Title of Invention

"A METHOD OF ANALYSING A SAMPLE AND APPARATUS THEREFOR"

Abstract Fluorescent labels are used as labels to investigate processes in biological samples without affecting their normal functioning. A method of analysing a sample (48) using fluorescent labels and apparatus including processing means (18) comprises the steps of: (a) irradiating (12) the sample (48) with excitation energy; (b) detecting (16) fluorescent radiation emitted by labels in the sample (48) when excited by the excitation energy; and (c) modifying the environmental conditions (42) of the sample with the apparatus in response to user input after the beginning of step (a) and/or in response to analysis by the processing means (18) of the results of the fluorescent radiation detection step (b). Thus modifications may be determined and executed interactively as an experiment progresses, and therefore directed towards specific structures and/or events identified as of particular interest in the course of the experiment.
Full Text

Field of the invention
This invention relates to a method and apparatus for analysing samples of biological
material. More particularly, it concerns the investigation of processes in samples such as
cells, in a way that may not interfere with cell functioning. Such investigation may
provide greater understanding of complex processes in life science and medicine, assist the
evaluation of new drug candidates, and enable the detection of disease or abnormal
processes.
Background
Examination of live samples provides access to the dynamics of cellular and sub-cellular
processes. The study of small-scale cellular processes has typically been carried out using
light microscopy.
In some cases, the complexity of the processes is such that tools are required to isolate
specific types of processes which can be studied in isolation, or in combination with a
small number of other processes. The use of labels (in the form of fluorescent molecules in
fluorescence microscopy) has provided tools of this nature.
A label may be extrinsic (that is, added to a sample) or intrinsic (such as a molecule which is present in the sample that is inherently distinctively coloured or fluorescent).

A fluorescent molecule has a specific excitation spectrum, being more strongly excited at
some wavelengths and less strongly excited at others. It also has a specific emission
spectrum, emitting more intensely at some wavelengths, and less intensely at others. The
excitation and emission spectra may range from the ultraviolet to the infrared.

A wide range of fluorescent labels have been developed from chemical molecules, such as
Rhodarnine and Fluorescein. Further fluorescent labels have been developed from
molecules found in luminescent organisms, for example the Aequorea Jellyfish which has
provided the Green Fluorescent Protein (GFP), and various corals providing DsRed and
HcRed. These have been termed AFPs (Aequorea Fluorescent Proteins).
The fluorescent labels may be associated with specific molecules of interest (DNA, RNA,
proteins, carbohydrates, antibodies, etc). Alternatively they may be made to-be sensitive to
certain characteristics (such as ionic concentration, pH, voltage potential, temperature, the
presence of a specific enzyme, the presence of specific enzyme substrates, force), altering
their fluorescent properties according to these characteristics. These labels may be
introduced into cells by passing through the cell membrane or by injection. Alternatively
they may be formed internally as part of the normal functioning of the cell, in the case of
the genetically encoded labels such as the AFPs.
In a known apparatus for imaging samples as shown in Figure la, a microscope 10 is fitted
with a light source 12 for illuminating a sample 4b. It may be in the form of a lamp, laser
or light emitting diode, for example. The microscope is fitted with optical filters 14 so
that the light emanating from the sample may be observed at selected wavebands.
Examination of the spatial and temporal distribution of light from the sample may provide
information on the structure and dynamics of the sample.
Phase contrast illumination may be employed to enhance imaging of thin samples.
Polarised light may be used to permit the visualisation of very thin samples with small
refractive index changes, for example using differential interference contrast (DIC)
techniques.
The microscope is fitted with an image acquisition system, comprising a light sensitive
detector 16 (sensitive from the ultraviolet to the infrared) such as a CCD camera, and
recording means such as a video recorder or computer 18 with a memory device 20, so
that dynamic behaviour of the sample may be captured and analysed offline. For example,
the velocity, distance travelled and path of moving parts of the sample may be monitored.

The system may employ a focus drive mechanism for altering the position of the imaging
focus plane. Various algorithms may be used to establish and maintain the optical focal
plane. Volumetric (XYZ) and volumetric time series (XYZT) data may be acquired using
deconvolution techniques. By selecting suitable excitation wavebands and/or selecting
suitable optical filter sets, volumetric multi-wavelength (XYWZ) and volumetric multi-
wavelength time series (XYWZT) data may be acquired.
Operations of the microscope are controlled by a microscope controller 24 in response to
commands from processing means in the form of computer 18. Instructions may be
entered into the computer by a user using keyboard 26, a mouse 28 and a display 30.
By way of example, Figure la shows control lines extending from the controller to the
microscope for aspects such as filter selection along line 32, selection of an objective lens
34, 34' along line 36, focus along line 22, a liquid dispenser 38 along line 40, an
environmental unit 42 along line 44 for modifying environmental conditions of a sample
46, and a sample transport unit 48 for moving the sample 46 relative to a lens of the
microscope by control via line 50.
The apparatus may also include an activation light source 52 for generating an activating
light beam 54 for use as discussed below. The direction of the activating light beam may
be adjusted by a guide 56.
The activating light beam 54 is incident on a dichroic mirror 60. The mirror directs the
beam towards an objective lens 34, and the beam is then incident on the sample 46. Light
emanating from the sample 46 passes back through the objective lens 34, but is not
diverted by mirror 60 so that is passes through a filter 14 before impinging on detector 16.
An output signal from the detector 16 is fed to the computer 18 along line 62.
The environmental unit 42 may control the temperature of the sample, and/or the
composition and flow of gas over the sample, for example.

Software loaded onto the computer enables it to acquire image data carried on the output
signal from the detector.
The software also allows the user to select parameters for the operation of various aspects
of the apparatus, such as the activating light beam, for example.
The controller allows the activating light beam to be directed by the setting of one or more
of the following parameters:
• the one or more regions of interest on the sample (location, size and shape)
• the wavebands
• the power level
•" the time of start of activation
• the duration of activation
• the number of repeat activation cycles
• the delay between activation cycles.
The computer permits recording of the image data from the detector via a digitiser at a
given data rate. The filter sets in the microscope and the waveband of the illuminating
light source 12 may be controlled to permit acquisition of data sets of one or more
wavelengths.
A known apparatus for imaging biological samples including fluorescent labels is shown in
Figure lb. It is similar to the apparatus shown in Figure la. However, instead of
iUumination light source 12, it includes an excitation light source 11, which generates a
light beam 55. This light source is capable of exciting one or more fluorescent labels
present in the sample by irradiating them with one or more specific wavebands. Filters 14
may be chosen so as to pass light emitted by labels at selected wavebands.
Figure 1c illustrates attachment of a fluorescent label 2, 2' to a component of interest 4, 4'
in a sample 6. Multiple labels may be used to provide information on the coincident

localisation of labelled components, revealing, for example, the organization of the
cytoskeleton of a cell.
The excitation light source 11 employed in the apparatus of Figure 1 may be pulsed and
the detector may be fitted with a gating device, so that the time taken for a fluorescent
label to emit light following the pulse may be used to distinguish between different labels
in a technique termed fluorescent lifetime imaging (FLIM) [ref. R. Cubedda, J. Phys. D:
Appl, Phys., 2002, Vol. 35, R61-R76].
An activating light beam 54 may enable a deeper understanding of the dynamic processes
in a sample such as a living cell (in many cases this may be based on the excitation light
source) to be obtained. The activating light beam may be directed to portions of the
sample in such a manner that the intense light from this beam alters the sample and/or
bleaches labels present in the sample and reduces their fluorescence. By observing the
subsequent changes in the light emanating from this region, and/or elsewhere in the
sample, information can be obtained on mechanisms of interaction and/or exchange of
various labelled components.
The beam may act to perforate a cell wall to allow the entry of an external agent, to dissect
all or part of the cell, to destroy all or part of the cell, or to change the environment of the
sample.
A graph plotting the intensity of fluorescence at a bleached region against time is shown in
Figure 1d.
For example, the rapid recovery of fluorescence at the bleached portion suggests to what
extent the label is free to return to the region. The mechanism, diffusion or local
synthesis, may be determined by estimation of diffusion rates and the mobile fraction [ref.
AxelRod,.Biophys. J., 1977, Vol. 18, pp. 129-131.].
Further analysis and experimentation may provide access to information about molecular
binding rates, assembly/disassembly and transport mechanisms [ref. J. Lippincott-Schwartz

et al, Nature Supp Imaging in Cell Biol, Sept 2003, S7-S14.]. Such techniques are popular
to explore the dynamics and regulation of processes in cells, for example protein
trafficking, lifetime and fate (recycling and breakdown).
Conventionally, such bleaching techniques may be applied as a "bleaching protocol"
comprising four phases as illustrated in Figure le:
(1) a pre-bleach phase in which the sample is imaged using normal fluorescence for a
period of time;
(2) a bleach phase during which the activating light beam is directed to iUuminate a
predefined region with light of a specific wavelength with a specified higher power
level for a certain period of time;
(3) a post-bleach phase in which the sample is again imaged using normal fluorescence
for a period of time; and
(4) an analysis phase in which the data which has been collected is processed to
determine parameters of interest, such as diffusion rate and mobile fraction
according to fluorescence intensity in various regions on the image at various time
points.
A set of related techniques are commonly referred to as FRAP (Fluorescence recovery
after photo-bleaching). Variants of the basic protocol may be used to further explore the
system, by repeated bleaching of the same region, and bleaching other portions etc (FLAP,
FLIP, iFRAP) [ref. J. Lippincott-Schwartz et al, see above; Phair et al, Nature, 2000,
Vol. 404, pp. 604-609.].
Alternatively, a label in the sample, the sample itself or its environment may comprise a
caged compound that releases an active group when ihuminated by the activating light
beam - a process known as uncaging [ref. J.C. Politz, Trend Cell Biol., 1999, Vol. 9, pp.
284-287.]. The active group may be a fluorescent label. Alternatively, the active group

may not be a fluorescent label, for example the active group may instead affect the pH of
the sample. The active group may have an indirectly visible or otherwise measurable
effect on the sample.
In addition a technique known as pattern photo-bleaching may be using to observe changes
in structure [ref. J. Ellenberg et al, Nature Supp Imaging in Cell Biol, Sept 2003,
S14-S19.]. In this technique, the bleaching process is used to introduce visually distinctive
landmark patterns on part of the sample, for example a grid, movement of which may be
tracked over time to understand, for example, mechanisms of membrane deformation or
shear.
Recent developments in fluorescent probes has resulted in new labels (photo-switchable
labels) which are sensitive to the incident light, in such as way that they change their
optical properties, altering their emission spectrum and/or their excitation spectrum.
Examples include the photo-activatable GFP (PA-GFP) and Kaede [ref. J. Lippincott-
Schwartz et al, see above.]. Furthermore, some of these probes allow their properties to be
altered reversibly or irreversibly. Such probes may be activated (made to emit more
intensely), or quenched (made to emit less intensely) in different wavebands. For example
the kindling FP KFP1 [ref. D.M. Chudakov et al, J. Biol. Chem., 2003, Vol. 278(9), pp.
7215-7219.].
Instrumentation has'been developed with an adapted activating light beam to excite photo-
switchable labels and force them to undergo the change in optical properties. This may, for
example, be useful in revealing or hiding labelled components during the course of an
experiment and in studying long term processes such as organism development [ref. D.M.
Chudakov et al, Nature Biotechnology, Feb 2003, Vol. 21, pp. 191-194.].
Hereinafter, the term photo-modification will be used to include photo-bleaching and its
variants (FRAP, iFRAP, FLIP, FLAP), photo-activation, photo-switching, and pattern
photo-bleaching.

Conventional control of an activating light beam involves setting up a number of activation
i
parameters and requires the user to be able to estimate the appropriate values. In general,
these are determined by trial and error using surplus material. It may be desired to set the
depth of activation to be say 30% (as is the case in the illustration of Figure Id) and the
appropriate laser power setting and duration must be determined to achieve this.
Similarly, the recovery time of the curve will determine the appropriate sampling regime
in the post-bleach period (number of images, interval between images) which in turn is
determined by the diffusion coefficient.
These activation parameters must be defined prior to the experiment. They cannot be
adapted to suit changing conditions. This means that the approach can only be applied to
changing samples by trial and error. Thus fast changes and/or rare events can be extremely
difficult to study.
In addition, in existing photo-bleaching procedures, the recovery after bleaching prevents
long time course experiments (10mins+) in the tracking of change since the labels recover
over shorter periods and thereafter there is no discernible bleached area.
Summary of the invention
The present invention provides a method of analysing a sample of biological material using
apparatus including processing means, comprising the steps of:
(a) irradiating the sample with electromagnetic and/or ionizing radiation;
(b) detecting electromagnetic radiation emanating from the sample and generating a
signal in response thereto; and
(c) modifying the sample, labels in the sample and/or the environmental conditions of
the sample with the apparatus in response to user input after the beginning of step (a)
and/or in response to analysis by the processing means of the signal generated in
radiation detection step (b).
The present invention may enable combination of imaging, measurement of one or more
characteristics in the images collected, and modification of the sample, labels in the

sample, and/or the environmental conditions of the sample, with manual and/or automatic
determination of the modification parameters according to the measurement made.
In comparison, conventional systems require the user to enter modification parameters
manually before the start of the experiment, and so prevent dynamic setting and
modification of those parameters, and thus interactive probing of complex systems.
Thus, in accordance with the invention, such modifications may be determined and
executed "in real time", interactively, as the experiment progresses. This flexibility
allows longer time course experiments to be conducted. It also allows rare, brief and/or
rapid events to be detected and acted upon, such as all fertilisation or mutation events.
Events that are difficult for a human operator to detect, such as coincident events can be
detected and acted upon.
Where the present method is carried out using a light microscope, the irradiation is
provided by an Ulumination light source. The electromagnetic radiation emanating from
the sample may in that case be in the form of light transmitted by the sample if ihuminated
from below, or reflected light if the same is illuminated from above.
In a preferred approach, the sample includes fluorescent labels, step (a) comprises
irradiating the sample with excitation energy, and step (b) comprises detecting fluorescent
radiation emitted by labels in the sample when excited by the excitation energy.
The excitation energy may be in the form of electromagnetic energy (preferably light) or
ionizing radiation, for example.
The modification step (c) may comprise irradiating at least a portion of the sample with an
energy beam. If labels are present in the sample, this beam may be selected such that the
fluorescence of labels in the portion is reduced or "bleached". In a particular embodiment,
steps (a) to (c) are-repeated after step (c), the period of time between the end of step (c)
and the start of step (a) and/or the parameters of step (c) when repeated being dependent
on said analysis by the processing means.

In a preferred embodiment, me apparatus operates so as to cycle between irradiation and
modification phases, and the resultant change in the appearance of the sample is detected.
In another approach, the modification step (c) comprises irradiating at least a portion of the
sample with an energy beam so as to modify the optical properties of photo-switchable
fluorescent labels in the portion.
For example, it may be desirable to measure intra-cell diffusion many times over an
extended period. Repeating a known FRAP experiment would have a cumulative photo-
bleaching effect; soon the cell would not be visible. Using photo-switchable labels, a
region can be switched rather than bleached. According to the present invention, a
diffusion (or other) process may be measured, and when it is determined by the processing •
means that the process and/or its measurement is complete, the photo-switchable labels
may be "quenched", returning them to their original state, before repeating the process.
By avoiding repeated photo-bleaching a sample can be imaged for longer, and the present
invention enables a process to be repeated and/or a series of measurements to be carried
out in response to detected fluorescent radiation over a prolonged period.
Avoidance of repeated photo-bleaching is also beneficial as an activating light beam is
often intense and its use may result in damage to the sample, or the creation of reactive
species such a singlet oxygen, which may perturb the process under study, requiring
sophisticated control experiments, and is thus not truly non-invasive. [Ref. D E Wolf, M
Edidin, P R Dragsten, 1980, and Proc. Natl. Acad. Sci. USA 77: 2043-2045.]
t.
After step (c), the environmental conditions of the sample may again be changed and the
light emanating from the sample thereafter detected for analysis.
In another embodiment, the environmental change is selected to cause labels in the sample
to release a substance. |

In a further embodiment, the environmental change is selected to modify the manner in
which the sample and/or labels in the sample respond to irradiation. For example, it may
change the colour of the sample or the excitation spectrum and/or emission spectrum of
labels in the sample. In particular the environmental change may be controlled activation
light.
The ability to modify the response of the labels in the course of a process may avoid a
need to physically introduce labels during the experiment. The procedures for introducing
the labels into the sample during an experiment are quite difficult to master, requiring
additional equipment, knowledge and skill on the part of the user. Experiments often have
to be repeated many times. In the case of microinjection, such procedures are also invasive
to the sample. It can be seen that interactive modification of labels already present in the
sample may not require physical introduction of additional labels. Labels may be activated
or "switched on" in a selected region.
In a further preferred approach, the modification step (c) comprises modifying the physical .
structure of at least part of the sample. For example, this may involve forming an opening
in a membrane of a selected cell in the sample to allow material to pass therethrough. The
opening may be formed using a localised energy beam such as a laser,, for example. In
this way, an individual cell may be selected in the sample which is exhibiting behaviour of
interest, and the user can form an opening in the cell wall to allow in material in response
to that behaviour. The intensity of the energy beam is preferably selected such that the cell
membrane is able to close or "heal" the opening shortly after its formation. Forming an
opening in a cell in this way to allow the ingress of genetic material is often referred to as
"transfection".
The opening may allow agents such as labels into the cell to interact wim or to highlight a
specific structure and/or process in the cell, or otherwise alter its optical properties. It
may allow genetic material to pass into the cell. Alternatively, material may be allowed to
pass into the cell so as to kill it.

In contrast to dispensing a liquid into a sample to alter the composition of the sample as a
whole, modification of an individual structure such as a cell is permitted using the
technique described above. It may also be employed in combination with liquid
dispensing, whereby the liquid dispensing step provides the material which passes through
the opening formed by the energy beam.
The environmental conditions of the sample to be changed may be its temperature, the
concentration of a certain ion, the pH of the sample, the voltage potential of the sample,
the presence of a specific enzyme, or the pressure of the sample. Labels in the sample
may be sensitive to these conditions and activate when a predetermined range is reached.
In another embodiment, the environment of the sample is changed by altering the
composition of the gas adjacent to the sample. For example, labels in the sample may be
activated by exposure to a predetermined gas or concentration of a predetermined gas,
such as oxygen.
In a further embodiment, the environment of the sample is changed by irradiating it with
activation energy.
The sample may be irradiated with an activation energy beam arranged to irradiate a
selected portion of the sample. The beam may define a pattern, such as a grid, for
example.
A parameter (or parameters) of the activation energy- irradiation determined by the
processor may be one or more of the following examples: the start time of the irradiation;
the portion of the sample to be irradiated; the waveband(s) of the energy; the power level;
the duration of the irradiation; the number of repeat cycles of the activation irradiation
energy; and the delay between repeat cycles.
The modification carried out in step (c) of a process embodying the invention may be
applied to a region of the sample selected in response to the user input and/or the analysis
by the processing means.

A parameter of the modification of step (c) may be determined by the processing means
with reference to data stored by the apparatus.
In a further implementation, the modification of step (c) is dependent on detection of the
occurrence of a predetermined event in the sample by the processing means.
Preferably, data generated by the analysis by the processing means is stored by the
apparatus so as to facilitate reference thereto in deterrrrirring a parameter of a subsequent
modification step.
The invention further provides apparatus for analysing a sample of biological material
including fluorescent labels, comprising:
irradiation means for irradiating the sample with electromagnetic and/or ionizing
radiation;
detection means for .detecting radiation emanating from the sample and outputtrng
an output signal in response thereto;
modification means for modifying the sample, labels in the sample,- and/or the
environmental conditions of the sample;
processing means for analysing the output signal from the detection means; and
control means for controlling the modification means in response to user input after
irradiation of the sample and/or in response to analysis by the processing means of the
output signal from the detection means.
In a preferred embodiment, the apparatus includes data storage means for storing data
generated by analysis by the processing means for reference thereto by the control means
in deterrnining a parameter of a modification by the modification means.
The apparatus may further include display means for presenting to a user an indication of
the electromagnetic radiation detected by the detection means, and input means for
enabling a user to control at least one parameter of a modification by the modification

means in response to detected radiation. Preferably, the input means enable a user to
identify a specific location in a sample for a modification by tiie modification means.
Detailed description of the invention »
Prior art and embodiments of the invention will now be described by way of example with
reference to the accompanying schematic drawings, wherein:
Figure la shows a block diagram of a known light microscopy apparatus for investigating
processes in a sample;
Figure lb shows a block diagram of a known fluorescence microscopy apparatus for
investigating processes in a sample;
Figure lc illustrates known use of fluorescent labels;
Figure Id shows a typical bleach recovery curve for a fluorescent label;
Figure le shows a flow diagram of a known bleaching protocol; and
Figures 2 to 7 show flow diagrams of modification protocols according to respective
embodiments of the invention.
The embodiments described with reference to Figures 2 to 7 concern photo-modification of
a sample and/or labels in a sample. It will be appreciated that the modification carried out
in accordance with the invention may take other forms.
Figures 2 to 7 represent diagrammatically protocols followed by processing means of a
suitable apparatus, together with associated inputs and control outputs fed to other parts of
the apparatus.
Figure 2 shows a first embodiment of the invention, which allows interactive control of
photo-modification. A user interface allows parameters to be set and/or modified during
the running of the experiment. The parameters control operation of the activating light
source and/or ancilliary devices such as the examples given in the Figure which may
modify the sample, labels in the sample and/or the immediate environment of the sample.

In an implementation in which the activating light beam bleaches fluorescent labels in the
sample, this protocol may be termed "FRAP on demand".
In the embodiment of Figure 2, the system allows the user to set the parameters of the
activating light beam comprising, the region of interest on the sample, the waveband(s),
the power level at those wavebands, and the duration of activation. The user is able to
image the sample and determine the start time of activation by a suitable action such as a
keypress.
The user may be able to determine one or more of the duration or end of activation, the
number of repeat cycles and the delay between cycles of activation, and the wavelength of
the activation by a suitable action such as a key press.
Furthermore, the user may be able to determine the location of activation and/or the size
and shape of the irradiated region by suitable action such as manipulation of an input
device such as a computer mouse.
A label may have a temperature sensitive domain, and the sample may be held on a holder,
the temperature of which may be controlled by the user via the computer to activate the
labels at an appropriate point in a process.
A label may be sensitive to gas such as oxygen, and the sample environmental unit
controlled by the user via the computer to activate the labels in the course of an
experiment.
Figure 3 shows a second embodiment that allows the use of a knowledge base to assist the
user in setting and/or modifying the parameters.
In the embodiment of Figure 3, the system is as Figure 2, and additionally allows a less
experienced user to set certain high level parameters of photo-modification parameters
such as the type of label to be bleached, and the system uses a knowledge base to

determine the low level parameters to be used by the hardware, such as the wavelength and
power level.
Additionally, a more experienced user may be able to modify the knowledge base.
In an implementation in which the activating light beam bleaches fluorescent labels in the
sample, this protocol may be termed "expert FRAP".
Figure 4a shows a third embodiment that uses information extracted from the images
collected during the pre-modification (observation) phase to set the photo-modification
parameters.
In an implementation in which the activating light beam bleaches fluorescent labels in the
sample, this protocol may be termed "clever FRAP".
Figure 4b shows the third embodiment implemented in the form of a state machine. Each
phase in the protocol is repeated until an exit condition is met. For the pre-modification
phase, the exit condition is when the image analysis module sends the start signal. For the
modification phase, the exit condition is when the image analysis module sends the stop •
signal. The additional parameters to send are not shown in this diagram.
In the embodiment of Figures 4a and 4b, me system allows the user to set certain
parameters of the activating light beam, whilst setting others on the basis of information
extracted from the data acquired during the pre-modification phase. In particular, the
image data may be analysed to detect an event such as the motion of a component in the
sample such as a cell in order to trigger the activating light beam.
The user may be able to determine the duration or end of activation by suitable action such
as a key press!
The data may be analysed to:

determine- location of a moving portion of the sample to direct the activating light
beam;
determine a change in intensity of part of the sample to direct the activating light
beam;
determine relative change in intensity of part of the sample to direct the activating
light beam;
determine relative change in fluorescent lifetime of part of the sample to direct the
activating light beam;
locate one or more substructures of interest, such as cellular components,
organelles, vesicles or other particles, where the location and/or size of the particle(s) is
used to direct the activating light beam;
locate one or more substructures of interest such as cellular components,
organelles, vesicles or other particles, where the location, motion, shape, speed, direction,
relative distance and/or size of the particle(s) is used to trigger the activating light beam;
detect key events in the cell cycle (such as cell division, cell death, viral invasion,
endoctyosis, exocytosis, tubulation, gene transfer etc.) and/or coincidence of more than
one event to trigger the activating light beam;
automatically control the instrument;
direct the setting of the irradiation source;
direct the setting of the emission wavelength. For example, by following changes
in emission wavelength and adjusting the wavelength of the irradiation light to maximise
the emission intensity of labels in a sample, it is possible to measure changes to
characteristics of the sample and do so more effectively;
direct the setting of the polarisation of the light;
direct the setting of the focus drive mechanism and thus the plane of focus of the
microscope;
direct a sample transport mechanism;
set the temperature of a sample heater; and
control a liquid dispensing mechanism.
The data may be preprocessed to remove distortions due to deformation or motion of all or
part of the sample.

Figure 5 shows a fourth embodiment in which an image analysis module processes the
image data acquired at each phase to determine the most appropriate parameter settings,
for example, the amount of light to in order to complete photo-modification.
In the embodiment of Figure 5, the system allows the user to set certain parameters of the
activating light beam, whilst the system sets others on the basis of information extracted
from the data acquired during the pre-bleach phase, during the bleach phase, and during
the post-bleach phase. In particular, the image data may be analysed to detect an event
such as the change of a component in the sample in order to end a phase.
Figure 6 shows a fifth embodiment in which the system moves from a monitor phase to a
modification phase according to the information extracted from the images collected. In an
implementation in which the activating light beam bleaches fluorescent labels in the
sample, this protocol may be termed "automated FRAP".
This is expected to be particularly useful when used in conjunction with photo-switching
labels which can be activated and quenched repeatedly and used to track the lifecycle of
individual components.
In the embodiment of Figure 6, the system allows the switching between a monitor phase
during which the sample is imaged using the excitation light source, and a modification
phase, during which the sample is imaged using the activating light source. The transition
between the two phases and parameters for the phase is determined by the analysis of the .
image data collected.
Figure 7 shows a sixth embodiment in which the system moves from a monitor phase to a
modification phase according to the information extracted from the images acquired and
collects the information about the sample behaviour to the activating beam at a number of
parameter settings so as to set up the knowledge base mentioned in Figure 3. In an
implementation in which the activating light beam bleaches fluorescent labels in the
sample, this protocol may be termed "autoconfig FRAP".

In the embodiment of Figure 7, the system allows the control of the pre-modification
phase, modification phase, post-modification phase and analysis phase as described in
relation to Figure 5. The parameters used at each stage are those that permit an exploration
of the possible parameters of the sample so as to quickly and efficiently determine the
hardware settings (laser power, modification duration, etc) by an iterative process, and
these settings are passed to knowledge base mentioned in connection with Figure 3. With
the appropriate filter sets, the system may be able to image during the modification phase,
for example during a bleach process, to monitor the parameters of the modification.
In a further embodiment, the sample contains one or more photo-switchable labels, so that
the activating light beam is able to activate, quench or switch the label as described above
in order to observe the labelled component over short (fast moving components), medium,
or long time scales (rare events or configurations).
The labels may comprise a naturally occurring fluorescent molecule such as NADH, a
calcium probe such as "Fura", and/or an antibody tagged with a fluorescent molecule and
bound to a selected species.
The labels may comprise a pair of fluorescent molecules with overlapping emission-
excitation spectra of the types used in fluorescence resonant energy transfer ("FRET"), and
the activating light beam may be set to photo-bleach the acceptor molecule in a region of
the sample, so that the FRET process may be confirmed in that region.
One of the FRET pair may be a photo-switching label, and the activating light beam may
be set to switch the photo-switching label in a region of the sample, so that the FRETing
process may be confirmed in that region.
The labels may comprise a caged compound that releases an active group when illuminated
by the activating light beam [ref. J.C. Politz, see above.].

In a further embodiment, the excitation light source 11 may emit one or more spectral
bands from a broadband light source such as a lamp. Furthermore, the excitation light
source may be controllable to provide excitation light at more than one distinct waveband
in order to distinguish between different labels. In some processes, it may be
advantageous for the excitation light source to be pulsed.
The excitation light source may be of the type employed in fluorescent speckle microscopy
[ref. M.C. Adams et al, Methods, 2002, Vol. 29, pp. 29-41.]).
In a further embodiment, the activating light beam may be derived from the same source as
the excitation light.
The activating light beam may be:
pulsed;
an optical trap or optical tweezer arrangement;
a laser dissection mechanism for removing a region of interest from the sample for
subsequent handling; or
a laser ablation mechanism for removing a region of interest from the sample for
subsequent handling.
A localised energy beam may be employed to form an opening in a structure, such as a
cell, in response to detected radiation emanating from a sample. Preferably, a laser is
used. This may be a Tirsapphire, argon-ion or frequency-shifted Nd:YAG laser for
example. Alternatively, a diode laser can be employed which may be less costly.
Exposing a cell to 0.3mW of violet light from a diode laser for 40ms has been found to
perforate a cell membrane. The beam was focussed onto the cell using'a xlOO microscope
objective lens to form a spot about 1 micron in diameter. This results in a power density
of around 1200MW/m2. The cell membrane was able to "heal" itself shortly after the
process without apparently suffering any long term damage or mutation [ref. Optics
Express, Vol.13, page 595].

The same light source may be used to generate the localised energy beam to form an
opening in a structure as is used to generate the activating light beam, and for the
excitation light beam.
In a further embodiment, the system may have one or more photo-detectors such as photo-
multiplier tubes and a scanning arrangement of the excitation light beam and/or me
emission light path to the detector and/or the detector to observe parts or all the sample.
The system may have more than one imaging detector fitted with different light filtering
components to distinguish between different labels.
The detector may be fitted with a gating device, and the excitation light source may be
pulsed, so that the time taken for the fluorescent label to emit light following the pulse may
be used to distinguish between different labels.
The controller may be implemented in hardware and/or software.
In a further embodiment, the system may use a confocal principle, collecting light at a
single image plane by the use of one or more pinholes and a scanning mechanism.
Alternatively, a structured iUumination technique may be utilised to section a sample
optically at different depths [ref. M.A.A. Neil, R. Juskaitis and T. Wilson, Optical
Letters, 22(24): 1905-1907, Dec. 15 1997]. This may be more cost effective than a laser
scanning confocal system, as it can be carried out using a conventional light microscope.
A total internal reflection (TIRF) principle may be employed in which the excitation light
source is directed to be internally reflected at the sample-sample carrier surface, thus
exciting only-parts of the sample close to the surface [ref. Y. Sako and T. Yanagida,
Nature Supp Imaging in Cell Biol, Sept 2003, SS1-SS5..].
Furthermore, the system may use a deconvolution principle, capturing a group of images
focused at different imaging planes, and applying a deconvolution process to allow

volumetric (XYZ) data to be acquired. Volumetric time series (XYZT) data, volumetric
multi-wavelength (XYWZ) data, or volumetric multi-wavelength time series (XYWZT)
data may be acquired.
A multi-photon microscope employing a long wavelength (800-1200nm) high speed pulsed
laser may form the excitation light source and/or as the activating light beam [ref. J.
Pawley, Handbook of Confocal Microscopy].
The light or fluorescence microscope may be replaced with a simplified arrangement of
sample holder, magnifying objective lens and dichroic mirror to direct light from the
irradiation light source and the activating light beam to the sample, and collect the emitted
light beam and to direct it to a detector.
Alternatively, the fluorescence microscope may be replaced by an endoscope.
The sample may be a homogenous population of cells such as a cell culture. Alternatively,
the sample may be a heterogenous multi-cellular assembly such as a cell culture; a multi-
cellular assembly such as a tissue culture; a stem cell sample; a tissue sample; an organ,
such as an eye or retina, or the inside of the gut or a blood vessel; a whole animal, such as
the worm C.elegans, an insect, fish, mammal or amphibian; a whole or part of a plant or
fungus, a dividing cell or an embryo; one of more samples held in a multi-site carrier such
as a slide, or a multi-well plate, which is scanned sequentially; or a material sample
undergoing some change such as diffusion of one or more species.

We claim
1. Apparatus for analysing a sample of biological material including
fluorescent labels, comprising:
- irradiation means (11, 52) for irradiating the sample with excitation
energy;
- detection means (16) for detecting fluorescent radiation emitted by
labels in the sample when excited by the excitation energy and outputting an
output signal in response thereto;
- processing means (18) for processing the output signal from the
detection means to create images of the sample;
- modification means (42) for modifying the environmental conditions of
the sample; and
- control means (18, 24) for controlling the modification means so as to
modify the environmental conditions of the sample, in response to user input
after irradiation of the sample and/or analysis by the processing means of said
images, wherein the modification is dependent on detection of the occurrence of
a predetermined event in the sample, and
wherein the modified environmental conditions are selected from: (i) the
sample temperature, (ii) the concentration of an ion in the sample, (iii) the pH of
the sample, (iv) the voltage potential of the sample, (v) the presence of an
enzyme, (vi) the pressure of the sample, and (vii) the composition of the gas
adjacent to the sample, and
in the case of items (i) to (vi), the labels in the sample are sensitive to the
selected environmental conditions and activate when a predetermined range is
reached, and
in the case of item (vii), the labels in the sample are activated by
exposure to a predetermined gas or predetermined concentration of a
predetermined gas.

2. Apparatus as claimed in Claim 1 including data storage means (20)
for storing data generated by analysis by the processing means for reference
thereto by the control means in determining a parameter of a modification by the
modification means.
3. Apparatus as claimed in Claim 1 or Claim 2 including display means
(30) for presenting to a user an indication of the fluorescent radiation detected by
the detection means, and input means (26, 28) for enabling a user to control at
least one parameter of a modification by the modification means in response to
detected fluorescent radiation.



ABSTRACT


A METHOD OF ANALYSING A SAMPLE AND APPARATUS THEREFOR
Fluorescent labels are used as labels to investigate processes in biological
samples without affecting their normal functioning. A method of analysing a
sample (48) using fluorescent labels and apparatus including processing means
(18) comprises the steps of:
(a) irradiating (12) the sample (48) with
excitation energy;
(b) detecting (16) fluorescent radiation emitted by labels in the sample (48)
when excited by the excitation energy; and
(c) modifying the environmental conditions (42) of the sample with the
apparatus in response to user input after the beginning of step (a) and/or in
response to analysis by the processing means (18) of the results of the
fluorescent radiation detection step (b).
Thus modifications may be determined and executed interactively as an
experiment progresses, and therefore directed towards specific structures and/or
events identified as of particular interest in the course of the experiment.

Documents:

00385-kolnp-2007-assignment.pdf

00385-kolnp-2007-correspondence-1.2.pdf

00385-kolnp-2007-correspondence1.1.pdf

00385-kolnp-2007-form-3-1.1.pdf

00385-kolnp-2007-general power of authority.pdf

00385-kolnp-2007-priority document.pdf

0385-kolnp-2007 abstract.pdf

0385-kolnp-2007 claims.pdf

0385-kolnp-2007 correspondence others.pdf

0385-kolnp-2007 description(complete).pdf

0385-kolnp-2007 drawings.pdf

0385-kolnp-2007 form-1.pdf

0385-kolnp-2007 form-3.pdf

0385-kolnp-2007 form-5.pdf

0385-kolnp-2007 international publication.pdf

385-KOLNP-2007-(19-11-2013)-ABSTRACT.pdf

385-KOLNP-2007-(19-11-2013)-ANNEXURE TO FORM 3.pdf

385-KOLNP-2007-(19-11-2013)-CLAIMS.pdf

385-KOLNP-2007-(19-11-2013)-CORRESPONDENCE.pdf

385-KOLNP-2007-(19-11-2013)-DESCRIPTION (COMPLETE).pdf

385-KOLNP-2007-(19-11-2013)-DRAWINGS.pdf

385-KOLNP-2007-(19-11-2013)-FORM-1.pdf

385-KOLNP-2007-(19-11-2013)-FORM-2.pdf

385-KOLNP-2007-(19-11-2013)-OTHERS.pdf

385-KOLNP-2007-(19-11-2013)-PA.pdf

385-KOLNP-2007-(19-11-2013)-PETITION UNDER RULE 137-1.pdf

385-KOLNP-2007-(19-11-2013)-PETITION UNDER RULE 137.tif

385-KOLNP-2007-(23-04-2014)-ABSTRACT.pdf

385-KOLNP-2007-(23-04-2014)-ANNEXURE TO FORM 3.pdf

385-KOLNP-2007-(23-04-2014)-CLAIMS.pdf

385-KOLNP-2007-(23-04-2014)-CORRESPONDENCE.pdf

385-KOLNP-2007-(23-04-2014)-DESCRIPTION (COMPLETE).pdf

385-KOLNP-2007-(23-04-2014)-DRAWINGS.pdf

385-KOLNP-2007-(23-04-2014)-FORM-1.pdf

385-KOLNP-2007-(23-04-2014)-FORM-2.pdf

385-KOLNP-2007-(23-04-2014)-OTHERS.pdf

385-KOLNP-2007-ASSIGNMENT.pdf

385-KOLNP-2007-CANCELLED PAGES.pdf

385-KOLNP-2007-CORRESPONDENCE OTHERS 1.3.pdf

385-KOLNP-2007-CORRESPONDENCE.pdf

385-KOLNP-2007-EXAMINATION REPORT.pdf

385-KOLNP-2007-FORM 13-1.1.pdf

385-kolnp-2007-form 13.pdf

385-KOLNP-2007-FORM 18-1.1.pdf

385-kolnp-2007-form 18.pdf

385-KOLNP-2007-GPA.pdf

385-KOLNP-2007-GRANTED-ABSTRACT.pdf

385-KOLNP-2007-GRANTED-CLAIMS.pdf

385-KOLNP-2007-GRANTED-DESCRIPTION (COMPLETE).pdf

385-KOLNP-2007-GRANTED-DRAWINGS.pdf

385-KOLNP-2007-GRANTED-FORM 1.pdf

385-KOLNP-2007-GRANTED-FORM 2.pdf

385-KOLNP-2007-GRANTED-FORM 3.pdf

385-KOLNP-2007-GRANTED-FORM 5.pdf

385-KOLNP-2007-GRANTED-SPECIFICATION-COMPLETE.pdf

385-KOLNP-2007-INTERNATIONAL PUBLICATION.pdf

385-KOLNP-2007-INTERNATIONAL SEARCH REPORT & OTHERS.pdf

385-KOLNP-2007-OTHERS.pdf

385-KOLNP-2007-PETITION UNDER RULE 137.pdf

385-KOLNP-2007-REPLY TO EXAMINATION REPORT.pdf

385-KOLNP-2007-TRANSLATED COPY OF PRIORITY DOCUMENT.pdf


Patent Number 262722
Indian Patent Application Number 385/KOLNP/2007
PG Journal Number 37/2014
Publication Date 12-Sep-2014
Grant Date 08-Sep-2014
Date of Filing 02-Feb-2007
Name of Patentee PERKINELMER SINGAPORE PTE LTD.
Applicant Address 47 AYER RAJAH CRESCENT, #06-12, SINGAPORE 139947
Inventors:
# Inventor's Name Inventor's Address
1 BLACKMORE, COLIN PERKINELMER LIFE SCIENCES, CHALFONT ROAD, SEER GREEN, BEACONSFIELD, BUCKINGHAMSHIRE, HP9 2FX
2 FITCH, ALISTAIR PERKINELMER LIFE SCIENCES, CHALFONT ROAD, SEER GREEN, BEACONSFIELD, BUCKINGHAMSHIRE, HP9 2FX
3 LADHA, SHAB PERKINELMER LIFE SCIENCES, CHALFONT ROAD, SEER GREEN, BEACONSFIELD, BUCKINGHAMSHIRE, HP9 2FX
4 COURTNEY, PATRICK PERKINELMER LIFE SCIENCES, CHALFONT ROAD, SEER GREEN, BEACONSFIELD, BUCKINGHAMSHIRE, HP9 2FX
PCT International Classification Number G01N 21/64
PCT International Application Number PCT/GB2005/003226
PCT International Filing date 2005-08-17
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 0512811.1 2005-06-23 U.K.
2 0419325.6 2004-09-01 U.K.