Alignment process for a single molecule detection instrument

Abstract

A Method and a system for aligning the optical components of a fiber- coupled, single molecule detection instrument for detecting protein concentrations in a prepared assay solution consisting of several components, the system comprising a digital autocollimator alignment telescope to thereby measure reference mirror angle and beam divergence, angle, and position, an instrument alignment frame to thereby constrain the instrument optical frame during the alignment procedure, a DeMux sorter focusing jig to thereby focus detector lenses in the DeMux sorter, objective inserts installed in place of objectives during the alignment procedure and multiple backlights and targets to thereby perform the optical element alignments.

Description

Alignment process for a single molecule detection instrumentBACKGROUND OF THE INVENTION

[0001] The disclosed invention relates to a method for aligning the optical cmponents of a system for a fiber-coupled, single molecule detection instrument.

[0002] The invention belongs to the technical fields of Protein Detection.

[0003] Instrumentation for detection of proteins can take a variety of forms and utilize a variety of techniques. In general, proteins are isolated from a sample and labeled for detection.

[0004] The highest sensitivity fluorescence detection instruments target single molecule accuracy, hence the single molecule detection (SMD) category. One approach for SMD is to limit the interrogated sample volume, often called the confocal volume, typically by use of a spatial pinhole filter. By reduction of the detection volume, out of focus light is blocked. This provides enhanced signal-to- noise and improved ability to identify single fluorescently labeled target molecules.

[0005] An example are the so called high-sensitivity ELISA assays which can quantify targets as low as 10 pg / mL concentrations. However, single-molecule detection instruments can typically achieve orders of magnitude lower sensitivity.

[0006] The SMCxPRO® and SMC® assay product line is the current state of the art platform for detection of ultra-rare protein targets using the single molecule detection technique. The SMCxPRO® utilizes assays created using proprietary preparation techniques, and along with its confocal detection architecture can resolve concentrations as low as 0.01 pg / mL.

[0007] This known state of the art still possess some disadvantages and limitations, like non-optimized signal-to-noise ratio, complex setup, and nonoptimized detection efficiency.

[0008] To comply with and enhance the state of the art a new and advanced SMC® instrument optical design for a SMC® next generation instrument has recently been introduced. It is a method and a detection system for detecting protein concentrations in a prepared assay solution consisting of several components, arranged after each after, the components being a fiber coupled laser for emitting laser light, a laser tube lens to focus the laser light, a laser cleanup filter for blocking non-laser wavelengths, a first dichroic filter for passing the laser light and reflecting fluorescence light, an objective lens to focus the laser light to a small excitation volume, the objective also collecting fluorescence light emitted from a sample, a pre-confocal and a post-confocal lens with a pinhole positioned between them to focus and re-focus the fluorescent light, a second dichroic filter, a focus photodiode for auto-focusing on the sample, at least one bandpass filter for spectrum filtering of the fluorescent light, at least one fiber focus lense for coupling the fluorescent light to an optical fiber, at least one fiber patch cables to couple fluorescently focused light to at least one computer- based detector for signal detection, wherein the sample is located after the objective lens, the second dichroic filter, the focus photodiode and the at least one bandpass filter are forming a De-Multiplexer for the incoming fluorescence light and the at least one bandpass filter, at least one fiber focus lense, at least one fiber patch cables and at least one computer-based detector always consist of same the number and one of each of them are located along a respective fluorescence light.

[0009] Furthermore all optical systems require a systematic methodology to align and secure elements relative to each other. The particular methodology used will depend on factors including cost, ease / skill of implementation, and accuracy / performance requirements.

[0010] The simplest methodology is often referred to as ‘drop in assembly’ in which optical elements and mechanical housings are assembled ‘blind’, with therelative alignment being determined solely by the accuracy and design of the combined components, e.g. mechanical bore dimensions, optical lens edge diameter / tilt / centration. This methodology can be precise, but requires high-cost components due to their stringent accuracy requirements, and even then the precision is limited by current manufacturing technologies.

[0011] A more advanced technique, referred to as ‘active alignment’, can take many forms but is essentially the use of various tooling, such as an alignment telescope, to provide feedback enabling active adjustment of each element, then locking / adhesion of components in the optical assembly. This form of alignment can be exceedingly precise and overcome manufacturing limitations on the accuracy of individual components. Even still, the steps and processes utilized to align and optimize each component need to be carefully thought through, and the opto-mechanical design should be optimized for ease of adjustment and maximum performance stability.

[0012] The mentioned SMC® next generation instrument now requires an optimal alignment procedure for alignment of its optical assembly in order to work properly. Since active alignment procedures provide the best results, it would be a big advantage if a specific active alignment procedure for this SMC® next generation instrument could be provided.BRIEF SUMMARY OF THE INVENTION

[0013] This task can be solved by a system for aligning the optical components of a fiber-coupled, single molecule detection instrument for detecting protein concentrations in a prepared assay solution consisting of several components, the system comprising a digital autocollimator alignment telescope to thereby measure reference mirror angle and beam divergence, angle, and position, an instrument alignment frame to thereby constrain the instrument optical frame during the alignment procedure, a DeMux sorter focusing jig to thereby focus detector lenses in the DeMux sorter, objective inserts installed in place of objectives during the alignment procedure and multiple backlights and targets tothereby perform the optical element alignments. Advantages for this alignment procedure are the high accuracy alignment of the optical assembly, far exceeding the accuracy of the optical elements and opto-mechanical components, e.g. lens and bore centration / tilt tolerances. The result is a system that can be aligned with highly reproducible performance. The optical efficiency is repeatable to less than 2-3% as measured by re-build I re-alignment testing and subsequent measurement using a Raman standard.

[0014] Advantageous and therefore preferred further developments of this invention emerge from the associated sub claims and from the description and the associated drawings.

[0015] One of those preferred further developments of the disclosed system comprise that the digital autocollimator alignment telescope provides separate autocollimator functions, like the measurements of reference mirror angle, and alignment telescope functions, like the measurements of beam divergence, angle, and position.

[0016] Another one of those preferred further developments of the disclosed system comprise that the an instrument alignment frame consists of a customized mechanical frame utilizing pneumatic components to constrain the instrument optical frame rigidly to avoid any drift during the alignment procedure.

[0017] Another one of those preferred further developments of the disclosed system comprise that the DeMux sorter focusing jig used to focus the detector lenses in the DeMux sorter consists of a customized mechanical frame and a jig.

[0018] Another one of those preferred further developments of the disclosed system comprise that the objective inserts installed in place of objectives comprise of an Insert base as insert tooling base with a correct entrance pupil diameter for the alignment; Insert mirror as an angular alignment reference to the objective axis; Insert pinhole as a position alignment reference to the objective axis Insert diffuser as a diffuser glass for coarse beam alignment; Insert neutral density filter for attenuation of high-intensity light; and Insert condenser for Kohler illumination with using a laser as source for the well plate alignment.

[0019] Another one of those preferred further developments of the disclosed system comprise that the multiple backlights and targets comprise of a General broadband backlight; Confocal, diffuse wide-angle, backlight; Pinhole target backlight as a reference point source with a precise location; Focal target backlight as a reference infinity focal source with a precise field angle; Sorter target - crosshair target for line-of-sight alignment in DeMux; and Well plate target as a reference well plate with target pinholes for the alignment.

[0020] Another solution to the given task is a method for aligning the optical components of a fiber-coupled, single molecule detection instrument system using the alignment system as previously described comprising of the following method steps of Setting the alignment telescope as master reference frame; Aligning a primary dichroic filter of the single molecule detection instrument system; Aligning a pre-confocal lens and pinhole filter of the single molecule detection instrument system; Aligning a post-confocal lens filter of the single molecule detection instrument system; Aligning a DeMux sorter dichroic filter of the single molecule detection instrument system; Focussing a DeMux detector and fiber focus lenses of the single molecule detection instrument system; Aligning a DeMux detector fibers of the single molecule detection instrument system; Aligning a laser to be used as a source for a Kohler illumination for a well plate alignment; Aligning the well plate to the master reference frame; and Performing a post-alignment bonding. This method uses the previously disclosed system to align the SMC® next generation instrument.

[0021] A preferred further development of the disclosed method comprise that the setting of the alignment telescope is done by using the insert mirror to align the telescope to the objective axis zero-degree field and the general backlight and insert pinhole to center the telescope to the objective entrance pupil. This step allows to set the telescope as a master reference frame, based on alignment to objective axis.

[0022] Another one of those preferred further developments of the disclosed method comprise that the aligning of a primary dichroic filter is done by providing a reference point source position at two locations on the optical bed via providingthe pinhole target backlight and by focusing the alignment telescope to each point and adjusting the dichroic position to a center image of each point. The method step enables to set the primary dichroic angle and position to a suitable instrument frame optical axis.

[0023] Another one of those preferred further developments of the disclosed method comprise that the aligning of a pre-confocal lens and pinhole filter is done by using a confocal backlight providing a diffuse illumination to pinhole, axially adjusting the pre-confocal lens to focus pinhole image to a minimum divergence and adjusting the pinhole to center the position in the master reference frame. The goal of theis method step is to set the pinhole to an optimal focus and align it to the center of the reference frame.

[0024] Another one of those preferred further developments of the disclosed method comprise that the aligning of the post-confocal lens filter is done by using a focal target backlight to provide an infinity reference target image, axially adjusting the post-confocal lens filter to focus the target image to minimum divergence and laterally adjusting the post-confocal lens filter to to center the target image to the master reference frame. This one allows to set the post- confocal lens to an optimal focus and align the field to an optical axis.

[0025] Another one of those preferred further developments of the disclosed method comprise that the aligning of the DeMux sorter dichroic filter is done by providing an optical line-of-sight measurement inside the DeMux sorter dichroic filter via a general backlight and sorter target and adjusting the DeMux sorter dichroic filter to align with an optical line-of-sight. The purpose of this step is to align the optical line-of-sight, meaning the fluorescence path, inside the DeMux sorter.

[0026] Another one of those preferred further developments of the disclosed method comprise that the focussing a DeMux detector and fiber focus lenses is done by setting up the DeMux sorter dichroic wing assemblies in the DeMux sorter focusing jig and axially adjusting the DeMux detector and fiber focus lenses by a using fiber backlight to minimum the divergence. This allows to pre-focus the detector and fiber focus lenses, meaning to focuse to the detector fibers.

[0027] Another one of those preferred further developments of the disclosed method comprise that the aligning of the DeMux detector fibers is done by installing the the DeMux sorter dichroic wing assemblies onto the DeMux detector, installing the DeMux Detector fiber assemblies and adjusting them laterally to center an image with the master reference frame. That allows to align the sorter detector fibers to the center of the reference frame.

[0028] Another one of those preferred further developments of the disclosed method comprise that the aligning the laser is done by installing the insert diffuser and adjusting the angle of the mirror to turn the laser for a coarse field angle alignment, installing the ND filter and focusing the laser fiber to minimize image divergence, and adjusting the mirror to turn the laser for a final field angle alignment to the master reference frame. The alignment of the laser enables to set the laser to an optimal focus and align its field to an optical axis to the reference frame.

[0029] Another one of those preferred further developments of the disclosed method comprise that the aligning the well plate to the master reference frame is done by installing the well target plate, inserting the laser illumination and condenser for backlighting, and measuring well plate target locations to create a correction matrix for the well plate alignment to the instrument coordinate frame. The purpose of this step is to align the well plate coordinate frame with the instrument coordinate frame.

[0030] Another one of those preferred further developments of the disclosed method comprise that the post-alignment bonding is done by mechanically locking the optical components into place after each alignment step and applying an adhesive at the optial components to permanently cement them at end of alignment procedure. The post-alignment bonding leads to a permanent locking of the optical assembly resulting in a low drift during the life of the instrument.BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

[0031] Figure 1 : A schematic of a SMC® next generation optical layout - up to detection multiplexed sorter (called DeMux in schematic).

[0032] Figure 2: A schematic of the interior of the DeMux of a detection multiplex sorter a SMC® next generation optical layout.

[0033] Figure 3: A CAD rendering of a DeMux sorter focusing jig, developed for optical alignment of the SMC® next generation instrument.

[0034] Figure 4: CAD renderings of an objective insert tooling, developed for optical alignment of the SMC® next generation instrument.

[0035] Figure 5: CAD renderings of a backlight target tooling, developed for alignment of the SMC® next generation instrument.

[0036] Figure 6: CAD renderings of a sorter and well plate tooling, developed for optical alignment of the SMC® next generation instrument.

[0037] Figure 7: A CAD rendering of SMC® next generation optical bed. Not all components are visible in this rendering.DETAILED DESCRIPTION OF THE INVENTION

[0038] The invention will be explained in more detail by presenting one preferred exemplary embodiment of the system and method of the invented alignment process for the next generation SMC instrument.

[0039] The primary tooling used for the preferred embodiment of instrument alignment is listed below and displayed in Figures 3 to 6.

[0040] The components of the preferred system embodiment are as follows:

[0041] 1 . A digital autocollimator alignment telescope comprising of an OEM digital alignment telescope with Autocollimator functions in form of measurements of reference mirror angle, and comprising of alignment telescope functions in form of measurements of beam divergence, angle, and position.

[0042] 2. An instrument alignment frame comprising of a custom mechanical frame utilizing pneumatic components to constrain the instrument optical frame rigidly with no drift during the alignment procedure.

[0043] 3. A DeMux sorter focusing jig comprising of a custom mechanical frame and jig used to focus detector lenses in DeMux sorter.

[0044] 4. Objective insertsin form of tooling installed in place of objective during alignment, comprising of an insert base in form of insert tooling base with correct entrance pupil diameter for alignment, comprising of an insert mirror in form of angular alignment reference to objective axis, comprising of an insert pinhole in form of position alignment reference to objective axis, comprising of an insert diffuser in form of diffuser glass for coarse beam alignment, comprising of an insert ND filter in form of neutral density filter for attenuation of high-intensity light, and comprising of an insert condenser in form of Kohler illumination, using laser as source, for well plate alignment.

[0045] 5. Backlights and targets in form of tooling used for optical element alignments, comprising of a general backlight in form of a broadband backlight, comprising of a confocal backlight in form of a diffuse wide-angle backlight, comprising of a pinhole target backlight in form of a reference point source with precise location, comprising of a focal target backlight in form of a reference infinity focal source with precise field angle, comprising of a sorter target in form of acrosshair target for line-of-sight alignment in DeMux, and comprising of a well plate target in form of a reference well plate with target pinholes for alignment.

[0046] The preferred embodiment of the alignment method comprises of the following alignment steps. The Figures 1 and 2 provide reference images of the SMC® next generation instruments optical layout and Figure 7 a CAD rendering of the required optical bed for the SMC® next generation instrument.

[0047] The method steps are:

[0048] 1 . Set alignment telescope as master reference frame with the goal to set telescope as master reference frame, based on alignment to objective axis and the steps of a) Insert mirror used to align telescope to objective axis zero-degreefield and b) General backlight and insert pinhole used to center telescope to objective entrance pupil.

[0049] 2. Alignment of primary dichroic with the goal to set primary dichroic angle and position to instrument frame optical axis and the steps of a) Pinhole target provides reference point source position at two locations on the optical bed and b) Telescope is focused to each point, dichroic position adjusted to center image of each point.

[0050] 3. Alignment of pre-confocal lens and pinhole with the goal to set pinhole to optimal focus and align to center of reference frame and the stepd of a) confocal backlight provides diffuse illumination to pinhole, b) Pre-confocal lens is adjusted axially to focus pinhole image to minimum divergence and c) Pinhole is adjusted to center position in reference frame.

[0051] 4. Alignment of post-confocal lens with the goal to set post-confocal lens to optimal focus and align field to optical axis and the steps of a) Focal target backlight provides infinity reference target image, b) Post-confocal lens adjusted axially to focus target image to minimum divergence and c) Post-confocal lens adjusted laterally to center target image to reference frame.

[0052] 5. Alignment of DeMux sorter dichroic with the goal to align optical I ine- of-sight (fluorescence path) inside DeMux sorter and the steps of a) General backlight and sorter target provide optical line-of-sight measurement inside DeMux and b) Dichroic adjusted to align optical line-of-sight.

[0053] 6. Focus of DeMux detector / fiber focus lenses with the goal to pre-focus detector / fiber focus lenses, focusing to detector fibers, and the steps of a) DeMux sorter wing assemblies are setup in sorter focusing jig and b) Detector / fiber focus lenses are adjusted axially using fiber backlight to minimum divergence.

[0054] 7. Alignment of DeMux detector fibers with the goal to align sorter detector fibers to center of reference frame and the steps of a) DeMux sorter wing assemblies are installed onto DeMux and b) Detector fiber assemblies are installed, adjusted laterally to center image with reference frame.

[0055] 8. Alignment of Laser with the goal to set laser to optimal focus and align field to optical axis, meaning the reference frame, and the steps of a) Insert diffuser installed, laser turning mirror angle adjusted for coarse field angle alignment, b) Insert ND filter installed, laser fiber is focused to minimize image divergence and c) Laser turning mirror adjusted for final field angle alignment to reference frame.

[0056] 9. Well plate alignment to master reference frame with the goal to align well plate coordinate frame with instrument coordinate frame and the steps of a) Well plate target installed, laser illumination and condenser insert used for backlighting and b) Well plate target locations measured and used to create correction matrix for well plate alignment to instrument coordinate frame.

[0057] 10. Post-alignment bonding with the goal to permanent locking of optical assembly for low drift during life of instrument and the steps of a) After each alignment step components are mechanically locked into place and b) At end of alignment procedure, adhesive is applied to permanently cement all components.

[0058] This alignment procedure and system allows a high accuracy alignment of the optical assembly, far exceeding the accuracy of the optical elements and opto-mechanical components, e.g. lens and bore centration / tilt tolerances. The result is a next generation instrument system that can be aligned with highly reproducible performance. The optical efficiency is repeatable to less than 2-3% as measured by re-build I re-alignment testing and subsequent measurement using a Raman standard.

Claims

CLAIMS1. A system for aligning the optical components of a fiber-coupled, single molecule detection instrument for detecting protein concentrations in a prepared assay solution consisting of several components, the system comprising a digital autocollimator alignment telescope to thereby measure reference mirror angle and beam divergence, angle, and position, an instrument alignment frame to thereby constrain the instrument optical frame during the alignment procedure, a DeMux sorter focusing jig to thereby focus detector lenses in the DeMux sorter, objective inserts installed in place of objectives during the alignment procedure and multiple backlights and targets to thereby perform the optical element alignments.

2. A system according to claim 1 , wherein the digital autocollimator alignment telescope provides separate autocollimator functions, like the measurements of reference mirror angle, and alignment telescope functions, like the measurements of beam divergence, angle, and position.

3. A system according to claim 1 , wherein the an instrument alignment frame consists of a customized mechanical frame utilizing pneumatic components to constrain the instrument optical frame rigidly to avoid any drift during the alignment procedure.

4. A system according to claim 1 , wherein the DeMux sorter focusing jig used to focus the detector lenses in the DeMux sorter consists of a customized mechanical frame and a jig.

5. A system according to claim 1 , wherein the objective inserts installed in place of objectives comprise of an• Insert base as insert tooling base with a correct entrance pupil diameter for the alignment;• Insert mirror as an angular alignment reference to the objective axis;• Insert pinhole as a position alignment reference to the objective axis• Insert diffuser as a diffuser glass for coarse beam alignment;• Insert neutral density filter for attenuation of high-intensity light; and• Insert condenser for Kohler illumination with using a laser as source for the well plate alignment.

6. A system according to claim 1 , wherein the multiple backlights and targets comprise of a• General broadband backlight;• Confocal, diffuse wide-angle, backlight;• Pinhole target backlight as a reference point source with a precise location;• Focal target backlight as a reference infinity focal source with a precise field angle;• Sorter target - crosshair target for line-of-sight alignment in DeMux; and• Well plate target as a reference well plate with target pinholes for the alignment.

7. A method for aligning the optical components of a fiber-coupled, single molecule detection instrument system using the alignment system according to the claims 1 to 6 comprising the following steps:• Setting the alignment telescope as master reference frame;• Aligning a primary dichroic filter of the single molecule detection instrument system;• Aligning a pre-confocal lens and pinhole filter of the single molecule detection instrument system;• Aligning a post-confocal lens filter of the single molecule detection instrument system;• Aligning a DeMux sorter dichroic filter of the single molecule detection instrument system;• Focussing a DeMux detector and fiber focus lenses of the single molecule detection instrument system;• Aligning a DeMux detector fibers of the single molecule detection instrument system;• Aligning a laser to be used as a source for a Kohler illumination for a well plate alignment;• Aligning the well plate to the master reference frame; and• Performing a post-alignment bonding.

8. A method according to claim 7, wherein, the setting of the alignment telescope is done by using the insert mirror to align the telescope to the objective axis zero-degree field and the general backlight and insert pinhole to center the telescope to the objective entrance pupil.

9. A method according to claim 7, wherein, the aligning of a primary dichroic filter is done by providing a reference point source position at two locations on the optical bed via providing thepinhole target backlight and by focusing the alignment telescope to each point and adjusting the dichroic position to a center image of each point.

10. A method according to claim 7, wherein, the aligning of a pre-confocal lens and pinhole filter is done by using a confocal backlight providing a diffuse illumination to pinhole, axially adjusting the pre-confocal lens to focus pinhole image to a minimum divergence and adjusting the pinhole to center the position in the master reference frame.

11. A method according to claim 7, wherein, the aligning of the post-confocal lens filter is done by using a focal target backlight to provide an infinity reference target image, axially adjusting the post-confocal lens filter to focus the target image to minimum divergence and laterally adjusting the post-confocal lens filter to to center the target image to the master reference frame.

12. A method according to claim 7, wherein, the aligning of the DeMux sorter dichroic filter is done by providing an optical line-of-sight measurement inside the DeMux sorter dichroic filter via a general backlight and sorter target and adjusting the DeMux sorter dichroic filter to align with an optical line-of-sight.

13. A method according to claim 7, wherein, the focussing a DeMux detector and fiber focus lenses is done by setting up the DeMux sorter dichroic wing assemblies in the DeMux sorter focusing jig and axially adjusting the DeMux detector and fiber focus lenses by a using fiber backlight to minimum the divergence.

14. A method according to claim 7, wherein,the aligning of the DeMux detector fibers is done by installing the the DeMux sorter dichroic wing assemblies onto the DeMux detector, installing the DeMux Detector fiber assemblies and adjusting them laterally to center an image with the master reference frame.

15. A method according to claim 7, wherein, the aligning the laser is done by installing the insert diffuser and adjusting the angle of the mirror to turn the laser for a coarse field angle alignment, installing the ND filter and focusing the laser fiber to minimize image divergence, and adjusting the mirror to turn the laser for a final field angle alignment to the master reference frame.

16. A method according to claim 7, wherein, the aligning the well plate to the master reference frame is done by installing the well target plate, inserting the laser illumination and condenser for backlighting, and measuring well plate target locations to create a correction matrix for the well plate alignment to the instrument coordinate frame.

17. A method according to claim 7, wherein, the post-alignment bonding is done by mechanically locking the optical components into place after each alignment step and applying an adhesive at the optial components to permanently cement them at end of alignment procedure.

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