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Formulatrix ROCK IMAGER RI 1000 User Manual

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ROCK IMAGER
User's Guide
Original Instructions
Applies to:
RI 1000, RI 182, RI 54, RI 2
FRAP Benchtop & SONICC Benchtop

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  • Page 1 ® ROCK IMAGER User’s Guide Original Instructions Applies to: RI 1000, RI 182, RI 54, RI 2 FRAP Benchtop & SONICC Benchtop...
  • Page 2 ROCK IMAGER User’s Guide Sixth Edition Revised January 2019 RIG-V37R019...
  • Page 3 Acknowledgements SONICC was invented by Garth Simpson's group at Purdue University and is exclusively licensed by FORMULATRIX. FRAP was developed in collaboration with Vadim Cherezov (TSRI) and the JCIMPT center led by Ray Stevens and supported by the NIH Common Fund in Structural Biology.

  • Page 4: Table Of Contents

    Table of Contents Table of Contents...................................iii Introduction....................................1 Symbols and Conventions....................................2 Safety Information...................................3 Regulatory Compliance......................................3 Equipment Safety Guidelines..................................4 Mechanical Hazards......................................4 Electrical Hazards........................................4 Laser Hazards..........................................5 Safety Labels..........................................5 Specifications....................................6 Dimensions ..........................................6 Computer Requirements....................................7 Environmental Requirements..................................7 Electrical Requirements.....................................9 Recommended Electrical Configurations............................10 Overview....................................11 Key Features..........................................11 Capabilities..........................................11 RI 1000............................................12 RI 182 and RI 54........................................13...
  • Page 5 User Interfaces........................................14 Imaging Methods........................................15 Components....................................16 RI 1000............................................16 RI 182 and RI 54........................................18 RI 2..............................................19 FRAP Benchtop........................................20 SONICC Benchtop........................................22 Accessories..........................................25 Imaging Methods..................................27 Visible Light..........................................27 SONICC............................................29 Ultraviolet Light........................................29 FRAP...............................................32 FACIT..............................................32 Multi-Fluorescence Imaging (MFI)................................33 UV Absorption Imaging....................................38 Installation and First Use..............................39 First Use............................................40 Using RI 1000, RI 182 &...
  • Page 6 Interpreting SONICC Results..................................54 SONICC Reference Plate Image Analysis............................56 Configuring FRAP Imaging Settings..........................57 Bleaching Options........................................57 Imaging Options........................................58 General Options........................................59 Manual Inspections................................60 Manually Inspecting a Plate..................................60 Manual Imaging Controls....................................62 About Auto Focus........................................66 About Drop Location......................................67 Using the Plate Type Editor.............................68 Improving Images.................................79 Setting Up Plate Definitions..................................79 Applying Plate Masks......................................81 Tuning Plates in ROCK IMAGER................................83...

  • Page 7: Introduction

    Your ROCK IMAGER must be used in the manner described in this user’s guide. Any other use may result in damage to your ROCK IMAGER or personal injury. FORMULATRIX, Inc. is not responsible in whole or in part for any damages caused by: improper use, unauthorized alterations, adjustments or modifications, failure to comply with the procedures outlined in the ROCK IMAGER User’s Guide, or...

  • Page 8: Symbols And Conventions

    ROCK IMAGER User's Guide | Chapter 1: Introduction Symbols and Conventions Convention Description This icon denotes a caution and advises you of precautions to take. This icon denotes a prohibited action. This icon denotes a compulsory action and advises you of actions you must take.

  • Page 9: Safety Information

    This device contains a laser radiation device within the barcode scanner. Do not stare into the beam or bodily injury may occur. The door should only be opened under the direction of trained FORMULATRIX personnel (applies to RI 1000, RI 182, RI 54, SONICC Benchtop, and FRAP High voltage components in the electronics drawer.

  • Page 10: Equipment Safety Guidelines

    ● Turn OFF power before inserting or removing power cables. ● Do not open the door to the ROCK IMAGER chassis without express instruction from trained FORMULATRIX personnel (applies to RI 1000, 182, 54, and SONICC Benchtop only). Mechanical Hazards ROCK IMAGER is a complex electromechanical device.

  • Page 11: Laser Hazards

    Do not stare into the beam or bodily injury may occur. Safety Labels Do not remove or alter any safety labels or warnings from FORMULATRIX products. The FRAP imager has one safety label affixed to the cover of the FRAP laser. This sticker is viewable when the FRAP door is open.

  • Page 12: Specifications

    Specifications Chapter 3 Dimensions RI 1000 Peltier Cooled 834 x 2197 x 1085 mm (33” x 87” x 43”) 387 kg (864 lbs) RI 1000 Compressor Cooled 898 x 2272 x 1117 mm (36” x 90” x 44”) 399 kg (880 lbs) RI 1000 Dual Imager 834 x 2197 x 1230 mm...

  • Page 13: Computer Requirements

    ROCK IMAGER User's Guide | Chapter 3: Specifications Environmental Requirements Ambient Temperature Range 0° C to 30° C. Operating Humidity 0 - 80% non-condensing. Storage Humidity 0 - 80% non-condensing. Ventilation Do not block the fan vents on the ROCK IMAGER chassis. Maintain at least 2 inches (50.8 mm) of free space between the vents and any obstruction or surface.
  • Page 14 ROCK IMAGER User's Guide | Chapter 3: Specifications ● Remote access (e.g. WebEx, TeamViewer, VPN) (Recommended) ● Email Server access (SMTP server credentials) ● Windows 7 32-bit or 64-bit, Windows 10 64-bit ● Optional: UPS unit ● 4 GB RAM ●...

  • Page 15: Electrical Requirements

    ROCK IMAGER User's Guide | Chapter 3: Specifications Electrical Requirements The power supply for the system must meet or exceed electrical requirements of the system to avoid damage and risk of fire. ROCK IMAGER 1000 – Peltier Cooled System ● Cooling Unit: 100-240 V, 50-60 Hz, 750 W max, 1 ph ●...

  • Page 16: Recommended Electrical Configurations

    ROCK IMAGER User's Guide | Chapter 3: Specifications ROCK IMAGER 2 ● Imager: 100-240 V, 50-60 Hz, 150 W max, 1 ph ● Computer: 525 W max, 1 ph SONICC Benchtop ● Imager: 100-240 V, 50-60 Hz, 1195 W max, 1 ph ●...

  • Page 17: Overview

    ● Visible light, UV (optional), FRAP (optional), and SONICC (optional) imaging capabilities ● Custom image scheduling ● Nightly diagnostic reports sent to FORMULATRIX for continued quality control ● ROCK IMAGERs are designed to minimize vibration to microtiter plates to prevent environmental disturbance ●...
  • Page 18 ROCK IMAGER User's Guide | Chapter 4: Overview Region of Interest (ROI). Specify a region of a drop to zoom into and ROCK IMAGER will automatically capture that area in high resolution. Multi-Capture. Capture multiple images of each drop with user adjustable settings for variables such as: exposure time, polarization state, and condenser aperture value.

  • Page 19: Ri 182 And Ri 54

    ROCK IMAGERUser's Guide | Chapter 4: Overview RI 182 and RI 54 ROCK IMAGERs 182 and 54 feature many of the same abilities as ROCK IMAGER 1000, although these imagers have limited plate capacity and no compressor-based cooling option. RI 182 ●...

  • Page 20: User Interfaces

    Your computer screen becomes the microscope’s eyepiece and physical optics and imager controls are replaced by on-screen controls. ROCK IMAGER Software All ROCK IMAGERs integrate with ROCK MAKER, FORMULATRIX’s crystallization experiment lifecycle management software. Automated imaging is scheduled and managed through ROCK MAKER. ROCK MAKER Software...

  • Page 21: Imaging Methods

    (<1 µm) in opaque solutions. It is available as a separate imaging system in a RI 1000 chassis, or as a standalone benchtop imaging system. FACIT (FORMULATRIX Advanced Contrast Imaging Technology). FACIT allows you to control the illumination pattern on a drop in order to see more crystal facets. Any light pattern can be projected onto a drop, including top-left, top-right, bottom-left, bottom-right, darkfield, and brightfield illumination.

  • Page 22: Components

    Components Chapter 5 RI 1000 ROCK IMAGER 1000 can include either SONICC or FRAP imaging methods in the base of the chassis, and either a visible light imager or a combined visible and UV imager in the upper portion of the chassis.
  • Page 23 ROCK IMAGER User's Guide | Chapter 5: Components A dual compressor, redundantly designed system can regulate temperature down to 4 °C with an ambient temperature of 10 °C to 30 °C. Load Port The load port is where you load and unload plates into the load port hotel. A hotel is composed of slots for several microplates.

  • Page 24: Ri 182 And Ri 54

    ROCK IMAGER User's Guide | Chapter 5: Components RI 182 and RI 54 ROCK IMAGERs 182 and 54 share the same style of chassis, only the ROCK IMAGER 182 chassis is deeper to allow for internal plate storage. Additionally, the robot hand in ROCK IMAGER 182 is capable of turning to place and retrieve plates from storage hotels along the inner back wall.
  • Page 25 ROCK IMAGER User's Guide | Chapter 5: Components RI 2 ROCK IMAGER 2 is our benchtop imager designed for visible and UV imaging. It can hold up to two SBS-footprint plates, or one Linbro plate with an optional adaptor. It is sold as either a visible light imager, a visible and UV light imager with separate light paths, or a single light path visible and UV imager.

  • Page 26: Frap Benchtop

    ROCK IMAGER User's Guide | Chapter 5: Components Barcode Scanner The barcode scanner allows for automated imaging and passes the images over to ROCK MAKER to be stored in a user-friendly viewing environment. FRAP Benchtop FRAP (Florescence Recovery After Photobleaching) imaging, useful for screening LCP protein crystallization conditions based on membrane diffusion rates, is a process by which a laser bleaches a dye-labeled protein in LCP.
  • Page 27 ROCK IMAGER User's Guide | Chapter 5: Components Door Sensor for Laser Interlock FRAP Optics Compound Zoom Turret Barcode Scanner Two Plate Imager Hand Motorized Condenser FRAP Benchtop Internal View Door Sensor for Laser For safety, the laser will automatically turn off when the door is opened; Interlock however, imaging will continue and your image results will not be affected.

  • Page 28: Sonicc Benchtop

    ROCK IMAGER User's Guide | Chapter 5: Components SONICC Benchtop SONICC imaging, which includes both SHG and UV-TPEF imaging methods, produces black and white images that makes protein crystal detection easy. Power Switches Main Door Load Port Door SONICC Benchtop Power Switches SONICC Benchtop has two power switches located on the right side of the chassis, one for the laser and one for the imaging optics.
  • Page 29 ROCK IMAGER User's Guide | Chapter 5: Components LBO Crystal Oven Laser Pulse Generator Door Sensor Laser Pulse Compressor Visible Imager SONICC Optics 12x Motorized Zoom SHG Objective Stage Barcode Scanner Photo Multiplier Tube (PMT) SHG Collection Optics Motorized Condenser SONICC Internal View LBO Crystal Oven The LBO crystal oven helps to generate the green femtosecond pulse...
  • Page 30 January 2018 to have 5x greater sensitivity than previous models, for dramatically increased image quality. Users with SONICC instruments manufactured prior to January 2018 who are interested in upgrading their PMT can contact sales@formulatrix.com to request a quote. SHG Collection Optics The transmission collection optics collect SHG signal while the epi/backwards collection optics collect UV-TPEF signal.

  • Page 31: Accessories

    ROCK IMAGER User's Guide | Chapter 5: Components Accessories Accessories are available for purchase online from the FORMULATRIX accessories store (www.formulatrix.com/store/home.php). Load Port Handle The load port handle is used with the RI 1000 chassis to add or remove load port hotels from the load port.
  • Page 32 ROCK IMAGER User's Guide | Chapter 5: Components LCP Plate Adaptor: Part No. LCPPA LCP Plate Adaptor Linbro Plate Adaptor for RI 2 Systems: Part No. 200547. EasyXtal Plate Adaptor for RI 2 Systems: Part No. 200544. RI 2 Plate Adaptor Barcode Printer Supplies 4000 Custom Labels for Barcode Printer: Part No.

  • Page 33: Imaging Methods

    Imaging Methods Chapter 6 Visible Light All ROCK IMAGER models feature a visible light camera that is used to take brightfield images of crystallization plates. The type of camera and capabilities offered depend on the ROCK IMAGER model you have. The advanced model includes continuous 12x motorized zoom option with a 5 megapixel, 2/3"...
  • Page 34 ROCK IMAGER User's Guide | Chapter 6: Imaging Methods Köhler Illumination with Motorized Aperture ROCK IMAGER uses the same light source as compound microscopes. With Köhler illumination, lenses are used to focus the light on the sample. The motorized aperture in the light path can bend the light from a cone to a column and vice versa for automated contrast adjustment.

  • Page 35: Sonicc

    UV light photography can help you more quickly differentiate protein crystals from salt crystals. FORMULATRIX's UV imaging technology uses the fluorescence of tryptophan under UV light to identify whether a crystal contains a protein or is a salt. Our UV solution uses 100% UV-optimized components, including UV-grade optics, a UV-sensitive camera and UV lighting.
  • Page 36 ROCK IMAGER User's Guide | Chapter 6: Imaging Methods ● Can distinguish between precipitate and crystals ● Available as an upgrade to ROCK IMAGER 1000 or as a bench top system that includes visible light optics SHG and UV-TPEF SONICC has two imaging methods, Second Harmonic Generation (SHG) and Ultraviolet Two-Photon Excited Fluorescence (UV-TPEF).
  • Page 37 ROCK IMAGER User's Guide | Chapter 6: Imaging Methods Nonlinear effects such as SHG require high electric fields, thus requiring the use of a femtosecond (fs) laser. The laser operates with a pulse width of 200 fs and has high peak powers resulting in nonlinear effects, but are short enough to reduce damage associated with localized heating.

  • Page 38: Frap

    FACIT — FORMULATRIX Advanced Contrast Imaging Technology — is a hardware option developed at FORMULATRIX in which a digital light projector modifies the light pattern used to illuminate your drops. Any pattern you can imagine can be projected onto a drop.

  • Page 39: Multi-Fluorescence Imaging (Mfi)

    ROCK IMAGER User's Guide | Chapter 6: Imaging Methods Multi-Fluorescence Imaging (MFI) FORMULATRIX’s MFI technology uses three different light wavelengths including ultraviolet (UV) and visible fluorescence that help you easily detect protein crystals, even those buried under precipitate. This option includes a full continuous zoom visible imaging optical setup and can be integrated into all ROCK IMAGER 2 and ROCK IMAGER 1000 models.
  • Page 40 UV imaging, and two visible fluorescence filter sets for detecting the fluorescence from Texas Red and Fluorescein dyes. If you would like to image fluorescence from other dyes and install a different filter module, contact support@formulatrix.com. MFI Schematic Drawing Finding protein-protein complexes With MFI, you can easily differentiate between crystals of a protein-protein complex and crystals of just one protein.
  • Page 41 ROCK IMAGER User's Guide | Chapter 6: Imaging Methods To find protein-protein complexes: 1. Label two suspected proteins or subunits with two different dyes. 2. Using the ROCK IMAGER software, image your drop at the two corresponding wavelengths. Crystals that fluoresce at both wavelengths are protein-protein complexes, whereas those that fluoresce at only one wavelength are of a single protein.
  • Page 42 ROCK IMAGER User's Guide | Chapter 6: Imaging Methods Imaging a Plate with MFI Although most imaging sessions are automated and executed according to the imaging schedule set in ROCK MAKER, it's possible to initiate an unscheduled imaging session from ROCK IMAGER. Prerequisites ●...
  • Page 43 ROCK IMAGER User's Guide | Chapter 6: Imaging Methods 3. Choose from the following options: ● To image an entire plate with the MFI imaging settings: in the Imager menu, point to Image, and click Plate(s). ● To image an entire plate with the imaging setting(s) of choice: in the Imager menu, point to Image, and click Plate(s) Using.

  • Page 44: Uv Absorption Imaging

    ROCK IMAGER User's Guide | Chapter 6: Imaging Methods UV Absorption Imaging Many samples, including RNA, DNA, and protein absorb UV, which can be used to create contrasting images. The UV absorption imager transmits UV light through the sample and then detects the same wavelength UV light on the camera.

  • Page 45: Installation And First Use

    Installation and First Use Chapter 7 The FORMULATRIX support team will perform the ROCK IMAGER installation in your laboratory or at your site; do not do this yourself. You will receive your instrument via a shipping service in a large crate (two extra crates for chillers if you purchase the ROCK IMAGER 4°C).

  • Page 46: First Use

    ROCK IMAGER User's Guide | Chapter 7: Installation and First Use First Use Once FORMULATRIX technicians have installed ROCK IMAGER, you may follow these steps to turn on and use your device: 1. Locate the power switch. ● RI 1000 – The power switches are in the electronics drawer. Open the electronics drawer and turn on the ROBOTICS switch and the COOLING switch.

  • Page 47: Using Ri 1000, Ri 182 & Ri 54

    Using RI 1000, RI 182 & RI 54 Chapter 8 ROCK IMAGERs 1000, 182, and 54 have the ability to image experiment plates in either Automatic Mode or Manual Mode. In Automatic Mode, a plate is imaged according to the schedule set in ROCK MAKER.

  • Page 48: Initiating An Imaging Session

    ROCK IMAGER User's Guide | Chapter 8: Using RI 1000, RI 182 & RI 54 ● To move the plate(s) in the load port to storage, go to the Load Port menu and select Store Contents. ROCK IMAGER will automatically scan the contents of the load port, and move the plate(s) to storage.

  • Page 49: Searching For Plates In Storage

    ROCK IMAGER User's Guide | Chapter 8: Using RI 1000, RI 182 & RI 54 Searching for Plates in Storage Once a plate is inside the imager, it could either be in the load port, under the imager, or in a storage hotel (if you have ROCK IMAGER 1000 or 182).

  • Page 50: Viewing A Plate's Automatic Imaging Schedule

    ROCK IMAGER User's Guide | Chapter 8: Using RI 1000, RI 182 & RI 54 Viewing a Plate’s Automatic Imaging Schedule Automatic imaging schedules are set in the ROCK MAKER software, but you can verify a plate's schedule or view it using the ROCK IMAGER software. To view a plate's imaging schedule and information, right-click a plate on any of the Plates sub-tabs and select Plate Information.

  • Page 51: Removing Plates

    ROCK IMAGER User's Guide | Chapter 8: Using RI 1000, RI 182 & RI 54 Removing Plates When all of the imaging for a certain plate has been completed, you can then remove the plate from ROCK IMAGER. Multiple plates can be moved at the same time to the load port, from which they may be removed from the system for external storage.

  • Page 52: Using Ri 2, Frap Benchtop & Sonicc Benchtop

    Using RI 2, FRAP Benchtop & SONICC Benchtop Chapter 9 FRAP Benchtop, SONICC Benchtop, and RI 2 are grouped together because these three imagers are the ones without storage. They also are the three ROCK IMAGERs that can perform Instant Imaging, which allows you to image plates without barcodes.

  • Page 53: Imaging A Plate With Frap Benchtop

    ROCK IMAGER User's Guide | Chapter 9: Using RI 2, FRAP Benchtop & SONICC Benchtop 4. Select which "slot" to image and scan, then click OK. In each imager, slot 1 is on the left and slot 2 is on the right. 5.
  • Page 54 ROCK IMAGER User's Guide | Chapter 9: Using RI 2, FRAP Benchtop & SONICC Benchtop ● To image an entire plate with the imaging setting(s) of choice, go to the Imager menu, point to Image, and select Plate(s) Using. ROCK IMAGER will display a dialog box that asks you to select available imaging settings (i.e.

  • Page 55: Imaging A Plate With Sonicc Benchtop

    ROCK IMAGER User's Guide | Chapter 9: Using RI 2, FRAP Benchtop & SONICC Benchtop Imaging a Plate with SONICC Benchtop This topic explains how to image a plate from start to finish using the SONICC Benchtop version of ROCK IMAGER. If you aren't using ROCK MAKER to track conditions, or you want to image a plate that does not have a barcode, please see Instant Imaging on page 46.

  • Page 56: Imaging A Plate With Ri 2

    The system will keep all skipped SONICC inspections in the imaging queue until the laser is repaired, at which time the inspections will be run automatically. Contact support@formulatrix.com for assistance. Imaging a Plate with RI 2 This topic explains how to image a plate with ROCK IMAGER 2. You have two options: If you are using barcode labels on your plates, then follow the instructions in this section.
  • Page 57 ROCK IMAGER User's Guide | Chapter 9: Using RI 2, FRAP Benchtop & SONICC Benchtop 3. Choose from the following options: ● To image an entire plate with the default imaging settings, on the Imager menu, point to Image, and click Plate(s). Then, select Slot 1 and/or Slot 2 in the Select Slots window shown below, and click OK.

  • Page 58: Viewing A Plate's Automatic Imaging Information

    ROCK IMAGER User's Guide | Chapter 9: Using RI 2, FRAP Benchtop & SONICC Benchtop ● To image a specific drop with your choice of imaging settings, on the Imager menu, point to Image, and click Drop Using. Then, select the applicable imaging settings from the Select Imaging Settings window.

  • Page 59: Interpreting Frap Offset Information

    ROCK IMAGER User's Guide | Chapter 9: Using RI 2, FRAP Benchtop & SONICC Benchtop Prerequisites ● You have loaded and scanned the plate. To view a plate's automatic imaging information: ● To view a plate's imaging schedule, go to the Imager tab and click More on Plate Information. Alternatively, right-click a plate on the Scheduling tab and select Plate Information.

  • Page 60: Interpreting Sonicc Results

    If the offset value differences are greater than ~30 µm, it may point to a problem with the stage or the image alignment algorithm. Email support@formulatrix.com assistance. FRAP Offset Values in ROCK MAKER Interpreting SONICC Results In general, the SHG imaging channel will provide the ability to quickly determine which wells contain protein crystals.
  • Page 61 ROCK IMAGER User's Guide | Chapter 9: Using RI 2, FRAP Benchtop & SONICC Benchtop If both the SHG and UV-TPEF channels show similar contrast then you can be certain that your sample is a protein crystal (unless you have fluorescent small molecules present). Note that all protein material, whether crystalline or not, will show up on the UV-TPEF channel, so it is important to compare to the SHG channel to determine if it is crystalline.

  • Page 62: Sonicc Reference Plate Image Analysis

    30% or more, or an increase of 100% or more, an email is automatically sent to FORMULATRIX support. A support engineer will then analyze your system to determine the cause of the degradation, fix any problems and/or replace the SONICC laser if necessary.

  • Page 63: Configuring Frap Imaging Settings

    FRAP imaging settings, and to only adjust the settings discussed in this section under the guidance of FORMULATRIX support. To access FRAP configuration settings, go to File and click FRAP Configurations. You may or may not be able to access the Servicing menu, as this menu is usually hidden in order to preserve the default FRAP settings.

  • Page 64: Imaging Options

    ROCK IMAGER User's Guide | Chapter 10: Configuring FRAP Imaging Settings Generally, it’s best to run High Throughput (HT) FRAP imaging first, and execute HT FRAP imaging once you identify interesting drops. ● Acquire images at fastest rate for. This setting defines how long, in seconds, the imager will take rapid pictures of your drop after laser bleaching is complete.

  • Page 65: General Options

    ROCK IMAGER User's Guide | Chapter 10: Configuring FRAP Imaging Settings General Options General FRAP options include settings related to the curve generated based on the FRAP data. ● Single Component Fitting. Fits the pixel intensity recovery data with one Bessel function. The result of this numerical fitting is a single diffusion rate.

  • Page 66: Manual Inspections

    Manual Inspections Chapter 11 You can use ROCK IMAGER's powerful optics to explore the contents of your plates at any time by switching from Automatic Mode to Manual Mode and using the manual imaging controls. In this section, we explain how to manually image a plate and how to use the associated optics and camera controls.
  • Page 67 ROCK IMAGER User's Guide | Chapter 11: Manual Inspections ● Select Automatically if you want the ROCK IMAGER to automatically revert to scheduled imaging after a certain amount of time. Type the desired number of minute(s) in the appropriate field shown in the orange box below, to set the time after which the Automatic Mode will resume.

  • Page 68: Manual Imaging Controls

    ROCK IMAGER User's Guide | Chapter 11: Manual Inspections Important: Leaving Live Image on for an extended period of time may cause thermal decomposition to samples. Tip: To save a picture of a live image, go to the File menu and select Save As. 7.
  • Page 69 ROCK IMAGER User's Guide | Chapter 11: Manual Inspections Well Control Well Controls are found on the Live Image sub-tab of the Imager tab, at the top of the right-hand part of your screen. Use the Well Controls to navigate around your plate to the drop of interest.
  • Page 70 ROCK IMAGER User's Guide | Chapter 11: Manual Inspections General Camera Control UV Camera Control Panel SONICC Camera Control Camera Controls Fluorescence Optics Controls Visible Light Optics Controls UV Optics Controls FRAP Optics Controls SONICC Optics Controls SLP Visible Light Optics SLP UV Optics Controls Optics Controls...
  • Page 71 ROCK IMAGER User's Guide | Chapter 11: Manual Inspections Leveling One of the buttons on the Camera Control panel is the Leveling button. Clicking it opens the Leveling Histogram, which you use to adjust the brightness and contrast of an image while you are viewing a drop in either Manual Mode or Automatic Mode.

  • Page 72: About Auto Focus

    ROCK IMAGER User's Guide | Chapter 11: Manual Inspections About Auto Focus When you click the Auto Focus button, you instruct ROCK IMAGER to execute the Auto Focus algorithm. Auto Focus locates objects in a drop along the Z-axis, which is especially important when imaging hanging drops on greased cover slides, or when zooming in on a region of interest.

  • Page 73: About Drop Location

    ROCK IMAGER User's Guide | Chapter 11: Manual Inspections About Drop Location ROCK IMAGER's Drop Location algorithm is extremely useful for hanging drop experiments, as the location of the drop varies in this type of experiment. When ROCK IMAGER executes drop location on a drop, the drop area is imaged with a large field of view, and the lighting is set in order to create shadowing within the drop.

  • Page 74: Using The Plate Type Editor

    Editor Chapter 12 The Plate Type Editor contains settings for all of your plate types. Upon install, the FORMULATRIX support team will configure the properties for optimal image quality and performance. If you need a new plate type added to your imager, then please contact support@formulatrix.com...
  • Page 75 Default definitions for each plate type are available by importing a plate definition .xml file (obtainable by support@formulatrix.com), or you can import a previously saved definition file. To import a plate definition file, click the Import from File button, select the desired plate definition (the plate definition should match the actual plate type you would like to use), and click Open.
  • Page 76 ROCK IMAGER User's Guide | Chapter 12: Using the Plate Type Editor Drop Positions The coordinates in the Drop Positions field correlate to the corner drops on the plate. From the coordinates of these corner positions, ROCK IMAGER uses linear interpolation to compute the locations of all drops on the plate.
  • Page 77 ROCK IMAGER User's Guide | Chapter 12: Using the Plate Type Editor Exposure ● Min Exposure — Determines the minimum exposure value used during plate imaging. To change the Min Exposure value, type the desired value in the corresponding box. ●...
  • Page 78 ROCK IMAGER User's Guide | Chapter 12: Using the Plate Type Editor ● Advanced Options on the Ultraviolet Imager Capture Levels ● Step Size (DoF %) — Determines the amount of depth of field overlap between slices. The default value is 40%, which means that 60% of a slice will be overlapped by the slice next to it.
  • Page 79 ROCK IMAGER User's Guide | Chapter 12: Using the Plate Type Editor ● Advanced Options on the SONICC Imager Capture Levels ● Step Size (DoF %) — Determines the amount of depth of field overlap between slices. The default value is 75%. To change the default value, type the desired value in the corresponding box.
  • Page 80 If you are using FRAP, you will see the Fluoro Light option which enables you to change the fluorescence signal intensity. If you are using ROCK IMAGER with FORMULATRIX Advanced Contrast Imaging Technology (FACIT), you can control the illumination pattern — such as top-left, top-right, bottom-left, darkfield, and brightfield —...
  • Page 81 ROCK IMAGER User's Guide | Chapter 12: Using the Plate Type Editor ● Gamma — Gamma affects the contrast of the midtones in the image. When you modify this setting, only the midtones are affected, without affecting black and white. ●...
  • Page 82 The Missed Zoom is available only when you select Locate as drop Location. If ROCK IMAGER failed locating a drop, it will use the Missed Zoom setting. If you are using ROCK IMAGER with FORMULATRIX Advanced Contrast Imaging Technology (FACIT), you can control the illumination pattern — such as top-left, top-right, bottom-left, darkfield, and brightfield —...
  • Page 83 ROCK IMAGER User's Guide | Chapter 12: Using the Plate Type Editor ● Exposure — Determines the amount of optical brightness. The higher the Exposure value, the brighter the image will be. ● Gain — Determines the amount of digital brightness. The higher the Gain value, the brighter the image will be.
  • Page 84 ROCK IMAGER User's Guide | Chapter 12: Using the Plate Type Editor For example, when the Focus threshold above value is set to .7, all slices that are above the best focus level, and are at least 70% in focus as the best focus level, will be saved. For the focus threshold values, a larger number will result in fewer slices acquired, and a smaller number will result in more slices.

  • Page 85: Improving Images

    Improving Images Chapter 13 FORMULATRIX technicians tune the settings for each type of plate you plan to use with your ROCK IMAGER during initial installation. However, if you start using new plate types, or you notice your imager isn't capturing drop images as well as it once was, you may want to fine-tune these settings to improve drop location accuracy using the Plate Type Editor.
  • Page 86 ROCK IMAGER User's Guide | Chapter 13: Improving Images 3. On the File menu, click Plate Type Editor. Make sure that Well Limits is set to Disabled and leave the Plate Type Editor open. Note: Do not click Apply or OK in the Plate Type Editor menu at this time.

  • Page 87: Applying Plate Masks

    ROCK IMAGER User's Guide | Chapter 13: Improving Images Drop A1 e. Return to the Plate Type Editor window, and click the button next to the well location to copy the coordinates. f. Repeat steps d and e for configuring the other three corner wells of the plate. 5.
  • Page 88 ROCK IMAGER User's Guide | Chapter 13: Improving Images Prerequisites ● You will be imaging the plate with UV or UV-TPEF imaging types. ● You want to improve ROCK IMAGER Processor’s Auto Scoring results. ● You have ROCK MAKER 2.7 or newer. ●...

  • Page 89: Tuning Plates In Rock Imager

    You can tune the drop locations saved in ROCK IMAGER for your plates to ensure clear and precise imaging. FORMULATRIX support will tune your plates when they install your ROCK IMAGER instrument, but if your drop images are off-centered or you add new plates to your system you can follow the tutorial below to tune the drop locations for your plate.
  • Page 90 Important: The following procedure requires that the ROCK IMAGER reference plate has already been tuned for both Visible and SONICC or FRAP imaging modes. The reference plate is tuned by FORMULATRIX support upon installation of your ROCK IMAGER system. If you see any message while performing this procedure that indicates that the reference plate has not been tuned, please contact support@formulatrix.com...
  • Page 91 ROCK IMAGER User's Guide | Chapter 13: Improving Images To tune the drop locations of a plate for SONICC or FRAP Imaging: 1. Make sure the plate you want to tune is under the Visible imager. 2. On the Imager Control panel, select Visible as the imager. 3.
  • Page 92 ROCK IMAGER User's Guide | Chapter 13: Improving Images 10. Then, click Apply. 11. To check whether the coordinates set by the Calculate from Visible button has directed the imager to the definite center point, go back to the ROCK IMAGER software interface and view the selected drop in Well A1 with live imaging.

  • Page 93: Clearing Drop Locations

    ROCK IMAGER User's Guide | Chapter 13: Improving Images 16. When the drop position is centered, go back to the Plate Type Editor and click in Well A1 to save the new drop position. The coordinates for the drop position in Well A1 will be generated in the X, Y, and Z columns.

  • Page 94: Rock Maker: The Crystallography Center

    ROCK MAKER: The Crystallography Center Chapter 14 ROCK MAKER is a complex, powerful laboratory information management system. Using ROCK MAKER, you can design screens and experiments; print barcode labels for plates and ingredient bottles; dispense plates (if ROCK MAKER is integrated with a liquid handling robot); view and score images;...

  • Page 95: Printing A Barcode Label For A Plate

    ROCK IMAGER User's Guide | Chapter 14: ROCK MAKER: The Crystallography Control Center Prerequisites ● You are running ROCK MAKER version 3.0 or newer. Creating a protein formulation: 1. In the ROCK MAKER main window, click the ROCK MAKER University button 2.

  • Page 96: Viewing And Scoring Images In Rock Maker

    ROCK IMAGER User's Guide | Chapter 14: ROCK MAKER: The Crystallography Control Center Viewing and Scoring Images in ROCK MAKER Once ROCK IMAGER has taken images, you view them with ROCK MAKER. Although you should refer to the ROCK MAKER help files for more details, this topic provides a quick overview about how to access and view your images with the ROCK MAKER software.
  • Page 97 ROCK IMAGER User's Guide | Chapter 14: ROCK MAKER: The Crystallography Control Center Using the Navigator ● To see each of the z-slices for a drop, go to the Drop tab and use the F11 and F12 keys on your keyboard. ●...
  • Page 98 ROCK IMAGER User's Guide | Chapter 14: ROCK MAKER: The Crystallography Control Center ● If your plate has multiple drops per well, you must change the Drop list on the Experiment Navigator. Drop List ● To adjust the brightness and contrast for your images, click the Tools tab in the Images tab set and use the Image Tuning controls.

  • Page 99: Faqs

    FAQs Chapter 15 If your question does not appear below, please email support@formulatrix.com and we will respond within 24 hours, Monday through Friday. How do I change the imaging schedule? Since schedules are defined in ROCK MAKER, to change the imaging schedule you need to use ROCK MAKER.
  • Page 100 How do I add a new plate type? Please contact us by sending an email to support@formulatrix.com that includes information about the plate type you wish to add. One of our support technicians will respond with a plate type definition file and explain how to add it to your plate library.
  • Page 101 If RIP is off, please start it. ● If RIP was installed on your local computer, there should be a shortcut on your desktop. You can also locate the FORMULATRIX folder and look for a file named RockImagerProcessor.

  • Page 102: Uv Faqs

    ROCK IMAGER User's Guide | Chapter 15: FAQs UV FAQs What plates and cover material are compatible with UV? Plates vary, and can even vary within a brand. The most confident answer is that polyolefin works fantastically, and polystyrene can vary from not good to OK. What is the smallest protein crystal that the UV camera can pick up? This answer is dependent upon your plate.
  • Page 103 ROCK IMAGER User's Guide | Chapter 15: FAQs ● UV doesn't make it to the crystal. This could be caused by media absorption, or (less likely) solution absorption. UV might not penetrate into a very, very deep well, which can happen in 5 mL drops.

  • Page 104: Sonicc Faqs

    ROCK IMAGER User's Guide | Chapter 15: FAQs SONICC FAQs Background What is SHG? SHG stands for Second Harmonic Generation and is a nonlinear optical process. In intense electric fields (i.e. in the presence of a femtosecond laser) the distance between the electrons and the nucleus are distorted (anharmonicity) resulting in nonlinear optical effects such as SHG where the frequency of the outgoing light is doubled that of the incident (i.e.
  • Page 105 ROCK IMAGER User's Guide | Chapter 15: FAQs What platforms is SONICC compatible with? All optically assessable platforms are compatible. Will the laser damage my crystals? Preliminary experiments show no detectable damage to protein crystals. In one experiment, a protein crystal was imaged on one half with excessive laser input.
  • Page 106 ROCK IMAGER User's Guide | Chapter 15: FAQs Active Pharmaceutical Ingredients (API) Applications Why use SONICC to detect APIs? Bioavailability is greatly decreased if the APIs crystallize and is therefore extremely important to monitor. Current methods of crystallization detection (thermal, optical, x-ray diffraction and spectrochemical techniques) have a 1% crystallinity limit of detection and are challenging in complex matrixes.

  • Page 107: Frap Faqs

    LCP-FRAP assay is able to prescreen crystallization conditions and identify positive conditions. Is LCP-FRAP integrated with other FORMULATRIX imaging products? FRAP is offered as a standalone system but can also be integrated with the ROCK IMAGER 1000. The ROCK IMAGER 1000 with FRAP maintains two independent imaging stations. You are able to run a FRAP experiment in parallel with bright field color imaging.

  • Page 108: Maintenance

    10 seconds after turning on the ROCK IMAGER to run the software. ● Make certain that each cable is properly connected to the computer. ● Please feel free to contact FORMULATRIX via email or phone with any questions. We are always happy to hear from our customers and provide assistance.
  • Page 109 2 years on-site service plan from the computer manufacturer and transfer this warranty to the customer. FORMULATRIX will assist the customer to diagnose any computer errors and re-install the software, but the customer must work with the computer manufacturer directly to resolve any hardware issues.
  • Page 110 Appendix A Optics Controls This table explains how to use each of the optics controls, which are used during manual inspections. Manual Inspections on page 60 for more information. Control Imaging Methods Description The Excitation list provides you with three different light wavelengths, including the standard package Ultraviolet 290 nm, Visible Fluorescence 485 nm, and Visible Fluorescence Excitation...
  • Page 111 ROCK IMAGER User's Guide | Appendix A: Optics Controls This table explains how to use each of the optics controls, which are used during manual inspections. Manual Inspections on page 60 for more information. Control Imaging Methods Description Available zoom settings depend on the options you purchased. In most cases the zoom selection will cause a mechanical change in the lens, which produces the desired zoom.
  • Page 112 ROCK IMAGER User's Guide | Appendix A: Optics Controls This table explains how to use each of the optics controls, which are used during manual inspections. Manual Inspections on page 60 for more information. Control Imaging Methods Description ● Visible On the visible imaging method, the illumination list provides you with various illumination patterns, including glancing, dark ●...
  • Page 113 ● FRAP By default, binning exposure compensation is enabled for all Visible images and disabled for all UV images for both PointGrey   SONICC and PixeLINK camera types. If you would like to disable binning exposure compensation, please contact support@formulatrix.com.
  • Page 114 ROCK IMAGER User's Guide | Appendix B: Camera Controls This table explains how to use each of the camera controls, which are used during manual inspections. Manual Inspections on page 60 for more information. Control Imaging Methods Description Clicking the EFI button captures the EFI of a specific drop using the current camera and optics settings.
  • Page 115 ROCK IMAGER User's Guide | Appendix B: Camera Controls This table explains how to use each of the camera controls, which are used during manual inspections. Manual Inspections on page 60 for more information. Control Imaging Methods Description This field affects the laser power at the sample. The On and Off radio buttons turn the Laser on and off.

  • Page 116: Index

    ROCK IMAGER User's Guide | Index: “Auto Exposure” to “Gamma” Index Auto Exposure................................... 72 Auto Focus..................................63, 105 Fixed Range (DoF Limited)............................64 Fixed Range (Interpolated)............................. 64 with Limits.................................... 64 Binning......................................104 Bright Field....................................103 Brightness....................................68 Calculate from Visible................................. 81 Capture Tune Drops................................
  • Page 117 ROCK IMAGER User's Guide | Index: “Hazards” to “Override Exposure” Hazards Electrical....................................4 Laser......................................5 Mechanical..................................... 4 Illumination....................................103 Image Masking..................................67 Image Size....................................104 imaging schedule................................... 91 Imaging schedule.................................. 90 Interpreting FRAP Offset Information........................51 Labeling Protein Sample..............................35 Laser..................................
  • Page 118 ROCK IMAGER User's Guide | Index: “Plate” to “Zoom” Plate Adding a new..................................91 Polarizer...................................... 103 Regulatory Compliance................................ 3 Requirements Computer....................................7 Electrical....................................8 Environmental..................................7 Safety....................................1, 4, 5, 21 Saving images................................... 92 Sensor......................................102 Sensors Crash......................................17 Plate Presence.................................. 17 Setting Up Plate Definitions............................

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