® DeltaVision Core and personalDV Restoration Microscopy System User's Manual Revision C ® built with precisionware AppliedPrecision ®
ii DeltaVision Core and personalDV User's Manual Legal Notices Revision C of the DeltaVision Core and personalDV Restoration Microscopy System User’s Manual. Part number 04-720104-000 Rev C. © 1999-2007 Applied Precision, LLC. All rights reserved. No part of this manual may be reproduced, transmitted, stored in a retrieval system, or translated into any language in any form by any means without the written permission of Applied Precision, LLC.
Contents iii Contents Preface .............................................................................................ix About This Manual................................................................................................................ix Document Conventions ......................................................................................................... x Lists .....................................................................................................................
iv DeltaVision Core and personalDV User's Manual Setting up a Personal Data Folder................................................................................26 Saving a Single, Multi-Channel Image ........................................................................27 Turning DeltaVision Off........................................................................................................28 4 Setting up and Running Experiments .......................................
Contents v 7 Facility Requirements & Components ......................................87 Electrical and Environmental Requirements .................................................................... 88 Electrical Requirements................................................................................................. 88 Environmental Requirements ...................................................................................... 89 Overview of Components ...........................................
vi DeltaVision Core and personalDV User's Manual 9 Maintenance ............................................................................133 Shutting Down and Starting the System .........................................................................133 DeltaVision Power Switches.........................................................................................134 Guidelines for Using Switches ...................................................................................
Contents vii Optics ............................................................................................................................. 182 Appendix E: Resolve3D and Keypad Options .......................... 183 The Resolve3D Window .................................................................................................... 184 The Resolve3D Menu................................................................................................... 185 The Resolve3D Toolbar .................
viii DeltaVision Core and personalDV User's Manual AppliedPrecision
Preface This manual shows how to use the DeltaVision system to acquire images. It also shows how to maintain the system. About This Manual describes the information in each chapter. Document Conventions explains the typography, notes, and other conventions used in this manual. Contacting Applied Precision, LLC provides information about how to contact customer support. About This Manual This manual provides instructions for scientists who are using DeltaVision to acquire data.
x DeltaVision Core and personalDV User's Manual Chapter 3, Getting Started, describes how to turn the system on, acquire an image, and run an experiment macro. Chapter 4, Setting Up and Running Experiments, shows how to set up experiment macros for 3D sectioning, Time lapse, Multiple wavelengths, Paneling (for stitching), and Point Visiting. Chapter 5, Acquiring Data From Live Specimens, provides information on how to use the DeltaVision Core system to collect images from live specimens.
Preface xi Notes, Warnings and Cautions Note Indicates information about the previous paragraph or step in a procedure. ! Important Indicates important or critical information about the previous paragraph or step in a procedure. Tip Indicates helpful advice. WARNING: Indicates important information regarding potential injury. WARNING: Indicates risk of explosion. WARNING: Indicates risk of shock. Indicates important information regarding potential damage to CAUTION: equipment or software.
xii DeltaVision Core and personalDV User's Manual Contacting Applied Precision, LLC If you have questions about DeltaVision, first refer to this manual or consult the online Help system. If you don’t find the information you need, contact us at one of the following addresses. Customer Service Hotline Phone: 800-862-5166 email: servicehotline@apllc.
1 1 Introduction This chapter provides an introduction to DeltaVision. What is DeltaVision? introduces the DeltaVision system and provides a history of its development. What Can You Use DeltaVision for? lists the supported imaging modes and summarizes the data acquisition options that are supported by DeltaVision. What should you know to use DeltaVision? summarizes the background and experience required to run the system.
2 DeltaVision Core and personalDV User's Manual History The original restoration microscopes were designed and developed in the laboratories of Dr. John W. Sedat and Dr. David A. Agard at the University of California, San Francisco. Their first working system actively collected images as early as 1983. At that time, a small deconvolution (128x128x64) required overnight processing on a million dollar mainframe computer.
Chapter 1: Introduction DeltaVision microscope was shipped to Bethe Scalettar at Lewis & Clark College, Portland, Oregon. All three of these systems are still active. The DeltaVision software has grown continuously since 1983, with contributions from scientific programmers, faculty, and graduate students at UCSF. Applied Precision’s contribution to the software started in earnest in 1994.
4 DeltaVision Core and personalDV User's Manual The following table summarizes the capabilities of the DeltaVision Core system. Capability Description Digital Microscopy Fluorescence Imaging. Also capable of Brightfield, Phase Contrast, and DIC imaging. Automated Optical Sectioning, Time-lapse, Point visiting Optical sectioning, filter changes, and shutters are coordinated by the controller. Quantitative Processing Image processing and 3-D reconstructions of multi-dimensional data files.
Chapter 1: Introduction Time-Lapse You can run macros to acquire time-lapse images and use the data to create timelapse movies. This is especially useful for studies of live samples or for experiments that use lasers. Point Visiting Point Visiting allows you to acquire data from several areas of interest during a single experiment. You can select which points to monitor on your sample and save them in a list that contains the exact stage coordinates of each point.
6 DeltaVision Core and personalDV User's Manual Reference Imaging is useful for acquiring reference images that can be used for Differential Interference Contrast (DIC) and other techniques. Laser Photo-bleaching and Photo-activation If your system has the Quantifiable Laser Module (QLM) hardware module, you can use DeltaVision to run and analyze laser photo-bleaching and photo-activation experiments.
2 2 Safety The precautions detailed in this chapter must be carefully observed to prevent possible personal danger: UV Exposure discusses potential for UV exposure from the xenon arc lamp. Bright Light Exposure warns about bright light exposure from the transmitted light source installed in the microscope. Burn provides guidelines for avoiding burns from the xenon arc lamp. (The arc lamp reaches very high temperatures.) Shock includes warnings about potential shock hazards.
8 DeltaVision Core and personalDV User's Manual UV Exposure Since the xenon arc lamp emits ultraviolet (UV) light, there is a danger of exposing your eyes and skin. Loss of eyesight could occur if unfiltered light from the xenon arc lamp reaches your eyes. To prevent UV exposure: • Open the shutter only when an excitation filter is engaged. • Do not open the xenon arc lamp housing during operation. See Chapter 9, Maintenance for detailed instructions on changing the xenon arc lamp bulb.
Chapter 2: Safety 9 Damage Prevention The following actions could damage the system: Moving the stage to the home position with the objective up could break or scratch the objective. The stage could be driven into the objective and potentially scratch the top lens or compress the lens housing, causing a leak, crack, or lens misalignment. Disconnecting cables before the system is completely shut down will damage one or more of the electronic circuit boards.
10 DeltaVision Core and personalDV User's Manual Caution or Warning Label This label indicates a danger of personal injury or possible damage to equipment. It is accompanied by an explanation of the specific danger. This label may be found on the microscope, the High Res camera, the Fast Camera, or the workstation.
3 3 Getting Started This chapter shows how to get started with DeltaVision. Before you start includes a checklist of things that you need to have before you can acquire images for your sample. Getting Familiar with DeltaVision describes the key controls that you will use to direct the light path, focus, and select filters. It also introduces the keypad and joystick. Turning DeltaVision on shows how to turn on the system.
12 DeltaVision Core and personalDV User's Manual Before you start Before you start, make sure that you: • Select the proper oil for your objective. The immersion oil kit includes 18 oils with refractive indexes that range from 1.500 to 1.534, in increments of 0.002. (For personalDV, the kit includes 6 oils that range from 1.512 to 1.522.) To calculate the best refractive index for your application, follow the instructions in Appendix A: The Immersion Oil Kit on Page 155.
Chapter 3: Getting Started 13 Analytical and Quantitative Light Microscopy, Sluder and Wolf Course Directors, Marine Biological Laboratory, Woods Hole, MA, May (annual).
14 DeltaVision Core and personalDV User's Manual Eyepiece Focus Fine Z Focus Focus Lock Coarse Z Focus Not currently used Not currently used Eyepiece Focus Use the Eyepiece Focus (on the left ocular) to focus the eyepiece. Focus Lock Use the Focus Lock to lock or unlock the Z focus. Fine Z Focus Use the Fine Z Focus knob to move the objective in very small increments. It is used to focus on the focal plane. Coarse Z Focus Use the Coarse Z Focus knob to move the objective in large increments.
Chapter 3: Getting Started 15 Eyepiece Filter Wheel Neutral Density Filter Emission Filter Excitation Filter DeltaVision provides five different types of filters for controlling the fluorescent light path: • Neutral Density filters reduce the amount of light that illuminates your sample when you are using fluorescence. DeltaVision provides six filters that block from 0 to 99.9% of all light.
16 DeltaVision Core and personalDV User's Manual Using the Keypad and Joystick The keypad and joystick are used to move the stage, open shutters, acquire images, and control other acquisition options. Key controls are shown below. Key controls on the Keypad Acquire Image Acquires an image and displays it on the monitor. Use this key when you are scanning through your sample and using the eyepiece to find a region of interest, or when you want to get a quick look at the specimen on the monitor.
Chapter 3: Getting Started 17 Turning DeltaVision On Use the following instructions to turn the system on for day-to-day use. For instructions that show how to turn on DeltaVision after a system shutdown, see Shutting Down and Starting the System on Page 133. To turn on DeltaVision: 1. Turn on the power strip bar. Note For personalDV, there is no power strip bar. Begin the power-up process with Step 2. 2. Turn on the IC/MIC.
18 DeltaVision Core and personalDV User's Manual 9. Release the Focus Lock by turning it clockwise (when facing the lock) until it is loose. 10. Lower the objective by turning the Coarse Z Focus knob away from you (clockwise) when facing the knob. Always lower the objective before you initialize the system CAUTION: to prevent damage to the objective lens. 11. A dialog box is displayed prompting you to lower the objective before continuing.
Chapter 3: Getting Started 19 The Resolve3D window, the Data Collection Window, and the Filter Monitor dialog box open on the desktop. Filter Monitor Resolve3D Acquisition Parameters Data Collection Window Resolve3D Stage Controls The Resolve3D window includes acquisition parameters and controls for moving the stage. The Data Collection window displays images as they are acquired and the Filter Monitor displays the filters that are selected.
20 DeltaVision Core and personalDV User's Manual 2. Rotate the objective turret to select an objective. Use the objective turret to select an objective Tip Use a high power objective (e.g., 60X) to find the sample. The approach for finding a sample with a fluorescent microscope is different than what you would use for a transmitted light microscope. With transmitted light, a typical approach is to find the sample with low powered objectives and then increase power after you find the sample.
Chapter 3: Getting Started CAUTION: Make sure that the objective is selected in the Resolve3D Lens list. 4. Rotate the dichroic filter wheel to select the appropriate dichroic mirror. For standard filters, use position 1. 5. In the Resolve3D Window, click the Info button to open the Lens Information window. Refractive Index Optical Conditions 6. In the Optical Conditions fields, enter the conditions for the sample.
22 DeltaVision Core and personalDV User's Manual 7. Note the displayed value in the Recommended Refractive Index field and use an oil with that refractive index. Before you start each data collection session, calculate the oil. CAUTION: Your oil selection should be the same if the sample and conditions are the same. 8. Place a drop of oil on the objective. Be sure to use the proper oil (see Page 12). CAUTION: Do not touch the glass dropper to the objective. 9. Place a drop of oil on the coverslip.
Chapter 3: Getting Started 23 The displayed colors match the wavelength of the filters. If a QLM module is installed (not available on personalDV), the Filter Monitor also displays the wavelengths of the lasers that are available on your system. Note You must move the filter wheel to initialize the Filter Monitor.
24 DeltaVision Core and personalDV User's Manual 3. Open the Excitation shutter by pressing the EX SHUTTER button in the lower left corner of the keypad. You should see light through the objective. The light on the stage should be the same color as the Excitation filter that you selected. (For example, if you selected DAPI, the light should be very deep violet. It may be hard to see.) Be sure the eyepiece filter position matches the desired excitation filter position.
Chapter 3: Getting Started 25 Acquiring an Image To acquire a DeltaVision image, you’ll need to direct light to the camera and work with the images that are displayed in the Data Collection window until you are satisfied. Then you can save the image as a DeltaVision file or create and run experiments, as shown in Creating and Running an Experiment Macro on Page 31. To acquire an image: 1. On the keypad, press EX Shutter to close the shutter. 2. Switch the beam selector to camera. 3.
26 DeltaVision Core and personalDV User's Manual Tip You can also acquire an image by clicking the ACQUIRE IMAGE button, right clicking on the stage view, or choosing File | Acquire on the Resolve3D menu. 7. To enlarge the thumbnail image that appears in the stage view, drag the mouse down over the zoom wheel. The Zoom Wheel Note Thumbnails appear only when the Show Stage Thumbnails option is selected on the Misc tab on the Resolve3D Settings window. 8. To clear the thumbnail image, click .
Chapter 3: Getting Started 27 2. In the Data folder field, enter the directory (e.g., /data1/myData) in which you want to save your images. To create a new folder, type the name of the folder after /data1 (e.g., /data1/myNewData) and select Save Settings. ! Important Store all files in the /data1 directory unless you are instructed otherwise by your system administrator.
28 DeltaVision Core and personalDV User's Manual Tip You can also open the Snapshot dialog by right clicking on the Resolve3D window to open a shortcut menu and choosing Snapshot. 2. In the Image File Name field, enter a file name. Then select which channels to save. (Snapshot uses the exposure conditions that are displayed in the Resolve3D window for those channels.) Note SoftWoRx adds the _R3D.dv extension to the file name.
Chapter 3: Getting Started 29 4. Log out of the workstation account. 5. Press the IC/MIC power switch to turn off the IC/MIC. Wait 30 – 60 seconds for the IC/MIC to power down. Note If the IC/MIC fails to turn off within 30 – 60 seconds after pressing the power switch, press and hold the IC/MIC power switch for 5 seconds to turn off the device. 6. Clean the objective with a clean, unused cotton swab or lens paper to remove all of the oil on the objective.
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4 4 Setting up and Running Experiments A DeltaVision experiment is a macro that runs a set of commands to collect image data. You will use experiments to acquire almost all of your data. This chapter shows how to set up and run experiments that use several DeltaVision data collection options.
32 DeltaVision Core and personalDV User’s Manual and include numerous options. After you create a macro, you can use it as a template to modify and create other macros. To create and run an experiment macro: 1. At the top of the Resolve3D window, click the Experiment button to open the Design/Run Experiment window. 2. Click the Design Experiment tab. 3. In the Experiment Name field, enter a name for the macro. Note By default, SoftWoRx uses Resolve3D as the experiment name.
Chapter 4: Setting Up and Running Experiments 6. In the Image file name field, enter the file name. Notes #1 Do not use file names that include spaces or special characters (e.g., %, /, \, “, !, $, #, ~, *, &, @, ’, +, = ). #2 The image file name has an _R3D.dv extension. This is the unprocessed (i.e., raw) Resolve3D image. A file with a .log extension is also created. The .
34 DeltaVision Core and personalDV User’s Manual When you set up a Z scan on DeltaVision, you normally begin by defining a focus range to use. This is accomplished by marking the top and bottom of the sample. You then focus on a plane of interest. The focus range is taken above and below the plane of interest. If you are new to 3D microscopy, you may have never had to deal with the specimen's three-dimensional size (except in relation to the rest of the specimen).
Chapter 4: Setting Up and Running Experiments 35 and displayed in the Data Collection window.) Drag the slider and acquire images until you are satisfied with the position. Then press the button to mark that location. Next, use the slider to find the bottom of the sample. When you are satisfied, press the button to mark that location. Note To reduce the risk of running the objective lens into the sample, the Z slider is limited to 5 μm at a time.
36 DeltaVision Core and personalDV User’s Manual 4. Specify the focus point of the microscope at the start of the experiment and move the stage to that focus point as follows: Choose one of the following *focus points in the Focus point when scan starts list. Then click the corresponding button in the Resolve3D window to move the stage to that focus point Top of the sample Middle of the sample Bottom of the sample * For most applications, the Middle of the sample provides the best results.
Chapter 4: Setting Up and Running Experiments 37 • To get the thickness of the specimen, as defined in Step 2, click Get thickness. softWoRx adjusts the number of sections to span the specimen thickness. • To specify the number of sections, enter the number of sections in the Number of optical sections field. 7. Save and run the experiment.
38 DeltaVision Core and personalDV User’s Manual Depth of Field Focal Plane Optical Section Spacing The Optical section spacing specifies the upper and lower boundaries for which information is collected. It is centered on the focal plane and includes the information above and below that plane. When you select an Optical section spacing, DeltaVision uses a Z step size that is the same distance as the Optical section spacing.
Chapter 4: Setting Up and Running Experiments 39 For best results, use at least three optical sections. Using one or two optical sections, however, will also provide acceptable results. The maximum number of optical sections is limited by memory and time. Applied Precision's benchmark deconvolution consists of 64 optical sections that have XY dimensions of 512x512.
40 DeltaVision Core and personalDV User’s Manual not absolute coordinates. All motion in Z is relative to the starting point and does not refer to an absolute Z position. Note The top and bottom can be viewed by pressing the button or the button respectively. The middle section can be viewed by pressing the button. Overscan Method: As the name implies, the overscan method involves using a Z range that is much greater than the range of interest.
Chapter 4: Setting Up and Running Experiments 41 Choosing which filters to use Your choice of filters will be determined by the types of fluorescent probes with which you have labeled your sample and the filters available on your system. The excitation and emission spectrum of your filters should match the spectra for the absorption and emission of the fluorescent probes you want to use. The following tables show which DeltaVision filters match each particular probe.
42 DeltaVision Core and personalDV User’s Manual the human eye at this wavelength. #4 The standard configuration for the six position filter wheels includes: DAPI, FITC, TRITC, CY-5, a polarizer, and a block position. For Instructions on to change filter wheel modules and calibrate filter wheels, see Changing Filter Wheel Modules on Page 114 and Calibrating the Filter Wheels on Page 121. To specify which filters to use: 1. On the Design/Run Experiment window, click the Design Experiment tab.
Chapter 4: Setting Up and Running Experiments enter a value in this field, the most recent exposure value is automatically set for each filter. 5. To select a different emission filter or to attenuate the intensity, choose a filter from the EM Filter or the %T lists. 6. You can choose which light source to use for each channel by selecting a shutter from the EX Shutter field.
44 DeltaVision Core and personalDV User’s Manual 2. Enter the desired time interval in the Time-lapse field. Note The minimum time interval is limited by acquisition time. If you specify a time that is less than the acquisition time, the experiment proceeds at the quickest possible rate. 3. Set up the remaining parameters for your experiment in one of the following ways: • Enter the desired number of time points in the Time Points field.
Chapter 4: Setting Up and Running Experiments Point Visiting monitors points that are in different fields of view To set up an experiment that visits points, you must: Mark the points to visit with the Keypad or with Resolve3D and save a point list. Edit the point list if necessary. Load the point list and specify which points to visit.
46 DeltaVision Core and personalDV User’s Manual The points marked using the keypad are automatically communicated to Resolve3D and displayed in the Stage view. If you open the Point List dialog, you will see the list of points. To mark points using the keypad and Resolve3D: 1. Use the arrows on the keypad or the joystick to move to the desired point. 2. Press the POINT MARK key on the keypad. The number of the marked point is displayed in the Stage view. Marked point in the Stage View 3.
Chapter 4: Setting Up and Running Experiments 47 The stage view after zooming in (left) and panning (right) 5. In the Resolve3D window click to open the Point List dialog box. 6. Click on a point to select it and then click Visit Point to move the stage to that point. 7. Use the XY Stage Controls on the Stage View to move the stage to the exact XY position. XY Stage Controls 8. Use the Fine Z Focus knob to focus while looking at the sample through the eyepieces.
48 DeltaVision Core and personalDV User’s Manual 2. Click Delete Point. To save the point list: 1. In the Point List dialog box, click Save List. 2. In the Prompt dialog, enter the desired name for the list, using eight characters or less. Since there is really no method of displaying a directory of these names, write the name of the list down to keep a record of it. 3. Press Enter or click OK.
Chapter 4: Setting Up and Running Experiments 2. Open the Design/Run Experiment window and click the Design Experiment tab. Then click the Point Visiting tab and check the Visit Point List option. The Point Visiting dialog box is linked to the point list that is open. 3. Enter the points that you would like to visit in the Visit Point List field. (For example, entering 1-3 specifies to visit points 1, 2, and 3. Entering 1-3, 5-7, 9 specifies to visit points 1-9 but not 4 or 8.
50 DeltaVision Core and personalDV User’s Manual 3. In the Image Display mode list, select Point Track. Collecting Panel Images Over Large Areas Panel collection macros are useful when you want to scan a large area with a relatively high magnification lens. You can use the panels as a means of reviewing a large area of a slide, or as data that you want to stitch together to form a single, large image.
Chapter 4: Setting Up and Running Experiments CAUTION: Do not try to reuse panel collection experiment macros. These macros are sensitive to microscope settings such as image size and magnification because the number of panels required depends upon many factors. Determining Border Rolloff Voxels Before you collect panel images, you must first determine the number of voxels in the Border Rolloff that is used in Deconvolution. Note Conceptually, a voxel is a three-dimensional pixel.
52 DeltaVision Core and personalDV User’s Manual 6. Click Get Start under the Panels tab. Then visit the other point that you marked in Step 1 and click Get End. (Alternatively, you can enter the coordinates that are displayed in the Point List dialog box.) 7. In the Overlap (pixels) field, enter at least twice the number of border rolloff voxels as you recorded when you determined Border Rolloff voxels (see Page 51).
Chapter 4: Setting Up and Running Experiments Conceptual view of Multiplexed Wavelength functionality Before you use the Multiplexed Wavelength option, you must first have it installed and configured correctly. Your Applied Precision representative will assist you in setting up this option and help you ensure that all hardware and software to support Multiplexed Wavelength functionality is installed properly.
54 DeltaVision Core and personalDV User’s Manual Settings The Resolve3D Settings window is displayed. 2. In the Resolve3D Settings window, click on the Misc tab. Misc tab 3. In the Resolve3D Settings window, select the EX and EM filter sets you want to use. When these fields are changed, the <<
Chapter 4: Setting Up and Running Experiments 55 Select the EX and EM filter sets Message displayed when filter wheel settings are changed 4. Click Activate Filter Sets. The following confirmation window is displayed. Note If you select filter sets for the Excitation filter wheel and Emission filter wheel fields and then click Done in this window, your selections are retained until you either activate the filter sets or exit Resolve3D.
56 DeltaVision Core and personalDV User’s Manual 6. Insert the selected secondary filter insert (mCherry is used in this example) into Filter Slot 1 of the secondary light path and click Next to continue. The system gathers the information for this window (in this case, “position 4 (500 LP)”) from the MXWSetup.ini file, not from the Instrument Controller. 7. Move the beam combiner to the appropriate position and click Next to continue.
Chapter 4: Setting Up and Running Experiments 57 At this point, you have completed activating the Multiplex Wavelength filter set. You should now continue with the steps in the next procedure for viewing a sample with the Multiplexed Wavelength operation. To view a sample using the Multiplexed Wavelength option: 1. Rotate the eyepiece filter wheel to the POL or BLANK position. 2. From the Resolve3D main menu, select the Excitation filter currently in the primary light path (CFP or GFP).
58 DeltaVision Core and personalDV User’s Manual 3. Use the EX button on the keypad to open the primary EX shutter and view the primary light path. 4. Use the EX2 (formerly CAMERA SHUTTER) button on the keypad to open the EX2 shutter and view the secondary light path. Note With the Multiplexed Wavelength option, you can view both selected wavelengths simultaneously by opening both EX shutters at the same time.
Chapter 4: Setting Up and Running Experiments 59 Multiplexed Tab The Multiplexed tab of the Design/Run Experiment window is displayed as shown. Select Checkbox 3. From the Multiplexed tab, select the Do Multiplexed Channel Imaging checkbox. If the currently active filter set is not Multiplex capable, the following window is displayed.
60 DeltaVision Core and personalDV User’s Manual You will need to change the active filter set to continue. Press OK to return to the Design/Run Experiment window. To change the active filter set for Multiplexed Wavelength experiments, see the procedure for activating a multiplexed wavelength filter set in “Setting Up the Multiplexed Wavelength Option.” (Also see “Setting Up Filter Wheels” in Chapter 8 for additional information on changing the active filter set.
5 5 Acquiring Data From Live Specimens This chapter shows how to use the following features to image live cells with DeltaVision. Set up experiment macros to automatically focus before acquiring each image. This is useful for cells that move during the experiment. Use Cell Tracking to follow cells as they move laterally and move the stage to keep them in the field of view. Use Optical Axis Integration (also referred to as Z Sweep Acquisition) to acquire a 2D projection.
62 DeltaVision Core and personalDV User's Manual Using Autofocus in Experiment Macros You can set up your experiment macro to automatically focus before each image is acquired. This is especially useful for long time-lapse experiments that are susceptible to changes in environmental conditions. Autofocus allows you to acquire focused images without closely monitoring the experiment.
Chapter 5: Acquiring Data From Live Specimens 2. Click the Design Experiment tab and then click the Time-lapse tab. 3. Select the Autofocus before imaging option. When this option is selected, the software automatically determines the parameters for the Autofocus process. 4. To change the various Autofocus parameters, click the Autofocus Options button. The Resolve3D Settings dialog box is displayed with the Autofocus tab selected. 5. Deselect the Automatically determine parameters option.
64 DeltaVision Core and personalDV User's Manual Time Point 1 Time Point 2 Time Point 3 Time Point 4 At Time Points 1 and 2, the cell is moving through the ROI (the inner square), but the center is still within the Move Threshold (the circle). At Time Point 3, once the center of the cell touches the threshold boundary, the stage moves to re-center the cell within the ROI. Note the new stage position displayed in the lower-right corner of Time Point 4. To use Cell Tracking: 1.
Chapter 5: Acquiring Data From Live Specimens 3. Click Cell Tracking Options. 4. Manually acquire an image for each channel and determine which single channel best identifies the features of your specimen. In the Reference Channel list, specify that channel. (The channels are numbered in the order that they are listed on the Design Experiment Channels tab.) Note DeltaVision uses the reference channel for image recognition. 5.
66 DeltaVision Core and personalDV User's Manual visiting experiment, make sure that the points that you are monitoring are centered before you start your experiment. 8. Click the Run Experiment tab and then click Start Scan to run the experiment.
Chapter 5: Acquiring Data From Live Specimens Channels are numbered as they appear in the Experiment Designer window. In the following example, DAPI is Channel 1 and FITC is Channel 2. Move Threshold The Move Threshold is the distance the center of the cell (as defined by the tracking method) must travel before the system resets the stage.
68 DeltaVision Core and personalDV User's Manual ROI Percent With rare exceptions, few live cells are isolated on the substrate. Most are genetically “programmed” to seek attachment and communication with other cells. When performing live cell microscopy, not only is it important to filter out unnecessary stage movement, it is also important to define an appropriate region of interest within the field of view.
Chapter 5: Acquiring Data From Live Specimens 69 Acquiring 3D Z Projections with OAI Continuous Stage Movement Multiple Stage Stops Optical Axis Integration (or OAI), also referred to as Continuous Z Sweep, is useful for acquiring 3D Z projections of live specimens. Instead of collecting an individual image at each focal plane, OAI collects and integrates one continuous image through an extended Z movement.
70 DeltaVision Core and personalDV User's Manual The following 2D image of endosomes in a HeLa cell (left) and a 3D Z projection (right) of the same area were acquired under similar conditions. The additional data in the 3D Z projection include objects that moved out of the depth-of-field of the 2D image during the data acquisition process. endosomes in a HeLa cell (left)and an instant 3D Z projection (right) of the same area Using Continuous Z Sweep To set up a Continuous Z Sweep Experiment: 1.
Chapter 5: Acquiring Data From Live Specimens 3. Click Experiment to open the Design/Run Experiment window. Then click the Sectioning tab. 4. Select the Z Sectioning and Enable OAI Scan options. 5. Click the Run Experiment tab and click Start Scan to start the experiment. Note When deconvolving OAI images, select the More Options button on the Deconvolve window, and then select the Deconvolve Projections option in the More Deconvolution Options dialog box.
72 DeltaVision Core and personalDV User's Manual You can specify to acquire the reference image at the top, middle, or bottom of the sample. Top of Sample Middle of Sample Bottom of Sample Note It is important to optimize the reference image settings prior to defining them in the Experiment Designer. To create a reference image: 1. Set up a 3D Sectioning experiment (see Sectioning Specimens for 3D Images on Page 33). 2.
Chapter 5: Acquiring Data From Live Specimens This experiment specifies using the DAPI and FITC filters to acquire images of each section and using the transmitted light to acquire a reference image in the middle of the sample. 6. Save and Run the Experiment. After DeltaVision acquires the data, it creates two image files. One file contains the reference image data and the other file contains all of the other data.
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6 6 Data Collection Techniques This chapter provides guidance and suggestions for: Finding a Specimen and Recording Its Position Finding Exposure Time Using Köhler and Critical Illumination Monitoring Data Acquisition Editing Experiment Macros Finding a Specimen and Recording its Position Recording the position of your slide is useful when you are conducting a point visiting experiment and you need to remove the slide before you are finished.
76 DeltaVision Core and personalDV User's Manual Note If you are using an oil immersion lens, you must apply immersion oil to the sample. See Appendix A: The Oil Immersion Kit, for more information about the oil calculator. To find and center the specimen: 1. Choose the objective by rotating the objective turret. Be sure to select the same objective in the Resolve3D window. 2. Secure the slide.
Chapter 6: Data Collection Techniques 8. Open the EX shutter and find the desired focal plane optically using either the focus knobs on the microscope or the Resolve3D Z stage controls. Note If you are using an Olympus or Nikon microscope, rotate the top of the focus knob toward you to move the objective up and away from you to move it down (when you are sitting in front of the microscope). 9.
78 DeltaVision Core and personalDV User's Manual 13. Record the position of the slide on the Position Indicator (the letter scale at the bottom of the Repeatable Slide Holder). The Position Indicator Finding Exposure Time The exposure time correlates with the signal intensity level for the image acquisition. Finding the appropriate exposure time for each filter in an experiment is crucial to acquiring the best image data. Many factors must be considered.
Chapter 6: Data Collection Techniques 79 The response of increasing the exposure time is roughly linear. Therefore, if an exposure time of 0.2 sec results in a maximum intensity level of 1000 counts, then an exposure time of 0.4 sec will result in a maximum intensity level of approximately 2000 counts. Use the following instructions to find the best exposure time for each excitation wavelength. This procedure must be performed for each excitation wavelength used in the experiment.
80 DeltaVision Core and personalDV User's Manual Using Köhler and Critical Illumination DeltaVision Core allows you to easily switch between two types of illumination: • Köhler Illumination is the most commonly used form of illumination. It provides very even specimen illumination across the field-of-view. The light uniformly drops off as the distance from the focal plane increases. • Critical Illumination directs the entire light source to the size of the detected area, and not the entire sample.
Chapter 6: Data Collection Techniques 81 To switch to Köhler Illumination: X Loosen the Locking Knob and push the Focus Control toward the stand until it snaps into place in the Köhler Illumination position. Then tighten the Locking Knob. Focus Control Köhler Illumination Spring Locking Knob In the Köhler Illumination position, the Fiber Optic Module is pushed in so that the Köhler Illumination Spring is in the groove.
82 DeltaVision Core and personalDV User's Manual To view previews of deconvolved images: 1. From the Resolve3D window, click Settings to open the Resolve3D Settings dialog box. Then click the Display tab. 2. Select the Deconvolve preview images option. Note This option does not provide a full iterative deconvolution, but it allows you to preview images as you collect them.
Chapter 6: Data Collection Techniques To select the display mode: 1. From the Resolve3D window, click Settings to open the Resolve3D Settings dialog box. Then click the Display tab. 2. On the Image display mode list, select a display mode. Table 1: Image display modes Mode Description None Displays images in the current window. Scratch Displays all images in the default Data Collection window (Window 21). Auto Grayscale Displays images in a separate window for each emission filter.
84 DeltaVision Core and personalDV User's Manual Displaying Statistics and the Histogram You can choose to display statistics and a histogram of the intensity values of each image as it is collected. These values are displayed at the bottom of the Resolve3D window. The Min, Max, and Mean values are the minimum, maximum, and mean intensity values. The histogram displays the intensity distribution. To display statistics and the histogram: 1.
Chapter 6: Data Collection Techniques 85 Status Area Commands list Macro Text Area The Experiment Macro Editor Menu Bar The menu bar File menu contains commands to open and save experiment macro files. The Save & Quit option saves the macro file with the current name. The Edit menu has commands for manipulating text. The Search menu provides search and replace capabilities and the Help menu displays additional information.
86 DeltaVision Core and personalDV User's Manual To edit a reference macro: 1. On the Resolve3D Experiment Macro Editor window, choose File | Open Reference Macro. 2. In the Open Experiment Macro File dialog box, select a macro file and click OK to open it. 3. Use the mouse to select the part of the reference macro that you want to copy. 4. On the Reference Macro menu, choose Edit | Copy. 5. On the Resolve3D Experiment Macro Editor, choose Edit | Paste to copy the macro into the macro editor. 6.
7 7 Facility Requirements & Components This chapter describes the main components of the DeltaVision System. It includes the following sections: Electrical and Environmental Requirements describes the DeltaVision operating and facility requirements. Overview of Components shows the location of the key system components. Optical Components describes the system light sources, cameras, and filters.
88 DeltaVision Core and personalDV User's Manual Electrical and Environmental Requirements An important aspect of collecting high quality images is meeting the proper electrical and environmental requirements for the system.
Chapter 7: Facility Requirements and Components Country-specific Requirements The following table shows the accredited agency and power cord set requirements for each country.
90 DeltaVision Core and personalDV User's Manual Temperature: 65 - 77 °F (18 - 25 °C), daily variation of no more than 3 °F (1.8 °C). The actual room temperature should be stable to within 1 degree (Fahrenheit or Celsius) per hour. Fluctuations in temperature will affect microscope optics, which can cause the specimen to drift approximately 1 μm per 0.1 degree Celsius. Humidity: Stable humidity levels under 50%, with daily variations of less than 10%.
Chapter 7: Facility Requirements and Components 91 Ambient Illumination For best results, minimize ambient illumination during data collection. A lighttight room is recommended. Ensure that there are no light sources pointed downward into the lens. A small desk lamp located near the workstation is recommended for preparing and monitoring experiments. You can press the BLANK SCREEN key on the keypad to darken the monitor for improved image quality.
92 DeltaVision Core and personalDV User's Manual personalDV Components Optical Components IC/MIC and Workstation Desktop Components Optical Components Xenon Arc Lamp White LED Trans Light Excitation Filters Neutral Density Filters Eyepiece Filters Camera Emission Filters Note Optical components are shown for a typical installation, which includes the Olympus IX71 Microscope. Other microscope configurations vary slightly. The Olympus IX71 is currently the only microscope available.
Chapter 7: Facility Requirements and Components 93 Fluorescence Microscope The microscope is an advanced research grade epifluorescence inverted microscope. Each objective is qualified by Applied Precision to guarantee the highest possible quality. The point spread function (PSF) is measured in order to uphold the image quality. The microscope supports a Differential Interference Contrast (DIC) module (this is an optional component).
94 DeltaVision Core and personalDV User's Manual personalDV comes standard with the CoolSNAP ES2 Camera. High-Speed Camera The High-Speed CCD camera (CoolSNAP HQ2 ) collects digital image data from the microscope. This camera is designed to collect image data at a high frame rate. At the fastest speed, the camera can collect 30 frames per second of a 64 × 64 pixel image. Increased acquisition speed is useful when collecting images of live cells that deteriorate over time.
Chapter 7: Facility Requirements and Components High-speed Camera Power Supply The High-speed Camera power supply provides electric power to the camera. It is housed on the bottom shelf of the cabinet. ES2 Camera The CoolSNAP ES2 CCD Camera is basically the same as the CoolSNAP HQ2 Camera, except the ES2 is not as deeply cooled. This makes it more affordable, but allows for slightly higher noise. However, the noise difference is virtually unnoticeable unless you are using long exposure times (~8s).
96 DeltaVision Core and personalDV User's Manual WARNING: The xenon arc lamp presents potentially harmful risks to the user, including the possibility of UV exposure to skin and eyes. Before operating the microscope, consult Chapter 2: Safety for important information regarding arc lamp operation. Note The illumination path alignment is critical to acquiring the highest possible image resolution. See Page Path on Page 146 for a complete description of the alignment procedure.
Chapter 7: Facility Requirements and Components Flat-Panel Display Monitor All DeltaVision systems are equipped with Flat-Panel LCD monitors. These monitors offer a very high level of performance in several areas pertaining to the quality of displayed images, the most critical of which is contrast ratio. Note For instructions that show how to adjust the monitor, see the Flat-Panel Display manual. CAUTION: DeltaVision is configured to work with the monitor that is included with the system.
98 DeltaVision Core and personalDV User's Manual Vibration Isolation Table Within the vibration isolation table is a breadboard surface that is supported by mechanical vibration isolators. These components provide optimal performance without an external air source. The isolators are sized for the system weight as delivered. If significant additional weight is added to the breadboard, higher capacity isolation may be required. Contact Applied Precision for more information.
Chapter 7: Facility Requirements and Components Note Configuration of cabinet components may vary slightly. Instrument Controller / Microscope Interface Chassis (IC/MIC) The Instrument Controller (IC) is the portion of this computer that interfaces with all of the microscope hardware (including the microscope stage motors, filter wheel motors, and cameras). It coordinates all activities related to positioning the stage and collecting images.
100 DeltaVision Core and personalDV User's Manual The Repeatable Slide Holder The Repeatable Slide Holder holds the slide on the stage. It also allows you to move the slide across the stage and to mark the slide position when you remove the slide. The ability to move the slide across the stage allows you to view the full slide on the 1" x 1" stage area. With the slide held against the three brass locators by the Return Spring, you can use the Slide Adjustment Knob to move the slide laterally.
Chapter 7: Facility Requirements and Components Calibration Kit The Calibration Kit is provided for calibration operations on the DeltaVision system. The kit includes the following six slides: • Three plastic fluorescent calibration slides – for flat-field calibration: • • • Blue plastic (EX 408nm, EM 440 nm) – Good for DAPI, Hoechst, etc. Orange plastic ((EX 488nm, EM 519nm) – Good for FITC, GFP, TRITC, CY-3®, etc. • Red plastic (EX 590nm, EM 650nm) – Good for CY-5®, etc.
102 DeltaVision Core and personalDV User's Manual Fiber Optic Module The Tool Kit A tool kit is provided that includes tools for maintaining the system.
Chapter 7: Facility Requirements and Components softWoRx softWoRx is the Linux software application that runs the acquisition workstation.
104 DeltaVision Core and personalDV User's Manual Optional Components You can purchase several optional DeltaVision components. Some options are available for both DeltaVision Core and personalDV, but many are available only for DeltaVision Core (as noted below): The Environmental Chamber provides a controlled temperature environment for live cell imaging. The chamber also supports CO2 injection, which allows you to control humidity and pH by maintaining a CO2 flow over the sample.
Chapter 7: Facility Requirements and Components The following subsections describe each of the optional DeltaVision components. The Environmental Chamber The Environmental Chamber (also called the Weather Station) includes a temperature controller, a CO2 humidifier, and a CO2 chamber enclosed in a Plexiglas covering. Note On some systems, the heater is on the other side of the Environmental Chamber.
106 DeltaVision Core and personalDV User's Manual The Live Cell Filter Module The Live Cell Filter Module includes the following filters: Filter Name Excitation CWL/BP Emission/Eyepiece CWL/BP CFP 430/24 470/24 YFP 510/20 535/30 mCherry 572/35 632/60 EGFP 470/40 525/50 Quantifiable Laser Module Components The Quantifiable Laser Module (QLM) adds a laser beam into the back aperture of the microscope objective to provide a focused illumination spot in the center of the optical field.
Chapter 7: Facility Requirements and Components 107 Total Internal Reflection Fluorescence (TIRF) Module The TIRF module includes the Olympus TIRF component and special adapters required to install it on a DeltaVision system. (TIRF requires a system with a QLM hardware module.) Note TIRF is not an available option for personalDV.
108 DeltaVision Core and personalDV User's Manual The addition of the Cascade II with Electron Multiplying CCD (EM-CCD) technology further improves the signal-to-noise-ratio (SNR) of the DeltaVision system. Unlike conventional CCD cameras, an Electron Multiplying CCD (EMCCD) is not limited by the readout noise of the output amplifier, even when operated at high readout speeds.
Chapter 7: Facility Requirements and Components Software softWoRx Explorer ® softWoRx Explorer is a cross-platform image viewer that is available for the following operating systems: For 32 bit systems Apple Mac OSX 10.2, 10.3, and 10.4 Microsoft Windows 2000 Professional, XP Home, or XP Professional Red Hat Enterprise Linux 2.1 - 4.0 with Gnome or KDE interface SuSe 8.2 - 9.2 For 64 bit systems Linux Red Hat Enterprise 3.0 - 4.0 SuSe 8.2 - 9.
110 DeltaVision Core and personalDV User's Manual The Colocalization feature enables users to easily isolate, visualize, and quantify regional overlap in 3D and 4D images. Results can be presented in two ways—as a new 3D or 4D channel or as a statistical report. softWoRx Data Management Solution (DMS) Support softWoRx DMS provides a functional infrastructure for the storage of biological images and their associated metadata in a centralized database.
8 8 Changing Cameras and Filters This chapter provides the following instructions for cameras and filters: Changing Cameras Using Live Cell or Custom Filter Wheel Modules Changing Filter Wheel Modules Calibrating the Filter Wheels Changing Cameras DeltaVision Core includes a High-speed Camera as a standard component. If you have the optional EM CCD camera, you can change cameras to better meet your imaging needs.
112 DeltaVision Core and personalDV User's Manual To change the camera: 1. Remove the camera cover by lifting it up and sliding it away from the microscope. 2. Remove the camera by pushing in and then pulling up on the camera end. You do not need to remove cables from the camera. 3. Remove the other camera from the camera tray and place the camera that you just removed on the camera tray. Always place the camera in the camera tray when it is not CAUTION: secured to the system.
Chapter 8: Changing Cameras and Filters 2. On the Imaging tab, click the Camera list and select the camera that is currently installed. Notes #1 The EM CCD camera is listed twice in the Camera list: CASCADE2_512 Conv./… sets the camera in Conventional mode. Cascade2_512 EMCCD/… sets the camera in electronmultiplication mode. #2 When the camera is changed, the binning number is reset to 1. #3 If the camera cables loosen or are accidentally disconnected, you may need to restart the IC software.
114 DeltaVision Core and personalDV User's Manual DeltaVision Filter Wheel Module Excitation filter wheel Emission filter wheel Eyepiece filter wheel A filter wheel module includes the Excitation, Emission, and Eyepiece filter wheels and can hold up to six filter sets. If your system has an alternate filter wheel module, you can swap modules to meet your imaging needs.
Chapter 8: Changing Cameras and Filters 115 WARNING: Before you start, make sure that the xenon lamp and the IC/MIC are turned OFF. To change the Emission filter wheel: 1. Turn off the IC/MIC. 2. Remove the camera cover. Then remove the camera by pushing in and pulling up on the camera end as shown below. 3. On the Emission filter wheel motor, disconnect the cable that connects the motor to the IC/MIC.
116 DeltaVision Core and personalDV User's Manual IC/MIC Cable Connector 6. Install the alternate Emission filter wheel. Tighten the Emission filter wheel set screw. 7. Reinstall the camera by sliding it into place and then pushing in and pressing down on the camera. 8. When you are finished changing filter wheels, restart the IC/MIC. 9.
Chapter 8: Changing Cameras and Filters 117 1. Ensure that the IC/MIC is turned off. 2. Turn off the xenon arc lamp and allow it to cool. 3. Loosen the two silver thumb screws at the top and bottom of the filter wheel housing. Thumb screws should loosen without the use of tools, but can also be removed with a flat-blade screwdriver if necessary. Loosening top thumb screw from filter wheel housing CAUTION: Do not bend the fiber optic cable into a coil with a diameter less than 24".
118 DeltaVision Core and personalDV User's Manual Removing the Excitation Filter Wheel housing from its mounted position 5. Disconnect the cable to the IC/MIC from the Excitation filter wheel and connect it to the alternate Excitation filter wheel. Disconnecting the IC/MIC cable from the Excitation Filter Wheel 6. Slide the alternate Excitation filter wheel housing back into its mounting position by pulling back on the Focusing Lens assembly as shown.
Chapter 8: Changing Cameras and Filters 119 7. Re-engage the Focusing Lens housing into the beveled support on the new Excitation filter wheel housing and align and tighten the two thumb screws at the top and bottom of the Excitation filter wheel housing. 8. Tighten the thumb screw on the Focusing Lens housing. 9. When you are finished changing filter wheels, restart the IC/MIC. 10.
120 DeltaVision Core and personalDV User's Manual 2. Holding the oculars in one hand, use the Olympus 3 mm hex key to loosen the set screw that holds the oculars to the Eyepiece filter wheel and set the oculars on the table. set screw Loosening the set screw and removing the oculars 3. Loosen the set screw that holds the Eyepiece filter wheel to the beveled mount on the stand and remove the Eyepiece filter wheel. set screw Removing the Eyepiece filter wheel 4.
Chapter 8: Changing Cameras and Filters 7. If the IC/MIC is on, click OK on the message box that indicates the cable is removed. When the message closes, the system is ready only if the position sensor is working. If the IC/MIC is off, you will need to manually update the filter set in the Resolve3D | Settings | Misc tab window. Calibrating the Filter Wheels Calibration initializes the filter positions and ensures that the filters are centered in the filter wheel openings.
122 DeltaVision Core and personalDV User's Manual a) If the Linux desktop is displayed, press Scroll Lock on the keyboard twice and then press the up arrow. b) Press the Esc key twice. c) When you are prompted to exit, type Y. 2. Double-click the Instrument Controller desktop folder to open it. 3. Double-click the Controller Configuration icon to open the ic540.ini file. 4. Create a backup file by renaming and saving the file.
Chapter 8: Changing Cameras and Filters 123 Home Position Offset = 0.0 Note You can cut and paste these settings from the .pdf file that includes these instructions to the ic540_dv.ini file. 8. Verify that the file includes the following Eyepiece filter settings for the Live Filter Set. These settings are typically below the Eyepiece Filter Wheel section: [Extra Eyepiece Filter Set 1] Filter Set Name = Live Cell MIC ID = 1 Number of Filters = 6 Filter Names = BLANK POL Filter Values 1 = 0.00 0.
124 DeltaVision Core and personalDV User's Manual Table 3: Examples of Filter Positions Filter Name Excitation CWL/BP* Emission/Eyepiece CWL/BP Blank CFP 436/10 465/30 Filter Positions Eyepiece Filter Wheel Label** 0 1 1 2 YFP 492/18 535/30 2 3 dsRED 580/20 630/60 3 4 EGFP 470/40 525/50 4 5 5 6 Blank CWL is wavelength and BP is bandpass ** The label on the Eyepiece Filter Wheel is offset by one number from the filter position that you use for the ic540_dv.ini file.
Chapter 8: Changing Cameras and Filters 125 Note Avoid contact between the filters and your fingertips. If necessary, wear latex gloves for this procedure. 2. On the Filter Wheel, find the position number for the filter that you are installing (as listed on your Customized Filter Table). Use a hex key to loosen the screw of the spring clip for that filter, and then remove the spring clip. 3. Grip the filter by its edges and carefully remove it from the filter wheel.
126 DeltaVision Core and personalDV User's Manual 5. Tighten the spring clip screw, making certain the spring clip arms are set properly over the filter as shown. 6. Repeat Steps 2 through 5 for each Excitation filter you are installing. 7. When you replace the filter wheel cover, be sure the hole in the cover lines up with the hole in the filter assembly. Then tighten the six hex screws.
Chapter 8: Changing Cameras and Filters 127 Holes must line up when filter wheel is assembled. To add emission filters: 1. Use a #1 Phillips screwdriver to loosen the six screws on the Emission Filter Wheel cover and remove the cover. Note Avoid contact between the filters and your fingertips. If necessary, wear latex gloves for this procedure. 2. On the Filter Wheel, find the position number for the filter that you are installing (as listed on your Customized Filter Table).
128 DeltaVision Core and personalDV User's Manual 3. Remove the spring clip and carefully remove the filter by its edges. 4. Place the new filter into the empty pocket, making certain the arrow on the edge of the filter is pointing up (toward the filter cover).
Chapter 8: Changing Cameras and Filters 129 Filter orientation arrow 5. Replace the spring clip and tighten the hex screw. Then, adjust the arms of the spring clip so that it slightly rides up on the metal ring of the filter as shown. 6. Repeat Steps 2 through 5 for each Emission filter you are installing. 7. When finished installing filters, replace the Emission Filter Wheel cover and tighten the six Phillips screws.
130 DeltaVision Core and personalDV User's Manual To add eyepiece filters: 1. Locate the 3mm hex key provided with the microscope. 2. Loosen the four screws that hold the module together and remove the front cover of the module. 3. Find the filter position number for the filter that you are installing.
Chapter 8: Changing Cameras and Filters 4 Drop the new filter into the empty filter holder at an angle so that the o-ring holds the filter in place. Be sure to orient the filter as follows: • For Chroma or excitation filters, the filter must be oriented so that the arrow points in to the direction of the light path (or down). • For Omega filters, the filter must be oriented so that the arrow points out from the direction of the light path (or up). 5. Repeat Step 4 for each filter that you are adding.
132 DeltaVision Core and personalDV User's Manual AppliedPrecision
9 9 Maintenance This chapter provides the following instructions for the basic maintenance of the system: Shutting Down and Starting the System Replacing the Xenon Bulb Replacing the Transmitted Light Aligning the Illumination Path Replacing IC/MIC Fuses Cleaning Moving the System Shutting Down and Starting the System Use the following instructions for shutting the system down during a power outage or other occasions that require total shutdowns.
134 DeltaVision Core and personalDV User's Manual DeltaVision Power Switches The main DeltaVision power switches are shown below. (The Master Switch on the isolation transformer that plugs into the wall is not shown.
Chapter 9: Maintenance 135 Guidelines for Using Switches Main Components Use this switch to turn power on and off for the DeltaVision Workstation, Instrument Controller and Microscope Interface Chassis (IC/MIC), the Fast Camera Power Supply, and the DeltaVision Microscope. Workstation, IC/MIC, and Monitor Leave these switches on except on rare occasions (such as power outages) when you need to shut down the entire system.
136 DeltaVision Core and personalDV User's Manual 1. Turn on the power strip bar. Note For personalDV, there is no power strip bar. Begin the power-up process with Step 2. 2. Turn on the IC/MIC. 3. Turn on the workstation. 4. Turn on the monitor. 5. Follow the instructions for turning on DeltaVision on Page 17. Replacing the Xenon Bulb WARNING: Ensure the xenon lamp is off and has had plenty of time to cool before starting this procedure.
Chapter 9: Maintenance 4. Loosen the two thumb screws on the opposite end of the xenon lamp housing. 5. Gently slide the internal lamp mechanism from the lamp housing. 6. Remove the center clip from the internal lamp mechanism as shown.
138 DeltaVision Core and personalDV User's Manual 7. Lift the bulb assembly (small black box) from the two supporting pins in the lamp mechanism. 8. Replace the xenon bulb assembly with a new one (Part #34-100390-000). 9. Replace the clip around the internal lamp assembly and gently slide the internal lamp mechanism into place within the lamp housing.
Chapter 9: Maintenance 139 10. Tighten the two thumb screws on the end of the lamp housing. 11. Place the open end of the lamp housing over the flange on the DeltaVision and tighten the three hex screws as shown.
140 DeltaVision Core and personalDV User's Manual 12. Turn the DeltaVision on as usual and start Resolve3D. 13. Before resetting the bulb age, write down the age of the bulb you just replaced. This will help you to keep track of when you may need to replace the next one. 14. Open the Imaging tab on the Settings window and reset the bulb age.
Chapter 9: Maintenance 141 hex screws 3. Carefully remove the transmitted light assembly from the housing. 4. Disconnect the other end of the attached cable from the Transmitted Illumination Source connection on the back of the IC/MIC (upper left-hand corner when facing the back of the unit; shown below).
142 DeltaVision Core and personalDV User's Manual Transmitted Illumination Source Connection (back of IC/MIC) 5. Re-insert the new transmitted light assembly (Part #52-851243-000) into the housing and tighten the two hex screws. 6. Re-connect the new transmitted light assembly cable to the Transmitted Illumination Source connection on the back of the IC/MIC (shown above).
Chapter 9: Maintenance 143 The Fiber Optic Module Use the Fiber Optic Module to align the light path from the fiber optic cable to the Fluorescence Illuminator. This module allows you to adjust the tilt, horizontal, and vertical orientation of the light path. Figure 7: The Fiber Optic Module Photo sensor Port Tilt Screws Critical Spring Köhler Spring Locking Knob Y Adjustment Screw X Adjustment Screw Adjustment or Lock Use to Tilt Screws Adjust the tilt of the Fiber Optic Module body.
144 DeltaVision Core and personalDV User's Manual Before You Check or Adjust Illumination Path Alignment Before you check or adjust alignment, remove all sources of blockage to the light path. Then verify the following settings to make sure that the various lenses and controls are in the correct positions (you can feel the controls snap into place when they are set in position). The Fluorescence Illuminator Slider on the back of the microscope is in an open position.
Chapter 9: Maintenance 145 Checking Illumination Path Alignment To provide an optimum light source, the tilt and position of the Fiber Optic Module must be aligned with the microscope. This procedure shows how to perform an “inside-outside” focus test to make sure that the module is properly aligned. It also shows how to perform “in focus” tests to make sure that the position of the module (distance from the microscope) is correct for Köhler Illumination, and if required, for Critical Illumination.
146 DeltaVision Core and personalDV User's Manual Middle Circle Inside Circle Focus on the outside edge of the middle circle After you focus on the outside edge of the middle circle, the outside edge of the inner circle should also be in focus and the centers of the inside and middle circles should be aligned. c. If the image is not if focus, go to Step Three: Set the Köhler Spring Position on Page 149. 9.
Chapter 9: Maintenance 147 2. Use the Eyepiece filter wheel to select the FITC eyepiece filter. In the Resolve3D window, set the other filters for alignment as follows: In this Field Select Excitation TRITC Emission FITC %T 0.1% 3. Press EX SHUTTER on the keypad to open the EX shutter. 4. Use the Z focus knob to focus on the scratches on the mirror slide. 5. Pull out the Field Stop lever all the way to close the Field Stop aperture. 6.
148 DeltaVision Core and personalDV User's Manual Step Two: Adjust the Tilt of the Fiber Optic Module Body 1. Make sure that the Beam Selector on the microscope base is set to direct the light to the eyepiece and verify that the 60X objective is set in its lowest position. The Z focus knob should be turned until it stops (with the objective fully lowered). 2. Remove the oil from the objective (See Cleaning on Page 153) and remove the Repeatable Slide Holder. 3.
Chapter 9: Maintenance 149 Step Three: Set the Köhler Spring Position 1. Move the Eyepiece Selection Wheel from the open (O) to the Centering Telescope (CT) lens. 2. Use the Centering Telescope Focus on the ocular to focus on the objective back aperture (the outside edge of the middle circle). Middle Circle Inside Circle Focus on the outside edge of the middle circle 3. Loosen the Locking Knob and use a 2.5 mm hex key to loosen the two Köhler Spring screws.
150 DeltaVision Core and personalDV User's Manual 5. Slide the Köhler Illumination spring so that it is in the groove on the Focus Control and tighten the Köhler spring locking screws to set the Köhler position. Step Four: Set the Critical Illumination Position 1. Move the Eyepiece Selection Wheel to Open (O) and fully open the field aperture. 2. Apply immersion oil to both the objective and the mirror slide and mount the mirror slide. 3. Loosen the Locking Knob.
Chapter 9: Maintenance 151 2. Adjust the center alignment in the Köhler position as follows: a. Slide the Focus Control to the Köhler position, set the eyepiece to CT, and use the Centering Telescope Focus to focus on the objective back aperture. You should see the area between the back aperture of the objective lens (the darker area) and the fiber tip (bright green). b. Use a 1.
152 DeltaVision Core and personalDV User's Manual 6. Fully close the Field Aperture and slowly rotate the microscope Z focus knob in and out of focus. The light should evenly expand around the aperture. If the light tends in one direction, call the API Customer Service Hotline (1-800-8625166). 7. Move the Fiber Optic Module between the Köhler and Critical position several times and make sure that the positions are repeatable (within about 1%).
Chapter 9: Maintenance Cleaning Most system surfaces are best cleaned with a lint-free cloth or lint-free swabs and spectroscopy-grade isopropyl alcohol or chloroform. Avoid contaminating the cleaning solution by never reusing the cleaning cloth or swabs. Operators should be trained in the handling of flammable liquids such as alcohol. Material Safety Data Sheets (MSDS) should be maintained for the cleaning solutions, as with any hazardous material.
154 DeltaVision Core and personalDV User's Manual AppliedPrecision
A Appendix A: The Immersion Oil Kit The immersion oil kit is a collection of oils with refractive indexes that range from 1.500 to 1.534. Use of the correct immersion oil decreases the spherical aberration in the image data. • For DeltaVision Core, the immersion oil kit includes eighteen oils that range from 1.500 to 1.534, in increments of 0.002. • For personalDV, the immersion oil kit includes six oils that range from 1.512 to 1.522, in increments of 0.002.
156 DeltaVision Core and personalDV User's Manual Distance from Coverslip to Specimen (microns) Establishes the distance from the surface of the coverslip to the desired focal plane. Temperature Defines the temperature of the specimen and the immersion medium. Specimen Refractive Index Defines the refractive index of the specimen, which is usually that of the mounting medium. In some cases, the specimen itself contributes significant refraction.
B Appendix B: Troubleshooting This appendix was designed to help you diagnose and correct the most common problems encountered on the DeltaVision system. It covers two types of troubleshooting tasks: Diagnosing System Problems Analyzing Reasons for Poor Image Quality If you are unable to correct a problem, fill out the DeltaVision Problem Report Form at the end of this appendix and either e-mail it to hotline@api.com or fax it to 425-557-1055, attn: Bio Service Hotline.
158 DeltaVision Core and personalDV User's Manual Table B-1: Controller Troubleshooting Chart Indication Cause Correction Encoder Error when initializing stage. Poor cable connection. Power down system, including IC/MIC. Reseat X, Y, and Z motor cables. Reseat motor cables on excitation module. Power up system. Stalled sequence of images. Computer misallocated memory. Exit the software and restart IC/MIC.
Appendix B: Troubleshooting Indication 159 Cause Correction on Page 136. Ensure that filter cube turret is locked in position on rail mount. Ensure shutter on filter cube is open. Ensure slider behind microscope is seated in an open position. Ensure proper filter cube is in position and seated in detent. Dim illumination. When fiber optic cable and focusing lens are removed, the projected light does not form a circle. Filter wheel(s) out of alignment. Dark, out of focus spots on image.
160 DeltaVision Core and personalDV User's Manual Table B-3: Image Quality Troubleshooting Chart Cont’d Indication Cause Correction Image has a traveling light or bubble. Air bubble in immersion oil. Clean front and back surfaces of objective and coverslip. Reapply immersion oil and restart experiment. Interference in image data. Possibly dirt, dust, oil, or air bubble. Clean front and back surfaces of objective and coverslip. Very bright image or camera saturation message. Camera saturation.
Appendix B: Troubleshooting 161 DeltaVision Problem Report Form Research Facility: Contact Person: Phone: System Serial Number: softWoRx version: (use Help → Software Versions to display) E-mail: Date: Problem Encountered: Please write a detailed description, answering as many of the following questions as possible.
162 DeltaVision Core and personalDV User's Manual AppliedPrecision
C Appendix C: Acquiring a PSF This appendix shows how to acquire a Point Spread Function (PSF) and convert it to an Optical Transfer Function (OTF). Acquiring a PSF shows how to measure a Point Spread Function. Converting PSF to OTF shows how to convert the Point Spread Function to the Optical Transfer Function that is required to process images.
164 DeltaVision Coreand personalDV User's Manual Note If you do not have the tools necessary to acquire a PSF, softWoRx includes a utility that allows you to calculate a theoretical OTF based on the numerical aperture of the camera lens, index of refraction, and emission wavelength. If the aperture of the lens is lower than 0.75 N.A., the calculated OTF may work as well, or even better, than a measured OTF; however, if the aperture is greater than 0.75 N.A.
Appendix C: Acquiring a PSF 165 5. Switch the beam selector to SP or SPL. Then adjust the exposure time and %T filter and click Acquire. Leave Data Collection window 21 open. 6. In the Image window, choose Tools | Measure Distance. Then set the Units to Pixels in the Measure Distance dialog box. 7. Draw a line across the image from a point on the top left square to a point in the same relative position on the top right square. 8. If the vertical delta is more than four pixels, re-align the slide.
166 DeltaVision Coreand personalDV User's Manual For example: If the desired lens is 40X/1.35 with ID=10403 (the third lens in the list), then the pixel size is 0.1656. MS_Lens_Names: 10X 20X 40X 60X 100X MS_Pixel_Sizes: 0.6680 0.3313 0.1656 0.1103 0.06631 MS_Lens_ID_Numbers: 10105 10205 10403 10602 10002 4. Mount a bead slide on the microscope and focus on the beads to obtain the maximum intensity. Find a bead that is located by itself. (Refer to “Finding Beads” located immediately after this procedure.
Appendix C: Acquiring a PSF Selecting the Correct Immersion Oil Accurate PSF measurements depend on the selection of the correct immersion oil. Our experience has shown that the oils recommended by microscope manufacturers are often not ideal for 3-D microscopy. We recommend that PSFs are measured with a minimal amount of spherical aberration. Inappropriate immersion oils yield asymmetric PSF measurements as a result of spherical aberration.
168 DeltaVision Coreand personalDV User's Manual Figure C-1: Flare from Immersion Oils (3-D Maximum Intensity Projections) X Z Correct Immersion Oil Immersion Oil Index Too Low Immersion Oil Index Too High Converting PSF to OTF The PSF to OTF program converts a measured point spread (PSF) to an optical transfer function (OTF). Essentially, the OTF is the Fourier transform of the PSF. The pixel size of the resulting image is given in cycles/μm.
Appendix C: Acquiring a PSF 169 Radial Resolution (XY) Figure C-2: Sample OTF Image Axial Resolution Figure C-3: PSF to OTF Conversion Each option in PSF to OTF Conversion is described briefly below. For additional information regarding these options, refer to the online Help. PSF File Defines the name of the PSF image file to be converted to an OTF. OTF File (sym) Displays the name of the resulting axially symmetric OTF.
170 DeltaVision Coreand personalDV User's Manual X Range Defines the start and end pixel numbers in X. Y Range Defines the start and end pixel numbers in Y. Z Range Defines the start and end pixel numbers in Z. T Range This field is not used for PSF to OTF conversion. Wavelengths Determined by PSF wavelength. Lens ID Specifies the lens identification number (e.g., 12004). Sub-Image: Center Specifies the central XYZ coordinates of the point spread.
Appendix C: Acquiring a PSF • Type the desired path and filename into the PSF File text box. 4. Click Do It. To place OTF into OTF Library If the objective used is in addition to those already present, you’ll need to modify softWoRx to use the new objective by adding the file to either /usr/local/softWoRx or dv2.10/config/system.dvrc as follows: 1. Log in to Linux as root. 2. Navigate to /usr/local/softWoRx/config/system.dvrc 3.
172 DeltaVision Coreand personalDV User's Manual AppliedPrecision
D Appendix D: Reference Information The appendix includes the following topics: Standard Filename Extensions lists the filename conventions used by DeltaVision. Standard Fluorescence Filters shows the excitation and emission peaks of the standard filters included with DeltaVision. Live Cell Filter Sets shows the excitation and emission peaks of the filters that are included in the optional Live Cell filter wheel module.
174 DeltaVision Core and personalDV User's Manual Standard Fluorescence Filters DeltaVision provides a four color filter set, a polarizer, and multiple sets of optional filters. These filters are designed to be used with many common fluorescent probes. If you are using fluorescent probes that are not well matched with the standard DeltaVision filters, contact Applied Precision for assistance. The excitation and emission peaks of the DeltaVision filters2 are provided in the following table.
Appendix D: Reference Information Reference List Selected references are provided on the following pages. Contact Applied Precision for the most recent list. If you notice omissions from the list, please inform Applied Precision. Microscopy Agard, D. A., Sedat J. W. (1983) Three-dimensional architecture of a polytene nucleus. Nature 302: 676-681. Agard, D. A. (1984) Optical Sectioning Microscopy: Cellular Architecture in Three Dimensions. Ann. Rev. Biophys. Bioeng. 13: 191-219. Agard D.A., Hiraoka Y.
176 DeltaVision Core and personalDV User's Manual Belmont A.S. and K. Bruce. (1994) Visualization of G1 chromosomes: a folded, twisted, supercoiled chromonema model of interphase chromatid structure. Journal of Cell Biology 127: 287-302. Charlton C.A., Mohler W.A., Radice G.L., Hynes R.O., Blau H.M. (1997) Fusion Competence of Myoblasts Rendered Genetically Null for N-Cadherin in Culture. Journal of Cell Biology 138: 331-336. Chen H., Sedat J.W., Agard D.A.
Appendix D: Reference Information Hartley W.J. How to Use a Microscope. Doubleday, 1964. Herman B., Jacobson K., ed. Optical Microscopy for Biology. Wiley-Liss, New York NY, 1980. Hiraoka Y., Agard D.A., Sedat J.W. (1991) Temporal and spatial coordination of chromosome movement, spindle formation and nuclear envelope breakdown during prometaphase in Drosophila melanogaster embryos. Journal of Cell Biology 111:2815-2828. (cover article) Hiraoka Y., Dernburg A.F., Parmalee S.J., Rykowski M.C., Agard D.A.
178 DeltaVision Core and personalDV User's Manual Jongens T.A., Ackerman L.D., Swedlow J.R., Jan L.Y., Jan Y.N., (1994) Germ cell-less functions early in the germ cell specification pathway of Drosophila. Genes Devel 8:2123-2136. Kam Z., Agard D.A., Sedat J.W. (1997) Three-dimensional microscopy in thick biological samples: a fresh approach for adjusting focus and correcting spherical aberration. Bioimaging 5:40-49. Kam Z., Chen H., Sedat J.W., Agard D.A.
Appendix D: Reference Information Ma X, Ehrhardt D. W., Margolin W. (1996) Colocalization of cell division proteins FtsZ and FtsA to cytoskeletal structures in living Escherichia coli cells by using green fluorescent protein. PNAS USA, 93: 12998-13003. Marshall W.F., Dernburg A.F., Harmon B., Agard D.A., Sedat J.W. (1996) Specific interactions of chromatin with the nuclear envelope: Positional determination within the nucleus in Drosophila melanogaster. Molecular Biology of the Cell, 7: 825842. Marshall W.
180 DeltaVision Core and personalDV User's Manual Paddy M.R., Belmont A.S., Saumweber H., Agard D.A., Sedat J.W. (1990) Interphase nuclear envelope lamins form a discontinuous network that interacts with only a fraction of the chromatin in the nuclear periphery. Cell 62:89-106. Paddy M.R., Chelsky D. (1991) Spoke: a 120-kD protein associated with a novel filamentous structure on or near kinetochore microtubules in the mitotic spindle. Journal of Cell Biology 113:161-171. Paddy M.R., Saumweber H., Agard D.
Appendix D: Reference Information Straight, A.F, Marshall W.F., Sedat J.W., Murray A.W. (1997) Mitosis in living budding yeast: Anaphase A but no metaphase plate. Science 277:574-578. Sullivan W., Minden J.S., Alberts B.M. (1990) daughterless-abolike, a Drosophila maternaleffect mutation that exhibits abnormal centrosome separation during the late blastoderm divisions. Development 110:311-323. Swedlow J.R., Agard D.A., Sedat J.W. (1993) Chromosome structure inside the nucleus.
182 DeltaVision Core and personalDV User's Manual Zimowska G., Aris J.P., Paddy M.R. (1997) A Drosophila Tpr protein homolog is localized both in the extrachromosomal channel network and to nuclear pore complexes. Journal of Cell Science 110: 927-944. Linux Operating System Siever, Ellen. Linux in a Nutshell, Third Edition. O’Reilly and Associates, 2001. Image Processing Bracewell R.N. The Fourier Transform and Its Applications. 2nd Edition, McGraw Hill, New York NY, 1986. Goodman J.W.
E Appendix E: Resolve3D and Keypad Options This chapter describes the following Resolve3D windows and dialogs: The Resolve3D Window is the main window for data acquisition. The Design/Run Experiment Dialog provides tools to select, design, edit, and execute experiment macros. The Experiment Designer is used to generate experiment command macros. The Settings Dialog used to control how images are displayed, select camera settings, and specify file output.
184 DeltaVision Core and personalDV User’s Manual The Resolve3D Window The Resolve3D window is the main data acquisition window. In addition to providing many of the acquisition options and controls, it provides access to the other dialogs and windows that are used for data acquisition. To open Resolve3D: X From the softWoRx menu, choose File | Acquire (Resolve3D).
Appendix E: Resolve3D and Keypad Options The Resolve3D Menu The Resolve3D menu has the following menu items: The File menu includes commands to acquire images and to open the key dialogs for setting up and running experiments. The View menu includes commands to manage marked points and to create a blank screen. The Options menu includes commands to open the Settings dialog box (where you can set display and image options) and to save settings. The Calibration menu opens the Calibration tool.
186 DeltaVision Core and personalDV User’s Manual Scratch File Creates a "scratch file" to which you can save individual image frames for later use. After a file is opened, clicking Save Current Image saves the most recently collected image frame. Clicking Close Scratch File or Done closes the file. Note that the image size cannot be changed while a scratch file is open. Experiment Opens the Design/Run Experiment dialog box.
Appendix E: Resolve3D and Keypad Options Command Line Interface Opens the Command Line dialog box that provides advanced users with the ability to issue individual Resolve3D commands to the system. CAUTION: The Command Line Interface should be used carefully because it can put the system in an unstable state. The Resolve3D Options Menu Use the Options menu to open the Settings dialog box (where you can set display and image options) and to save configuration settings and state information.
188 DeltaVision Core and personalDV User’s Manual Read Opens the Read Calibration Files dialog box that you can use to read calibration tables. Manage Opens the Manage tool that you can use to designate which calibration tables are active and to remove tables from the system's session memory. The Resolve3D Help Menu Use this menu to turn ToolTips on or off, get help on the Resolve3D window, or find out which version of softWoRx you are using.
Appendix E: Resolve3D and Keypad Options Experiment Opens the Design/Run Experiment dialog box that you can use to select a previously created experiment, open the Experiment Designer window to design a new experiment, or open the Experiment Macro Editor to create or edit an experiment macro. Settings Opens the Settings dialog box that allows you to control display, imaging, and file output options.
190 DeltaVision Core and personalDV User’s Manual Excitation Specifies an excitation filter. When this filter is selected, a corresponding emission filter is automatically selected and the following parameters are set to the last values that were used for that excitation filter. Exposure time Neutral Density filter Active illumination shutters Number of frames to average Target intensity Camera gain You can choose Save Settings from the Options menu to store the configuration.
Appendix E: Resolve3D and Keypad Options Image Size Specifies image size (pixels or CCD detector elements) for acquired images. The pull-down list contains predefined sizes for convenience. Special sizes may be entered by choosing Other... from the pull-down list. (Image size must be a multiple of four.) Lens Specifies the objective lens name. The pull-down list contains the lenses that are known to be part of the microscope system.
192 DeltaVision Core and personalDV User’s Manual Figure E-9: The Resolve3D Stage Position Control Stage Control and Display Tools Center Object Centers the stage on an object selected in an Image Window. The pixel size must be correct in order for object centering to work properly, which means that the correct lens and auxiliary magnification setting must be selected. An image will be acquired after the object is centered.
Appendix E: Resolve3D and Keypad Options Note All scans that are set up using the Experiment Designer scan in Z, using relative coordinates. The Mark Top and Mark Bottom buttons are most helpful in determining the thickness of the sample to be scanned. When an experiment is started, the scan region is determined by the current focus point and the thickness of the sample.
194 DeltaVision Core and personalDV User’s Manual Marked Points list Opens the Point List dialog box to manage the list of marked points. Zoom Tool Controls zoom. Drag the thumbwheel down to zoom in; up to zoom out. The button under the thumbwheel returns the zoom to 1:1. Z Slider Moves the stage up or down. dX Specifies the X step size, in microns. dY Specifies the Y step size, in microns. Stage Trails Window The blue box represents the current stage location.
Appendix E: Resolve3D and Keypad Options Image Intensity and Scale Values The Resolve3D Intensity values show the statistics for the intensity values of the image in numerical and graphical formats. The scale of the image is also shown. Figure E-10: The Resolve3D Image and Intensity and Scale Values Min, Max, Mean Displays the minimum, maximum, and mean intensity values of the most recently acquired image. For a 12 bit CCD camera, these values range between 0 and 4095.
196 DeltaVision Core and personalDV User’s Manual Resolve3D Shortcuts You can right-click anywhere in the Resolve3D window to open a shortcut menu that allows you to acquire images, mark points, and create a blank screen (for imaging under very light-sensitive conditions). Figure E-11: Resolve3D Shortcut Menu Acquire Collects and displays an image from the microscope. The current settings are used to collect the image. This image is only displayed in the Data Collection window.
Appendix E: Resolve3D and Keypad Options Figure E-12: Design Experiment Experiment name Specifies the name of the experiment macro. Enable Fast Acquisition Enables fast acquisition experiments (see Page 198). Sectioning Specifies sectioning for 3D images (see Page 198). Channels Specifies channels (filters) and exposure time (see Page 200). Time-lapse Specifies criteria for time-lapse experiments (see Page 201). Point Visiting Specifies a list of marked points (see Page 202).
198 DeltaVision Core and personalDV User’s Manual Experiment name and Enable Fast Acquisition Experiment name Specifies the name of the file that is generated by the Experiment Designer. A file extension of .exp will be added to the name. Enable Fast Acquisition Enables fast image acquisition of 2D images. (This is only available when you are using an interline camera.) This data acquisition mode should be used carefully.
Appendix E: Resolve3D and Keypad Options 199 Figure E-13: Design Experiment Sectioning Setup Options Focus point when scan starts Specifies the Z location of the sample when the experiment starts. The recommended option is Middle of Sample. Optical section spacing Specifies the spacing (in microns) between each optical section. The focal point will be changed by this value after each image is collected.
200 DeltaVision Core and personalDV User’s Manual Channels Setup Use the Design Experiment Channels tab to select wavelengths (filters) and to specify an exposure time for each filter. Figure E-14: Design Experiment Channels Options Refresh exposure conditions Updates the filter and exposure settings to those last used in the main Resolve3D window. Active Wavelength Toggle Buttons Enable the exposure time, filters, and display settings for specific wavelengths.
Appendix E: Resolve3D and Keypad Options %T Filter Specifies the Neutral Density filter to use. The % value indicates light transmittance. A value of 100 % indicates that no light is blocked (or no filtering). Ex Shutter Specifies the excitation (illumination) shutter to use for each channel of the image. Reference Image Specifies to use an alternate filter or the transmitted light to acquire a reference image that can be combined with other images.
202 DeltaVision Core and personalDV User’s Manual Time Points Specifies the number of time samples to collect in a time-lapse experiment. Enable Cell Tracking Moves the stage laterally to follow cells as they move during a time-lapse experiment. With the Enable Cell Tracking option selected, DeltaVision automatically keeps cells in the field of view. Cell Tracking Options Opens the Cell Tracking Options dialog box that allows you to set the parameters for cell tracking.
Appendix E: Resolve3D and Keypad Options Visit Point List Specifies the list of points, described by number, to visit during the experiment. All sectioning and wavelength procedures are repeated at each of the listed points. A point list can be entered as a series of numbers separated by commas or dashes. Separating two numbers with a dash '-' (as shown in the following example) indicates that all point numbers in between should also be visited.
204 DeltaVision Core and personalDV User’s Manual Collect Panels Specifies to use a panel collection macro. Overlap (pixels) Specifies the amount of overlap (in pixels) between adjacent panels. Start Coordinates Specifies the XYZ coordinates at which to start collecting panels. Use the Get Start button to obtain the current XYZ stage coordinates. End Coordinates Specifies the XYZ coordinates at which to finish collecting panels. Use the Get End button to obtain the current XYZ stage coordinates.
Appendix E: Resolve3D and Keypad Options Image file name Specifies the file name to use for the image in this text field. If you do not provide a file name, you will be asked to provide one when the experiment starts running. If you have the Auto-increment file names setting (in the Settings dialog box) turned on, you will only need to provide a name once for each session. The names will have incrementing numbers appended to them automatically.
206 DeltaVision Core and personalDV User’s Manual Estimated Finish Displays the estimated clock time in which a running time-lapse experiment will finish. Current command: Reports each macro command as it is executed. Start Scan Starts the selected macro to run an experiment. Cancel Scan Terminates a scan while the experiment is running. (The images that are collected before the experiment is cancelled are automatically saved.) Help Opens the Help for the Design/Run Experiment dialog box.
Appendix E: Resolve3D and Keypad Options Settings Dialog Box Display Options The Settings dialog box Display tab options allow you to control how the images are displayed. Image Display Mode Specifies which window and wave are used for image display. Five modes are currently available: Model Description None Displays images in the current window and wave. Scratch Displays all images in the specified window, Wave 1. Auto Grayscale Displays images in a separate window for each Emission (EM) filter.
208 DeltaVision Core and personalDV User’s Manual Auto intensity scale Provides automatic scaling of the image intensity between the minimum and maximum brightness. (This switch applies only to the appearance of the image, not the actual data.) Acquire after point visit Automatically acquires an image when the Visit Point option is selected. Settings Dialog Box Imaging Options The Settings dialog box Imaging options allow you to select camera settings.
Appendix E: Resolve3D and Keypad Options Current temperature Displays the current temperature. Resolve3D monitors the temperature and updates this value occasionally. If you want to force a request for the current temperature, click the Refresh button. Xe Lamp bulb age (hours) Displays a count for the current number of hours on the xenon bulb. Use photosensor Enables use of the Photo sensor.
210 DeltaVision Core and personalDV User’s Manual value reverts back to the global softWoRx Data Directory as specified in User Parameters. Use this option in environments where several users are running the microscope and using a single system login. Data folders can be created for each user under a common parent folder. As users start Resolve3D, they can choose to use their own folders. In most cases, you should not use this option if each user has their own system login in your environment.
Appendix E: Resolve3D and Keypad Options Channel for Autofocus This setting indicates the wavelength to use for Autofocus. Contrast calculation method This setting determines the polarity of the contrast calculation. There are three choices for image contrast calculation methods: • Auto – The instrument controller usually can determine which contrast calculation method to use, but not always.
212 DeltaVision Core and personalDV User’s Manual Allow Lost Motion Compensation (LMC) Enable Lost Motion Compensation to remove the effect of hysteresis in the stage. This option should be selected for most applications. When it is turned off, the Z series range may shift. Clear this option if you want to improve speed at the expense of position repeatability. Show stage trails Display the path of stage movement on the Stage View.
Appendix E: Resolve3D and Keypad Options Keypad/Joystick Operation Many of the functions accessible through Resolve3D are also available on the keypad/joystick. This section describes each key on the keypad/joystick. Note Some buttons on your keypad may not be active. Figure E-23: Keypad/Joystick RESET Clears communication buffers, closes shutters, stops all motors, and clears encoder errors. Use the RESET button when you suspect that the workstation and controller are not synchronized.
214 DeltaVision Core and personalDV User’s Manual LMC RESET ON/OFF Executes a Lost Motion Compensation (LMC) move. Tip You can disable LMC on the Resolve3D Settings dialog. (For more information, see the online Help.) REMOVE TRAIL Clears the stage movement history from memory. KEYLIGHT ON/OFF Turns the keypad backlight on and off. QUIT Quits the controller program when pressed twice. (To restart the Instrument Controller program, double-click the Instrument Controller icon on the desktop.
Appendix E: Resolve3D and Keypad Options 215 Neutral Density filter is at the maximum value and you push the increase arrow ( ), the wheel moves to engage the minimum value filter. RETRACE ARROWS Retrace ( ) moves the stage one position back in the stage movement history buffer. Retrace ( ) moves the stage one position forward in the stage movement history buffer. EX ARROWS Changes the excitation filter to the previous ( ) or next ( ) filter on the filter wheel.
216 DeltaVision Core and personalDV User’s Manual EX SHUTTER Toggles the excitation shutter between open and closed. TRANS SHUTTER Toggles the shutter for the transmitted light source between open and closed. POINT MARK Adds the stage position to the marked points list.
Index %T Filter field, 217 3D images, 4, 35–42 3D Z projections, 75–77 Acquire Image button, 17, 233 Acquire Image setting, 209 Acquire images, 20–25, 26–27 Acquire Mode arrows, 232 Active Wavelength buttons, 216 Agard, Dr. David A.
218 optional software, 121–22 photo sensor, 107 repeatable slide holder, 111–13 software, 114 softWoRx Workstation, 111 TIRF module, 119 tool kit, 113 transmitted light source, 107 Xenon lamp, 106 Configuring filter wheels, 134–35 Consumable parts, 122 Contact Applied Precision, xii Continuous Acquire, 201 Contrast calculation polarity, 228 Control Mode button, 231 Controlling the stage, 207–11 Conventions, document, x Converting PSF to OTF, 183–86 Counter space, for system, 98 Country-specific requirement
Appendix E: Resolve3D and Keypad Options point visiting, 47–53 running, 33–55 setting up, 33–55 time-lapse, 42–47, 69–74 Explorer, 121 Exposure time, 84–86 Eyepiece filter wheel, 24, 131–33 filter wheel and safety, 9 filters, 16 focus, 15 Eyepiece filters installing, 142–44 Fast button, 232 Fiber Optic Cable, 10 Fiber optic module, 113, 156 adjust tilt of, 161–62 checking alignment \r, 158 Field stop aperture, 160–61 File system full error, 172 Filename extensions, 188 Filter sets, 15–17 Filters activating
220 collecting panels for, 53–55 Exposure time, 85 marking points for, 48–50 monitoring, 88–93 point visiting, 47–53 troubleshooting, 173–75 with continuous Z sweep, 75–77 Image control fields, 205–7 Image Display and Analysis, 4 Image Fusion tool, 79 Image intensity, 211–12 Images 2D in 3 wavelengths, 15–17 3D, 35–42 acquiring, 20–25, 26–27 and optical section spacing, 39–42 calibrate, 206 collecting panels for, 53–55 display mode, 223 distortion, 173 intensity values, 211–12 live cell, 5, 66–79 modes for
Appendix E: Resolve3D and Keypad Options Microscope Interface Chassis, 110 Middle of sample, 39 Middle of the sample option, 38 Minimum intensity, 211 Monitor switch, 147 Monitoring data acquisition, 88–93 Monitoring point visiting, 52 Move threshold, 73 Moving DeltaVision, 167 Multiplexed wavelength experiment design, 61–64 filter set activation, 56–60 option, 55–64, 120 overview, 56 ND arrows, 232 Network connection, 100 Neutral density filters, 16 No button, 232 Number of optical sections field, 39 OAI.
222 Replacing the tran light, 153–55 Requirements altitude, 100 country-specific, 98 electrical, 97–98 environmental, 98–101 humidity, 100 line, 97 power cord, 97 temperature, 100 Reset button, 231 Resolution ratio, 170 Resolve3D %T setting, 206 Acquire Image, 201 Autofocus options, 227–28 Aux Mag setting, 207 Bin setting, 207 bulb icon, 30, 148 calibrate images, 206 Calibration menu, 203–4 cell tracking options, 218 Channels tab, 216–17 Clear Stage Thumbnails, 202 Clear Stage Trails, 202 Command Line Inte
Appendix E: Resolve3D and Keypad Options Spherical aberration, minimizing, 182 Stage controls, 207–11 Stage speed, 83 Standard filename extensions, 188 Standard filter sets, 104 Standard microscope slide, 82 Start Coordinates setting, 220 Statistics, 93 Step Decrease and Increase buttons, 233 Stopping DeltaVision, 29–31 Sub-image, 185 Symmetric flare, 182 Target temperature display, 225 Technical support, xii, 100 Temperature requirements, 100 Theoretical OTF.
