LS Reloaded Versatile automation-friendly scanning solutions for many microarray formats in research

Transcription

1 LS Reloaded Versatile automation-friendly scanning solutions for many microarray formats in research 1

2 Automated fluorescence laser scanning for advanced research use Three solutions offering the very latest developments in sample scanning Accelerated and reliable analysis With its automation-ready design, parallel dual-channel detection capability, and its ability to operate up to 4 excitation lasers and 24 emission filters, the LS Series Laser Scanner greatly accelerates fluorescence laser scanning. Increased throughput and multiplexing capabilities for array imaging and analysis mean savings in both time and resources. The possibility of having three different grades of confocal measurements together with an autofocus system gives required flexibility and the high reproducibility in sample imaging for diverse applications and format sizes. Spotting Quality Control (QC) by scattered light detection after printing even without the use of dyes permits chips of insufficient quality to be excluded prior to hybridization, saving time and money. LaserCheck installation and operation quality control tool kit provides up to date reports on every LS unit regarding mechanics, electronics and optics. Different formats one solution The introduction of new applications in the post-genomic era is giving rise to novel microarray formats and sizes, including new protein and tissue arrays. The LS Series Laser Scanner provides flexible scanning technology that allows researchers to automatically scan many different sample formats for fluorescence such as glass slides, microplates, membranes, gels, blots, colony plates, and even encapsulated features in micro channels all on one instrument. Increased sensitivity The LS Reloaded from Tecan is the first fluorescence laser scanning system to support dual color Evanescence Resonance measurements. This unique chip scanning technology maximizes sensitivity by about 10-fold for dual laser applications (532 to 633 nm excitation). Seamless integration The universal 4-slide adapter seamlessly interfaces with Tecan s automated HS 400 Pro and HS 4800 Pro slide hybridization stations as well as the Connect autoloader and each of the LS Reloaded laser scanners without further slide manipulation. Array-Pro Analyzer provides every conceivable image analysis tool to characterize the measurement system and achieve significant signal/noise ratios, repeatability of results, and quantitative accuracy. 3x1 inch microscope slide on universal 4-slide adapter. The LS Reloaded and Array-Pro Analyzer are seamlessly linked to provide an indispensable tool for microarray and high-throughput screening research. Such advances are driving the revolution in Genomics, Proteomics, Drug Discovery, and Bioinformatics. The Quality Control, sensitivity, automation and throughput improvements provide reliable sample imaging and analysis for appropriate statistics and downstream data mining. In addition, with its unprecedented flexibility, the LS Reloaded Series of Laser Scanners and the Array-Pro Analyzer guarantee that existing and future requirements of analysis in the rapidly growing field of micro-feature formats and bioinformatics will be met. Confocal upside-down scanning of microarrays in 384- and 96-well glass bottom microplates by LS Reloaded 2

3 Innovative Features for today s demanding Applications Adjustable Angle of Incidence Higher sensitivity with Evanescence Resonance technology from Novartis offers the advantage of detecting expression of low abundance genes from limited amounts of sample, such as in a biopsy assay. The continuously adjustable angle of incidence of laser beam enables the fast scanning of slides supporting evanescence resonance technology, even when more than one laser/dye combination is used. Therefore, more genes are accessible from smaller amounts of sample by increasing optical sensitivity (SNR) up to 20-fold. In addition, for samples with three dimensional (3D) structures such as wells, pillars, or chambers, the angle of incidence can be optimally adjusted to avoid undesired shadowing or reflection effects. LS Reloaded support Novartis Evanescence Resonator glass chip technology: Evanescence Resonance depends on angle of incidence of the excitation laser beam. The adjusted angle of incidence enables signal amplification (background subtracted) up to a factor of 20. Adjustable incidence of angle of laser beam: Support of Evanescence Resonance Field generation and optical requirements of other special three dimensional formats. Autofocus Sample Positioning Gain of biological information by enabling Evanescence Resonance with LS Reloaded: More spots are detectable (SNR >3) by improved scanning optics covering 4.5 logs of dynamic range. Each sample can be automatically focused by an intrinsic pre-autofocus system. With its sophisticated 5-axis transport system, the LS Reloaded accurately corrects for height and 2 tilt angles to compensate for variable samples and geometric tolerances in order to assure low variations in inter-slide and intra-slide scan measurements. Automatic sample position adjustments to the required focal plane by autofocus and 5-axis transport table of LS Reloaded achieve maximal intra-slide uniformity and inter-scan reproducibility. 3

4 Options to increase sensitivity LS Reloaded automatically distinguishes between different surfaces of a sample by using the difference of refraction indices of the composite layers. The resulting total reflection is used in a pre-autofocus z-scan to correct automatically for planar geometry of the chosen reference surface (one out of three at the same time) in the focus. This enables, for example, top reading through glass or plastics (inverted slide loading) to increase sensitivity by 2-fold due to higher photon collection efficiency on glass slides. Although 20 Hz linear resonance scanning is fast, especially for moving optics characterized by a high working distance of 6.5 mm, it is possible to achieve high sensitivity. This is done by generating and collecting more photons from the sample via the option for multiple average scanning and optimizing dwell (integration) time in the software. This option increases sensitivity again by 1.4 to 3 times due to prolonged duration of laser exposure and photon collection time. Increased photon collection efficiency: Option for scanning of fluorescence through glass by LS Reloaded. Larger pinhole: non-confocal Confocal and Non-confocal The confocal mode of the LS Reloaded not only provides better background suppression, but also better discrimination between bright and dark signals in the sample. The LS Reloaded offers three user-selectable pinhole sizes, which give the user three grades of confocal measurements. While handling samples of considerable optical depth (e.g. flow-through chips, gels) or non-flat surfaces, the instrument can be easily switched from confocal to non-confocal mode. Small pinhole: confocal For bulky Samples Non-confocal scan achieves optimized signal and homogeneity on bulkysamples Filter or Flow-through matrix. Confocal scan non-confocal CCD image Better signal to background ratio on planar surfaces of glass and plastics. QC-Gel with holder: Fluorescence labelled cdna scanned by LS Reloaded. 4

5 Simultaneous 2-color scanning High throughput sequencing, single nucleotide polymorphism (SNP) detection, comparative genome hybridization, gene expression analysis and many other applications often demand for multiplexed labeling. The option of simultaneous dual-channel detection allows two dyes to be measured at the same time for maximum Autoloading: 4-slide adapter or inverted microplate loading on LS x00 Series Laser Scanner by Tecan s Connect Slide-QC: Scattered Light Detection imaging throughput. A single dye calibration step is sufficient to give the required specificity for each measurement. Unwanted signals from dyes in either channel are easily accounted for rapid recalculations using accompanied software. Sequential scanning of three and more dyes or batch scanning with different gain settings for extended dynamic range can be performed by customized definition of simple scripts. For quick and easy quality checks of spots and entire arrays before hybridization has been performed, the LS Reloaded has an additional standard absorbance filter for scattered light detection. Independent of the laser, the spotting-buffer deposition (shape) as well as surface artifacts on slide can be detected in a QC scan after array production: No additional use of dyes and no bleaching effects. Defective or inhomogeneous coating, irregular or missing spots as well as artifacts (e.g. scratches, dust) on the slide are detectable before valuable labeled sample is applied. An automated batch run of scan and image analysis for local and global background, spot intensity, spot morphology, and spot position, can be applied. A report and flag of passed or failed spots and slides ensure that only slides that have passed QC test are hybridized. Operates up to 4 lasers and 24 filters Each LS Reloaded is equipped with 633 nm laser. Instruments equipped with two up to four lasers (594 nm, 532 nm and 488 nm) are available as additional options or can be refit. A huge selection of additional fluorescence emission filters is available. A collection of up to 24 emission filters can be used for one instrument at a time. Batch Processing A 4-slide holder with the same outside dimensions as a standard (SBS) microplate allows automatic sample loading with standard stacking devices such as Tecan s Connect autoloader. Even without automatic loading, up to four standard microscope slides can be processed with no further user intervention in one run. In addition, almost any user-defined method can be applied to up to 200 slides or 50 microplates in a single run allowing fully automated image acquisition and analysis. QC of array printing prior to hybridization: Scattered light measurement by LS Reloaded detects irregularities on the surface and misprinting. 5

6 Hybridization, scanning, analysis: seamlessly interfaced suite Tecan s Microarray Suite covers the automation of the process from sample preparation to image analysis. Effective sample handling is critical for generating reproducible data. Therefore, slide handling can be less critical if the slides are fixed in a 4-slide adapter, which avoids further touching of the slides after being removal from the box. Hybridization, washing and drying of the slides can be performed automatically on Tecan s HS 400 Pro and HS 4800 Pro Hybridization Stations. Slides processed on the HS series are ready for direct scanning on the LS Reloaded without further processing. From Images to Solution: MediaCybernetics Array-Pro Analyzer 4.5 highly sophisticated image processing software is implemented and fully integrated with instrument control. HS x00 Pro series of Hybridization Stations. Image Analysis Software MediaCybernetics Array-Pro Analyzer 4.5 patented automatic spot-finding algorithms, wizard guided grid-tofeature alignment and state-of-the art statistical tools (normalization tools various techniques to characterize background and signal) are available for spot quantification and qualification. These tools are fully implemented in the instrument control of the LS Reloaded and can be applied automatically. Procedures can be configured by, or for the user to specific laboratory standards and requirements. The individually defined method can be applied to large batch processes for rapid automated data creation. The open software structure with macro programming language (Visual Basic) enables highly sophisticated creation of methods to automate all processes of spot and image analysis. Interactive and dynamic-linked graphics and data tables with user-defined selection rules for filtering and automatic spot flagging provide a complete overview at a glance. Many efficient image-processing tools enable optimized image display. The software also allows results to be stored, retrieved and printed at any time. File formats like *.GAL, *.txt or *.csv supporting gene annotations (ID) can be directly imported. Direct URL link to public databases as well as interactive share of data with cluster analysis software such as GeneMaths XT from Applied Maths enables sophisticated data mining. Determination: Different global or local referencing methods of signal, background, and normalization can be permutated for method optimization. Statistical Feedback: Extensive statistical user feedback to establish the quality of the data and the experiment by calibrating standards, controls and replicates. Free application of any userdefined selection rule for automatic spot flagging. 6

7 Integrated System: Workstation Tecan develops and supports customized system integration to the highest possible level. This covers almost any kind of automated process from liquid handling and detection up to analysis and fully automated lab systems. The Freedom Evo provides the flexibility to arrange devices not only on the worktable but also around it and even underneath the worktable. One example is the complete integration of the LS Series Laser Scanner in an optimized workflow system driven by a Tecan FACTS driver. Such a system is suitable for high sensitive integrated microplate fluorescence scanning and tagged expressing colony (hit) picking. LS 400 integration in Genesis Freedom molecular biology workstation for colony hit picking. Continuously increased functionality in soft- and hardware Automatic gain control Even in a batch of similarly processed slides the quality and level of fluorescence signal can differ over a wide range. Tecan is therefore offering Automatic Gain Control AGC to automatically adjust for the PMT settings during a batch mode. Each individual slide is scanned appropriately using an optimised dynamic range without saturation effects. Interactive Pre-scan Options Individual areas of interest (spotted area) require for fast pre-scanning of a specific array type or other samples in order to reduce scanning time and size of image files in subsequent routine. The precise scan area definition as well as barcode scan region, the reference area for gain adjustment, and positioning for autofocus can be defined by the user with simple mouse clicks in the appropriate prescan images. To scan fluorescing sample with and without focusing on a reference surface the optimal focus position can be determined directly in a z-scan image as a visual interface. This provides an optimal manual focusing on special formats with fluorescing samples, which are not directly located on a planar and reflecting surface. A constant offset of the fluorescing sample from a reference surface can be determined interactively in the z-scan image without further calculation needed. This enables advanced manual as well as automatic focusing. Increasing numbers of different formats are used on our already installed systems. New formats are becoming available every year. To support customer convenience using our laser scanner system, Tecan improves the customized format definition process to maximized ease-ofuse in seconds. Multiplex scanning of more than 3000 colonies per plate for fluorescence-tag hit picking. LaserCheck Customized file serving Increasing number of customers are using microarrays as a high throughput screening assay for analyzing a vast number of samples. Tecan is recognized as a leading provider of automation solutions worldwide. The LS Series of Laser Scanners is no exception enabling flexibility and appropriate performance. To support customers Laboratory Information Management System (LIMS) with user-defined file management settings, Tecan provides further flexibility without compromising safety. LaserCheck plate (IQ/OQ) Tecan provides a check tool, which enables convenient automatic quality control routine of LS x00 and LS Reloaded Series Laser Scanners in customer labs with performance tests beyond so called fluorescing standards. 7

8 LS Reloaded specifications General Possible Laser Sources: 7 mw 633 nm HeNe, 10 mw 532 nm solid state, (power tunable) 20 mw 488 nm solid state, 3.5 mw 594 nm HeNe Detectors 1 or 2 PMTs (for optional simultaneous dual color detection) Emission Filters 1 or 2 filter slides, space for 4 filters each 1 Laser System: CY5 (692/40) filter, 2-4 Laser System: CY5, ROX (635/35), CY3 (575/50), FITC (535/25) standard filters Additional filters can easily be added. Up to 24 filters can be applied per system and registered by LS Software. Substrates as microscope slides, microplates or any other substrate up to the size of microplates (15x85x127 mm) Scan area slides 22x75 mm one run; 1-96 scan areas over an area up to microplate footprint (automated stitching) Autofocus for transparent or not-transparent surfaces with or without segmentations performed in advance to each individual scan Depth of Focus (100-80% intensity) 90, 300, 800 µm by 3 software selectable pinhole sizes (0.35 to 3.0 mm). Working Distance 6.5 mm (focus) NA 0.6 Angle of laser beam 25-0 for evanescence resonance scanning and three-dimensional surfaces Interface to PC serial and high-speed data acquisition connection Power V auto-sensing, 50 /60 Hz, 250 VA Dimensions 812 mm (W) x 566 mm (D) x 355 mm (H) Weight 60 kg Performance Signal to electronic noise 5 orders of magnitude Sensitivity <0.1 Fluorophore equivalent/µm 2 <0.01 Fluorophore equivalent//µm 2 by adjusted laser beam angle for evanescence resonance slide scanning Intrascenic Dynamic Range 4 orders of magnitude Gain Adjustment 350 V V enables 5 orders of magnitude signal amplification Pixel Resolution 6, 10, 20, 40 µm Reading speed 4 minutes for a full slide, 25 minutes a SBS plate (dual color, 10 µm pixel resolution), PC (minimum configuration delivered with instrument) Pentium IV 2,6 GHz, 2 GByte RAM, 80 GByte Hard Disk, CD-ROM /DVD Writer, Network card, Analog Modem, ArrayPro USB protection key (single site or network), 17" Color TFT Monitor, Microsoft Windows 2000, Office Software package, PCanywhere, WinZip Options Barcode reader for top- or alternative bottom-reading of microscope slides Additional 4-slide adapter for 4 (25x75x1 mm) slides LaserCheck quality control tool kit (SBS format) Gel-adapter with lid e.g. for (72x108x5 mm) gels Adapter for larger (75x116x1 mm) glass plates Additional filter slide carrier for 4 emission filters Additional fluorescence (e.g.520/10 nm) and absorbance filter from filter stock Connect (LS configuration) autoloader for automated 4-slide adapter and inverted MTP stacking and loading Tecan Group Ltd. is making all efforts to include accurate and up-to-date information into this publication. Yet, it cannot be ruled out that omissions or errors might have occurred. Therefore, Tecan Group Ltd. cannot make any representations or warranties, expressed or implied, as to the accuracy or completeness of the information provided in this publication. Changes in this publication can be made any time without notice. All mentioned trademarks are protected by law. For technical details and detailed procedures of the specifications provided in this document please contact your Tecan representative. 2006, Tecan Trading AG, Switzerland, all rights reserved. Tecan is in major countries a registered trademark of Tecan Group Ltd., Männedorf, Switzerland. Genesis Freedom, Freedom Evo are registered trademarks, HS 400 Pro, HS 4800 Pro,Connect, LS x00, LS Reloaded, FACTS, LaserCheck are trademarks of Tecan Group Ltd., Männedorf Switzland. Array-Pro Analyzer is a registered trademark of MediaCybernetics, Silver Springs, USA. GeneMaths XT is a registered trademark of Applied Maths BVBA, Sint-Mertens-Latem, Belgium Tecan Asia (Pte) Ltd., T , Tecan Sales Austria GmbH, T , Tecan Sales International GmbH, T , Tecan Benelux B.V.B.A., T , Tecan Benelux B.V.B.A., T , Tecan Group Ltd., Beijing Rep. Office, T , Tecan Deutschland GmbH, T , Tecan France S.A.S., T , Tecan Italia S.r.l., T , Tecan Sales International GmbH, T , Tecan Japan Co. Ltd, T , Tecan Nordic AB, T , Tecan Nordic AB, Rep. Office Denmark, T , Tecan Portugal, T , Tecan Sales Switzerland AG, T , Tecan Iberica Instr. S.L., T , Tecan UK Ltd., T , Tecan US Inc., T V.1.0,