Olympus Fluoview-1000 User Manual
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Olympus Fluoview-1000 User Manual

Olympus Fluoview-1000 User Manual

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V.M. Bloedel Hearing Research Center, Core for Communication Research
Center on Human Development and Disability, Digital Microscopy Center
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May 11, 2011
Olympus Fluoview-1000 User's Guide
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Summary of Contents for Olympus Fluoview-1000

  • Page 1: Table Of Contents

    Olympus Fluoview-1000 User’s Guide V.M. Bloedel Hearing Research Center, Core for Communication Research Center on Human Development and Disability, Digital Microscopy Center Quick Start Guide Start-Up Shut-Down Quick Guide to Getting an Image ......................Place the sample on the microscope ......................
  • Page 2 Olympus Fluoview-1000 User’s Guide V.M. Bloedel Hearing Research Center, Core for Communication Research Center on Human Development and Disability, Digital Microscopy Center Scan mode and sequential capture ..................... Using Channel Groups, Figure 9A......................Adjusting the channels with sequential imaging ..................
  • Page 3 Olympus Fluoview-1000 User’s Guide V.M. Bloedel Hearing Research Center, Core for Communication Research Center on Human Development and Disability, Digital Microscopy Center Bleedthrough............................Reducing bleedthrough ........................Procedure to minimize bleedthrough ....................Noise..............................Signal Noise (Shot Noise)......................... Detector Noise.
  • Page 4 Olympus Fluoview-1000 User’s Guide V.M. Bloedel Hearing Research Center, Core for Communication Research Center on Human Development and Disability, Digital Microscopy Center FV-1000 Optical Specifications ......................No Image! ..............................Start-up problems........................... Scans, but no image..........................The confocal image appears unevenly illuminated: ................

  • Page 5: Quick Start Guide

    A. Start-up: All items are lettered in the order in which they are to be turned on. 1. Enter your name and session into the Olympus FV-1000 web calendar; 2. Turn on items labeled with letters: (A) Turn on mercury lamp, if needed - power supply is on shelf over monitor;...

  • Page 6: Quick Guide To Getting An Image

    Olympus Fluoview-1000 User’s Guide V.M. Bloedel Hearing Research Center, Core for Communication Research Center on Human Development and Disability, Digital Microscopy Center 1. Quick Guide to Getting an Image Examples of the software control windows are on the following pages. The text contains numbers that refer to specific numbered controls in figures.

  • Page 7: Place The Sample On The Microscope

    Olympus Fluoview-1000 User’s Guide V.M. Bloedel Hearing Research Center, Core for Communication Research Center on Human Development and Disability, Digital Microscopy Center 1.1 Place the sample on the microscope 1. Make sure the objective lens is not extending above the level of the specimen holder;...

  • Page 8: Figure 2. Enlarged View Of Fluoview Windows

    Olympus Fluoview-1000 User’s Guide V.M. Bloedel Hearing Research Center, Core for Communication Research Center on Human Development and Disability, Digital Microscopy Center a. Image Acquisition Control Window>Click on transmitted light, Figure 4.1; b. Image Acquisition Control Window>adjust brightness (TR Lamp) (click or scroll), Figure 4.4;...

  • Page 9: Brightfield Vs. Dic

    Olympus Fluoview-1000 User’s Guide V.M. Bloedel Hearing Research Center, Core for Communication Research Center on Human Development and Disability, Digital Microscopy Center 1.3 Brightfield vs. DIC The default setting for plain brightfield imaging is with the condenser DIC prism in place, “DICT”.

  • Page 10: Finding The Sample - Confocal

    Olympus Fluoview-1000 User’s Guide V.M. Bloedel Hearing Research Center, Core for Communication Research Center on Human Development and Disability, Digital Microscopy Center Mirror Button Function Mode Visualization Confocal Imaging Confocal Confocal only, automatic DAPI UV excitation Epi-fluorescence Eyepieces only FITC...

  • Page 11: Setting Up For Confocal Microscopy

    Olympus Fluoview-1000 User’s Guide V.M. Bloedel Hearing Research Center, Core for Communication Research Center on Human Development and Disability, Digital Microscopy Center 4.6 XY Repeat 4.7 Acquisition scan 4.5 Fast scan 4.8 Stop scan 4.1 Tungsten Lamp 4.2 Fluorescence shutter 4.3 Dye List...

  • Page 12: Changing The Dye Selection

    Olympus Fluoview-1000 User’s Guide V.M. Bloedel Hearing Research Center, Core for Communication Research Center on Human Development and Disability, Digital Microscopy Center Dye names are written into the image file metadata. However, fluorophores with broadly similar excitation and emission spectra will use the same laser line and filters. For example, FITC, EGFP, Alexa488 and Mitotracker Green will all use the 488 nm laser and a bandpass filter of 500 nm to 550 nm 2.1.1 Changing the Dye Selection...

  • Page 13: Settings For The Fluorescence Detectors

    Olympus Fluoview-1000 User’s Guide V.M. Bloedel Hearing Research Center, Core for Communication Research Center on Human Development and Disability, Digital Microscopy Center 7. Confocal Aperture, 4.10 – Controls the diameter of the pinhole. 8. Kalman Averaging, 4.11 – Allows averaging signals from multiple scans to reduce noise.

  • Page 14: Laser Controls

    Olympus Fluoview-1000 User’s Guide V.M. Bloedel Hearing Research Center, Core for Communication Research Center on Human Development and Disability, Digital Microscopy Center 5.6 Channel 5.7 Active if 5.5 Channel group checked 5.1 PMT Voltage 5.2 Amplifier Gain 5.4 Laser Settings 5.3 Offset (black level)

  • Page 15: Laser Power Selector, Figure 7.3

    Olympus Fluoview-1000 User’s Guide V.M. Bloedel Hearing Research Center, Core for Communication Research Center on Human Development and Disability, Digital Microscopy Center 2.5.3 Laser Power Selector, Figure 7.3. These controls regulate the percentage of laser power allowed to pass through the acoustico-optical tunable filter (AOTF) located in the scanhead.

  • Page 16: Scan Controls

    Olympus Fluoview-1000 User’s Guide V.M. Bloedel Hearing Research Center, Core for Communication Research Center on Human Development and Disability, Digital Microscopy Center Scan Controls 2.6.1 Scan Mode, Figure 6.1 1. Unidirectional - Standard scan mode for routine imaging. Each line is scanned from left to right, the laser beam is blanked while the scanner jumps back to the left side to scan the next line.

  • Page 17: Scan Regions, Figure 6.3

    Olympus Fluoview-1000 User’s Guide V.M. Bloedel Hearing Research Center, Core for Communication Research Center on Human Development and Disability, Digital Microscopy Center Dwell Time Box Size Scan Time Seconds Time for Set per Pixel (P:) Per Line (L:) per Frame (F:) (S:) 2.0 µs...

  • Page 18: Box Size Effect On Resolution

    Olympus Fluoview-1000 User’s Guide V.M. Bloedel Hearing Research Center, Core for Communication Research Center on Human Development and Disability, Digital Microscopy Center 2.6.6 Box Size effect on resolution The resolution of an image is determined by dividing the scanned field by the box size to set the area represented by each pixel in the final image.

  • Page 19: Scan Zoom, 8.4

    Olympus Fluoview-1000 User’s Guide V.M. Bloedel Hearing Research Center, Core for Communication Research Center on Human Development and Disability, Digital Microscopy Center 2.7.4 Scan Zoom, 8.4. This controls the size of the scanned region and the resolution. Click in the slider area to increment scan zoom by 1X increments or click on the arrows to increment by 0.1X increments.

  • Page 20: Figure 9. Sequential Imaging Control

    Olympus Fluoview-1000 User’s Guide V.M. Bloedel Hearing Research Center, Core for Communication Research Center on Human Development and Disability, Digital Microscopy Center 9.3 Kalman Averaging 9.2 Groups 9.1 Sequential Line vs. Frame 9.3 Channels Figure 9 – Sequential Acquisition and Averaging Controls 9.2A...

  • Page 21: Use Of Sequential, Simultaneous And Averaging

    Olympus Fluoview-1000 User’s Guide V.M. Bloedel Hearing Research Center, Core for Communication Research Center on Human Development and Disability, Digital Microscopy Center Use of Sequential, Simultaneous and Averaging. Simultaneous Capture versus Sequential Capture. Simultaneous capture collects a multi-channel image by scanning the sample with the laser lines for all selected channels at the same time.

  • Page 22: Scan Mode And Sequential Capture

    Olympus Fluoview-1000 User’s Guide V.M. Bloedel Hearing Research Center, Core for Communication Research Center on Human Development and Disability, Digital Microscopy Center 3.2.3 Scan mode and sequential capture 1. ‘Scan X2’, ‘Scan X4’ or ‘XY Repeat’ - click on the desired channel for Frame scanning. That channel will be scanned until the user clicks on ‘Stop’...

  • Page 23: Collecting A Z-Series

    Olympus Fluoview-1000 User’s Guide V.M. Bloedel Hearing Research Center, Core for Communication Research Center on Human Development and Disability, Digital Microscopy Center 1. Live Window is set to gray scale (control-h); 2. Set Live Window for tile view (each detector is viewed in its own pane);...

  • Page 24: Figure 10. Z-Series Control Window

    Olympus Fluoview-1000 User’s Guide V.M. Bloedel Hearing Research Center, Core for Communication Research Center on Human Development and Disability, Digital Microscopy Center 10.1 Lens Objective 10.2 Move Focus to: Start Middle 10.3 Current Focus Level 10.5 Set Focus to 0 10.4 Objective Resolution...

  • Page 25: Settings For Z-Series Collection

    Olympus Fluoview-1000 User’s Guide V.M. Bloedel Hearing Research Center, Core for Communication Research Center on Human Development and Disability, Digital Microscopy Center Settings for z-series collection. 1. Determine the axial extent of volume needed (how far you need to focus through the sample);...

  • Page 26: Vertical Sections

    Files: Saving and Transferring Saving Files Olympus created 2 proprietary file formats for images collected on their confocal microscopes. Both formats record all meta-data, regions of interest and preserve the original 12-bit (4096) image intensity values. Each format has its benefits and drawbacks.

  • Page 27: Where Are Your Files On The Confocal

    Some commercial software applications will open the Olympus .oib and .oif formats, e.g. Imaris, Slidebook and Fluoview. The Huygens software will only open the .oif format. Any image processing application can import the tiff files found in the .oif format. See the document on importing Olympus files on our website, http://cajal.oto.washington.edu/corec/imageprocessing.php.

  • Page 28: Olympus Free File Viewer

    5.3.1 Olympus free file viewer Olympus has a free file viewer for Windows that can open .oif and .oib files. However, it only allows you to view files and export the images to other formats, such as TIFF or JPEG. This application may be copied and installed on computers in your lab.

  • Page 29: File Import From The Oif.files Directory

    Olympus Fluoview-1000 User’s Guide V.M. Bloedel Hearing Research Center, Core for Communication Research Center on Human Development and Disability, Digital Microscopy Center 5.4.4 File Import from the OIF.Files directory. ImageJ can import each channel of 16-bit TIFFS as a separate stack from the ‘Image>Import>Image Sequence...’...

  • Page 30: Recording To Cd-Rom And Dvd-Rom On The Mac Pro

    Olympus Fluoview-1000 User’s Guide V.M. Bloedel Hearing Research Center, Core for Communication Research Center on Human Development and Disability, Digital Microscopy Center Flash drives are OS independent. Hard drives formatted for Windows will not work on the Mac unless you have switched to Windows with Fusion. Click on the Fusion icon in the OSX dock to start Windows7.

  • Page 31: Optimizing Image Collection

    Olympus Fluoview-1000 User’s Guide V.M. Bloedel Hearing Research Center, Core for Communication Research Center on Human Development and Disability, Digital Microscopy Center Optimizing Image Collection The definition of “best” image will depend on the purpose(s) of the image and the nature of the specimen.

  • Page 32: Labeling Controls

    Olympus Fluoview-1000 User’s Guide V.M. Bloedel Hearing Research Center, Core for Communication Research Center on Human Development and Disability, Digital Microscopy Center 3. Set the Live Window to ‘Tile Display’; 4. Press ‘control-h’ to set the look-up tables (LUT) to gray scale;...

  • Page 33: Bleedthrough

    Olympus Fluoview-1000 User’s Guide V.M. Bloedel Hearing Research Center, Core for Communication Research Center on Human Development and Disability, Digital Microscopy Center disk. An iris set to this size passes approximately 80% of incident emissions and achieves a 20% increase in lateral and axial resolution, relative to the widefield microscope. The ‘Auto CA’ button (Figure 4.10) sets the aperture to 1 Airy disk diameter for the middle wavelength for multi-channel...

  • Page 34: Noise

    Olympus Fluoview-1000 User’s Guide V.M. Bloedel Hearing Research Center, Core for Communication Research Center on Human Development and Disability, Digital Microscopy Center b. optimize the laser and channel parameters 4. Repeat step 3 with each negative control, adjust laser power and channel settings;...

  • Page 35: Detector Noise

    Olympus Fluoview-1000 User’s Guide V.M. Bloedel Hearing Research Center, Core for Communication Research Center on Human Development and Disability, Digital Microscopy Center 6.6.2 Detector Noise. The HV setting on the PMT controls the likelihood photo-electrons will be accumulated and the degree of signal amplification.

  • Page 36: Using Zoom

    Olympus Fluoview-1000 User’s Guide V.M. Bloedel Hearing Research Center, Core for Communication Research Center on Human Development and Disability, Digital Microscopy Center Increased laser power – stronger excitation of fluorophores, with disadvantages of photobleaching, bleedthrough and autofluorescence. Longer dwell time - increases the signal by exciting each sample point in the specimen for a longer period of time, but provides similar disadvantages as higher laser power;...

  • Page 37: Obtaining A Z-Series

    Olympus Fluoview-1000 User’s Guide V.M. Bloedel Hearing Research Center, Core for Communication Research Center on Human Development and Disability, Digital Microscopy Center resolved by that objective. Using a zoom setting to create lateral resolution in beyond Nyquist will not improve the resolution of a confocal image. However, the exception is collecting images at 2X Nyquist, or 4.6 times the objective resolution, can be useful for deconvolution.

  • Page 38: Sampling Frequency And The Nyquist Theorem

    Olympus Fluoview-1000 User’s Guide V.M. Bloedel Hearing Research Center, Core for Communication Research Center on Human Development and Disability, Digital Microscopy Center Since NA determines the smallest resolvable object, and the magnification magnifies that object, a pair of objectives with the same NA, e.g. 20X and 40X, will provide the same resolution. Magnification can be compensated by use of zoom and box size.

  • Page 39: Size Of Pixels

    Olympus Fluoview-1000 User’s Guide V.M. Bloedel Hearing Research Center, Core for Communication Research Center on Human Development and Disability, Digital Microscopy Center 5. Box size increases resolution while maintaining the field of view. Increasing the zoom without changing the box size will decrease the field of view.

  • Page 40: Figure 12. Dic And Focus Controls, Lower Half Of The Microscope

    Olympus Fluoview-1000 User’s Guide V.M. Bloedel Hearing Research Center, Core for Communication Research Center on Human Development and Disability, Digital Microscopy Center wavelength(nm) Theoretical lateral resolution: 2(NA) Wavelength = emission, in nanometers, usually the central wavelength of the emission band of the barrier filter.

  • Page 41: Microscope Controls

    Olympus Fluoview-1000 User’s Guide V.M. Bloedel Hearing Research Center, Core for Communication Research Center on Human Development and Disability, Digital Microscopy Center Microscope controls. There are a few manual controls Focus. The IX-81 has a single focus knob, Figure 12.1, on each side of the stand. It is used for both coarse and fine focus.

  • Page 42: Dic Polarizer

    Olympus Fluoview-1000 User’s Guide V.M. Bloedel Hearing Research Center, Core for Communication Research Center on Human Development and Disability, Digital Microscopy Center 13.3 Field Stop 13.4 DIC Polarizer slider (out position) 13.2 Condenser focus knob 13.5 Centering screws 13.1 Condenser Aperture Figure 13.

  • Page 43: Condenser Position

    Olympus Fluoview-1000 User’s Guide V.M. Bloedel Hearing Research Center, Core for Communication Research Center on Human Development and Disability, Digital Microscopy Center effective in finding your sample without subjecting it to fluorescence. Focus of the condenser may need to be re-adjusted if changing the level of focus in the sample or after switching objectives.

  • Page 44: Photoactivation Methods

    Olympus Fluoview-1000 User’s Guide V.M. Bloedel Hearing Research Center, Core for Communication Research Center on Human Development and Disability, Digital Microscopy Center Photoactivation Methods. Several imaging procedures involve the application of light in various combinations of wavelength, intensity, duration and region of interest (ROI) in order to promote a reaction. Common applications include photoactivation of fluorescent proteins, photobleaching fluorophores and photoablation of cells.

  • Page 45: Figure 15. The Stimulus Setting Window For Photoactivation

    Olympus Fluoview-1000 User’s Guide V.M. Bloedel Hearing Research Center, Core for Communication Research Center on Human Development and Disability, Digital Microscopy Center Select scanner Choose entire field or ROI selections Set dwell time Choose laser line and power level for...

  • Page 46: Avoid Spontaneous Photoactivation

    Olympus Fluoview-1000 User’s Guide V.M. Bloedel Hearing Research Center, Core for Communication Research Center on Human Development and Disability, Digital Microscopy Center 5. Start a photobleach operation by setting the XY Scan button to “time” then begin an XYT operation;...

  • Page 47: Set 'Darkroom' Color Mode

    Olympus Fluoview-1000 User’s Guide V.M. Bloedel Hearing Research Center, Core for Communication Research Center on Human Development and Disability, Digital Microscopy Center 8.4.1 Set ‘Darkroom’ color mode. 1. Move the cursor along the toolbar located just below the menus in Fluoview 2.1c;...

  • Page 48: Technical Information

    Olympus Fluoview-1000 User’s Guide V.M. Bloedel Hearing Research Center, Core for Communication Research Center on Human Development and Disability, Digital Microscopy Center Technical Information Caring for Objectives 1. Check the turret before placing your sample in the holder or activating the Multi-Area TimeLapse module.

  • Page 49: Table 6. Dichroic Mirrors And Barrier Filters

    Olympus Fluoview-1000 User’s Guide V.M. Bloedel Hearing Research Center, Core for Communication Research Center on Human Development and Disability, Digital Microscopy Center Table 6. Dichroic Mirrors and Barrier Filters Dichroic Mirrors Dichroic Mirrors Dichroic Mirrors Dichroic Mirrors Barrier Filters Barrier Filters...

  • Page 50: No Image

    Olympus Fluoview-1000 User’s Guide V.M. Bloedel Hearing Research Center, Core for Communication Research Center on Human Development and Disability, Digital Microscopy Center No Image! 10.1 Start-up problems. • Fluoview starts to load but stalls with the Fluoview workspace open - Is the microscope controller turned on? See Start-up protocol.

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