Transcript
Date of Issue: August 2009 Revision: 9 Date of Revision: Oct 10, 2012
Olympus FV1000 Standard Operating Procedure
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TABLE OF CONTENTS DISCLAIMER ................................................................................ iv ACKNOWLEDGEMENTS ..................................................................... v 1.
INTRODUCTION........................................................................ 1 1.1 Purpose of the Standard Operating Procedure ............................. 1 1.2 Emergency Contact Information .............................................. 1 1.3 User Fees and Instrument Booking ........................................... 2 1.4 Basic Theoretical Background ................................................. 2 1.4.1 Sample Preparation Suggestions......................................... 3 1.5 Instrumentation ................................................................. 5 1.5.1 Overview .................................................................... 5 1.5.2 Lasers and Filters .......................................................... 5 1.5.3 Objectives .................................................................. 5 1.5.4 Scan Head................................................................... 6
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POTENTIAL HAZARDS ................................................................. 7
3.
PERSONAL PROTECTIVE EQUIPMENT ............................................... 8
4.
ACCIDENT PROCEDURES ............................................................. 8
5.
WASTE DISPOSAL PROCEDURES ..................................................... 8
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PROTOCOL ............................................................................. 9 6.1 Start-up........................................................................... 9 6.1.1 Viewing sample with transmitted light and Kolher illumination .. 10 6.1.2 Epifluorescence Imaging ................................................ 11 6.2 Preparation for Imaging ........................................................ 1 6.3 XY Image Acquisition ........................................................... 3 6.4 Z-Series or 3-D Stack Image Acquisition ..................................... 4 6.5 T-Series Image Acquisition..................................................... 4 6.6 Image Analysis ................................................................... 5 6.7 Saving and Viewing Final Images ............................................. 9 6.8 Cleaning Objectives .......................................................... 10 6.9 Shutdown ....................................................................... 10
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TROUBLESHOOTING ................................................................ 10
8.
PREVENTATIVE MAINTENANCE .................................................... 11 8.1 Monthly ......................................................................... 11 8.2 Annually or as Required ...................................................... 11 8.2.1 Replace the Mercury Burner ........................................... 11
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QUICK REFERENCE GUIDE .......................................................... 12
REFERENCES .............................................................................. 13 APPENDIX 1: FLUOROCHROME PEAK EXCITATION AND EMISSION WAVELENGTHS 14 APPENDIX 2: USER LOG .................................................................. 20 APPENDIX 3: PREVENTATIVE MAINTENANCE LOG .................................... 22
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DISCLAIMER The materials contained in this document have been compiled from sources believed to be reliable and to represent the best opinions on the subject. This document is intended to serve only as a starting point for good practices and does not purport to specify minimal legal standards. No warranty, guarantee, or representation is made by Laurier as to the accuracy or sufficiency of information contained herein, and Laurier assumes no responsibility in connection therewith.
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ACKNOWLEDGEMENTS The following individuals of Laurier contributed to the writing, editing, and production of this manual: Gena Braun (Instrumentation Technician); Dr. Diano Marrone (Psychology); Stephanie Kibbee (Environmental/Occupational Health and Safety Office).
This manual was prepared for Laurier. Any corrections, additions or comments should be brought to the attention of the Instrumentation Technician at 519-884-0710 ext. 2361.
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1. INTRODUCTION 1.1 Purpose of the Standard Operating Procedure This standard operating procedure (SOP) is NOT a substitute for training and/or reading the appropriate manuals before use. All principle investigators and supervisors must document that training has been received by students and staff who will be using the Olympus FV1000 confocal laser scanning microscope. A list of authorized users will be maintained by the Instrumentation Technician. This SOP is intended to promote consistent and safe use of the Olympus FV1000. This SOP covers the potential hazards, personal protection requirements, spill and accident procedures, waste disposal considerations, and instrument operation for the Olympus FV1000.
1.2 Emergency Contact Information The Olympus FV1000 contains several embedded class 3B lasers, and is therefore regulated under the Laurier Laser Safety Program. The Laser Safety Manual is available at the EOHS website1. The manual provides information on: Responsibilities of the laser operator, and Laser Safety Officer Laser registration requirements Training requirements Sign and labeling requirements Eyewear requirements For more information on the specific lasers and hazards relevant to this instrument, see sections 1.5 (Instrumentation) and 2.0 (Potential Hazards) in this SOP. Emergency contacts for situations involving the Olympus FV1000 lasers are as follows: Principle Investigator: Technicians: Laser Safety Office:
Dr. Diano Marrone ext. 2990 Gena Braun/Jiangxiao Sun ext 2361 or 519-500-4548 Sarah Lamb ext 3108
Immediately notify the Laser Safety Officer at extension 3108 for all laserrelated injuries and near-misses. If the contacts listed above cannot be reached, or in the case of a more general emergency, call 9-911 from any campus phone and/or Special Constable Service at ext. 3333.
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http://www.wlu.ca/documents/42977/Laser_Safety_Manual_2010.pdf
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1.3 User Fees and Instrument Booking To recover the operating costs of the FV1000, the following user fees have been established: Internal users:
Hourly: $ 20 Annually: $ 1500 (Internal users do not pay a training fee)
External users:
Training Fee: $ 55/hour (2-3 hours) Assisted Use: $ 40/hour Unassisted Use: $ 30/hour
Following training, users may book the confocal as follows: 1. Go to instrument booking website http://www.supersaas.com/schedule/login/WLU_Instruments/ 2. Use your user name and password to log in. Log in information is provided by the Research Instrumentation Technician following training. 3. Click on the day that you wish to book. Enter the time slot in “When” and “To” boxes. 4. Make sure you select “Confocal Microscope” in the “Instruments” drop down box. 5. If you want to book the same time slot in other days, select from “Repeat” drop down box for “daily, weekly” etc. 6. Click on “Create reservation”. 7. Please make sure to cancel your reservation if you will not be using the microscope, so that others can book the instrument if they need.
1.4 Basic Theoretical Background The first commercially available laser scanning confocal microscope (LSCM) appeared on the market in 1987, and has since become a very valuable tool in biological research. LSCM is based on the same fundamentals as standard widefield microscopy and epifluorescence, with a few distinct advantages. The critical advantage arises from the use of simultaneously in focus (confocal) pinholes to direct both excitation and emission light. As a result, only a thin section of the specimen is excited and emits fluorescence that can pass through the pinhole and be detected by the photomultiplier tube (PMT) (see Figure 1-1). Exclusion of fluorescence from sample planes that are not in focus greatly reduces the background and increases the sensitivity of LSCM over other techniques. Different planes within the specimen can also be sequentially brought into focus and used to generate a 3-D image. Confocal microscopy is not recommended for all specimen types because the high laser intensity causes rapid photobleaching when compared other widefield techniques. CLSM is highly useful for examining thick specimens,
Olympus FV1000 Standard Operating Procedure
producing 3-D reconstructions to assess cell structure, studying the spatial distribution of labeling, imaging of live cells or other applications that require simultaneous and rapid multi-channel imaging, and studying multi-labeled specimens.
Figure 1-1: Basic illustration of confocal imaging; only the signal that is confocal with the emission pinhole is detected, reducing out-of focus background signal and improving the image significantly.
1.4.1 Sample Preparation Suggestions A number of factors can affect the quality of a confocal image, including sample preparation. Special attention should be paid to the selection of the appropriate dye(s) and mounting medium. When selecting a dye or set of dyes, keep the following in mind: - Each dye should have an absorption maximum close to one of the available laser lines (405, 458, 488, 515, 543, 643). - For multi-colour labeling, each dye should have relatively narrow and well separated absorption and emission maxima to avoid bleed-through (excitation light reaching the detector). - The dye should be resistant to photobleaching. - Each dye should have high quantum efficiency, meaning a large number of photons are produced with minimal excitation power. There are a few families of dyes with specific benefits or drawbacks, so research your selected dye carefully. For example, fluorescein isothiocyanate (FITC), a very popular and common dye, is easily influenced by environment
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(i.e. pH) and has a relatively broad emission spectrum, so it is not ideal for studies that require dual or triple labeling. Appendix 1 lists several dyes and their excitation/emission wavelengths. The appropriate mounting medium depends on the nature of the specimen (fixed or live), and the magnification to be used. For fixed slides, the medium should contain anti-fade reagents to minimize photobleaching. Conversely, anti-fade reagents can be toxic to living organisms and are typically not recommended for live specimens. The medium and anti-fade reagent must also be compatible with the selected dye; some anti-fade compounds can cleave the fluorescent molecules of certain dyes and cause the signal to degrade with in a few days or less. If you plan to image using the 60x or the 100x oil immersion lenses, your mounting medium must have a refractive index that is similar to oil. Consider using 50-80% glycerol or 2,2-thiodiethanol in your mounting medium to improve the image (see Staudt et al., 2007). Again, confirm that your dyes are compatible with all the components of your mounting medium before preparing the specimen. All mounting media must be completely dry before the specimen is viewed on the FV1000. All sample preparation should be done in a separate lab, NOT in the room with the FV1000. Additional information on mounting media and anti-fade reagents has been summarized by Tonny Collins (Wright Cell Imaging Facility, Toronto) at: http://www.uhnresearch.ca/facilities/wcif/PDF/Mountants.pdf
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1.5 Instrumentation 1.5.1 Overview The FV1000 inverted CLSM can be used to examine both fixed and live specimens. Light is produced by a several lasers, and passes through the appropriate filters and mirrors and a pinhole before being scanned across the image in a raster patter. High speed imaging (up to 16 frames/sec for a 256 x 256 image) and rapid spectral scanning (100 nm/sec) are carried out using galvo scanning mirrors. Wavelengths can be resolved to 2 nm on this system, and all filters are ion-sputter coated to provide improved transmission efficiency and allow imaging at lower laser intensity. Fluorescence produced by the specimen passes back through mirrors and filters and a pinhole to be detected by the appropriate photomultiplier tube (PMT). The available lasers and optics are described in more detail below.
1.5.2 Lasers and Filters The FV1000 is equipped with a multi-line argon laser, a green helium-neon (HeNe) laser, two diode lasers, and a mercury lamp. The argon laser produces excitation light at 458, 488, and 515 nm; the green He-Ne at 543 nm; the blue diode at 405 nm; and the red diode at 635 nm. The mercury lamp must be on for at least 30 minutes before it can be switched off; after turning it of, it must cool down for ~15 minutes before being turned back on. The lamp and lasers should not be switched on and off frequently as this shortens the lifetime. The light source for transmitted light is a halogen bulb, which is connected to the microscope via a fiber optic cable. Brightfield and differential interference images using transmitted light can be viewed through the oculars or collected via the computer at the same time as laser scanning. Epifluorescence illumination is conducted using the mercury lamp, and three filter cubes for DAPI, FITC, and TRITC are available.
1.5.3 Objectives The terms that follow the objective magnification indicate the corrections applied to the lens, as follows: - “Plan” indicates a flat field correction, which corrects for field curvature produced by the lens. - “S-Apo” indicates apochromatic correction which is the highest degree of correction for spherical and chromatic aberration. - “Fluor” indicates fluorite aberration correction, which is multi-colour chromatic and spherical aberration. - “NA” is the numerical aperture of the lens, which determines resolution and depth of field. - “WD” is the working distance.
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There are 3 air objectives, and two oil-immersion objectives available on the FV1000 confocal microscope: - 10x (Plan S-Apo, NA 0.4, WD 3.1 mm) - 20x (Plan S-Apo, NA 0.75, WD 0.65 mm) (DIC) - 40x (Plan Fluor, NA 0.6, WD 2.7-4.0 mm, correction collar) (DIC) - 60x oil (Plan Apo, NA 1.42, WD 0.15 mm) (DIC) - 100x oil (Plan S-Apo, NA 1.4, WD 0.12 mm) (DIC) The correction collar on the 40x objective can be adjusted to account for variation in coverslip thickness. All of the other objectives must be used with a 0.17 mm (#1) coverslip. Extra care must be taken when using oil objectives: - An oil immersion objective must be cleaned before adding any more oil to view a new slide. If it is not cleaned, excess oil can run down the side of the objective and into the microscope. - DO NOT use any of the air objectives (10x, 20x, or 40x) immediately after using an oil immersion (60x or 100x) objective. The 20x and 40x objectives in particular have a very small working distance, and can easily come into contact with any oil left on the slide. The slide must be cleaned before switching to the air objectives.
1.5.4 Scan Head The microscope scan head contains the optics required to accept, filter, and detect the laser and fluorescence signals, and to scan in a raster or bidirectional pattern across the specimen. The FV1000 scan head contains 3 internal photomultiplier tubes (PMTs) for fluorescence detection, and one external PMT to detect transmitted light. As a result, up to three fluorescent signals and a transmitted light signal can be collected simultaneously. The signal for each channel passes though a single pinhole, which can be adjusted in size from 50 to 800 m, in 5 m increments. Scanning is done using two galvo-mirrors, and can cover a standard square, a slice, a line, or a user selected region of interest. Multi-dimensional scans can be completed for Zseries (3-D) or T-Series (time series) experiments; the minimum increment for a Z-scan is 0.01 m.
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2. POTENTIAL HAZARDS The FV1000 is a class 3B laser system and uses a variety of high power lasers which present laser radiation and heat hazards. The laser radiation from this instrument can cause serious eye damage if direct or reflected laser light enters you eyes. Follow all warning labels on the equipment. Never look directly at or touch a slide while scanning. Never switch objectives while scanning.
The FV1000 operates under high voltage. Never tamper with any of the connections or electrical cables, and contact the Instrumentation Technician immediately if any of the cables appear damaged. Do not expose your hand or finger to the laser beam output from the objective mount hole, objective tip or condenser lens, or your skin may be damaged. Never attempt to output the laser beam outside the system by inserting a mirror or a similar object in the light path. The laser beam may enter your eyes, and this is extremely hazardous. Make sure that the slide is laying flat on the stage. If the specimen inclines, the laser beam may reflect into your eyes, which is extremely hazardous. Do not bend or pull any of the laser fiber cables. If the laser fiber cable is damaged, the laser light may leak outside it and create a hazardous situation. Should such an event occur, immediately turn off the laser power and contact the Instrumentation Technician. The air outlet of the laser cooling fan blows out warm air and the mercury lamp housing gets hot. Do not place a flammable or non-heat-resistant object near these items. The cleaning fluids (mixture of alcohol or ether) used to clean the optics are highly flammable. Take special care in handling. The FV1000 rests on a floating table. The table dampens any vibrations to the system, and is connected to a cylinder of compressed air. Do not adjust the regulator on the compressed air cylinder or attempt to change an empty cylinder; the cylinder is maintained and replaced by the Instrumentation Technician.
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3. PERSONAL PROTECTIVE EQUIPMENT Gloves and a lab coat are encouraged for any type of lab work. Safety glasses are not required for the FV1000 because the laser path is enclosed within the live cell chamber, but never look directly at the laser light; always look through the protective pane above the microscope oculars.
See the WLU Laboratory Health and Safety Manual for additional information on personal protective equipment: http://www.wlu.ca/documents/23120/Laboratory_Health_%26_ Safety_Manual__Feb_2007_Final.pdf.
4. ACCIDENT PROCEDURES All incidents must be reported to the Instrumentation Technician and if applicable, a student’s supervisor. All accidents, incidents and near misses must be reported to the Environmental/Occupational Health and Safety (EOHS) Office via the WLU Employee Accident/Incident/Occupational Disease Report form (www.wlu.ca/eohs/forms). To meet regulatory requirements, these forms must be submitted to EOHS within 24 hours of occurrence, with the exception of critical injuries, which must be reported immediately to the EOHS Office by telephone. Critical injuries include any of the following; place life in jeopardy, produce unconsciousness, result in substantial loss of blood, involve fracture of a leg or arm but not a finger or toe, involve amputation of a leg, arm, hand or foot, but not a finger or toe, consist of burns to a major portion of the body, or cause the loss of sight in an eye. Additional details regarding incident reporting can be found in the WLU Accident Incident Procedure (www.wlu.ca/eohs). The WLU Laboratory Health and Safety Manual provides detailed instructions for dealing with major and minor spills. Before using ANY hazardous materials, make sure you understand the proper clean-up procedure. All sample preparation and disposal should be done in a separate lab, NOT in the room with the FV1000.
5. WASTE DISPOSAL PROCEDURES If any hazardous chemicals are used for sample analysis or preparation, they must be disposed of properly, as outlined in the WLU Laboratory Health and Safety Manual.
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6. PROTOCOL Anyone using the confocal microscope must receive hands on training. This document is a summary of the procedure and is only intended to help you remember the various steps.
6.1 Start-up You only need to turn on the components that you plan to use. Turning lasers and the mercury lamp on and off and running them affects their lifespan, so if you are sure you won’t be using a given lamp or laser, don’t turn it on. You must turn on all the lasers you might potentially need at the start, because you should not turn lasers on once the computer is on. The 405+635 diode laser power supply also runs the laser combiner, so this component must always be turned on, regardless of the wavelengths you are interested in viewing. If you just need the computer to look at data or transfer it to a CD/DVD, turn only the computer and monitors on. If you will be using the microscope to collect z-stacks or time series images, it is recommended to turn the system on, including the temperature controller, up to two hours before imaging. This will allow all of the components to reach thermal equilibrium and reduces z-drift during imaging. ALL mounting media or other substances used to secure coverslips (i.e. nail polish) must be COMPLETELY DRY before the slide is viewed on the confocal. Mounting media may otherwise leak onto the objective and is very difficult to remove. 1. TURN ON THE FAN. 2. Make sure the window shade is down all the way. 3. Sign-in in the user log book and fill in mercury lamp hours at start, and lasers to be used. 4. Turn on the microscope components according to the numbers on labels: 1. U-RFL-T: Mercury burner power supply – required for epifluorescence observation. 2. Power bar. This will turn on the following: a. FV10-MCPSU: Power supply for the diode lasers (405 and 643 nm). b. Prior ProScan II: Stage controller. c. IX2-UCB: Microscope controller power supply (on the windowsill). d. Scan head power supply. 3. Only turn on if required for your dye: Melles Griot argon (458, 488, and 515 nm ) laser power supply. Turn on the power switch and then the key. 5. Turn on the computer and monitors. 6. Log on to the computer: a. Login: Administrator b. Password: fluoview.
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7. Double click on the Olympus FV10-ASW 1.7 software icon and log on using your username and password. 8. Wait for the software and microscope to initialize. 9. Click on Device, then Microscope Controller.
6.1.1 Viewing sample with transmitted light and Kolher illumination Before viewing specimen with transmitted light (brightfield) the microscope condenser should be properly aligned to provide even and bright illumination (commonly called Kolher). If you do not need to view or collect brightfield or differential interference contract (DIC) images, Kolher illumination is not necessary, and you can skip to section 6.1.2. Refer to figures 6-1 and 6-2 for hardware and software diagrams. 1. In the Acquisition Setting window, select the 10x objective (drop down list, #6 in Figure 6-2). 2. Manually move the objective down by turning the knob clockwise. 3. Mount your slide on the microscope, coverslip down. 4. Make sure the polarizer is in the optical path (above the stage – manual slider, #4 in Figure 6-1)). 5. Align the DIC/Wollaston prism in the light path as well (below the stage) by sliding it in until in comes to a stop (#7 in Figure 6-1). 6. Select “DICT” from the Microscope Controller window, and the DIC cube will be rotated into place automatically (#15 in Figure 6-2). 7. Use the software to select Transmitted Light mode (turn on Trans Lamp, upper button by #8 in Figure 6-2). 8. Focus on the specimen using the microscope oculars. a. Adjust the brightness using the buttons on the front of the microscope. b. The focus mechanism can be set to fine using either the F/C button below the right focus knob, or in the Micoscope Controller window. i. Close your left eye and focus on the specimen using the fine focus knob. ii. Close your right eye and focus on the specimen using the diopter ring on the left ocular. iii. Open both eyes and confirm that the focus is comfortable. c. Turn the shear knob on the DIC prism (just under the stage, #7 in Figure 6-1) to adjust the contrast. d. Adjust the DIC prism focal plane by pulling out or pushing in the small L-shaped knob just beside the shear knob. (Push it in or pull it out until the best image is observed). 9. Completely open the condenser aperture diaphram (just above the stage, #6 in Figure 6-1) and close the field diaphragm (top diaphram – outside of the live cell case, #1 in Figure 6-1) enough so that you can see the diaphragm edges in the eyepieces.
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10. Focus the condensor knob (#5 in Fugre 6-1) so that the edges of the diaphragm AND the speciman appear very sharp (do not use the fine/course focus for the objectives). 11. If nessicary, center the condensor using the centering screws (it helps to open the field diaphragm so that the edges of the diaphragm almost fill the field of view, #3 in Figure 6-1). 12. When the condensor is focused and centered, open the field diaphragm so that it completely fills the eye pieces (#1 in Figure 6-1). 13. If you need DIC images only, proceed to 6.2, otherwise proceed to section 6.1.2 for epifluorescence imaging.
6.1.2 Epifluorescence Imaging Examining your specimen using epifluorescence before collecting a confocal image allows focusing and objective/magnification selection without the photobleaching risks associated with high intensity laser light. Use epifluoresence to find the region of interest and focus at the desired magnification as follows before proceeding to laser scanning. Refer to figures 6-1 and 6-2 for hardware and software diagrams. 1. Close the manual shutter on the mercury lamp. 2. If you do not plan to acquire DIC images in addition to confocal images, pull the Wollaston prism out using the shear knob (below the objective turret) until it comes to a stop (not completely) (#7 in Figure 6-1). 3. Click on the Epi Lamp button at the top left corner of the Acquisition Setting window (#8 in Figure 6-2). 4. Choose the appropriate filter cube (mirror) for your speciman in the Microscope Controller window (DAPI, FITC, or TRITC) (#15 in Figure 6-2). 5. Open the shutter on the mercury lamp to illuminate your sample, and use the joystick and fine focus to locate your region of interest. a. Note: If you don’t see any light, check to make sure that the shutter on the mercury lamp is open. The mercury lamp brightness can also be adjusted using this shutter. 6. When you have found the region of interest and focused with the 10x objective, move sequentially up to the high powered objectives: switch to the 20x, focus, then switch to 40x if desired, and focus again. (Only use the 60x and 100x oil objectives if you have been trained by the Instrumentation Technician). 7. When you have found the region of interest and focused with the desired objective, close the shutter on the mercury lamp to avoid bleaching your sample.
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Figure 6-1: Inverted microscope components (live cell chamber not shown)
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Figure 6-2: Software windows and basic description of controls for the FV-ASW software
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6.2 Preparation for Imaging 1. Turn off brightfield or epifluorescence illumination by clicking the relevant button at the top left of the Acquisition Setting window (#8 in Figure 6-2). The microscope then automatically sets up for laser scanning (LSM). 2. Change the focus mechanism to fine (using the green buttons beside the focus knob or using the drop down list - #16 in Figure 6-2). 3. Click on the Dye List button on the left in the Image Acquisition Control window (#9, Figure 6-2). a. Double click on the dyes that you have in your sample to add them to the Selected Dyes box. Up to three dyes can be selected in addition to transmitted light. b. If the dye you are using is not in the list, it can be added by clicking on Tools Maintenance User Settings. Click on the Dye tab, and then click the “add” button on the far right side of the window. Type in the name of the dye and then enter the excitation and emission wavelengths near the bottom left corner of the screen. Select the laser that most closely corresponds to the excitation wavelength. Press “save and close”. c. Click Apply. The appropriate filters will automatically move into place. d. Close the Dye List window. 4. The fourth detector chanel, TD1, is for DIC or brightfield image collection only. If you plan to collect a DIC image as well, slide the Wollastin prism back into the light path (#7, Figure 6-1), then click the box beside TD1 in the Acquisition Control window. You can select any laser to correspond with the TD1 channel except the 405. 5. Set up your scanning parameters in the Acquisition Setting window: a. Mode (#1, Figure 6-2): one way raster scanning (normal) or bidirectional (high speed. Bidirecitonal is best for live cell imaging but can cause slight image abberation. This image can be corrected using the Image Adjust button, next to the scanning speed slider). b. The scanning area must be normal (rectangle) to start. i. After an image has been collected, different regions of interest (ROIs) can be selected using the buttons in this section: clip scan (diamond button; select rectangle, ellipse, or polygon), line (line button; straight or curved line), or point (point button). More than one area can be scanned at a time using the ROI manager button in the 2D Control Panel window, and laser parameters can be set individually for each area. c. Image acquisition speed (#2, Figure 6-2): Select fast scanning speed for image acquisition initially (a slower speed will give a better signal, but also causes higher photobleaching). Make sure “AutoHV” is not selected at this point.
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d. Size (#3, Figure 6-2): intially set at 512 x 512 pixels, and adjusted later to optimize resolution. e. Area (#4, Figure 6-2): leave at 1:1 initally (no zoom). f. Laser (#5, Figure 6-2): lasers are selected automatically for fluorescence according to the dye that you select. Do not adjust the laser powers at this point. g. Microscope (#6, Figure 6-2): make sure the correct objective is selected from the drop down menu. 6. In the Image Acquisition Control window: a. If you are using more than one dye, check the Sequential box to minimize bleedthrough. i. Line sequential scan minimizines the time difference between images for each channel by scanning only a line in each channel at a time. This is typically the best choice. ii. Frame sequential scan takes one full image on a given channel before scanning the next channel. b. Click on XY repeat to initiate sample scanning (#10, Figure 6-2). Alternatively, the Focus x2 or Focus x4 buttons can be used to obtain a very rough scan that skips lines, doubles or quadrupoles the scanning speed, and minimizes photobleaching. Remember that while the image is being scanned, the specimen is prone to photobleaching – avoid scanning for any longer than neccesary. c. The focus between what is seen with epi (though the oculars) and what is seen on the screen is slightly different. You wil need to move the objective up VERY CAREFULLY (while in FINE FOCUS, turn the knob very slowely counter clockwise) to bring the image into focus. Only a very small amount of adjustment should be required. If the image does not come in to focus easily, switch back to a lower magnification and/or switch back to epiflourescence mode to focus again. d. Adjust the brightness of the image: i. If the signal is low in all channels, slow the scan speed down while scanning until signal can be observed in all channels. ii. Press Ctrl+H to show the high and low limits of detection. If the signal is too high and saturating the detector, it will show up as red. Any blue on the image indicates zero signal detection. iii. Adjust HV on each of the channels (#12, Figure 6-2) independantly until just a few pixels of red can be seen (avoid setting HV higher than 700 as this will increase background instrument noise). (Note: The HV for DIC in the TD1 channel is usually much lower – try starting around 200). If the HV is quite high and the signal is still too dim, try adjusting the C.A. (confocal aperture); an increased CA will increase brightness, but also decrease resolution, and increase cross-section thickness. The scan rate can also be
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decreased futher, or the laser power can be adjusted as a last resort. Typical starting laser powers as as follows: 405, 458, 488, and 635: 0-5% 515: 10-20% 543: 45-55% iv. Avoid adjusting the gain or offset if possible (#13 and #14, Figure 6-2), however; if HV is high and the image is still too dim, the gain can be increased. Increasing the offset will turn the background darker. v. Check for bleedthrough by switching off (unchecking) all but one laser then make sure that there is only signal in the relevant channel. Do this for each laser in turn. (If you are already doing sequential scanning, this is unnessicary). vi. Kalman integration can be used to collect several images and average them to decrease S/N. This slows scanning, can dim an image, and increases photobleaching. vii. Note: for very weak signals, the photon-counting mode provides better sensitivity. e. Press Ctrl+H to return to regular viewing mode. f. If desired, optimize the resolution (Nyquist) by pressing “i” in the Image Acquisition Control window. To achieve optimal resolution the pixel size should be aprox ½ of the optical resolution. Adjust the size of the image (number of pixels) and the zoom or change objectives to optimize resolution. i. When setting the pixel size of your image, keep in mind your final format; journal, poster, etc. An image that will be blown up for a poster will need a high pixel count to avoid becoming pixelated. An image for a journal should have the dots-per-inch required by that journal, which can be determined based on image size and pixel number (i.e. the Journal of Microscopy requires 300 dpi). g. The fine focus can be adjusted again slightly to obtain the brightest image. 7. When adjustements for brightness and senstivity are complete, press the Stop button. 8. The imge is now optimized; a. to collect a single XY scan, proceed to section 6.3. b. to collect a 3-D image, proceed to section 6.4. c. to collect a time series image, proceed to section 6.5.
6.3 XY Image Acquisition 1. If you are satisfied with the image, click the XY button to do a single XY scan (#11, Figure 6-2), and then press Stop. 2. When image acquisition is complete, the image will appear in the 2D View window. This window can be used to manipulate the 2D image in a number
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of ways; see Section 6.6 for instructions on image analysis, manipulation, and saving your files.
6.4 Z-Series or 3-D Stack Image Acquisition 1. Optimize your image as described in Section 6.2. 2. Right below the XY button (#11, Figure 6-2) in the Image Acquisition Control window, select the Depth button. XYZ should now be bolded in the button. 3. Scan rapidly using XY Repeat or Focus x2 or Focus x4. 4. In the Image Acquisition window (z-stack controls indicated by #7 on Figure 6-2): a. Use the down button (or fine focus knob) to move the focus to the bottom (or just past the bottom) of your sample. Press the Set button below Start to indicate the start position. b. Use the up button (or fine focus knob) to move to the top of the sample, or to the last XY image that you want to take. Press the Set button below End. c. Click the “go” button next to Center to go the middle of your Zseries. Adjust the brightness as needed by changing the HV or laser powers. d. Stop the XY Scanning. e. Set your step size as desired, or press “Op” to the right of the Step Size box. This will automatically set the slice thickness to Nyquist/2, which garuntees that you will oversample the Z-series and capture all possible detail. Keep in mind that more sampling leads to longer scanning and increased photobleaching. The “S” at the top of the Acquisition Setting window will indicate the total time to acquire the stack. If you require a very small step size you can also increase the scan speed or decrease the depth of the stack to decrease scanning time. 5. Start the scan using the XYZ scan button. 6. Click on Series Done if you are satisfied with the stack collected, or Append Images to add additional frames. 7. If you are satisfied with the image, proceed to section 6.6 for image analysis.
6.5 T-Series Image Acquisition 1. Optimize your image as described in Section 6.2. 2. Right below the XY button in the Image Acquisition Control window, select the Time button. XYt should now be bolded in the button. 3. Set the rest interval (in the format hh:mm:ss.ms) this is the time between one acquisition start and the next) and the scanning time. Note: you can do regular LSM or visual observation during rest time (time between acquisition sets). 4. Re-start XY scanning, and if the image is acceptable, press XY(t). 5. When image acquisition is complete, press the Series Done button, or Append next to add additional frames to the image.
Olympus FV1000 Standard Operating Procedure
5
6. If you are satisfied with the image, proceed to section 6.6 for image analysis.
6.6 Image Analysis When image acquisition is complete, the image will appear in the 2D View window. This window, along with the 2-D control panel, can be used to manipulate the 2D image in a number of ways, and the various tools are described in Table 6-1 and 6-2. The image can also be saved and viewed in different ways (Table 6-3), and processed (Table 6-4).
6.6.1 Add a scale bar to your image: 1. In 2D view window, click on the Pencil button, and then click on the ruler at the bottom. 2. In the area of interest, click and drag the ruler to show a scale bar. 3. Proceed to Section 6.7 to save the image.
Olympus FV1000 Standard Operating Procedure
6
Table 6-1: 2D-View Window Tool button LUT (look up table)
Single/Panel/Tile Zoom, 1:1, Fit to Window Slider Active Overlay Numbered buttons down the side of the window (1, 2, etc) Pencil button
Animation arrows 3-D button
Intensity profile button Measurement button Histogram button
Series Analysis
Line Series Colocalization
Function Use the LUT to change the gamma, intensity, contrast, and pseudocolour of any channel to enhance dim images or highlight certain features with a different colour. Change the view of the sample, or look at more than one image at once. Channels can be viewed separately or overlaid using the Tile button. These buttons adjust the size of the image on the screen. Use to scan through the frames collected for this speciman (primarily for Z- or T-series images). Add text to the image describing Z position, time, or wavelength. Use to select the desired channels displayed in the image. Allows access to region of interest (ROI) buttons and other drawing tools (i.e. text, scale bar, grid). After a ROI is selected, imaging can be conducted on that area specifically to avoid unnecessary bleaching of entire speciman in light path. Used to play an animation or scroll through Z- or T-series. Allows viewing of a Z-series from any angle, selection of cross sections, rotation along a given axis and creation of animation (using the More arrow). Displays the intensity profile for a selected ROI. This profile can be used to determine the size of a given structure, for example. Measure the size, area, etc., of selected ROI. Also accessible from the Analysis Menu. Displays an intensity profile (intensity vs. frequency) for the selected ROI. Also accessible from the Analysis Menu. Displays intensity vales for a given ROI and each channel for each frame in a series (i.e. can use it to determine which frame best displays a given feature/ROI). Also accessible from the Analysis Menu. Illustrates the change in intensity of a line as a series (Z or T) progresses. Graph displays extent of colocalization, and statistics page calculations Pearson's coefficient, overlap and colocalization indexes.
Olympus FV1000 Standard Operating Procedure
7
Table 6-2: 2D Control Panel Window Tool button Digital Zoom ROI Format and Manager buttons Load Scan data button Stepping box Tile box Intensity profiles
Function Adjust digital zoom. Change the text and line formatting for labeling ROIs, and view information on ROIs selected. If accessible, the acquisition conditions for the image can be read. Allows stepping through Z, time, or animation series frames. Determines the organization of the tiles from a set of images. Click to display the vertical or horizontal intensity distribution along a line.
Table 6-3: Main Fluoview Window Tool button Properties ( “i” ) Report, Thumbnail, Thumbnail + property Various buttons to organize how windows are displayed Darkroom colour button
Function Display image information in Data Manager Window. Change how files are displayed in the Explorer window.
Assists in organizing the screen-view when many windows are open.
Dims the monitor display to minimize stray light.
Olympus FV1000 Standard Operating Procedure
8
Table 6-4: Processing Menu Menu Item
Filter Setting… and Filter
Threshold
Image Calculation Correcting Pixel Gaps
Correcting Z Gaps
Ratio/Concentration
Edit Experiment
Function Mathematical filters can be used to improve image clarity or emphasize certain characteristics. The filters available are: sharpen, average, DIC, sobel, median, shading, lapacian. Sharpen: highlights the edges of images, improves blurred images, but also increases noise. Sobel: Emphasizes contours. Lapacian: emphasizes intensity changes. Press “Preview” at bottom of Filter window first, then select various filters to observe result. Select the Single or Series button, if doing a single image, enter the desired frame in the Image Position box. If you want to save the filtered image, press New Image. This feature is used to make the image binary (two colour). The C and Z indicate the channel and frame to display. The thresholds can be adjusted by clicking on the channel in the table below threshold, and manually entering new values, or by moving the bars on the graph Used to subtract/add/divide images or constants. This window can be used to correct for image shift between channels. Image position can be used to select the desire frame. The white box in the Preview area can be dragged to view other areas of the image. To correct for Z position shift between channels. Moving the yellow bar changes the Z-slice image. The Ratio tab is used to look at changes in intensity between two channels. Use the drop down menu to select the desired operation, and the equation for that operation will be displayed in the Equation box. Equations include baseline/background subtraction, ratio of channels, FRET image comparison (photo bleached image required). The Concentration tab is used to analyze a change in ion intensity over time. This feature requires knowledge of intensity values with and without ion, and the dissociation constant (in nM). See the Help menu for an explanation of F values. Combine single channels, append or extract series, from two different images to create a new image or series
Olympus FV1000 Standard Operating Procedure
9
Additional Tools: Virtual channel: Useful if fluorescent signals from two reagents overlap. Delay image acquisition: After a set time has passed, acquisition can be started or terminated using XYt imaging. Image noise reduction: Can be done using Kalman filtering (takes several scans and averages them). Live plot window: plots change in intensity in ROI over time. First outline ROI in Live View window by clicking on the “pen” tool. Then select Live Plot from Live menu. Live Tiling: allows viewing of the specimen as time elapses while the image is being acquired (button in Live View window has the image of a wrench).
6.7 Saving and Viewing Final Images 1. When you are finished collecting and analyzing your image, select File Save As and save it to your folder as a .oif or .oib file (automatically saved in D:\FV10-ASW\users\yourusername\Image). Make sure “Include all ROI” (or selected ROIs) are checked before you save the image so that scale bars and other additions to the image are included. a. You can also right click on the image to export as a TIFF, JPEG, or other image format. b. An OIF format saves the images as TIFFs in a folder, and creates a file with the sample information. Both the file and the TIFFs are required to open the image. If you save this data to a memory key or CD, you need to copy both the .oif files and the associated sub-folder containing the images. c. An OIB format saves all of the images in a set of files. This format is recommended if analyzing the images in Metamorph or a similar program. 2. Manipulations can be done using the fluoview software on the confocal computer, or another program or fluoview software on a different computer (recommended). a. A free version of the Fluoview software can be downloaded from:. https://support.olympus.co.jp/cf_secure/en/lisg/bio/download/ ga/fv10_asw/ Our serial number is 7M10. b. ImageJ (http://rsb.info.nih.gov/ij/) and VOXX (http://www.nephrology.iupui.edu/imaging/voxx/) software can also be used to view .oif files.
Note: DATA IS CLEARED FROM THE CONFOCAL COMPUTER PERIODICALLY, so SAVE YOUR DATA ELSEWHERE (disk, another computer) for safekeeping.
Olympus FV1000 Standard Operating Procedure
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6.8 Cleaning Objectives NEVER USE KIMWIPES OR OTHER TISSUE PAPER TO CLEAN OBJECTIVES. USE ONLY LENS PAPER, supplied in SR314. If you notice oil on the non-oil objectives (10x, 20x and 40x), DO NOT try to clean them. Inform the Instrumentation Technician as soon as possible and note the problem in the log book. 1. Using clean lens paper gently blot off the oil from the lens. Do NOT drag the paper across the lens, just dab off the oil. The front lens of the objective is very delicate and must be protected from scratching. 2. Wipe any oil off of the objective barrel. 3. Put a drop of ethanol on a clean piece of lens paper, and gently draw it across the lens (DO NOT RUB).
6.9 Shutdown 1. Switch back to the 10x objective, then manually lower the objective by turning the knob clockwise. 2. Remove your slide. 3. If you used the oil objectives, make sure the oil has been removed as per section 6.8. 4. Make sure you have saved your data and then exit the Fluoview software. Close the FL (mercury lamp) shutter as recommended by the Caution message. 5. If the argon laser (#3) it turned on, turn it’s key to the upright position, but DO NOT TURN OFF THE POWER SWITCH YET. This allows the fan to continue to blow until the laser has cooled down. 6. Turn off power bar (#2) 7. Turn off the Mercury power supply (#1). 8. When the fan has stopped running on the argon laser, the power can be turned off (#3). 9. Transfer your data to a CD or memory stick if desired (recommended). 10. Shutdown the computer. 11. Cover the microscope. Make sure the cover DOES NOT touch the mercury lamp, which is hot and may melt the cover. 12. Clean up anything you have left in the room. 13. Fill in the rest of the log book.
7. TROUBLESHOOTING For troubleshooting tips, see the Help menu in the Fluoview software.
Olympus FV1000 Standard Operating Procedure
8. PREVENTATIVE MAINTENANCE Users are not to perform maintenance. Unless noted otherwise, these procedures are carried out by the Instrumentation Technician.
8.1 Monthly -
If the system has not been used for a month or more, it will be run for ~ 8 hours to ensure long-term laser stability Objective lenses will be checked and cleaned if required Check the 5% CO2 and humidity canister for the Weather Station chamber Check the CO2 for the anti-vibration table
8.2 Annually or as Required 8.2.1 Replace the Mercury Burner The mercury burner should be replaced when the lamp hours reach 350 hours or when the epifluorescence images appear unstable due to lamp failure.
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Olympus FV1000 Standard Operating Procedure
12
9. QUICK REFERENCE GUIDE 1. 2. 3. 4. 5. 6. 7. 8.
Remove the cover from the microscope. Sign in in the user log book and fill in the relevant details. Turn on the microscope components according to the numbers on lables: Log on to the computer and the software (FV10-ASW 1.7) Wait for the software and microscope to initialize. Cafully place your slide on the stage. Set up Kohler. View the image using epifluorescence and the appropriate filter cube to fine-tune the focus. 8. When you have found the region of interest and focused with the desired objective, close the shutter on the mercury lamp to avoid bleaching your sample. 9. Turn off brightfield or epifluorescence illumination. The microscope then automatically sets up for laser scanning (LSM). 10. In the Image Acquisition Control window, select desired dyes. 9. Set up your image parameters in the Acquisition Setting window: a. One way scanning (normal) or bidirectional. b. Select fast scanning speed for initial image acquisition initially c. Set the size to 512 x 512 pixels d. Leave zoom at 1:1 initally. e. Set the laser powers should be set as low as possible to minimize bleaching (except the 543 laser, which should be at at least 50%). f. Select the correct objective from the drop down menu. 10. In the Image Acquisition Control window: a. If you are using more than one dye, check the Sequential box to minimize bleedthrough. b. Click on XY repeat or Focus x2 or Focus x4 to initiate scanning c. Use the fine focus or the up and down button in the Acquisition Setting window to align the microscope with the cross section to be observed. d. Adjust HV on the active channels until the image can be observed (do not set higher than 700). e. Adjust the brightness of the image using HV, CA, scan speed, or laser powers, and check for satration (Ctrl + H). f. If desired, optimize the resolution (Nyquist) by pressing “i” in the Image Acquisition Control window. 11. When adjustements for brightness and senstivity are complete, press the Stop button. 12. The image is now optimized. 13. Collect a single XY scan, or create a Z- or T-series. 14. When image acquisition is complete, the image will appear in the 2D View window. 15. Adjust the image as nessicary, then save as an .oif or .oib file. Transfer to a CD or memory key for long term storage on your personal computer.
Olympus FV1000 Standard Operating Procedure
REFERENCES Laboratory Health and Safety Manual. 2007. Wilfrid Laurier University Environmental/Occupational Health and Safety Office. Olympus website: http://www.olympusfluoview.com. Pawley, JB (Ed.). 2006. Handbook of Biological Confocal Microscopy, 3rd Edition. Springer Science + Business Media: Singapore. Staudt, T, Lang, MC, Medda, R, Engelhardt, J, & Hell, S.W. 2007. 2,2'thiodiethanol: a new water soluble mounting medium for high resolution optical microscopy. Microsc Res Tech 70: 1-9. Texas A&M University. Microscopy and Imaging Center. Olympus FV1000. http://microscopy.tamu.edu/instruments/light-microscopy/olympusfv1000-confocal-microscope.html. Accessed May 26, 2008.
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Olympus FV1000 Standard Operating Procedure
APPENDIX 1: FLUOROCHROME PEAK EXCITATION AND EMISSION WAVELENGTHS The following list of fluorochromes and wavelengths is from the Olympus website (http://www.olympusmicro.com/primer/techniques/fluorescence/ fluorotable2.html) Fluorochrome
Excitation Wavelength
Emission Wavelength
Acid Fuchsin
540
630
Acridine Orange (Bound to DNA)
502
526
455-600
560-680
Acridine Yellow
470
550
Acriflavin
Acridine Red
436
520
AFA (Acriflavin Feulgen SITSA)
355-425
460
Alizarin Complexon
530-560
580
Alizarin Red
530-560
580
Allophycocyanin
650
661
ACMA
430
474
AMCA-S, AMC
345
445
Aminoactinomycin D
555
655
7-Aminoactinomycin D-AAD
546
647
Aminocoumarin
350
445
Anthroyl Stearate
361-381
446
Astrazon Brilliant Red 4G
500
585
Astrazon Orange R
470
540
Astrazon Red 6B
520
595
Astrazon Yellow 7 GLL
450
480
Atabrine
436
490
Auramine
460
550
Aurophosphine
450-490
515
Aurophosphine G
450
580
BAO 9-(Bisaminophenyloxadiazole)
365
395
BCECF
505
530
Berberine Sulphate
430
550
Bisbenzamide
360
600-610
BOBO-1, BO-PRO-1
462
481
Blancophor FFG Solution
390
470
Blancophor SV
370
435
15
Olympus FV1000 Standard Operating Procedure Bodipy Fl
503
512
Bodipy TMR
542
574
Bodipy TR
589
617
BOPRO 1
462
481
Brilliant Sulphoflavin FF
430
520
Calcein
494
517
Calcien Blue
370
435
Calcium Green
505
532
Calcium Orange
549
576
Calcofluor RW Solution
370
440
Calcofluor White
440
500-520
Calcofluor White -ABT Solution
380
475
Calcofluor White- Std Solution
365
435
548(low pH) 576(high pH)
587(low pH) 635(high pH)
6-Carboxyrhodamine 6G
525
555
Cascade Blue
400
425
Catecholamine
410
470
450-490
515
504(low pH) 514(high pH)
587(low pH) 540(high pH)
Coriphosphine O
460
575
Coumarin-Phalloidin
387
470
CY3.18
554
568
CY5.18
649
666
CY7
710
805
DANS (1-DimethylAminoNaphthaline-5-Sulphonic Acid)
340
525
340-380
430
Dansyl NH-CH3 in water
340
578
DAPI
350
470
DiA
456
590
Diamino Phenyl Oxydiazole (DAO)
280
460
5-(and 6-)carboxy SNARF-1 indicator
Chinacrine CL-NERF
DANSA (DiaminoNaphthylSulphonic Acid)
Di-8-ANEPPS
488
605
310-370
520
DiI [DiIC18(3)]
549
565
DiO [DiOC18(3)]
484
501
Diphenyl Brilliant Flavine 7GFF
430
520
DM-NERF
497(low pH) 510(high pH)
527(low pH) 536(high pH)
Dopamine
340
490-520
Dimethylamino-5- Sulphonic Acid
16
Olympus FV1000 Standard Operating Procedure ELF-97 alcohol
345
530
Eosin
525
545
Erythrosin ITC
530
558
Ethidium Bromide
510
595
Euchrysin
430
540
FIF (Formaldehyde Induced Fluorescence)
405
435
375-530
612
Fluorescein
494
518
Fluorescein Isothiocyanate (FITC)
490
525
Fluo 3
485
503
FM1-43
479
Flazo Orange
598
Fura-2
363(low [Ca ]) 335(high [Ca2+])
512(low [Ca2+]) 505(high [Ca2+])
Fura Red
472(low [Ca2+]) 436(high [Ca2+])
657(low [Ca2+]) 637(high [Ca2+])
Genacryl Brilliant Red B
520
590
Genacryl Brilliant Yellow 10GF
430
485
Genacryl Pink 3G
470
583
Genacryl Yellow 5GF
430
475
Gloxalic Acid
405
460
Granular Blue
355
425
530-560
580
Hoechst 33258, 33342 (Bound to DNA)
352
461
3-Hydroxypyrene-5,8,10-TriSulfonic Acid
403
513
7-Hydroxy-4methylcoumarin
360
455
380-415
520-530
Indo-1
350
405-482
Intrawhite Cf Liquid
360
430
Leucophor PAF
370
430
Leucophor SF
380
465
Leucophor WS
395
465
Lissamine Rhodamine B200 (RD200)
575
595
Lucifer Yellow CH
425
528
Lucifer Yellow VS
430
535
Haematoporphyrin
5-HydroxyTryptamine (5-HT)
2+
17
Olympus FV1000 Standard Operating Procedure LysoSensor Blue DND-192, DND-167
374
425
LysoSensor Green DND-153, DND-189
442
505
384(low pH) 329(high pH)
540(low pH) 440(high pH)
LysoTracker Green
504
511
LysoTracker Yellow
534
551
LysoTracker Red
577
592
Magdala Red
524
600
Magnesium Green
506
531
Magnesium Orange
550
575
Maxilon Brilliant Flavin 10 GFF
450
495
Maxilon Brilliant Flavin 8 GFF
460
495
Mitotracker Green FM
490
516
Mitotracker Orange CMTMRos
551
576
MPS (Methyl Green Pyronine Stilbene)
364
395
Mithramycin
450
570
NBD
465
535
LysoSensor Yellow/Blue
NBD Amine
450
530
Nile Red
515-530
525-605
Nitrobenzoxadidole
460-470
510-650
340
490-520
Noradrenaline Nuclear Fast Red
289-530
580
Nuclear Yellow
365
495
Nylosan Brilliant Flavin E8G
460
510
Oregon Green 488 fluorophore
496
524
Oregon Green 500 fluorophore
503
522
Oregon Green 514 fluorophore
511
530
Pararosaniline (Feulgen)
570
625
Phorwite AR Solution
360
430
Phorwite BKL
370
430
Phorwite Rev
380
430
Phorwite RPA
375
430
Phosphine 3R
465
565
Phosphine R
480-565
578
Pontochrome Blue Black
535-553
605
POPO-1, PO-PRO-1
434
456
Primuline
410
550
Procion Yellow
470
600
18
Olympus FV1000 Standard Operating Procedure Propidium Iodide Pyronine
536
617
410
540
540-590
560-650
Pyrozal Brilliant Flavin 7GF
365
495
Quinacrine Mustard
423
503
R-phycoerythrin
565
575
Rhodamine 110
496
520
Rhodamine 123
511
534
Rhodamine 5 GLD
470
565
Rhodamine 6G
526
555
Pyronine B
Rhodamine B
540
625
523-557
595
Rhodamine B Extra
550
605
Rhodamine BB
540
580
Rhodamine BG
540
572
Rhodamine Green fluorophore
502
527
Rhodamine Red
570
590
Rhodamine WT
530
555
Rhodol Green fluorophore
499
525
Rose Bengal
540
550-600
Serotonin
365
520-540
Sevron Brilliant Red 2B
520
595
Sevron Brilliant Red 4G
500
583
Sevron Brilliant Red B
530
590
Sevron Orange
440
530
Sevron Yellow L
430
490
SITS (Primuline)
395-425
450
SITS (Stilbene Isothiosulphonic Acid)
365
460
Sodium Green
507
535
Stilbene
335
440
Snarf 1
563
639
Sulpho Rhodamine B Can C
520
595
Sulpho Rhodamine G Extra
470
570
SYTOX Green nucleic acid stain
504
523
SYTO Green fluorescent nucleic acid stains
494 ± 6
515 ± 7
SYTO Green fluorescent nucleic acid stains
515 ± 7
543 ± 13
SYTO 17 red fluorescent nucleic acid stain
621
634
Tetracycline
390
560
TRITC (Tetramethyl Rhodamine
557
576
Rhodamine B 200
19
Olympus FV1000 Standard Operating Procedure Isothiocyanate) Texas Red
596
615
Thiazine Red R
510
580
Thioflavin S
430
550
Thioflavin TCN
350
460
Thioflavin 5
430
550
370-385
477-484
Thiozol Orange
453
480
Tinopol CBS
390
430
TOTO 1, TO-RRO-1
514
533
TOTO 3, TO-PRO-3
642
661
True Blue
365
420-430
Ultralite
656
678
Uranine B
420
520
Uvitex SFC
365
435
X-Rhodamine
580
605
Xylene Orange
546
580
XRITC
582
601
YOYO-1, YOYO-PRO-1
491
509
YOYO-3, YOYO-PRO-3
612
631
Thiolyte
For additional lists of fluorescent dyes and their properties, see: Interactive database of fluorescence spectra: http://www.mcb.arizona.edu/ipc/fret/default.htm Olympus fluorchrome data tables: http://www.olympusmicro.com/primer/techniques/fluorescence/fluorotable2. html Molecular Probes dye spectra viewer: http://probes.invitrogen.com/resources/spectraviewer/
Olympus FV1000 Standard Operating Procedure
APPENDIX 2: USER LOG
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Olympus FV1000 Standard Operating Procedure
DATE & TIME IN
NAME & EXTENSION
SUPERVISOR
SAMPLE AND DYES USED
MERCURY LAMP HOURS AT START
HE-NE LASER USED (Y/N)
OBJECTIVES USED (AIR: 10, 20, 40, OIL: 60 OR 100)
MERCURY LAMP HOURS AT END
TIME OUT
PROBLEMS / COMMENTS
Olympus FV1000 Standard Operating Procedure
APPENDIX 3: PREVENTATIVE MAINTENANCE LOG
22
23
Olympus FV1000 Standard Operating Procedure
DATE
NAME
EXT #
TYPE OF MAINTENANCE
FREQUENCY OF MAINTENANCE (I.E. WEEKLY)
PROBLEMS / COMMENTS
