Patent Application: US-15979007-A

Abstract:
methods and apparatus relating to the imaging of biological samples are provided . more particularly , they relate to the detection of light emanating from fluorescent species present in a sample in order to study the structure and dynamics of such a sample . such a method of analysis comprises irradiating the sample with a pulse of excitation energy causing fluorescent species in the sample to fluoresce ; detecting light emanating from the sample during a predetermined period of time after the pulse ; generating and storing data recording at least the wavelength of the detected light against time ; and analysing the data with reference to the respective lifetimes of the fluorescent species to detect the presence of the respective emissions from three or more different fluorescent species which emit light simultaneously during at least part of said predetermined period , which are indistinguishable from each other on the basis of their wavelength or lifetime alone .

Description:
an embodiment of the invention is presented by way of illustration in fig6 , where species a , b , c , d , e and f may be discriminated in the combined wavelength and lifetime domain employing the techniques described herein . a wavelength - only measurement would separate a / e / d from b / c / f but be unable to separate a from e from d , and likewise for b , c and f , while a lifetime - only would separate a / f from b / e from c / d , but again be unable to separate the between these pairs . in fact there is no combination of simple wavelength - only or lifetime - only way of distinguishing these 6 species . the figure clearly shows the large area free to be populated by species allows much greater multiplexing ( within limits of detection , time etc ). this offers an improved signal to noise due to low background . interfering auto - fluorescence typically appears in short lifetime region (& lt ; 2 ns ) and may be gated out . the detection can be extended by scanning excitation and / or emission wavelengths and polarisation for estimation of optical rotation ( fig7 ), and combinations thereof . an embodiment of apparatus for fluorescence detection is shown in fig8 . it comprises : an excitation light source ( 1 ) which may be modulated at least in time and from which an excitation band may be selected ; a delivery means ( 2 ) to deliver this excitation energy to a sample ; a sample ( 3 ) in a sample holder containing at least one species ( 3 a ) with the potential to exhibit at least fluorescence ; a delivery means ( 4 ) to deliver emission energy from the sample towards a detector ; one or more detectors ( 5 ) onto which emission energy is directed sensitive to some specific aspect of the emission energy including at least time and wavelength using combination of time gating and filters ; a means to capture ( 6 ) the signal from the detector / s into a digital storage device such as a computer memory for further processing , analysis and display ( 7 ). in a preferred embodiment , the excitation light source ( 1 ) is agile — that is — it may be programmed to emit specific pulse protocols in which the duration of pulses , waveband and power of each pulse , and timing between pulse may be specified and changed quickly . this may be used to selectively excite or saturate particular species in the sample according to the sensitivity of that species to particular pulse types and decay lifetimes , over a wide range , for example combining long ms lifetime lanthanide with short ns lifetime small molecule species thus overcoming dynamic range limitations of existing instruments as well as improving capture speed . in a further embodiment , the detector ( 5 ) is configured to have regions sensitive to different wavebands so that all photons may be captured . incoming photons are first separated into two beams according to their wavelength band by a dichroic mirror . the photons are then projected onto one of two time - gated detectors . this ensures that every photon within a particular time window is captured by one or other of the detectors . this permits collection of both wavelength and lifetime data and thus more efficient and faster operation with less bleaching . in another embodiment , the detector ( 5 ) is replaced by one which is sensitive to both photon energy and timing [ fraser 2003 ]. this permits collection of both wavelength and lifetime data and thus more efficient and faster operation with less bleaching . the excitation light source ( 1 ) may comprise one or more lasers , diode laser , dpss , light emitting diode / s , and / or a supercontinuum source . the delivery means ( 2 ) may comprise one or more lens assemblies , optical fiber , mirror , dichroics , etc . the sample ( 3 ) may comprise a cell , adherent cell layer , suspension of cells , or assembly of beads . the sample holder may include a mechanism for transport along the z axis , carriage and transport in xy , and incubator for live cells , means for dispensing of fluids , control of temperature . the fluorescent species ( 3 a ) may include one or more small fluorescent molecules such as fluoroscein , genetically encoded fluorescent molecules such as gfp , intrinsic species such as fad and nadh , puretime dyes , eu - chelates , biosensors such as fura , quantum dots etc . the detector ( 5 ) may comprise one or more pmts , a spatially sensitive detector , a gating element such as an mcp , an imaging detector , a spatially partitioned imaging detector , an array of wavelength sensitive detectors , combined with one or more selection mechanisms including time window , waveband and scanning mechanisms for imaging . the data capture and processing system ( 6 , 7 ) may include means for analysis such as deconvolution and other transforms , and means for collecting a time course series to calculate kinetic data . the present method enables robust fret experiments to be carried out using the fret - flim method and to allow multiple fret systems to be operated at once to determine for example the sequence of events within a particular cellular pathway . as shown in fig9 , one fret process may be studied by observing the change in lifetime of a donor d 1 at a particular waveband , while another fret process may be studied by observing the change in lifetime of a donor d 2 at another waveband . some further examples of applications utilising the present method are illustrated in fig1 a to 10 c . they show the advantage of using combined emission and lifetime to identify and localise characteristics of interest . these examples are only illustrative and it will be appreciated that other combinations are possible with different labels , including intrinsic ( naturally present ) fluorophores . possible characteristics of interest include ( 1 ) biological function — for example protein - protein interaction ( as indicated by a fret signal ), ( 2 ) micro - environment , for example ion concentration such as ph , and ( 3 ) presence of a structural or biochemical species , for example dna or rna . these three characteristics are often important in drug discovery to understand the state of a live cell as it responds to a stimulus . fig1 a shows the use of a combined emission and lifetime analysis system for an excitation waveband around 488 nm , comprising : a functional probe d which represents the donor of a fret pair e . g . gfp from a gfp - yfp fret system with emission at 505 - 530 nm and lifetime between 1 . 5 ns and 2 . 5 ns according to the status of the interaction ( fret / no fret ); a probe sensing the micro - environment , where b and f represents the extremes of the ph probe resorufin with emission around 580 nm and a lifetime from 0 . 5 to 3 ns ; a structural probe c such as the nuclear stain bodipy with emission at 500 - 520 nm with a 6 ns lifetime . furthermore , e represents a functionalised quantum dot probe with an emission at 625 nm and a complex lifetime . fig1 b shows another example , also using an excitation waveband around 488 nm , in which d is a nucleic acid probe such as yoyo - 1 with emission at 510 - 550 nm and a lifetime of 1 . 5 ns when bound to at - rich dna and 4 . 1 ns when bound to gc rich dna ; c is a functional probe based on the puretime22 dye with emission at 560 nm with a 22 ns lifetime ; b and f are extremes of an environmental probe such as the lip order dye di - 4 - aneppdhq with emission around 570 - 630 ns and a lifetime from 1 . 8 to 3 . 6 ns ; and e represents a functionalised quantum dot probe with an emission at 700 nm and a complex lifetime . the benefit of such a scheme is that several types of qualitative (‘ is the structure present ?’) and quantitative (‘ what is the ph ?’) question may be asked of the system at the same time , in comparison to conventional approaches which only allow a very small number of qualitative questions . the examples are for an excitation wavelength of 488 nm or similar . the examples can clearly be extended for other and multiple excitation wavelengths . for example , the excitation band may be switched from 405 nm for a gfp2 - yfp fret pair , to 488 nm for an egfp - yfp fret pair . in a further example ( fig1 c ), an excitation waveband around 360 nm was used , a functional probe b could be used based on a coumarin derivative with a emission at 450 nm and a lifetime of 3 ns ; the presence of chloride ions could be sensed using a probe c such as mqae with an emission band at 460 nm and a characteristic lifetime of 25 ns ; a structural component d such as the nucleus could be sensed using the dapi probe with emission at 460 nm and a lifetime of 0 . 2 - 2 ns . the same system can also simultaneously utilise a series of probes ( e ) based on functionalised quantum dots which are also excited at 360 nm and each emit in a waveband to be selected according to the quantum dot , typically from 500 nm to 800 nm and beyond . r . neher and e . neher , optimizing imaging parameters for the separation of multiple labels in a fluorescence image , j . microscopy , 213 ( 1 ), january 2004 , pp . 46 - 62 . s . vikström , m . korte , p . pulli , p . hurskainen and c . gripenberg - 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