Confocal Microscope

Date of Issue: August 2009
Revision: 9
Date of Revision: Oct 10, 2012
Olympus FV1000 Standard Operating Procedure
ii
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
2.
POTENTIAL HAZARDS ................................................................. 7
3.
PERSONAL PROTECTIVE EQUIPMENT ............................................... 8
4.
ACCIDENT PROCEDURES ............................................................. 8
5.
WASTE DISPOSAL PROCEDURES ..................................................... 8
6.
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
7.
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
9.
iii
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
iv
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.
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.
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.
http://www.wlu.ca/documents/42977/Laser_Safety_Manual_2010.pdf
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,
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
(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
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.
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.
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.
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 students 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.
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 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, 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.
10
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.
11
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 dont 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.
Figure 6-1: Inverted microscope components (live cell chamber not shown)
12
Figure 6-2: Software windows and basic description of controls for the FV-ASW software
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.
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
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 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
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.
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.
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.
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.
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
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.
10
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 its 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.
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.
11
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.
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.
13
14
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

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

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

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
503
522
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

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

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/
APPENDIX 2: USER LOG
20
21
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
APPENDIX 3: PREVENTATIVE MAINTENANCE LOG
22
23
DATE
NAME
EXT #
TYPE OF MAINTENANCE
FREQUENCY OF MAINTENANCE
(I.E. WEEKLY)
PROBLEMS / COMMENTS

Confocal Microscope

Hochgeladen von

Dokumentinformationen

Copyright

Verfügbare Formate

Dieses Dokument teilen

Dokument teilen oder einbetten

Freigabeoptionen

Stufen Sie dieses Dokument als nützlich ein?

Sind diese Inhalte unangemessen?

Copyright:

Verfügbare Formate

Confocal Microscope

Hochgeladen von

Copyright:

Verfügbare Formate

Date of Issue: August 2009

Olympus FV1000 Standard Operating Procedure

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

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

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

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

PREVENTATIVE MAINTENANCE .................................................... 11

Olympus FV1000 Standard Operating Procedure

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

Olympus FV1000 Standard Operating Procedure

Olympus FV1000 Standard Operating Procedure

Olympus FV1000 Standard Operating Procedure

1.2 Emergency Contact Information

Dr. Diano Marrone

Olympus FV1000 Standard Operating Procedure

1.3 User Fees and Instrument Booking

Training Fee: $ 55/hour (2-3 hours)

Following training, users may book the confocal as follows:

1.4 Basic Theoretical Background

Olympus FV1000 Standard Operating Procedure

producing 3-D reconstructions to assess cell structure, studying the spatial

1.4.1 Sample Preparation Suggestions

Olympus FV1000 Standard Operating Procedure

Olympus FV1000 Standard Operating Procedure

1.5.2 Lasers and Filters

Olympus FV1000 Standard Operating Procedure

1.5.4 Scan Head

Olympus FV1000 Standard Operating Procedure

Olympus FV1000 Standard Operating Procedure

3. PERSONAL PROTECTIVE EQUIPMENT

5. WASTE DISPOSAL PROCEDURES

Olympus FV1000 Standard Operating Procedure

Olympus FV1000 Standard Operating Procedure

6.1.1 Viewing sample with transmitted light and Kolher illumination

Olympus FV1000 Standard Operating Procedure

6.1.2 Epifluorescence Imaging

Olympus FV1000 Standard Operating Procedure

Olympus FV1000 Standard Operating Procedure

Olympus FV1000 Standard Operating Procedure

6.2 Preparation for Imaging

Olympus FV1000 Standard Operating Procedure

Olympus FV1000 Standard Operating Procedure

decreased futher, or the laser power can be adjusted as a

6.3 XY Image Acquisition

Olympus FV1000 Standard Operating Procedure

of ways; see Section 6.6 for instructions on image analysis, manipulation,

6.4 Z-Series or 3-D Stack Image Acquisition

6.5 T-Series Image Acquisition

Olympus FV1000 Standard Operating Procedure

6.6 Image Analysis

6.6.1 Add a scale bar to your image:

Olympus FV1000 Standard Operating Procedure

Table 6-1: 2D-View Window

Intensity profile button

Olympus FV1000 Standard Operating Procedure

Table 6-2: 2D Control Panel Window

Table 6-3: Main Fluoview Window

Assists in organizing the screen-view when many windows

Dims the monitor display to minimize stray light.

Olympus FV1000 Standard Operating Procedure

Table 6-4: Processing Menu

Olympus FV1000 Standard Operating Procedure

6.7 Saving and Viewing Final Images

Note: DATA IS CLEARED FROM THE CONFOCAL COMPUTER PERIODICALLY, so