Confocal Microscope

7/24/2019 Confocal Microscope

1/41


2/41

Olympus FV1000 Standard Operating Procedure ii

TABLE OF CONTENTS

DISCLAIMER ................................................................................ iv

ACKNOWLEDGEMENTS ..................................................................... v

1. INTRODUCTION ........................................................................ 11.1 Purpose of the Standard Operating Procedure ............................. 11.2 Emergency Contact Information .............................................. 11.3 User Fees and Instrument Booking ........................................... 2

1.4 Basic Theoretical Background ................................................. 21.4.1 Sample Preparation Suggestions ......................................... 3

1.5 Instrumentation ................................................................. 51.5.1 Overview .................................................................... 51.5.2 Lasers and Filters .......................................................... 51.5.3 Objectives .................................................................. 51.5.4 Scan Head ................................................................... 6

2. POTENTIAL HAZARDS ................................................................. 7

3. PERSONAL PROTECTIVE EQUIPMENT ............................................... 8

4. ACCIDENT PROCEDURES ............................................................. 8

5. WASTE DISPOSAL PROCEDURES ..................................................... 8

6. PROTOCOL ............................................................................. 96.1 Start-up ........................................................................... 9

6.1.1 Viewing sample with transmitted light and Kolher illumination .. 10


3/41

Olympus FV1000 Standard Operating Procedure iii

9. QUICK REFERENCE GUIDE .......................................................... 12

REFERENCES .............................................................................. 13

APPENDIX 1: FLUOROCHROME PEAK EXCITATION AND EMISSION WAVELENGTHS 14APPENDIX 2: USER LOG .................................................................. 20APPENDIX 3: PREVENTATIVE MAINTENANCE LOG .................................... 22


4/41

Olympus FV1000 Standard Operating Procedure iv

DISCLAIMER

The materials contained in this document have been compiled from sourcesbelieved to be reliable and to represent the best opinions on the subject. Thisdocument is intended to serve only as a starting point for good practices anddoes not purport to specify minimal legal standards. No warranty, guarantee,or representation is made by Laurier as to the accuracy or sufficiency ofinformation contained herein, and Laurier assumes no responsibility inconnection therewith.


5/41

Olympus FV1000 Standard Operating Procedure v

ACKNOWLEDGEMENTS

The following individuals of Laurier contributed to the writing, editing, andproduction of this manual: Gena Braun (Instrumentation Technician); Dr. DianoMarrone (Psychology); Stephanie Kibbee (Environmental/Occupational Healthand Safety Office).


6/41

Olympus FV1000 Standard Operating Procedure 1

1. INTRODUCTION

1.1 Purpose of the Standard Operating Procedure

This standard operating procedure (SOP) is NOT a substitute for trainingand/or reading the appropriate manuals before use. All principleinvestigators and supervisors must document that training has beenreceived 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 OlympusFV1000. This SOP covers the potential hazards, personal protectionrequirements, 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 istherefore regulated under the Laurier Laser Safety Program. The Laser SafetyManual 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


7/41


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: $ 20Annually: $ 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 Whenand To boxes.

4. Make sure you select Confocal Microscope in the Instruments dropdown box.

5. If you want to book the same time slot in other days, select fromRepeat 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 themicroscope, so that others can book the instrument if they need.
http://www.supersaas.com/schedule/login/WLU_Instruments/http://www.supersaas.com/schedule/login/WLU_Instruments/http://www.supersaas.com/schedule/login/WLU_Instruments/


8/41


producing 3-D reconstructions to assess cell structure, studying the spatialdistribution of labeling, imaging of live cells or other applications that requiresimultaneous and rapid multi-channel imaging, and studying multi-labeled

specimens.

Figure 1-1: Basic illustration of confocal imaging; only the signal that isconfocal with the emission pinhole is detected, reducing out-of focusbackground signal and improving the image significantly.

1.4.1Sample Preparation Suggestions

A number of factors can affect the quality of a confocal image including


9/41


(i.e. pH) and has a relatively broad emission spectrum, so it is not ideal forstudies that require dual or triple labeling. Appendix 1 lists several dyes andtheir 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 mediumshould contain anti-fade reagents to minimize photobleaching. Conversely,anti-fade reagents can be toxic to living organisms and are typically notrecommended for live specimens. The medium and anti-fade reagent must alsobe compatible with the selected dye; some anti-fade compounds can cleavethe fluorescent molecules of certain dyes and cause the signal to degrade within a few days or less.

If you plan to image using the 60x or the 100x oil immersion lenses, yourmounting medium must have a refractive index that is similar to oil. Considerusing 50-80% glycerol or 2,2-thiodiethanol in your mounting medium to improvethe image (see Staudt et al., 2007). Again, confirm that your dyes arecompatible with all the components of your mounting medium before preparing

the specimen. All mounting media must be completely dry before thespecimen is viewed on the FV1000.

All sample preparation should be done in a separate lab, NOT in the roomwith the FV1000.

Additional information on mounting media and anti-fade reagents has beensummarized by Tonny Collins (Wright Cell Imaging Facility, Toronto) at:

http://www.uhnresearch.ca/facilities/wcif/PDF/Mountants.pdf
http://www.uhnresearch.ca/facilities/wcif/PDF/Mountants.pdfhttp://www.uhnresearch.ca/facilities/wcif/PDF/Mountants.pdf


10/41


1.5 Instrumentation

1.5.1

OverviewThe FV1000 inverted CLSM can be used to examine both fixed and livespecimens. Light is produced by a several lasers, and passes through theappropriate filters and mirrors and a pinhole before being scanned across theimage in a raster patter. High speed imaging (up to 16 frames/sec for a 256 x256 image) and rapid spectral scanning (100 nm/sec) are carried out usinggalvo scanning mirrors. Wavelengths can be resolved to 2 nm on this system,

and all filters are ion-sputter coated to provide improved transmissionefficiency and allow imaging at lower laser intensity. Fluorescence produced bythe specimen passes back through mirrors and filters and a pinhole to bedetected by the appropriate photomultiplier tube (PMT). The available lasersand optics are described in more detail below.

1.5.2Lasers and Filters

The FV1000 is equipped with a multi-line argon laser, a green helium-neon (He-

Ne) laser, two diode lasers, and a mercury lamp. The argon laser producesexcitation light at 458, 488, and 515 nm; the green He-Ne at 543 nm; the bluediode at 405 nm; and the red diode at 635 nm. The mercury lamp must be onfor at least 30 minutes before it can be switched off; after turning it of, itmust cool down for ~15 minutes before being turned back on. The lamp andlasers should not be switched on and off frequently as this shortens thelifetime.

The light source for transmitted light is a halogen bulb, which is connected tothe microscope via a fiber optic cable. Brightfield and differential interferenceimages using transmitted light can be viewed through the oculars or collected


11/41


There are 3 air objectives, and two oil-immersion objectives available on the FV1000confocal 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 incoverslip 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 theobjective and into the microscope.

- DO NOT use any of the air objectives (10x, 20x, or 40x) immediately afterusing an oil immersion (60x or 100x) objective.The 20x and 40x objectives inparticular have a very small working distance, and can easily come into contactwith any oil left on the slide. The slide must be cleaned before switching to the

air objectives.

1.5.4Scan Head

The microscope scan head contains the optics required to accept, filter, anddetect the laser and fluorescence signals, and to scan in a raster or bi-directional pattern across the specimen. The FV1000 scan head contains 3internal photomultiplier tubes (PMTs) for fluorescence detection, and oneexternal PMT to detect transmitted light. As a result, up to three fluorescentsignals and a transmitted light signal can be collected simultaneously. Thesignal for each channel passes though a single pinhole, which can be adjusted


12/41


2. POTENTIAL HAZARDS

The FV1000 is a class 3B laser system and uses a variety of high powerlasers which present laser radiation and heat hazards. The laser radiationfrom this instrument can cause serious eye damage if direct or reflectedlaser 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 theconnections or electrical cables, and contact the Instrumentation Technicianimmediately if any of the cables appear damaged.

Do not expose your hand or finger to the laser beam output from the objectivemount hole, objective tip or condenser lens, or your skin may be damaged.Never attempt to output the laser beam outside the systemby inserting amirror or a similar object in the light path. The laser beam may enter youreyes, and this is extremely hazardous.

Make sure that the slide is laying flat on the stage. If the specimen inclines, thelaser 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 isdamaged, 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


13/41


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 laserpath is enclosed within the live cell chamber, but never lookdirectly at the laser light; always look through the protectivepane above the microscope oculars.

See the WLU Laboratory Health and Safety Manual for additionalinformation on personal protective equipment:http://www.wlu.ca/documents/23120/Laboratory_Health_%26_Safety_Manual__Feb_2007_Final.pdf .

4.

ACCIDENT PROCEDURESAll incidents must be reported to the Instrumentation Technician and ifapplicable, a students supervisor. All accidents, incidents and near missesmust be reported to the Environmental/Occupational Health and Safety (EOHS)Office via the WLU Employee Accident/Incident/Occupational Disease Reportform (www.wlu.ca/eohs/forms). To meet regulatory requirements, these formsmust be submitted to EOHS within 24 hours of occurrence, with the exceptionof critical injuries, which must be reported immediately to the EOHS Office bytelephone. Critical injuries include any of the following; place life in jeopardy,produce unconsciousness, result in substantial loss of blood, involve fracture ofa leg or arm but not a finger or toe, involve amputation of a leg, arm, hand orf b fi i f b j i f h b d
http://www.wlu.ca/documents/23120/Laboratory_Health_%26_Safety_Manual__Feb_2007_Final.pdfhttp://www.wlu.ca/documents/23120/Laboratory_Health_%26_Safety_Manual__Feb_2007_Final.pdfhttp://www.wlu.ca/documents/23120/Laboratory_Health_%26_Safety_Manual__Feb_2007_Final.pdfhttp://www.wlu.ca/eohs/formshttp://www.wlu.ca/eohs/formshttp://www.wlu.ca/eohs/formshttp://www.wlu.ca/eohs/formshttp://www.wlu.ca/documents/23120/Laboratory_Health_%26_Safety_Manual__Feb_2007_Final.pdfhttp://www.wlu.ca/documents/23120/Laboratory_Health_%26_Safety_Manual__Feb_2007_Final.pdf


14/41


6. PROTOCOL

Anyone using the confocal microscope must receive hands on training. Thisdocument is a summary of the procedure and is only intended to help youremember the various steps.

6.1 Start-up

You only need to turn on the components that you plan to use. Turning lasersand the mercury lamp on and off and running them affects their lifespan, so ifyou are sureyou wont be using a given lamp or laser, dont turn it on. You

must turn on all the lasers you might potentially need at the start, becauseyou 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 alwaysbeturned on, regardless of the wavelengths you are interested in viewing. Ifyou just need the computer to look at data or transfer it to a CD/DVD, turnonly 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 temperaturecontroller, up to two hours before imaging. This will allow all of thecomponents to reach thermal equilibrium and reduces z-drift during imaging.

ALL mounting media or other substances used to secure coverslips (i.e. nailpolish) must be COMPLETELY DRYbefore the slide is viewed on the confocal.Mounting media may otherwise leak onto the objective and is very difficult toremove.

1. TURN ON THE FAN.2. Make sure the window shade is down all the way.


15/41


7. Double click on the Olympus FV10-ASW 1.7 software icon and log on usingyour username and password.

8. Wait for the software and microscope to initialize.

9. Click on Device, then Microscope Controller.

6.1.1Viewing sample with transmitted light and Kolher illumination

Before viewing specimen with transmitted light (brightfield) the microscopecondenser should be properly aligned to provide even and bright illumination(commonly called Kolher). If you do not need to view or collect brightfield ordifferential 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 manualslider, #4 in Figure 6-1)).

5. Align the DIC/Wollaston prism in the light path as well (below the stage) bysliding 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 willbe 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 foc s mechanism can be set to fine sing either the F/C


16/41


10.Focus the condensor knob(#5 in Fugre 6-1) so that the edges of thediaphragm AND the speciman appear very sharp (do not use the fine/coursefocus for the objectives).

11.If nessicary, center the condensor using the centering screws (it helps toopen the field diaphragm so that the edges of the diaphragm almost fill thefield of view, #3 in Figure 6-1).

12.When the condensor is focused and centered, open the field diaphragm sothat 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 section6.1.2 for epifluorescence imaging.

6.1.2

Epifluorescence Imaging

Examining your specimen using epifluorescence before collecting a confocalimage allows focusing and objective/magnification selection without thephotobleaching risks associated with high intensity laser light. Useepifluoresence to find the region of interest and focus at the desiredmagnification 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 AcquisitionSetting window (#8 in Figure 6-2).

4. Choose the appropriate filter cube (mirror) for your speciman in theMicroscope Controller window (DAPI, FITC, or TRITC) (#15 in Figure 6-2).

5. Open the shutter on the mercury lamp to illuminate your sample, and usethe jo stick and fine foc s to locate o r region of interest


17/41



18/41


Figure 6-2: Software windows and basic description of controls for the FV-ASW software


19/41


6.2 Preparation for Imaging

1. Turn off brightfield or epifluorescence illumination by clicking the relevantbutton 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 thefocus 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 Controlwindow (#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 selectedin addition to transmitted light.

b. If the dye you are using is not in the list, it can be added byclicking on Tools Maintenance User Settings. Click on theDye tab, and then click the add button on the far right side ofthe window. Type in the name of the dye and then enter theexcitation and emission wavelengths near the bottom left corner

of the screen. Select the laser that most closely corresponds tothe 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 collectiononly. If you plan to collect a DIC image as well, slide the Wollastin prismback 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 withthe 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


20/41


d. Size (#3, Figure 6-2): intially set at 512 x 512 pixels, and adjustedlater 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 forfluorescence according to the dye that you select. Do not adjustthe laser powers at this point.

g. Microscope (#6, Figure 6-2): make sure the correct objective isselected 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 sequentialscan minimizines the time difference

between images for each channel by scanning only a line ineach channel at a time. This is typically the best choice.

ii. Frame sequentialscan takes one full image on a givenchannel 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 quadrupolesthe scanning speed, and minimizes photobleaching. Rememberthat while the image is being scanned, the specimen is prone tophotobleachingavoid scanning for any longer than neccesary.

c. The focus between what is seen with epi (though the oculars) andwhat is seen on the screen is slightly different. You wil need tomove 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 berequired. If the image does not come in to focus easily, switchback to a lower magnification and/or switch back to epi-flourescence mode to focus again


21/41


decreased futher, or the laser power can be adjusted as alast 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 toodim, the gain can be increased. Increasing the offset willturn the background darker.

v. Check for bleedthrough by switching off (unchecking) allbut one laser then make sure that there is only signal in therelevant channel. Do this for each laser in turn. (If you arealready doing sequential scanning, this is unnessicary).

vi. Kalman integration can be used to collect several imagesand 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 modeprovides better sensitivity.

e. Press Ctrl+H to return to regular viewing mode.f. If desired, optimize the resolution (Nyquist) by pressing iin theImage Acquisition Control window. To achieve optimal resolutionthe pixel size should be aprox of the optical resolution. Adjustthe size of the image (number of pixels) and the zoom or changeobjectives to optimize resolution.

i. When setting the pixel size of your image, keep in mindyour final format; journal, poster, etc. An image that will

be blown up for a poster will need a high pixel count toavoid becoming pixelated. An image for a journal shouldhave the dots-per-inch required by that journal, which canbe determined based on image size and pixel number (i e


22/41


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 Acquisition1. 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 thebottom (or just past the bottom) of your sample. Press the Setbutton below Start to indicate the start position.

b. Use the up button (or fine focus knob) to move to the top of thesample, or to the last XY image that you want to take. Press theSet button below End.

c. Click the go button next to Center to go the middle of your Z-series. 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 toNyquist/2, which garuntees that you will oversample the Z-seriesand capture all possible detail. Keep in mind that more samplingleads to longer scanning and increased photobleaching. The S atthe top of the Acquisition Setting window will indicate the total

time to acquire the stack. If you require a very small step size youcan also increase the scan speed or decrease the depth of thestack to decrease scanning time.

5 St t th i th XYZ b tt


23/41


6. If you are satisfied with the image, proceed to section 6.6 for imageanalysis.

6.6

Image AnalysisWhen image acquisition is complete, the image will appear in the 2D Viewwindow. This window, along with the 2-D control panel, can be used tomanipulate the 2D image in a number of ways, and the various tools aredescribed in Table 6-1 and 6-2. The image can also be saved and viewed indifferent 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 atthe 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.


24/41


Table 6-1: 2D-View Window

Tool button Function

LUT (look up table)Use the LUT to change the gamma, intensity, contrast,and pseudocolour of any channel to enhance dim imagesor highlight certain features with a different colour.

Single/Panel/TileChange the view of the sample, or look at more than oneimage at once. Channels can be viewed separately oroverlaid using the Tile button.

Zoom, 1:1, Fit to

Window

These buttons adjust the size of the image on the screen.

SliderUse to scan through the frames collected for thisspeciman (primarily for Z- or T-series images).

Active OverlayAdd text to the image describing Z position, time, orwavelength.

Numbered buttonsdown the side of the

window (1, 2, etc)

Use to select the desired channels displayed in the image.

Pencil button

Allows access to region of interest (ROI) buttons and otherdrawing tools (i.e. text, scale bar, grid). After a ROI isselected, imaging can be conducted on that areaspecifically to avoid unnecessary bleaching of entirespeciman in light path.

Animation arrows Used to play an animation or scroll through Z- or T-series.

3-D button Allows viewing of a Z-series from any angle, selection ofcross sections, rotation along a given axis and creation ofanimation (using the More arrow).

Displays the intensity profile for a selected ROI This


25/41


Table 6-2: 2D Control Panel Window


Digital Zoom Adjust digital zoom.

ROI Format andManager buttons

Change the text and line formatting for labeling ROIs, andview information on ROIs selected.

Load Scan data

button

If accessible, the acquisition conditions for the image can

be read.Stepping box

Allows stepping through Z, time, or animation seriesframes.

Tile boxDetermines the organization of the tiles from a set ofimages.

Intensity profilesClick to display the vertical or horizontal intensitydistribution along a line.

Table 6-3: Main Fluoview Window


Properties ( i ) Display image information in Data Manager Window.

Report, Thumbnail,Thumbnail + property

Change how files are displayed in the Explorer window.

Various buttons to


26/41


Table 6-4: Processing Menu

Menu Item Function

Filter SettingandFilter

Mathematical filters can be used to improve image clarityor emphasize certain characteristics. The filters availableare: sharpen, average, DIC, sobel, median, shading,lapacian.Sharpen: highlights the edges of images, improves blurredimages, but also increases noise.Sobel: Emphasizes contours.Lapacian: emphasizes intensity changes.

Press Preview at bottom of Filter window first, thenselect various filters to observe result. Select the Singleor Series button, if doing a single image, enter the desiredframe in the Image Position box. If you want to save thefiltered image, press New Image.

Threshold

This feature is used to make the image binary (twocolour). The C and Z indicate the channel and frame to

display. The thresholds can be adjusted by clicking on thechannel in the table below threshold, and manuallyentering new values, or by moving the bars on the graph

Image Calculation Used to subtract/add/divide images or constants.

Correcting Pixel Gaps

This window can be used to correct for image shiftbetween channels. Image position can be used to selectthe desire frame. The white box in the Preview area canbe dragged to view other areas of the image.

Correcting Z GapsTo correct for Z position shift between channels. Movingthe yellow bar changes the Z-slice image.

The Ratio tab is used to look at changes in intensity


27/41


Additional Tools:

Virtual channel: Useful if fluorescent signals from two reagents overlap.

Delay image acquisition: After a set time has passed, acquisition can be startedor terminated using XYt imaging.

Image noise reduction: Can be done using Kalman filtering (takes several scansand averages them).

Live plot window: plots change in intensity in ROI over time. First outline ROI inLive View window by clicking on the pen tool. Then select Live Plot from Livemenu.

Live Tiling: allows viewing of the specimen as time elapses while the image isbeing acquired (button in Live View window has the image of a wrench).

6.7

Saving and Viewing Final Images1. 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 savedin D:\FV10-ASW\users\yourusername\Image).Make sure Include all ROI(or selected ROIs) are checked before you save the image so that scalebars and other additions to the image are included.

a.

You can also right click on the image to export as a TIFF, JPEG, orother image format.

b.

An OIF format saves the images as TIFFs in a folder, and creates afile with the sample information. Both the file and the TIFFs arerequired to open the image. If you save this data to a memoryk CD d t b th th if fil d th


28/41


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 toclean them. Inform the Instrumentation Technician as soon as possible and notethe problem in the log book.

1. Using clean lens paper gently blot off the oil from the lens. Do NOT drag thepaper across the lens, just dab off the oil. The front lens of the objective isvery 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 byturning 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 Cautionmessage.

5. If the argon laser (#3) it turned on, turn its key to the upright position,but DO NOT TURN OFF THE POWER SWITCH YET.This allows the fan tocontinue to blow until the laser has cooled down.


29/41


8. PREVENTATIVE MAINTENANCE

Users are not to perform maintenance. Unless noted otherwise, theseprocedures 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% CO2and humidity canister for the Weather Station chamber- Check the CO2for the anti-vibration table

8.2 Annually or as Required

8.2.1Replace the Mercury Burner

The mercury burner should be replaced when the lamp hours reach 350 hoursor when the epifluorescence images appear unstable due to lamp failure.


30/41


9. QUICK REFERENCE GUIDE

1. Remove the cover from the microscope.

2. Sign in in the user log book and fill in the relevant details.3. Turn on the microscope components according to the numbers on lables:4. Log on to the computer and the software (FV10-ASW 1.7)5. Wait for the software and microscope to initialize.6. Cafully place your slide on the stage.7. Set up Kohler.8. 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 desiredobjective, close the shutteron the mercury lamp to avoid bleaching yoursample.

9. Turn off brightfield or epifluorescence illumination. The microscope thenautomatically 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 initiallyc. Set the size to 512 x 512 pixelsd. 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 tominimize bleedthrough.

b. Click on XY repeat or Focus x2 or Focus x4 to initiate scanningU th fi f th d d b tt i th A i iti


31/41


REFERENCES

Laboratory Health and Safety Manual. 2007. Wilfrid Laurier UniversityEnvironmental/Occupational Health and Safety Office.

Olympus website: http://www.olympusfluoview.com.

Pawley, JB (Ed.). 2006. Handbook of Biological Confocal Microscopy, 3rdEdition. 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 resolutionoptical microscopy. Microsc Res Tech 70:1-9.

Texas A&M University. Microscopy and Imaging Center. Olympus FV1000.http://microscopy.tamu.edu/instruments/light-microscopy/olympus-fv1000-confocal-microscope.html. Accessed May 26, 2008.


32/41


APPENDIX 1: FLUOROCHROME PEAK EXCITATION AND EMISSIONWAVELENGTHS

The following list of fluorochromes and wavelengths is from the Olympuswebsite (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

Acridine Red 455-600 560-680

Acridine Yellow 470 550

Acriflavin 436 520

AFA (AcriflavinFeulgen SITSA)

355-425 460

Alizarin Complexon 530-560 580Alizarin 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 445Anthroyl Stearate 361-381 446

Astrazon BrilliantRed 4G

500 585


33/41


Bodipy Fl 503 512

Bodipy TMR 542 574

Bodipy TR 589 617

BOPRO 1 462 481

Brilliant Sulpho-flavin 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

5-(and 6-)carboxy SNARF-1indicator

548(low pH)576(high pH)


6-Carboxyrhodamine 6G 525 555

Cascade Blue 400 425Catecholamine 410 470

Chinacrine 450-490 515

CL-NERF504(low pH)514(high pH)


Coriphosphine O 460 575

Coumarin-Phalloidin 387 470

CY3.18 554 568

CY5.18 649 666

CY7 710 805

DANS (1-DimethylAmino-340 525


34/41


ELF-97 alcohol 345 530

Eosin 525 545

Erythrosin ITC 530 558

Ethidium Bromide 510 595

Euchrysin 430 540

FIF (FormaldehydeInduced Fluorescence)

405 435

Flazo Orange 375-530 612

Fluorescein 494 518

Fluorescein Iso-

thiocyanate (FITC)490 525

Fluo 3 485 503

FM1-43 479 598

Fura-2363(low [Ca +])335(high [Ca2+])

512(low [Ca +])505(high [Ca2+])

Fura Red472(low [Ca +])436(high [Ca2+])

657(low [Ca +])637(high [Ca2+])

Genacryl Brilliant

Red B 520 590Genacryl BrilliantYellow 10GF

430 485

Genacryl Pink 3G 470 583

Genacryl Yellow5GF

430 475

Gloxalic Acid 405 460

Granular Blue 355 425

Haematoporphyrin 530-560 580

Hoechst 33258, 33342(Bound to DNA)

352 461


35/41


LysoSensor BlueDND-192, DND-167

374 425

LysoSensor Green

DND-153, DND-189442 505

LysoSensor Yellow/Blue384(low pH)329(high pH)


LysoTracker Green 504 511

LysoTracker Yellow 534 551

LysoTracker Red 577 592

Magdala Red 524 600

Magnesium Green 506 531Magnesium Orange 550 575

Maxilon BrilliantFlavin 10 GFF

450 495

Maxilon BrilliantFlavin 8 GFF

460 495

Mitotracker Green FM 490 516

Mitotracker Orange

CMTMRos551 576

MPS (Methyl GreenPyronine Stilbene)

364 395

Mithramycin 450 570

NBD 465 535

NBD Amine 450 530

Nile Red 515-530 525-605

Nitrobenzoxadidole 460-470 510-650

Noradrenaline 340 490-520

Nuclear Fast Red 289-530 580

Nuclear Yellow 365 495


36/41


Propidium Iodide 536 617

Pyronine 410 540

Pyronine B 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

Rhodamine B 540 625

Rhodamine B 200 523-557 595

Rhodamine B Extra 550 605

Rhodamine BB 540 580

Rhodamine BG 540 572

Rhodamine Greenfluorophore

502 527

Rhodamine Red 570 590Rhodamine 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 590Sevron Orange 440 530

Sevron Yellow L 430 490


37/41


Isothiocyanate)

Texas Red 596 615

Thiazine Red R 510 580

Thioflavin S 430 550

Thioflavin TCN 350 460

Thioflavin 5 430 550

Thiolyte 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

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:


38/41


APPENDIX 2: USER LOG


39/41


DATE &TIMEIN

NAME &EXTENSION

SUPERVISOR SAMPLE ANDDYES USED

MERCURY LAMPHOURS AT

START

HE-NELASER

USED (Y/N)

OBJECTIVES USED(AIR:10,20,40,OIL:60OR 100)

MERCURYLAMP HOURS

AT END

TIME OUT PROBLEMS /COMMENTS


40/41


APPENDIX 3: PREVENTATIVE MAINTENANCE LOG


41/41


DATE NAME EXT # TYPE OF MAINTENANCE FREQUENCY OF MAINTENANCE(I.E.WEEKLY)

PROBLEMS /COMMENTS

Documents

Confocal Microscope