U.S. patent application number 13/702839 was filed with the patent office on 2013-07-04 for method and apparatus for characterizing biological objects.
This patent application is currently assigned to CELLTOOL GMBH. The applicant listed for this patent is Karin Schutze, Raimund Schutze. Invention is credited to Karin Schutze, Raimund Schutze.
Application Number | 20130171685 13/702839 |
Document ID | / |
Family ID | 44508493 |
Filed Date | 2013-07-04 |
United States Patent
Application |
20130171685 |
Kind Code |
A1 |
Schutze; Raimund ; et
al. |
July 4, 2013 |
METHOD AND APPARATUS FOR CHARACTERIZING BIOLOGICAL OBJECTS
Abstract
In order to quantitatively characterize biological objects, for
example individual cells, a stimulus is applied to a biological
object in a contactless fashion. A measurement and a further
measurement are performed on the biological object in order to
ascertain a response of the biological object to the stimulus,
wherein the measurement and the further measurement comprise
detecting Raman scattering on and/or in the biological object
and/or capturing data using digital holographic microinterferometry
(DHMI). The biological object is characterized according to a
result of the measurement and is sorted if needed. The stimulus can
be applied by means of a laser beam that creates optical tweezers
or an optical trap, by means of ultrasonic waves or an electric or
magnetic radio frequency field.
Inventors: |
Schutze; Raimund; (Tutzing,
DE) ; Schutze; Karin; (Tutzing, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Schutze; Raimund
Schutze; Karin |
Tutzing
Tutzing |
|
DE
DE |
|
|
Assignee: |
CELLTOOL GMBH
Tutzing
DE
|
Family ID: |
44508493 |
Appl. No.: |
13/702839 |
Filed: |
June 9, 2011 |
PCT Filed: |
June 9, 2011 |
PCT NO: |
PCT/EP2011/002839 |
371 Date: |
March 22, 2013 |
Current U.S.
Class: |
435/34 ;
435/288.7 |
Current CPC
Class: |
C12Q 1/04 20130101; G01N
15/1468 20130101; C12Q 1/025 20130101; G03H 1/0443 20130101; G01N
2015/149 20130101; G01N 21/65 20130101; G01J 3/44 20130101; G01N
15/1484 20130101; G01N 21/453 20130101 |
Class at
Publication: |
435/34 ;
435/288.7 |
International
Class: |
C12Q 1/04 20060101
C12Q001/04; C12Q 1/02 20060101 C12Q001/02 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 9, 2010 |
DE |
10 2010 023 099.5 |
Claims
1. A method for characterizing a biological object, the method
comprising: applying a stimulus to the biological object in a
contactless fashion; carrying out plural measurements on the
biological object to ascertain a response of the biological object
to the stimulus, wherein, to ascertain the response, a measurement
which comprises a detection of Raman scattering is performed on the
biological object before application of the stimulus, and a further
measurement which comprises a further detection of Raman scattering
is performed on the biological object after or during application
of the stimulus; performing a comparison of results of the
measurement and the further measurement; and characterizing the
biological object as a function of the comparison of the results of
the measurements.
2. The method according to claim 1, wherein the measurement and the
further measurement comprise both the detection of a Raman
scattering and data acquisition by means of DHMI.
3. The method according to claim 1, wherein a Raman spectrum is
captured in the measurement and a further Raman spectrum is
captured in the further measurement, wherein a difference spectrum
between the Raman spectrum and the further Raman spectrum is
determined, and wherein the biological object is characterized
based on the difference spectrum.
4. The method according to claim 3, wherein a first excitation beam
which is irradiated onto the biological object in the measurement
for detecting the Raman spectrum and a further excitation beam
which is irradiated onto the biological object for detecting the
further Raman spectrum in the further measurement have different
polarizations.
5. The method according to claim 1, wherein a background Raman
spectrum of a substrate on which the biological object is arranged
is detected, and wherein the background Raman spectrum is
subtracted from a Raman spectrum detected in the measurement and
from a further Raman spectrum detected in the further
measurement.
6. The method according to claim 1, wherein the stimulus is applied
without use of a marker.
7. The method according to claim 1, wherein electromagnetic
radiation is irradiated onto the biological object to apply the
stimulus.
8. The method according to claim 1, wherein a laser beam or plural
laser beams are irradiated onto the biological object to apply the
stimulus.
9. The method according to claim 8, wherein the laser beam or the
plural laser beams are irradiated onto the biological object in a
pulsed fashion, wherein a repetition rate of the laser beam or of
the plural laser beams is varied to excite a resonance vibration of
the biological object.
10. The method according to claim 8, wherein the laser beam is
scanned over plural different channels of a microfluidic system to
apply the stimulus to plural biological objects.
11. The method according to claim 8, wherein a deformation of the
biological object is induced using the laser beam, wherein a power
density of the laser beam at the biological object is selected such
that the biological object is not destroyed.
12. The method according to claim 8, wherein the laser beam holds
the biological object at a position in a fluid flow, and wherein
the laser beam and a flow speed of the fluid flow are set such that
the fluid flow induces a deformation of the biological object.
13. The method according to claim 1, wherein the stimulus is
applied using ultrasonic waves or using an electric or magnetic
high frequency field.
14. The method according to claim 1, wherein a volume and/or a
shape of the biological object in response to the stimulus is
determined by means of DHMI.
15. The method according to claim 1, wherein, by detecting the
Raman scattering on the biological object an agglomeration of
molecules and/or a molecular composition in a nucleus and/or a
cytoplasm of a cell is respectively determined.
16. The method according to claim 1, wherein the measurement and
the further measurement are performed using a measurement laser
beam which is scanned over different positions; and wherein the
measurement laser beam is scanned such that the measurement and the
further measurement are performed on plural portions of the
biological object.
17. (canceled)
18. The method according to claim 1, wherein the measurement and
the further measurement are performed on the biological object
while the biological object is positioned in a fluid channel of a
microfluidic system.
19. The method according to claim 18, wherein the microfluidic
system comprises a closed fluid loop in which biological objects
are transported before the measurement is performed.
20. The method according to claim 1, wherein a result of the
comparison of results of the measurement and the further
measurement is automatically matched with a data base which has
entries for different types of biological objects.
21. The method according to claim 1, wherein the biological object
is automatically sorted as a function of the result of the
comparison.
22. The method according to claim 1, wherein the method is
performed automatically with aid of a computer.
23. An apparatus for characterizing a biological object,
comprising: a device for generating a stimulus on the biological
object; and a measurement device for performing a measurement on
the biological object to ascertain a response of the biological
object to the stimulus, the measurement device being configured for
data acquisition by means of a detection of Raman scattering on the
biological object, the measurement device being configured, for
ascertaining the response of the biological object to the stimulus,
to perform a measurement comprising a detection of the Raman
scattering before application of the stimulus, and to perform a
further measurement comprising a further detection of the Raman
scattering after or during application of the stimulus; and an
evaluation logic which is configured to compare results of the
measurement performed before application of the stimulus and
results of the further measurement performed after or during
application of the stimulus, and to characterize the biological
object as a function of a comparison of the results of the
measurement and of the further measurement.
24. The apparatus according to claim 23, wherein the measurement
device comprises a Raman device for detecting a Raman scattering on
the biological object and a DHMI device for capturing a DHMI image
of the biological object.
25. The apparatus according to claim 24, wherein the apparatus
comprises an optical microscope, and wherein the Raman device is
configured to controllably move a focus of an excitation beam of
the Raman device in three orthogonal spatial directions
independently of a focus of a beam path of the optical
microscope.
26. The apparatus according to claim 25, wherein the Raman device
comprises a lens and a positioning device coupled to the lens and
configured to controllably move the lens in three orthogonal
spatial directions in a controllable manner, to controllably move
the focus of the excitation beam of the Raman device in three
orthogonal spatial directions independently of a focus of the
optical microscope.
27. The apparatus according to claim 23, comprising an
electronically controllable beam deflection device for scanning a
laser beam of the device for generating the stimulus and/or a
measurement beam of the measurement device over a plurality of
positions.
28. The apparatus according to claim 27, wherein the microfluidic
system comprises a closed fluid loop for transporting biological
objects before the measurement is performed.
29. (canceled)
30. The method according to claim 1, wherein applying the stimulus
comprises exposing the biological object to chemical agents, active
substances or drugs.
31. The method according to claim 15, wherein, by detecting the
Raman scattering on the biological object an agglomeration of a
protein composition in a nucleus and/or a cytoplasm of a cell is
respectively determined.
32. The method according to claim 19, wherein the closed loop of
the microfluidic system transports the biological objects
continuously before the measurement is performed.
33. The apparatus according to claim 28, wherein the closed fluid
loop of the microfluidic system transports biological objects
continuously before the measurement is performed.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Technical Field
[0002] The invention relates to a method and an apparatus for
characterizing biological objects, for example individual cells.
The invention relates in particular to a method and an apparatus
which allow biological objects in a microfluidic system to be
characterized in a contactless fashion and to then be sorted.
[0003] 2. Background Information
[0004] Various techniques for observing and manipulating biological
objects are known, by means of which the targeted manipulation and
observation was made possible for ever smaller objects, for example
individual cells. Various techniques for analyzing biological
objects use a targeted stimulation of the biological objects. The
manipulation of individual cells for measuring their elasticity
properties using a scanning force microscope as described in M.
Radmacher et al., "Measuring the Viscoelastic Properties of Human
Platelets with the Atomic Force Microscope", Biophys. J. 70, 556
(1996) belongs to such techniques. Traditionally, such techniques
are frequently used to gain knowledge on the structure, the
functionality and the properties of biological objects.
[0005] DE 10 2005 036 326 A1 and WO 2007/014622 A1 describe a
method and an apparatus for analyzing biological objects. In this
case, a change in shape or volume of a biological object is
monitored by means of digital holographic microinterferometry
(DHMI). However, such measurements do not provide information on
changes in the interior of the biological object.
[0006] Raman spectroscopy may be used to determine properties in
the interior of a cell. For characterizing and sorting cells, one
can make use of the Raman spectrum of dead cells being different
from the Raman spectrum of living cells, for example. Based on the
Raman spectrum, it can thus be determined whether a cell is dead or
alive. Techniques in which Raman spectroscopy is performed on
biological objects are conventionally limited to only one Raman
spectrum being captured for a biological object. This may impede a
characterization in cases in which, for example, different
biological objects have similar or identical Raman spectra under
the present measurement conditions.
[0007] The gentle, contactless and if possible marker-free
characterization of biological objects becomes increasingly
important, for example in biological research, in stem cell
research, in genetic research, in medical diagnosis or in forensic
sciences. Time is an important factor in such applications. For
practical applications, it is important to obtain a sufficient
throughput of biological objects in the characterization. If
biological objects are mechanically stimulated one by one, it may
be difficult to obtain a sufficient throughput. Setting a stimulus
may also be performed using biochemical substances. However, this
may have the effect that the biological object becomes useless for
a further analysis, depending on the respective biological object
of interest and the substance which is used.
[0008] For practical applications there is moreover a need to
perform the characterization of biological objects with even
greater accuracy. Additionally, a large degree of automation is
desirable. Using conventional methods and apparatuses, an automatic
recognition of the desired biological objects is frequently not
possible or is possible with great effort only. This is partly due
to the fact that, in conventional methods and apparatuses, only one
monitoring technique is used or three-dimensional objects are
represented only two-dimensionally, for example. When using
conventional techniques, such as a laser scanner, a
three-dimensional imaging requires a time-consuming scanning of
various planes and a subsequent digital combination of the image
data. This impedes a more extensive automation of the
characterization of cells.
BRIEF SUMMARY OF THE INVENTION
[0009] It is an object of the invention to provide a method and an
apparatus for characterizing biological objects, wherein a simple,
fast and accurate characterization of individual biological objects
is possible. In particular, it is an object of the invention to
provide such a method and such an apparatus in which a stimulus is
applied to the biological object and which is not only based on
using two-dimensional images of an external change of the
biological object, but which provides information on processes in
an interior of the cell.
[0010] According to the invention, this object is attained by a
method and an apparatus as defined in the independent claims. The
dependent claims define embodiments of the invention.
[0011] According to the invention, a stimulus is applied to a
biological object and a response of the biological object is
measured using Raman scattering and/or digital holographic
microinterferometry (DHMI) to characterize biological objects. In
the art, DHMI is also known as digital holographic microscopy.
[0012] To ascertain the response of the biological object [0013] a
DHMI measurement for determining a refractive index and/or a
detection of Raman scattering is performed before application of
the stimulus, and [0014] a further DHMI measurement for determining
a refractive index and/or a further detection of Raman scattering
is performed after or during application of the stimulus, with the
biological object being characterized based on a comparison of the
results of the measurements.
[0015] In particular, the intrinsic response to the stimulus may be
observed in the method and the apparatus. Advantageously, the
response to the stimulus may be determined both with regard to
extrinsic parameters, for example the shape or the volume of the
object, and with regard to intrinsic parameters, for example the
local molecular composition or the local refractive index within
the cell. The method and the apparatus allow a more comprehensive
characterization of cells. For illustration, external changes may
not only be imaged in two dimensions, but in three dimensions. By
means of DHMI and/or Raman spectroscopy, the intrinsic responses to
the stimulus may be analyzed.
[0016] The characterization of the biological objects is made based
on a comparison of the measurement results which are determined
before application of the stimulus and during or after application
of the stimulus. Greater flexibility in the characterization of
biological objects is thereby attained. Even if different
biological objects have similar Raman spectra under the given
measurement conditions, for example, a change in the Raman spectrum
can be detected using the method and the apparatus according to
embodiments. This allows objects to be discriminated which have
similar Raman spectra under certain measurement conditions, while
the Raman spectra show different changes in response to application
of the stimulus. For example, during application of the stimulus a
shift of a Raman spectrum, of individual spectral lines or spectral
weights may occur, which is characteristic for the biological
object. Different biological objects may also show different return
characteristics after application of the stimulus. These may be
monitored as a function of time in the Raman spectrum and may be
used for the characterization.
[0017] Analogously, the response of the biological object to a
stimulus may be monitored using DHMI for determining a refractive
index. In this case, the refractive index may be detected at plural
times to monitor return characteristics of the biological object
after application of the stimulus which can be used for
characterization.
[0018] Biological objects may show significantly different response
characteristics after application of a stimulus even when they have
a similar morphology or topology and thus cannot be easily
discriminated using conventional 2D images. By means of Raman
scattering, the scattering characteristics of the biological object
may be detected. Based on the frequency shifts of the light
scattered on the biological object, a characteristic behaviour of
the object, for example with regard to vibration spectra of
molecules and thus with regard to the molecules which are present,
can be detected. Since plural spectral lines of a vibration
spectrum or of plural vibration spectra may be detected in
measuring Raman scattering, a characteristic vibration spectrum may
be detected after application of the stimulus to the biological
object. For example, individual molecules may be identified.
Chemical changes may be shown by detecting the Raman spectrum,
which chemical changes are attributed to pathological cells in
tumor tissue, for example. Thereby, tumor cells may be
discriminated from healthy cells, for example. Raman spectra may
also provide information on the differentiation stage of cells and
may thereby discriminate stem cells from body cells.
[0019] Digital holographic microinterferometry (DHMI) is a
holographic measurement technique. Two phase-coherent partial
beams, an object beam and a reference beam, are superimposed. The
resulting interference image is captured by an electronic image
sensor, such as a CCD sensor. Different object planes can be
reconstructed from a single DHMI image, so that additional
information is available for the characterization of cells. By
means of DHMI, molecular changes in an interior of the individual
cells may be imaged indirectly, so that the cell types can be
characterized quickly and in a contactless fashion according to
their morphological properties such as layer thickness, volume,
micro-movements or deformations. Due to these characteristics,
dynamic processes after application of a stimulus to the biological
object may be monitored by means of DHMI data acquisition and may
be used for characterization of the biological object. A reliable
analysis of the biological object is possible also in the presence
of optical blur, for example upon movement of the object in the
depth direction during data acquisition, because a focus correction
may be performed automatically and with the aid of a computer. A
mechanical focus correction may be omitted. This allows a very fast
data acquisition without time-consuming scanning and thereby helps
increasing the throughput.
[0020] In an embodiment, a DHMI data acquisition may be performed
on a cell to determine a ratio of nucleus to cytoplasm.
Alternatively or additionally, typical nucleus patterns may be
quantified according to their different refractive characteristics.
Thereby, the method and the apparatus according to embodiments of
the invention may be used for cytometry.
[0021] In the methods and apparatuses, the stimulus is
advantageously applied such that the biological object is not
destroyed or damaged. The power irradiated onto the biological
object for Raman spectroscopy and/or DHMI is set such that it does
not lead to a destruction of the biological object. Thus, the
biological object remains an intact biological object both before
and after application of the stimulus. Both when using Raman
spectroscopy and when using DHMI, characteristic properties of
biological objects can be determined without a marking of the
biological object being required for this purpose.
[0022] With the method according to the invention and the apparatus
according to the invention, a contactless and marker-free
characterization of a biological object is possible which does not
destroy the biological object. In this process, biological objects
may be characterized according to their response to a stimulus
which is measured by Raman spectroscopy and/or DHMI.
[0023] The further Raman spectroscopy and/or the further data
acquisition for DHMI for determining a refractive index is
performed after the stimulus was applied or while the stimulus is
being applied. In this manner, the return characteristics of a
biological object, such as a cell, may be monitored after a change
was induced by the stimulus. It is also possible that more than two
Raman spectra are detected in a time-sequential manner to monitor
the response of the biological object as a function of time.
[0024] A difference spectrum of a Raman spectrum captured in a
measurement and of a further Raman spectrum captured in a further
measurement may be computed. The difference spectrum may be
compared to reference difference spectra which are stored in a data
base to characterize the biological object.
[0025] A laser light source which is used for the Raman
spectroscopy may also be used to generate a resonant vibration of
the biological object. In this case, light output by the laser
light source is first irradiated onto the biological object in a
pulsed fashion with a repetition rate. The repetition rate may be
adjusted to excite a resonance vibration of the biological object.
By monitoring the 3D shape it can be controlled whether the
resonance has been reached. Alternatively or additionally, reaching
the resonance may be monitored based on a captured Raman spectrum.
Upon reaching the resonance, the laser intensity may be reduced for
example to avoid destruction of the biological object. While the
biological executes resonance vibrations, changes in the interior
of the biological object may be observed by means of DHMI for
determining a refractive index and/or by means of Raman
spectroscopy.
[0026] The measurement performed on the biological object may
comprise both a Raman spectroscopy and a data acquisition by means
of DHMI.
[0027] By combining Raman spectroscopy and DHMI for measuring a
shape, both an external change of the biological object, such as a
change in shape or volume, and a change in the interior of the
biological object may be determined. The determined change in the
interior which is also referred to as intrinsic change may be a
change in the refractive index monitored by DHMI or a change in the
scattering characteristics in the Raman spectrum in response to the
stimulus. When compared to techniques which only determine changes
in the shape of the object, additional information may thus be
obtained which may be used for characterizing the object.
Additionally, shape or volume changes after application of the
stimulus to the biological object can be determined using DHMI.
When a Raman spectroscopy is also performed, information on changes
of the captured Raman spectrum and thus of the vibration spectrum
may also be obtained. A change in the protein or molecular
composition may be determined, for example. The results obtained by
DHMI and Raman spectroscopy may be correlated to characterize or
identify the biological object more accurately.
[0028] The stimulus may be applied in a marker-free manner. Thereby
it can be ensured that the biological object is available for
further study after its characterization. This is in particular
advantageous when the characterization is performed for sorting
living cells.
[0029] The stimulus may be applied to the biological object using
optical radiation. The stimulus may be applied such that a
deformation of the biological object is induced. Various techniques
may be used for this purpose. According to an embodiment at least
one optical tweezer or trap is generated using one or plural laser
beams. In an embodiment an optical tweezer is generated using at
least one laser beam which holds the biological object in a fluid
flow having a finite flow velocity. In this case, the laser power
and thus the depth of the trap potential and the flow velocity of
the fluid flow may be set such that the shear forces applied by the
fluid onto the biological object lead to a deformation of the
biological object. In this manner, a deformation of the biological
object may be attained by the interplay of fluid forces and optical
forces. The laser beam may be directed along a fluid channel or
counter the fluid channel. In this process, plural biological
objects may be held simultaneously along a longitudinal direction
of the fluid channel.
[0030] In embodiments, the optical tweezer which holds the object
at a desired position may be constituted by the beam which is
irradiated onto the object for performing the Raman
spectroscopy.
[0031] According to another embodiment, a pair of optical tweezers
can be used for applying the stimulus, by means of which forces may
be generated on opposite sides of the biological object such that a
deformation of the object is realized, for example. The two optical
tweezers may be moved laterally to induce stretching of the object,
for example. In another embodiment, a laser beam or a pair of
essentially counter-propagating laser beams may be used, the power
and profile of which are set such that forces are applied onto the
biological object which compress or stretch the biological object
in a propagation direction of the laser beams. In another
embodiment, the cell may be deformed using a micro beam or two
counter-propagating micro beams. In this process, a laser beam with
which light is irradiated onto the biological object for a short
time only is advantageously used as a micro beam. For example, a
pulsed laser may be used to generate the micro beam. Alternatively,
the beam of a continuously operating laser may be affected, for
example using an electro-optical element, such that it impinges
onto the biological object for a short time span as a micro beam. A
local disturbance on the membrane and/or in the cytoplasm of a cell
may also be induced using a micro beam, e.g. by optoperforation and
optoporation, respectively. The resultant change in the protein
composition may be detected using Raman spectroscopy and/or the
change in the morphology and the refractive index, respectively, of
the cell may be detected using DHMI.
[0032] In further embodiments, a stimulus may be applied without
inducing deformation of a biological object, which stimulus
comprises local heating, for example. The response thereto can be
detected by DHMI and/or Raman spectroscopy.
[0033] In each one of these embodiments, the beam path of the laser
beam or the laser beams, respectively, may be scanned over
different positions. In this manner, stimuli may be applied to
biological objects which are held at different measurement
positions. A deflection device may be provided which scans the
laser beam or the laser beams, respectively, in a controllable
manner. In an embodiment, an optical element, e.g. a mirror, may be
adjusted in a controlled manner for this purpose. In another
embodiment, a laser beam may also be scanned over different
positions by means of a SLM (spatial light modulator) crystal. In
an embodiment, the scanning may be performed such that the laser
beam or the laser beams, respectively, are directed onto different
fluid channels in a time-sequential manner. Alternatively or
additionally, the scanning may be performed such that the laser
beam or the laser beams, respectively, are directed onto different
measurement positions which are spaced along a longitudinal
direction or a transverse direction of a channel to apply the
stimulus to various biological objects. In an embodiment, the
scanning is performed in two dimensions, with the laser beam(s)
being adjusted both between different fluid channels and along the
longitudinal direction of the channels between different positions
at which a stimulus is to be applied to biological objects. If at
least two laser beams are directed onto a biological object for
applying a stimulus, the two laser beams are scanned synchronously.
A parallelisation or multiplexing, respectively, in characterizing
biological objects may be attained by use of the mentioned
procedures, which allows the throughput to be increased
further.
[0034] A data acquisition by means of DHMI may be performed after a
deformation of a biological object was induced using optical
methods. A data acquisition is additionally performed before
applying the stimulus in order to be able to judge the response of
the biological object. Thereby, cells or other biological objects
and their changes may be reconstructed in three dimensions and the
response to the stimulus may be quantified. Various cross sections
of the biological object may be reconstructed from a single DHMI
image. Upon deformation in response to an optically induced
stimulus there generally results a change not only in one cross
section of the biological objects, but typically a change in three
dimensions. For a non-homogeneous object, such as a cell, the
response to compressive or tensile forces has different degrees in
the three dimensions. Using 3D imaging by means of DHMI more
accurate information on the elasticity characteristics in the
various directions can be obtained. Digital refocusing upon
performing a computer-aided evaluation of the DHMI data allows one
to forgo a mechanical focus correction. Moreover, the complete cell
volume may be captured with a single snapshot in DHMI, such that
scanning is not required. In this manner, the throughput in the
characterization may be increased.
[0035] The refractive index of a cell or of components of the cell,
such as the cell nucleus, respectively, is determined in a
quantitative manner by DHMI data acquisition. Plural DHMI data
acquisitions may be performed to determine the refractive index,
with the cell being rotated or changed in its position between the
various DHMI data acquisitions. A rotation of the cell may be
induced by the laser beam or the laser beams, respectively, which
are also used for applying the stimulus to the cell. A laser beam
profile which is not rotationally symmetric may be used to induce a
rotation of the cell. This profile may be generated by using an
optical fibre having an elliptic cross section, for example. It is
also possible that laser beams are used which have a helical wave
front. For illustration, counter-propagating laser beams may be
used which have a Laguerre-Gauss-profile and a helical wave front
such that the Poynting vector varies in a spiral shape along the
axis which defines the propagation direction. Such beams transfer a
torque onto the biological object with which the biological object
can be rotated. Alternatively or additionally, optical fibres may
be used which provide radiation with which the biological object
can be rotated. The optical fibres may have a cross section which
is not rotationally symmetric. The fibres may have an elliptic
cross section, for example.
[0036] Laser beams may also be used to hold the biological object
in a solution at a desired position. When a deformation of the
biological object is induced using a laser beam, an optical tweezer
or optical trap for the object may also be generated by this laser
beam. This allows a holographic data acquisition to be performed on
an individual cell in a solution.
[0037] A Raman spectroscopy may be performed before application of
a stimulus and after or during application of the stimulus. For
example, the Raman spectroscopy may be performed both before
application of the stimulus and after or during the stimulus is
applied using optical or other techniques. In this manner, a change
in the cellular activity during an induced stress may be monitored
in the Raman spectrum, for example. Additionally, the deformation
of the biological object may be quantitatively determined by
computer-aided processing of conventional 2D images. In addition to
changes in the Raman spectrum information on the degree of
cross-linking of structural molecules, such as the microtubule
network or the actin network can be obtained by quantitative
measurement of the elasticity.
[0038] When a Raman spectroscopy is performed a measurement laser
beam which is irradiated onto the biological object for the Raman
spectroscopy may be scanned between different positions. A scanning
may be performed along a fluid channel of a microfluidic system
and/or between different fluid channels of the microfluidic system.
In this manner one can attain that the average duration of a
measurement process is not limited by the time scale which is
required to position a new biological object such that a Raman
spectroscopy can be performed thereon. A beam path for the DHMI
data acquisition may also similarly be adjusted between different
locations in a controlled manner. This may in particular be
advantageous when the different biological objects on which
measurements are to be performed in parallel have a distance which
is greater than a diameter of the object wave for DHMI data
acquisition in an object plane. If plural laser beams are
irradiated onto the biological object for the corresponding
measurement, e.g. in a Raman spectroscopy in which the light of the
excitation laser for the Raman spectroscopy also generates the trap
potential for the biological object, the plural laser beams are
scanned synchronously.
[0039] When a Raman spectroscopy is performed in methods and
apparatuses according to embodiments, it is not required that the
complete Raman spectrum be detected. For example, only a part of
the spectrum, e.g. individual spectral lines, may be detected. This
may be sufficient for a comparison with a data base in which
characteristic spectral lines of the Raman spectrum or the shift
thereof in response to the stimulus are stored. The spectral range
in which the Raman spectroscopy is performed may be selected as a
function of the biological objects which are to be
characterized.
[0040] The stimulus onto the biological object does not necessarily
have to be applied using optical radiation. Rather, ultrasonic
waves or electromagnetic fields, in particular high-frequency
fields, may also be used. The stimulus may also be applied
chemically, for example by administration of active substances or
drugs. The stimulus may also be applied mechanically, for example
by moving the cells over structured surfaces or by relocating cells
onto surfaces.
[0041] In all embodiments, conventional microscopy may also be
performed in addition to a Raman spectroscopy and/or a DHMI data
acquisition. For illustration, a phase contrast microscopy, a
fluorescence microscopy or another microscopy may be used to enable
microscopic observation of the object which is being
characterized.
[0042] Preferably, a microfluidic system is used in the embodiments
of the invention. Biological objects may be kept in solution using
the microfluidic system. This allows a characterization to be
performed on living cells. Advantageously, the biological object to
be characterized, for example a cell, is transported in the
microfluidic system. For performing the measurement on the
biological object, the biological object may then be brought to a
desired measurement position for example by using optical tweezers
or traps or a diversion pulse of a laser micro beam with which the
biological object may be removed from a circulating fluid flow or
by using microfluidic valves or other adjusting elements.
[0043] In the various embodiments, the Raman spectroscopy and/or
DHMI data acquisition may advantageously also be performed on the
object in a fluid channel. Both the measurement before application
of the stimulus and the further measurement which is performed
during or after application of the stimulus may be performed on the
object in the fluid channel.
[0044] The microfluidic system may have a closed fluid loop in
which biological objects are transported until the measurement is
performed thereon. Sinking of biological objects may thereby be
reduced. The sample in solution may be supplied to the
characterization to a large extent.
[0045] In the methods and apparatuses according to various
embodiments, it is possible that not only one stimulus is applied,
but plural stimuli having various intensity and/or of various type
may be applied. The response of the biological object to the
various stimuli may be determined using Raman spectroscopy and/or
DHMI to characterize the biological object. Accordingly, an
apparatus may have plural devices which allow stimuli of different
types to be applied. For example, a device for applying a stimulus
using optical techniques, a device for applying a stimulus using
ultrasonic waves and/or a device for applying a stimulus using
high-frequency fields may be provided. In this manner, the
behaviour of the object in response to different stimuli may be
determined. Extrinsic and/or intrinsic changes may respectively be
detected. In an embodiment, the behaviour of the cell after
application of at least two different stimuli is determined using
both DHMI and Raman spectroscopy, with the two different stimuli
being respective applied using one of laser radiation, ultrasound
or electromagnetic high-frequency fields. A deformation of a cell
may give rise to a rearrangement of proteins in addition to changes
in shape and volume. By using ultrasound a local molecular
composition may be changed and/or heat may be generated, for
example. By using electromagnetic high-frequency fields, an
oscillation of molecules and thus an intrinsic change of the object
may be induced. By data acquisition using DHMI and/or Raman
spectroscopy a biological object may be characterized with regard
to its external parameters but also with regard to processes in the
interior of the object.
[0046] The characteristics of Raman spectroscopy and DHMI allow the
characterization of biological objects to be automated to a large
degree. In particular, the spectra obtained by Raman spectroscopy
and/or the information on shape, volume and/or refractive index
obtained from DHMI are suitable for a largely automatic
characterization. The acquired Raman spectra and/or information on
volume and shape of the biological object may be compared with a
data base, for example, to automatically sort cells. In this
manner, healthy cells may be discriminated from tumor cells, for
example, and may be automatically directed to different collection
vessels. In further embodiments, certain cells, cell cycles or
clones may be recognized by evaluating the response upon
application of a respective stimulus.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0047] In the following, the invention will be explained with
reference to the drawing by means of preferred embodiments.
[0048] FIG. 1 shows a schematic representation of a system for
characterizing and sorting biological objects according to an
embodiment.
[0049] FIG. 2 shows a schematic representation of an apparatus for
characterizing biological objects according to an embodiment.
[0050] FIG. 3 shows a schematic representation of a device for
Raman spectroscopy for an apparatus according to an embodiment.
[0051] FIG. 4 shows a schematic representation of an apparatus for
characterizing biological objects according to another
embodiment.
[0052] FIG. 5 shows a schematic representation of a microfluidic
system for performing measurements in parallel in apparatuses
according to an embodiment.
[0053] FIG. 6 shows a schematic representation of an apparatus for
characterizing biological objects according to another
embodiment.
[0054] FIG. 7 shows a schematic representation of a cell for
explaining the operation of the apparatus of FIG. 6.
[0055] FIG. 8 shows a schematic representation of an apparatus for
characterizing biological objects according to another
embodiment.
[0056] FIG. 9 is a flow diagram representation of a method
according to an embodiment.
[0057] FIG. 10 illustrates a data acquisition and data processing
in a method according to an embodiment.
DETAILED DESCRIPTION OF THE INVENTION
[0058] FIG. 1 is a schematic representation of a system 1 for
sorting biological objects according to an embodiment. The system 1
comprises an apparatus 2 for characterizing biological objects and
a computing device 3 coupled to the apparatus 2. The apparatus 2
performs a Raman spectroscopy and/or a digital holographic
microinterferometry (DHMI) on a biological object to quantitatively
determine its response to a stimulus. Measurement results which are
acquired thereby are output to the computing device 3 via an
interface. The computing device 3 has a data base 4 which has
information regarding the behaviour of different biological
objects. For example, the data base 4 may comprise information on a
Raman spectrum after application of the stimulus, information on
shifts of spectral lines of the Raman spectrum in response to the
stimulus, information on shape or volume after application of the
stimulus or information on changes in the shape or changes in the
volume for various types of cells (healthy cells or tumor cells),
for different stages of a cell cycle or similar. Depending on a
comparison of the acquired and processed data, which may represent
a change in a Raman spectrum or a refractive index for example, to
a threshold value or a comparison of the acquired data with the
data base 4, the computing device 3 may output a control command
for sorting the biological object to the apparatus 2, may output
information on the object over a display device 5 or may offer
various options for a further processing of the object to the user
which the user may select over a user interface 6.
[0059] The apparatus 2 is configured to characterize cells in a
contactless manner and quickly. The apparatus 2 has a microfluidic
system 11 which is supported by a carrier 10. The microfluidic
system 11 comprises a closed fluid loop 12. A continuous fluid flow
may be maintained in the closed fluid loop 12 during operation of
the apparatus 2 using suitable microfluidic devices to prevent
sedimentation of biological objects which are transported in a
solution in the fluid loop 12. The characterization of cells is
performed in the microfluidic system 11. This allows measurements
to be performed on living cells or cell clusters. The microfluidic
system 11 has a fluid channel 13. To characterize biological
object, e.g. cells, the cells 8, 9 are positioned in the fluid
channel 13. Although FIG. 1 exemplarily shows a separate fluid
channel 13 into which the cells may be displaced from the closed
fluid loop 12, for example using microfluidic devices or an optical
tweezer, the fluid channel 13 may also be a portion of the closed
loop 12. A laser pulse is also suitable to transport the cell from
a laminar flow into a neighbouring channel where the measurement is
performed.
[0060] For performing a characterization, the cell 8 is positioned
in a measurement region 18 and the cell 9 is positioned in a
measurement region 19. The measurement regions 18, 19 are spaced
along the longitudinal axis of the fluid channel 13. The
measurement regions 18, 19 may be defined by the focal area of
laser beams, for example, which form an optical trap or an optical
tweezer for the cells 8 and 9, respectively. A fluid flow in the
fluid channel 13 may be stopped when a measurement is to be
performed on the cells 8, 9. A centering in the channel may be
performed by hydrodynamic focussing, for example. A positioning
along the channel occurs automatically, for example upon performing
a Raman spectroscopy, when the cells 8, 9 are drawn into the focal
area of the laser beam which is irradiated for performing the Raman
spectroscopy.
[0061] While a configuration according to an embodiment is shown in
FIG. 1 in which plural measurement regions 18, 19 are spaced in a
longitudinal direction of the channel in the channel 12 various
other configurations may be realized in each one of the embodiments
described herein. For example, the measurement may respectively be
performed on an individual object only so that the parallel
execution of measurements may be forgone. In another embodiment,
different biological objects on which a measurement is performed
may be spaced in a transverse direction of the channel. For this
purpose, plural biological objects may be positioned in
transversally spaced portions of a laminar flow in a channel or in
plural transversally spaced laminar flows. It is also possible that
plural biological objects are positioned in channels which are
transversally spaced when the measurement is performed.
[0062] The apparatus 2 has a device 21 for generating a stimulus to
the cells 8, 9. The device 21 may comprise a laser source, a source
for electromagnetic radiation, in particular in a high-frequency
range, or a device for generating ultrasonic waves. For
illustration there is shown a configuration in which the device 21
has a laser source for generating a micro beam which may be
irradiated onto the cells 8 and 9 in the measurement regions 18 and
19, respectively, via a beam splitter 26 and a lens 29. A power of
the laser source 21 may be set such that a local disturbance is
induced on the cell membrane or/and in the cytoplasm of the cells
8, 9 by the micro beam. Other configurations of the device are also
possible which allow a stimulus to be applied using a laser beam.
In particular, the device 21 may be configured such that it
generates a laser beam or plural laser beams having an energy
density, a profile and a direction which induce a deformation of
the cell 8, 9. It is also possible that two counter-propagating
pulsed laser micro beams are used, with a deformation of the cell
being induced by a laser pulse or a sequence of laser pulses. In
this case, the two beams are pulsed such that they simultaneously
provide light power to the biological object. It is also possible
that an excitation beam for Raman spectroscopy is irradiated onto
the biological object in a pulsed manner. A repetition rate of the
pulses may be varied to excite a resonance vibration of the
biological object. The occurrence of the resonance may be detected
and controlled by monitoring the 3D shape, for example by DHMI. A
closed loop control may be provided to adjust the laser light power
upon occurrence of the resonance such that a destruction of the
biological object may be prevented.
[0063] The apparatus 2 further has at least one of a device 22 for
performing a Raman spectroscopy and a device 24a, 24b, 24c for
performing a DHMI. With this device or these devices, respectively,
the behaviour of the cell 8, 9 in response to the stimulus is
determined. The apparatus 2 of the system 1 of FIG. 1 comprises
both the device 22 for performing the Raman spectroscopy and a
device 24a, 24b, 24c for performing DHMI. Depending on the
application, only one of these devices may be present. A
controllable deflection device 23 is provided to deflect an
excitation beam of the device 22 for performing the Raman
spectroscopy such that the spectrum of the cell 8 or of the cell 9
is selectively measured. The excitation beam of the Raman device 22
is directed via a beam splitter 27 and a lens 29, e.g. the
objective of a microscope, onto the cell 8, 9. A control and
evaluation logic 25 is coupled to the Raman device 22, the
deflection device 23 and the DHMI device 24a, 24b, 24c to control
these devices for performing a data acquisition and to evaluate the
data captured by the Raman device 22 and the DHMI device 24a, 24b,
24c. The Raman device 22 and the DHMI device 24a, 24b, 24c
respectively perform a data acquisition before, during or after
application of the stimulus on the respective cell 8, 9 to
ascertain the response of the cell to the stimulus. By comparing
the DHMI and Raman signals captured before application of the
stimulus and after or during application of the stimulus,
information on the behaviour of the cells in response to the
stimulus can be obtained.
[0064] Similarly to solids, biological objects also have vibration
spectra which are dependent on cell activity and stages of a cell
cycle, for example. Accordingly, changes induced by the stimulus
may be monitored. For performing the Raman spectroscopy on living
biological objects the Raman device 22 may comprise a diode laser
having a wavelength of 785 nm, for example. The laser beam may be
focussed onto the cells 8, 9. Cells and bacteria are not damaged at
this wavelength. The scattered Raman signals are measured by means
of a spectrometer. The spectrometer may be provided with a CCD
sensor optimized for near infrared, for example.
[0065] An adjustment of the laser beam irradiated onto the cells
for Raman spectroscopy using the deflection device 23 may also be
performed such that the biological object, for example the cell, is
sampled at plural points. Thereby, a Raman spectrum may be captured
for each one of the various measurement points. Conclusions on the
chemical or biochemical composition of the biological object may be
drawn from the various spectra after application of the stimulus.
This information may be determined with a spatial resolution by
performing a scan over the biological object.
[0066] The Raman device 22 may be configured such that a position
of a focus of the excitation beam may be adjusted in all three
spatial directions for performing the Raman spectroscopy. When the
apparatus 2 comprises an optical microscope, the Raman device 22 is
advantageously configured such that the position of the focus of
the excitation beam for performing the Raman spectroscopy may be
controlled independently of a focus of the microscope beam
path.
[0067] The detection of Raman spectra may be performed such that
before or after capturing the Raman spectra on a biological object
a Raman spectrum of a substrate on which the biological object is
positioned is also captured. This background spectrum may be used
to computationally subtract signal components which are caused by
the substrate from the data acquired by Raman spectroscopy on the
biological object. The position and height of peaks in the
background spectrum may be compared to the position and height of
peaks in the Raman spectra detected on the biological object to
determine a multiplicative factor which specifies the weight of the
background signal in the Raman spectra captured on the biological
object and which is taken into account in the computational
subtraction of the background spectrum. For example, the background
spectrum may be multiplied by a multiplicative factor which
specifies the weight before it is subtracted from the Raman spectra
captured on the biological object. Subtraction of the background
spectrum may in particular be advantageously employed when the
biological object is positioned directly on a solid substrate.
[0068] The DHMI device is configured such that a laser beam is
split into two partial beams, which form an object wave and a
reference wave which is phase coherent thereto. The object wave and
the reference wave may be directed via suitable optical components
such that the object wave is directed onto the object, with signals
of the object wave which are scattered or reflected on the object
impinging onto a two-dimensional image sensor, such as a CCD
sensor. The reference wave impinges onto the two-dimensional image
sensor without being reflected or scattered on the object. In FIG.
1, the DHMI device has a component 24a which is schematically shown
and which outputs the object wave. The object wave is directed via
a beam splitter 28 and a condenser lens 30 onto one of the cells 8,
9 or is simultaneously directed onto both cells 8, 9. The reference
wave is directed from a component 24b via a beam splitter onto a
two-dimensional image sensor 24c, such as a CCD sensor. The
interference pattern which results from the phase differences
between the object wave and the reference wave at the image sensor
24c allows the shape of the biological object to be
reconstructed.
[0069] The DHMI device having components 24a, 24b, 24c provides a
contactless, marker-free and quantitative phase contrast imaging.
Various object planes can be reconstructed from a single hologram
captured using the DHMI device 24a, 24b, 24c. A minimally invasive
dynamic detection of deformations and movements of living cells in
three dimensions is also possible in addition to a marker-free
analysis of topography and morphology, respectively. A digital
refocusing may also be performed by computationally evaluating the
signal captured by the image sensor 24c. Stability may thereby be
attained even when the cell moves in a depth direction.
Deformations of the biological objects may thereby be detected in
three dimensions for characterizing a cell.
[0070] Using the apparatus 2 the response of a biological object
may be examined both with regard to changes in the topography or
morphology and with regard to changes in the Raman spectrum. The
measurement of the response to a known stimulus allows the
behaviour of the biological object to be captured both with regard
to changes in the morphology and topography, respectively, and with
regard to changes in the Raman spectrum. By comparison with a
corresponding data base a determination can be made, for example,
which cell type is present, in which stage of a cell cycle the cell
is, etc. Both Raman spectroscopy and DHMI are fast measurement
techniques which can capture the essential characteristics, i.e.
the spectrum and hologram, respectively, in a single shot. In
embodiments, only a part of the Raman spectrum is detected to be
able to carry out a comparison with a data base. Individual lines
of a Raman spectrum may be detected, for example, based on which a
comparison with known data sets can be performed. In this manner a
further increase of the throughput can be attained. The relevant
lines may be measured once before application of the stimulus and
once more during or after application of the stimulus. Differences
of the spectral weights, i.e. of the peaks in the detected spectrum
may be determined for the relevant lines of the spectrum to
characterize the biological object based on the differences.
[0071] The biological objects may be processed further because the
characterization is performed marker-free and without destruction
of the biological object in the apparatus 2 of FIG. 1. To this end,
a signal may be provided by the control and evaluation logic 25 or
by the computing device 3 according to which the cell 8, 9 is
selectively sorted into one of plural output channels 14, 15 of the
microfluidic system 11.
[0072] The block diagram representation of the various components
of the apparatus 2 is to be regarded as being a schematic
representation. For illustration, components of the Raman device
22, 23 may also be provided such that the excitation beam is
coupled in via the lens 30. Alternative configurations of the
device 21 with which the stimulus is applied are also possible.
[0073] FIG. 2 is a schematic representation of an apparatus 32 for
characterizing a cell according to another embodiment. Components
of the apparatus 32 which correspond in terms of function and/or
construction to components of the apparatus 2 are designated with
the same reference numerals as in FIG. 1.
[0074] The apparatus has components of a conventional microscope in
addition to the modules 24a and 24b of the DHMI device which
provide an object wave and reference wave for DHMI and in addition
to the Raman device 22. The object and reference wave for DHMI data
acquisition and the excitation beam for the Raman spectroscopy may
be coupled into the beam path of the microscope. The microscope has
an illumination device 34. Other components known from microscopy,
which are schematically shown at 35, may be provided. A
two-dimensional image sensor 36, such as a CCD sensor, may be
provided both for image acquisition for the conventional microscopy
on the biological objects and for data acquisition in DHMI. Tube
lenses 37a, 37b may be provided in a microscope body 33.
[0075] The apparatus 32 has both a device 24a, 24b, 38 for DHMI
data acquisition and a device 22 for capturing a Raman spectrum. An
object beam 45 for DHMI is coupled into the beam path of the
microscope via the beam splitter and is directed onto a fluid
channel of a microfluidic system 11 (only schematically shown) via
the condenser lens 30. A biological object 8 such as a cell is
positioned in the fluid channel for characterization. A reference
beam 46 for DHMI is directed onto the image sensor 38 via a beam
splitter 36 and a tube lens 37b. An excitation beam for Raman
spectroscopy is coupled into the beam path of the microscope via a
controllable deflection device 23 and a beam splitter 26. The
excitation beam is focussed onto the biological object 8 via the
objective 29. The controllable deflection device 23 allows Raman
spectra to be detected in a spatially resolved manner on a
biological object, e.g. on a cell. Optionally, a scanning over
objects on various locations may be provided to detect Raman
spectral lines of objects in a time-sequential manner. Light
scattered on the object is guided to a spectrometer. For example,
the scattered light may be guided via the beam splitter 26 to a
Raman spectrometer which is provided in the Raman device 22.
[0076] The apparatus 32 has a device 40 for applying a stimulus to
a biological object 8. The device comprises two fibres 42, 44 with
which an optical signal having a high intensity may be guided to a
measurement region of the fluid channel in which the biological
object is positioned for performing the measurement. The fibres 42,
44 are coupled to one or plural sources 41, 43 for the optical
signal. A laser 41 may be provided, with the laser beam being
coupled into the first fibre 42 via suitable optical components and
being coupled into the second fibre 44 via optical components 43.
The optical fibres 42, 44 may be arranged such that beams which
exit from their ends are essentially counter-propagating. Thereby
the stimulus may be applied to the biological object 8 by optical
means. The stimulus may be applied by a short laser pulse with a
Raman spectrum and a DHMI image of the object 8 being captured
before application of the laser pulse and/or during the laser pulse
and/or after termination of the laser pulse. The stimulus may also
be applied over a longer time period to induce a longer lasting
deformation of the object 8, for example. A Raman spectrum and a
DHMI image of the object 8 may be detected while light is
irradiated from the ends of the fibres 42, 44 onto the object. The
optical stimulus may also be applied in that a pulsating force is
applied onto the biological object which causes resonance
vibrations of the biological object. Corresponding data
acquisitions may also be performed before the stimulus is applied
or when the irradiation of light from the ends of the fibres 42, 44
was terminated.
[0077] The object 8 may be characterized based on the Raman
spectrum and the DHMI data which are captured before application of
the stimulus and which are captured during or after application of
the stimulus. For this purpose, a comparison with a data base may
be made for example to determine a cell type or a stage of a cell
cycle. If no matching entry can be determined for the captured
data, the data base may be extended by an entry in which the
characteristic properties of the measured Raman spectrum and the
information on volume and shape of the object 8 which are
reconstructed from the DHMI data are assigned to an object type. In
this manner the data base for automatic characterization may be
created and extended, respectively.
[0078] FIG. 3 is a schematic representation of a Raman device which
may be used as a device for capturing a Raman spectrum in the
apparatuses according to various embodiments.
[0079] The Raman device 22 comprises a laser source 51. An output
beam of the laser source 51 is directed via an orifice plate 52, a
lens 53 and beam splitter 54 to the biological object as an
excitation beam 55 for Raman scattering. Scattered light 56 is
directed via the beam splitter 54, a Raman edge filter 57 and an
orifice plate 58 to a Raman spectrometer. The Raman spectrometer
comprises a grating 60 and a one-dimensional or a two-dimensional
image sensor 62, such as a CCD sensor. Lenses 59 and 61 are
provided between the orifice plate 38 and the grating 60 as well as
between the grating 60 and the image sensor 62, respectively. The
scattered light which is separated into its spectral components by
the grating 60 is thereby directed onto different positions 63, 64
on the image sensor 62 and is advantageously focussed thereon. The
different spectral lines of the Raman spectrum are thus captured on
the image sensor 62. When scanning the excitation beam 55 over
various points of the object surface plural Raman spectra which are
associated with different points or areas on the object surface may
be detected in a time-sequential manner.
[0080] The Raman device 22 may be configured such that a position
of a focus of the excitation beam 55 may be controllably moved in
all three spatial directions. The Raman device 22 may be configured
such that the position of the focus of the excitation beam 55 for
the Raman spectroscopy may be controlled independently of a focus
of the beam path of an optical microscope of the apparatus in which
the Raman device 22 is used. For this purpose there may be provided
a positioning device 57, for example, which is coupled to the lens
53 to displace the same in three orthogonal directions (x, y, z).
The position of the focus of the excitation beam 55 may thereby be
controlled.
[0081] FIG. 4 is a schematic representation of an apparatus 72 for
characterizing a cell according to another embodiment. Components
of the apparatus 72 which correspond in terms of function and/or
construction to components of the apparatus 2 of FIG. 1 or to
components of the apparatus 32 of FIG. 2 are designated with the
same reference numerals as in FIG. 1 and FIG. 2, respectively.
[0082] In the apparatus 72 a stimulus onto a biological object is
generated using optical radiation. In this case one dispenses of
optical fibres for guiding the laser light up to a measurement
region. The device for generating a stimulus has a component 73
which outputs a laser beam 79 to a controllable deflection device
74. The laser beam is directed to a condenser lens 30 via the
deflection device 74 and a beam splitter 77. The device for
generating a stimulus has a component 75 which outputs a further
laser beam 80 to a controllable deflection device 76. The further
laser beam 80 is directed to the objective 29 via the deflection
device 76 and a beam splitter 78. The components 73, 75 may be ends
of optical fibres, for example, using which laser light is guided
from a laser to the deflection devices 74, 76. Alternatively, the
laser beam may be guided to the deflection devices 74, 76 via
suitable deflection mirrors 73, 75. The controllable deflection
device 74, 76 may comprise an adjustable element, such as a
deflection mirror which is adjustable using a motor, or an
electro-optical component, such as a spatial light modulator (SLM).
The deflection devices 74, 76 allow the laser beams 79, 80 to be
scanned over different positions. In this manner stimuli may be
applied to plural biological objects, for example, such as plural
cells which are positioned along the longitudinal axis of a fluid
channel or in different fluid channels of the microfluidic system.
This has the effect that plural biological objects may be
characterized in parallel.
[0083] The power and the profile of the laser beams 79 and 80 may
be set such that a deformation of the biological object 8 is
induced. In an implementation the power and the profile of the
laser beams 79 and 80 are set such that they exert forces onto the
biological object such that the object is compressed in the
propagation direction of the beams 79 and 80, i.e. in a vertical
direction in FIG. 6. In another implementation the power and the
profile of the laser beams 79 and 80 may be set such that they
exert forces onto the biological object such that the object is
stretched in the propagation direction of the beams 79 and 80, i.e.
in a vertical direction in FIG. 6.
[0084] The deflection devices 74, 76 are controlled such that the
laser beams 79 and 80 are synchronously scanned over various
positions. In this manner the stimuli known in the art, such as
deformation by compression or stretching along the propagation
direction of the counter-propagating beams 79 and 80 may be
realized also with scanning over plural positions. The scanning of
the excitation beam of the Raman device 22 may advantageously also
change between the different measurement regions at the same rate
as the beams 79, 80 for applying the stimulus. It is however not
required that the excitation beam of the Raman device 22 impinges
onto a biological object to be characterized simultaneously with
the beams 79, 80.
[0085] An implementation of the apparatus for characterizing
biological objects in which the laser beams are not guided using
optical fibres up to the fluid channel allows a simple and fast
scanning of the laser beams. The construction of the apparatus may
be simplified.
[0086] While deflection devices 74, 76 are shown in FIG. 4, the
deflection devices 74, 76 may also be omitted in other
embodiments.
[0087] FIG. 5 is a schematic representation of a microfluidic
system 81 in which measurements may be performed in parallel in
plural fluid channels. A microfluidic system having the
configuration shown in FIG. 5 may for example be used when the
beams for applying the stimulus or the excitation beam for the
Raman spectroscopy can be scanned.
[0088] The microfluidic system 81 has a closed loop 12 and plural
fluid channels 82, 86 in which measurements may be performed on
biological objects. A biological object 8 which is located in a
measurement region 85 in the fluid channel 82 during data
acquisition for characterization may be selectively sorted into one
of plural output channels 83, 84 depending on a result of the
characterization. Similarly, a biological object 90 which is
located in a measurement region 89 in the fluid channel 86 during
data acquisition for characterization may be selectively sorted
into one of plural output channels 87, 88 depending on a result of
the characterization.
[0089] When optical radiation which is used for applying the
stimulus and/or for measuring a response to the stimulus is scanned
between the measurement regions 85 and 89 in the different fluid
channels a characterization of biological objects may be performed
in parallel in the two fluid channels 82 and 86.
[0090] FIG. 6 is a schematic representation of an apparatus 92 for
characterizing a cell according to another embodiment. Components
of the apparatus 92 which correspond with respect to their function
and/or construction to components of the apparatus 2 of FIG. 1, to
component of the apparatus 32 of FIG. 2 or to components of the
apparatus 72 of FIG. 4 are designated with the same reference
numerals as in FIG. 1, FIG. 2 and FIG. 4, respectively.
[0091] In the apparatus 92, a stimulus onto a biological object is
generated using optical radiation. Similarly to the apparatus 72 of
FIG. 4 laser beams are irradiated via the lens 30 or the objective
29, respectively, onto the biological object 8 for applying the
stimulus. The device for generating a stimulus has a component 93
which outputs a laser beam 103 which is directed to the lens 30 via
a beam splitter 77. A further component 95 is provided which
outputs a laser beam 105 which is directed to the objective 80 via
a beam splitter 78. Here, the components 93, 95 are configured such
that the laser beams 103 and 106 act as a first optical tweezer for
a biological object 8. The device for generating the stimulus also
has components 94, 96 with which a second optical tweezer for the
same biological object 8 is generated. The component 94 outputs a
laser beam 104 which is directed to the lens 30 via the beam
splitter 77. The component 96 outputs a laser beam 106 which is
directed to the objective 29 via the beam splitter 78. The various
components 93-96 may be fed from the same laser light source.
[0092] The device for generating the stimulus can be configured
such that the optical tweezer generated by the laser beams 103 and
105 and the further optical tweezer generated by the laser beams
104 and 106 have a distance which is approximately equal to a
diameter of the biological object or can be adjusted to have such a
distance. In this manner forces which give rise to a stretching of
the biological object may be applied to the biological object.
[0093] FIG. 7 exemplarily shows this action of a pair of optical
tweezers. The focal areas of the pair of laser beams 105, 107 and
of the pair of laser beams 104, 106 are spaced from each other.
Cell portions are drawn into the volume areas having maximum light
energy due to the dipole trap effect. If both optical tweezers are
set to a suitable distance, for example by a lateral movement of
the tweezers away from each other, a stretching of the biological
object may be induced as illustrated with arrows 109 and 110.
[0094] The apparatus 92 may also be provided with deflection
devices (not shown in FIG. 6) to allow one of the pairs 103, 105 or
104, 106 of laser beams or both pairs of laser beams to be
adjusted. By adjusting at least one of the pairs of laser beams
which form an optical tweezer the apparatus 92 is configurable such
that it can induce a change in shape also on biological objects
which have clearly different dimensions. The rate at which
biological objects can be characterized may be increased by a
combined scanning of both optical tweezers.
[0095] While embodiments have been described in which a
transmission light configuration is used for DHMI data acquisition,
a reflection light configuration may also be used in each one of
the embodiments.
[0096] FIG. 8 is a schematic representation of an apparatus 112 for
characterizing a cell according to another embodiment. Components
of the apparatus 112 which correspond with respect to their
function and/or construction to components of the apparatus 2 of
FIG. 1, to components of the apparatus 32 of FIG. 2, to components
of the apparatus 72 of FIG. 4 or to components of the apparatus 92
of FIG. 6 are designated with the same reference numerals as in
FIG. 1, FIG. 2, FIG. 4 and FIG. 6, respectively.
[0097] The apparatus 112 has a device for DHMI data acquisition
having a source 24a for the object wave 45 and a source 24b for the
reference wave 46. The interference pattern resulting from the
superposition is captured on the two-dimensional image sensor 38.
The apparatus 112 further has a device 22 for performing the Raman
spectroscopy. The apparatus 112 has a device 113 for generating a
laser pulse or plural laser pulses. A controllable deflection
device 114 may optionally be provided such that the laser pulse or
the laser pulses may selectively be directed onto one of plural
different biological objects or onto different portions of a
biological object. Using the micro beam generated by the device 113
a local disturbance on the cell membrane and/or in the cytoplasm of
a cell may be generated, for example. The response of the cell to
the disturbance may be detected for characterizing the cell. As
described with reference to FIGS. 1-7 a DHMI data acquisition and a
Raman spectroscopy may be performed to this end before the
disturbance was created. A further DHMI data acquisition and a
further Raman spectroscopy are performed while the disturbance is
being generated or after the disturbance was generated.
[0098] Changes in intrinsic properties of biological objects in
response to a stimulus may be monitored using the apparatuses. The
biological object may be characterized depending on the change in
the intrinsic properties. Since the application of the stimulus and
the observation can be performed in a destruction-free manner the
biological object may be characterized without destruction and may
subsequently be subjected to a further manipulation or use.
[0099] FIG. 9 is a flow diagram representation of a method 120
according to an embodiment. The method may be performed using the
apparatus or the system according to any one of the embodiments
described with reference to FIGS. 1-8.
[0100] In step 121a measurement is performed on a biological
object. In this step, a DHMI measurement for determining a
refractive index is performed and/or a Raman spectrum is captured.
The measurement at 121 is performed before application of a
stimulus.
[0101] In step 122 a stimulus is applied. For applying the stimulus
different techniques may be used which were already described and
which may include the use of laser light, electromagnetic
alternating fields, electromagnetic radiation, ultrasound or
similar, for example.
[0102] In step 123 at least one further measurement may be
performed during or after application of the stimulus on the same
biological object on which the measurement was performed in step
121. In this step, a further DHMI measurement for determining the
refractive index is performed and/or a further Raman spectrum is
captured.
[0103] In step 124 the measurement results obtained in 121 and 123
are compared. To this end, a difference between the measurement
result obtained at 121 and the measurement result obtained at 123
may be computed, for example. If a Raman spectrum was respectively
captured at 121 and 123 a difference spectrum may be computed at
124. If more than two measurements were performed on the biological
object a correspondingly greater number of measurement results may
be compared. For example, plural Raman spectra which were captured
during or after application of the stimulus in a time-sequential
manner may respectively be compared to a Raman spectrum which was
determined on the same biological object before application of the
stimulus.
[0104] In step 125 the biological object is characterized based on
the comparison of measurement results obtained before application
of the stimulus and during or after application of the stimulus.
For this purpose a difference spectrum which was determined in step
124 may be compared to a reference difference spectrum stored in a
data base, for example.
[0105] FIG. 10 illustrates the execution of the method according to
an embodiment. States 131-134 of a biological object at different
times t1, t2, t3 and t4 are shown. Raman spectra 141-143 are
detected at times t1, t2 and t3. A difference spectrum 152 between
the Raman spectrum 142 detected at t2 and the Raman spectrum 141
detected t1 may be determined from the Raman spectra 141 and 142. A
difference spectrum 153 between the Raman spectrum 143 detected at
t3 and the Raman spectrum 141 detected at t1 may be determined from
the Raman spectra 141 and 143.
[0106] As schematically shown the biological object may be
stimulated such that it is made to vibrate. At time t1 the
biological object, e.g. a cell, has a state 131. With a Raman
spectroscopy performed at time t1 information on intrinsic
properties of the biological object cell may be obtained, such as
information on a density, amount or a type of certain molecules 135
in a.
[0107] A pulsating force may be applied to the biological object to
excite the biological object to vibrate. A stimulus is applied to
the biological object by exciting a vibration, in particular by
exciting a resonance vibration. For this purpose an excitation beam
of the Raman spectrometer may be irradiated onto the biological
object in a pulsed manner with a repetition rate, for example. The
repetition rate may be varied until a resonance is reached.
[0108] States of the biological object at different times t2, t3
and t4 of the vibration cycle are shown at 132-134. A deformation
vibration is shown for illustration. A change in the interior of
the biological object is induced by the vibration which can occur
in addition to a change in volume and/or shape of the biological
object. For example, a density and/or arrangement of the molecules
135 in the interior of the cell may change as a function of time.
It is also possible that a type of the molecules 135 in the
interior of the cell changes. Such changes lead to a shift of
spectral lines and/or to a change in spectral weights of the Raman
spectra 142, 143 captured during the vibration at t2 and t3
compared to the Raman spectrum 141 captured at t1. Alternatively or
additionally excitation beams having different polarizations may be
used in the Raman spectroscopy which is performed at different
times.
[0109] While embodiments have been described with reference to the
drawings modifications may be implemented in other embodiments.
While apparatuses have been described in detail in which both a
DHMI data acquisition and a Raman spectroscopy are performed to
determine a response of a biological object to a stimulus, in other
embodiments performing the DHMI data acquisition or performing the
Raman spectroscopy may be omitted. For example, in further
applications it may be sufficient to characterize the biological
object with regard to the change in its Raman spectrum in response
to the stimulus. In this case the Raman spectroscopy may provide
all data required for characterization.
[0110] While embodiments have been described in which the
characterization of biological objects after a data base comparison
is used for a further action such as for sorting the biological
objects, in other embodiments the characterization using the
methods and apparatuses described herein may also be used to
generate a data base for the automatic sorting or to gain
information on the structure of biological objects.
[0111] While embodiments have been described in which various
optical signals, such as the excitation beam for the Raman
spectroscopy are scanned over different positions, no scanning is
required in other embodiments.
[0112] While embodiments have been described in which a stimulus
was generated using optical radiation, in other embodiments the
stimulus may also be generated in other ways, for example by
ultrasound, high-frequency pulse, electromagnetic radiation or
administration of active substances and drugs, respectively. The
stimulus may also be generated by the interplay of optical
radiation and fluid flow. For example shear forces which are
applied by a fluid flow onto an object which is trapped in an
optical tweezer may induce deformation of the object. A data
acquisition using DHMI and/or Raman spectroscopy may be detected in
response to different stimuli. Additionally or alternatively, the
response to the irradiation of ultrasound or of an electric or
magnetic high-frequency field may be determined.
* * * * *