastest. It is preferable to align the time portion of the signal responsive to the varying intensity prism ' s wedge direction with the object 's trajectory. If the light from the second polarizer may be processed to extract desired spectral resolution at wavelength à is N /N , N fringes first information about the object, e . g ., the optical spectrum must be recorded as the object traverses across the field of of the light emanating from the object. The second portion view . If the travel distance of the object or the image of the 30 of the signal responsive to the time varying light caused by
object is L along the wedge direction of the Wollaston prism
interaction of the emanating light with the spatialmask may
with birefringence An , the wedge angle of the Wollaston should be a NN2LAn . The maximum spectral bandwidth is
be processed to extract second information about the object, such as velocity and / or size of the object. The second
limited by the sample rate of the detector signal, which must bandwidth is also limited by the size of the object. If the object diameter is d , then the maximum number of fringes that can be recorded by the detector is approximately L /2d , so the minimum detectable wavelength is hæ4adan .
information may be used to apply a correction to the first calibrate the wavelength scale of the optical spectrum . According to some implementations , the output signal can be analyzed without time demultiplexing the signal 152 into
that the object's spectral information is encoded in the frequency domain of the time varying signal. Using these
also lie within a certain bandwidth . If this is the case, then a portion of the emanating light can be imaged onto the
with a single output to obtain the emanating light spectrum , and there is no significant loss of optical throughput with
as shown , e . g ., half the emanating light falling on the spatial mask , while simultaneously half of the emanating light falls
increasing spectral resolution as occurs with conventional
on the first polarizer as shown . The intensity variations
spectroscopy . In some implementations, the outputs of two
caused by the spatialmask lie outside of the frequency range
respects to the optical device 103 of FIG . 1. Optical device
the spatial mask can be combined onto the same detector
through the first polarizer 131 which polarizes the light
mask in the output signal 152 can be separated in software .
be fast enough to record the observed fringes . The spectral 35 information , such as using object velocity information to
the first and second portions. This scenario may occur when According to the approaches discussed herein , the 40 the optical bandwidth of the light 105 emanating from the object’ s motion is used to encode spectral information in the object 101 is known not to exceed a certain range so that the time varying signal of at least one single -pixel detector such fringes of the interferogram detected at the detector should approaches, it is possible to use one single -pixel detector 45 spatialmask which may be placed adjacent to the waveplate
or more single pixel detectors are combined to enhance 50 of the interferogram fringes generated by the polarizer / waveplate /polarizer structure . Thus , light falling on the signal to noise ratio of the combined signal. FIG . 3 shows optical device 305 that is similar in some polarizer /waveplate /polarizer structure and light falling on
305 includes a waveplate 140 sandwiched between first and 150 . The electrical output signal component responsive to second polarizers 131 , 132 . The emanating light 105 from 55 the polarizer/waveplate/ polarizer structure and the signal moving object 101 traveling along trajectory 111 passes component responsive to light modulated by the spatial along a first polarization direction . The light polarized by the first polarizer is received by a waveplate 140 that has an
This separation is possible because the light from the
polarizer /waveplate /polarizer structure lies in a frequency
optical retardance that varies as a function of position along 60 range that is substantially orthogonal to the spatially modu
the trajectory direction 111 .
At each wavelength , the phase delay in the emanating
lated light from the spatial mask .
In some embodiments , each of the first and second
light caused by the waveplate 140 creates a varying polar- polarizers can optionally be replaced by a polarizing beam ization that is dependent on the wavelength . The varying splitter. In some embodiments, two (or more ) single pixel polarization of light is converted to a varying light intensity 65 detectors can be used to detect light having different polar by the second polarizer 132 . The light from the second izations. FIG . 4 illustrates an optical device 405 that polarizer falls on detector 150 . In some configurations, the includes a polarizing beamsplitter 406 in the place of the
US 10 ,048 , 192 B2 second polarizer shown in FIG . 3. The first polarizer 131 polarizes the light emanating 105 from the object 101 along a first polarization direction . The polarizing beamsplitter 406 splits the light from the waveplate 140 into light 411 polarized along a second polarization direction and light 412 5 polarized along a third polarization direction . For example ,
Approaches described herein provide for detecting the spectra at high spectral resolution of light emanating from one or more moving objects . These approaches use a single pixel detector and are absent of tradeoffs between optical throughput and spectral resolution that affect conventional forms of spectroscopy . These approaches involve using the
' s motion to generate an interferogram and to convert one of the polarization axes of the polarizing beamsplitter object the interferogram to an electrical signal using a single pixel 406 could be chosen to lie at 45 degrees with respect to a . In some implementations only one single pixel slow axis of the waveplate 140 as viewed along the opticalà 10 detector detector is used and in some cases multiple single pixel axis . Detector 150 is arranged to detect light 411 polarized detectors are used . Spectral information of the emanating along the second direction . Detector 420 is arranged to light is encoded in the frequency domain of the electrical detect light 412 polarized along the third polarization direc signal. The spectral information can be extracted from the tion . Optional components 145 , 410 may be respectively output signal of the detector using a time domain to fre disposed in the light path between the polarizing beam beam 15 quency domain transformation such as a Fourier transform . splitter 406 and detector 150 and/or in the light path between As in other forms of Fourier spectroscopy , light at all polarizing beam splitter 406 and detector 420 . In some wavelengths is recorded simultaneously , so there is no embodiments, the optional components 145 , 410 comprise tradeoff between spectral resolution and optical throughput. optical filters that optically filter light 411 and 412 . In some In various embodiments, the optical device and /or pro embodiments , component 145 and /or component 410 may 20 cessor shown and described , for example , in FIGS. 1 -5 may
be or comprise a spatial mask . Components 145 , 410 can additionally include optics that image the emanating light
from the object onto the spatial filters. The outputs 153, 421
of the detectors 150 , 420 are both coupled to processor 400 .
be implemented in a flow cytometer that can be configured to analyze objects and/or analytes that are bound to the
objects present in a sample. In these embodiments, the
processor may have the capability of processing the electri
The processor 400 may be configured to analyze outputs to 25 cal signal using one ormultiple processing channels wherein enhance optical throughput and signal to noise ratio of the each processing channel corresponds to a particular fluores optical device . cence channel of the flow cytometer. The processing chan
In embodiments in which components 145 and /or 410
nels may be electrical hardware channels and /or may be
comprise spatialmasks, the processor 400 can extract addireconfigurable software channels . For example , in some tional information about the objects , such as object speed 30 implementations, the emanating light from objects moving and / or size , based on time variation in the electrical signal in the flow channel of the flow cytometer is detected ,
caused by the spatial mask . Interaction of the emanating the electrical output signal 153 , 421 of a detector 150 , 420
light with a spatial mask generates frequency components in
transformed to an electrical signal, sampled , and stored . Information may be extracted from each processing channel by passing the electrical signal through one or more analog
that can be discriminated from the frequency components 35 and / or digital electronic filters . For example , each process
that result from varying light intensity caused by a time
ing channel may correspond to a spectral slice ( frequency
FIG . 5 is a flow diagram of a method of operating the
processor can be programmed to automatically determine
optical detector and processor discussed herein . Light ema-
the processing channels to be used , e. g., the number and/or
dependent variation in polarization .
range of the electrical signal . In some implementations, the
nates 510 from one or more objects moving along a trajec - 40 frequency ranges of channels that are optimal for the optical
tory . The emanating light is polarized 520 along a first
signal, based on the expected and/ or observed spectral
polarization direction . A varying polarization of the light is
components of the electrical signal. In some embodiments ,
created 530 by a waveplate having a variable retardance
the processor may determine optimal filter coefficients and/
along the trajectory of the object. The varying polarization
or configurations for the channel filters . The channel filters
is transformed 540 into a time varying intensity of light. A 45 can be modified before and / or during operation of the flow
time varying electrical signal is generated 550 in response to trical signal includes information about one or more char acteristics of the object. For example , the information may
cytometer, e .g., either manually or automatically . In some embodiments , the processor can be configured to determine an optimal number of processing channels and/or optimal frequency ranges of the processing channels using a
from the object . The time varying electrical signal is ana lyzed 560 to extract information about the spectrum of the
to determine the optimal channels, the processor analyzes the electrical signal and identifies groups of different object
Traditionally this has been accomplished using filter wheels
be modified either manually or automatically during opera
to define the individual fluorescence channels that are fixed
the time varying intensity of light. The time varying elec -
be encoded in the fluorescence spectrum of light emanating 50 clustering algorithm . According to some implementations,
populations that are clustered based on spectral information . light emanating from the object. In some implementations, such as flow cytometry , it is These clustered groups are then used to determine the useful to have the capability of detecting light in individual 55 optimal number and /or frequency ranges of the processor channels across a relatively wide optical bandwidth range . channels . In some implementations, the electronic filters can
by the hardware . Another technique has used a prism to
As previously discussed , in some implementations , more
disperse light and a photomultiplier tube array to detect the 60 than one single pixel detector may be used wherein each
dispersed light. Still other techniques rely on the use of detector detects a different polarization of the emanating spatial modulation of the emanating light that is resolved light. The electrical signals from each of the detectors and /or into channels by a linear variable filter. Drawbacks to these time domain to frequency domain transformations of these techniques include inflexibility due to hardware limitations, signals may be stored , combined , and /or used to analyze the expensive detector arrays , and/or loss of spectral resolution 65 spectra of light emanating from the one ormore objects . For and / or optical throughput. Approaches described herein can example , if optical signals of opposite polarizations were
be employed to mitigate these factors.
detected by the same detector, the oppositely polarized
US 10 ,048 , 192 B2 optical signals would tend to cancel each other out because the interferogram measured at one polarization has the opposite phase as the interferogram measured at an orthogonal polarization . Using two detectors , the signals from each of the detectors can be processed before combining so that 5 the signals are in phase and thus additive rather than
subtractive. For example , the time varying signal from one
10 colors (optical spectrum ranges ) of interest without the need to process other frequency ranges that are not of interest . In some embodiments , the processing makes use of adaptable filters having parameters that can be automatically adjusted by the processor and/or can be semi-automatically adjusted based on some input from the user. For example, in one scenario , the processor may determine which frequency
of the detectors may be phase inverted , or the signals from components are present in the electrical signal and deter both detectors may be transformed from the time domain to mine the number and frequency ranges of the filters used . In the frequency domain , prior to combining. The combination 10 another scenario , a user may input information related to of the two signals can increase the optical throughput of the
optical device relative to a single signal and, correspond
expected frequency ranges of the electrical signal (or fre quency ranges of interest ) and the processor may initially
polarizing beamsplitter. Each waveplate or Wollaston should
frequency ranges of object populations and to determine the
respect to a polarization axis of the beamsplitter when viewed from the waveplate or Wollaston 's optical axis . The wedge axis of the waveplate or Wollaston , i.e ., the direction transverse to the waveplate or Wollaston ' s thickness direc tion along which the optical retardance varies , should be 25
sample based on variation of these frequency ranges. In some embodiments the clustering algorithm may be or comprise a principal component analysis of the electrical signal. For example , consider the scenario wherein the clustering algorithm identifies M different object types cor
ingly, the signal to noise ratio of the combined signal is use those frequency ranges to set up the filters and may increased . In some scenarios, a polarizing beamsplitter is used in place of each of the first and second polarizers . In 15 automatically make adjustments . The processor may determine the frequency range (s ) in these scenarios, it is necessary to place two waveplates or Wollaston prisms after the first polarizing beamsplitter, so the electrical signal corresponding to optical spectra of there is one waveplate for each polarization exiting the first interest using a clustering algorithm to group frequencies or
be oriented so that a slow axis is about 45 degrees with 20 highest fidelity separation between object types present in a
about parallel with the object trajectory . A polarizing beam -
responding to M optical spectra and M principal components
splitter and a pair of detectors is then placed after each waveplate , with a polarization direction parallel to one of the
of the electrical signal. The processor can then detect the object types having any of those M optical spectra of interest
polarization directions of the first polarizing beamsplitter. by performing a dot product of each of the M principal Thus, in total, there are three polarizing beamsplitters and 30 components with the electrical signal in the time domain .
four detectors, each detector arranged to detect one of the
The results of the dot product comprise a vector in a
two light polarized light beams produced by the second or third polarizing beamsplitters. By detecting all polarization
subdivided M - dimensional space , whereby the presence of the vector in one region of that space may indicate that an
combinations of light, this configuration with four detectors
pixel detector configuration .
object of a particular type has been detected . Fourier trans utilizing this technique.
In some embodiments , the processormay first detect and
In some embodiments , the processor is capable of cor
trigger on the presence of an object in the flow path based on characteristics of the electrical signal, such as whether the
recting for errors caused by physical properties of the waveplate and /or other optical components of the optical
has up to four times the optical throughput as the single - 35 formation of the electrical signalmay not be necessary when
electrical signal' s amplitude lies above a threshold value . 40 device . For example , consider that the number of fringes is The triggered portion of the signal, perhaps including pre - dependent on wavelength and on birefringence An . How trigger and posttrigger samples , may be selected for further ever, birefringence is also dependent on wavelength , thus a processing , such as transformation to the frequency domain . correction factor is needed to accurately relate the frequency
The spectral information associated with each detected
of the electrical signal to the optical spectrum of the object.
object is encoded in the frequency components of the 45 Otherwise, calibration at one frequency of the electrical
electrical signal. When the frequency components associ-
signal ( Fourier component) would not be applicable across
ated with the optical spectra of light emanating from differ ent types of objects differ , the processor can discriminate
other frequencies/Fourier components . This correction fac tor may be determined and applied for in the processor, for
objects of different types based on transformations of the example using the known wavelength dependence of the 50 birefringence . electrical signal. In some embodiments , the analysis may only include In general, light from the object incident on the waveplate transformation of certain frequency ranges or portions of the will be substantially collimated . However, software correc electrical signal, rather than a full transformation across a tion may also be needed to account for the difference in wide frequency range . In these embodiments, optical filters optical retardance as a function of angle of the light incident may be placed before the detectors, or the processor may use 55 on the waveplate . The changing angle is caused by a change
software and /or hardware digital and /or analog filters , to
remove or reduce frequency components in the signal that are not of interest prior to performing the time domain to frequency domain transformation . Or, the processor may use
in position of the moving object as it or its image moves
laterally across the waveplate surface .
In various embodiments , all or part of the optical device and/ or processor may be implemented in opto - electronic
software and / or hardware digital and /or analog filters as a 60 hardware . In some exemplary embodiments , functions of the
means to directly pick out frequency components of interest in the electrical signal, in some cases obviating further
processor may be implemented in firmware , software run ning on a microcontroller or other device , or any combina
tion of hardware , software and firmware .
Approaches described herein allow flexibility in process-
The foregoing description of various embodiments has ing the entire frequency range of the electrical signal or 65 been presented for the purposes of illustration and descrip processing only specified portions of the frequency range tion and not limitation . The embodiments disclosed are not ( frequency slices ) of the electrical signal that correspond to intended to be exhaustive or to limit the possible implemen
US 10 ,048 ,192 B2 tations to the embodiments disclosed . Many modifications
and variations are possible in light of the above teaching The invention claimed is : 1 . A method , comprising :
receiving emanating light from an object or image of the
response to a specified polarization of light; and combining the time varying output signals to form a
object ( object/object image ) moving along a trajectory relative to a waveplate of a spectral encoder, the object/object image traversing a distance , L , along the
trajectory within a field of view of the spectral encoder, 10 the object/object image having a diameter , d , along the trajectory ; obtaining spectral information for the object /image point by generating a position -dependent polarization inter ferogram from the emanating light including generat- 15
ing N interference fringes based on motion of the object/object image, the N interference fringes gener ated from light at wavelength à emanating from the object/object image as the object/object image moves the distance L along the the trajectory, wherein Lz2dN , 20
wherein generating the N interference fringes com prises: polarizing light along a first polarization direction , the light emanating from the object/object image mov ing along the trajectory ; after polarizing the light along the first direction , caus ing a time dependent variation in polarization of the light that is dependent on an optical spectrum of the
light andmovement of the object/object image along the trajectory , the time dependent variation in polar- 30 ization of the light provided by a waveplate having
an optical retardance that varies as a function of
position along the trajectory and having a slow axis at an angle to the first polarization direction; and
converting the time dependent variation in polarization 35
of the light to a time varying intensity of the light by
polarizing the light received from the waveplate along a second polarization direction ; and
wherein obtaining spectral information comprises detect
ing the time varying intensity of the light and gener - 40
ating a time varying electrical signal in response to the time varying intensity of the light, the time varying electrical output signal encoding spectral information
about the object/object image with spectral resolution
8. The method of claim 1, further comprising identifying an object type based on the time varying electrical signal . 9 . The method of claim 1 , wherein : generating the time varying electrical signal comprises generating multiple time varying electrical signals in combined output signal; mation about the object/object image. 10 . A system , comprising : a spectral encoder configured to receive emanating light from an object or image of an object (object/object
processing the combined output signal to determine infor
image) as the object/object image moves along a tra jectory relative to a waveplate of the spectral encoder,
the spectral encoder comprising :
a first polarizer configured to polarize light received by the spectral encoder along a first polarization direc tion ;
the waveplate which is configured to cause a time dependent variation in polarization of the light that is dependent on an optical spectrum of the light and movement of the object /object image along the tra jectory , the waveplate having an optical retardance that varies as a function of position along the trajec
tory and having a slow axis at an angle to the first polarization direction ; a second polarizer configured to convert the time
dependent variation in polarization of the light to a time varying intensity of the light by polarizing the
light received from the waveplate along a second
polarization direction ,
wherein the spectral encoder generates a position dependent polarization interferogram that includes N interference fringes based on motion of the object/ object image, the N interference fringes generated from light at wavelength à emanating from the object /object image as the object/object image tra verses the distance L within a field of view of the spectral encoder, wherein the object/ object image has a diameter, d , along the trajectory and Lz2dN ; and
at wavelength à of about WN . 45 circuitry configured to : 2 . The method of claim 1 , further comprising analyzing obtain spectral information for the object/object image the time varying electrical signal using an algorithm to from the spectral encoder; and determine a number of processing channels and /or fre generate a time varying electrical output signal in quency ranges of the processing channels based on a trans response to the time varying intensity ofthe light, the formation of the electrical signal. time varying electrical output signal encoding spec 3. The method of claim 1, further comprising imaging the tral information about the object/object image with light emanating from the object/object image on the wave spectral resolution at wavelength 2 of about NN . plate . 11. The system of claim 10 , wherein : 4 . The method of claim 1, further comprising spatially optical device and processor are components of a flow modulating a portion of the light emanating from the object/ 55 thecytometer ; and object image . further comprising a processor configured to determine 5. The method of claim 4 , further comprising determining presence and/ or amount of one or more of objects and a velocity of the object based on the portion of the light analytes based on the time varying electrical output emanating from the object/object image that is spatially signal. modulated . 12 . The system of claim 11, wherein the processor com 6 . Themethod of claim 1 , further comprising : prises multiple digital filters, each digital filter associated processing the time varying electrical signal; and determining information about the object based on the with a channel of the flow cytometer. 13 . The system of claim 10 , wherein : time varying electrical signal. 7 . Themethod of claim 1 , further comprising determining 65 the circuitry comprises multiple detectors , each detector an optical spectrum of the light emanating from the object/ generating a time varying electrical output signal in response to a specified polarization of light; and object image based on the time varying electrical signal.
US 10,048 , 192 B2 13
the circuitry further comprises a processor configured to
combine the time varying electrical output signals of
the multiple detectors and to process the combined output signal.
14 . The system of claim 10 , further comprising a spatial 5
mask , wherein a portion of the light emanating from the object/object image is spatially modulated by the spatial mask and the circuitry is arranged to receive the spatially modulated light.
15 . The system of claim 10 , wherein the circuitry further 10
comprises a processor configured to determine an optical spectrum of the light emanating from the object/object image based on the time varying electrical output signal. 16 . The system of claim 10 , wherein the circuitry further
comprises a processor configured to identify an object type 15
of the object/ object image based on the time varying elec trical output signal.
17 . The system of claim 10 , wherein the circuitry further
comprises a processor configured to determine velocity of the object based on the portion of the detector output signal 20 responsive to the spatially modulated light. 18 . The system of claim 10 , wherein the circuitry com prises a processor configured to transform the time varying
electrical output signal from a time domain signal to a the object/object image from the frequency domain signal. 19 . The system of claim 10 , wherein the circuitry includes
frequency domain signal and to extract information about 25
a processor configured to process the time varying electrical output signal to correct for optical properties of one ormore components of the spectral encoder . 30 *