SHINE ATE Regional Center – NSF DUE# 1204279
Number: _____11__________________
WORK INSTRUCTION BREAKDOWN SHEET
Operation: Laser Scanning Confocal Microscope Operations
Instrument: Olympus Fluoview FV10i Laser Scanning Confocal Microscope
IMPORTANT STEPS
A logical segment of the
operation when
something happens to
advance the work.
Gather necessary
resources.
Gather necessary
resources.
KEY POINTS
1Anything
in a step that
might—
1. Make or break the
job
2. Injure the worker
3. Be a Cultural
Consideration
4. Make the work
easier to do, i.e.
“knack”, “trick”,
special timing, bit of
special information
REASONS WHY
Reasons for each key
point
Sample Stage Preparation
If using small cylindrical
sample holders, use the
setup to the right. Notice
the different types of
“sample locks” (which
hold the samples firmly in
place) and “sample sites”
(where the sample is held
on the stage during
imaging).
If using glass slides, use
the setup to the right.
Notice the “oil deposition
site” (where oil is
deposited before using
the 60x Objective) and
the “stage key” (used to
align to microscope when
inserting stage).
Adapted from Graupp, P. & Wrona, R. (2006) The TWI Workbook: Essential Skills for Supervisors. New York, NY. Productivity
Press.
SHINE ATE Regional Center – NSF DUE# 1204279
Place sample site on the
stage (small cylindrical
samples).
Once sample slide is in
place, secure it using
sample locks.
Place sample site on the
stage (glass slides).
For small cylindrical
sample holders, place the
sample onto the sample
site and lock it in with the
sample locks.
For class slides, place
sample into sample site
so that the area of
interest is located near
the “3” marker on the
stage.
This is good practice
because the machine will
start imaging at this
center point.
Ensure that the cover slip
side of the glass side is
facing down and away
from the Operator.
Once sample slide is in
place, secure it using
sample locks.
Open microscope hood
and insert stage.
Align the stage key hole
with the cylindrical piece
of metal on the
microscope that fits
inside it. Close the hood,
and now the sample is
ready to be imaged.
One aspect is essential;
the Operator must make
sure that the cover slip
has a height equal to or
less that a #1.5 cover slip,
otherwise the sample or
the instrument can be
damaged.
Using the “Hood Open”
button. Restart of the
application is required for
any sample holder
change.
They will line up and
snuggly fit together.
Adapted from Graupp, P. & Wrona, R. (2006) The TWI Workbook: Essential Skills for Supervisors. New York, NY. Productivity
Press.
SHINE ATE Regional Center – NSF DUE# 1204279
Dye Selection
Select “Set from Dye Data
Base” tab, near the top
left of the yellow
highlighted window
above.
Identify the type of dye of
which the sample is
strained.
Select a dye name on the
list and then click
“Apply”.
If no dye is given, select a
wavelength of light that is
known to excited
fluorophores in that
sample. If that
information is unknown,
then the Operator must
first test the four basic
excitation wavelengths
provided (Blue, Green,
Red and FarRed).
No more than four and at
least two wavelengths
must be selected before
imaging can begin.
The dye name will now
be registered on the
“Selected Dyes” list
(denoted by the green
highlighted window in
the image above).
Adapted from Graupp, P. & Wrona, R. (2006) The TWI Workbook: Essential Skills for Supervisors. New York, NY. Productivity
Press.
SHINE ATE Regional Center – NSF DUE# 1204279
Click “Start” near the
bottom center of the
screen (denoted by the
red highlighted window
in the image above).
Acquisition of Map Image (refer to “Map Image” tab)
When three or more dyes
have been selected then
the Operator must choose
a combination of two for
the initial image
mapping.
This is done by clicking
on the desired
combination and then
clicking on “OK” at the
bottom of the window.
Select container of
interest.
For glass slides, the
number on the stage of
which the glass slide has
been placed corresponds
to the container number.
Whichever container,
indicated by the light grey
box in the image to the
left, is selected will be the
container that is mapped.
Other parts of the glass
slide can be looked at by
switching containers.
Click “Register” to mark
down all the containers to
be mapped; use the
“Delete” button to remove
registered containers.
Start map acquisition:
Adapted from Graupp, P. & Wrona, R. (2006) The TWI Workbook: Essential Skills for Supervisors. New York, NY. Productivity
Press.
SHINE ATE Regional Center – NSF DUE# 1204279
Using the window to the
right, adjust the
brightness of the map
image.
Under the “Scanning
Order” section, select to
either image the current
container, containers that
have already been
registered, or manually
select the sections of the
container to be manually
imaged.
If the Operator chooses to
manually select sections,
simply double click on the
acquisition screen inside
one of the large squares
and the machine will
raster that site.
The Operator is now
ready to acquire the map
image. Click “Start”
locate at the bottle left of
the window to the right.
During map acquisition:
Once the Operator has
selected “Start”, the
progress bar can be
observed at the bottom of
the screen, which
indicates the machine is
conducting autofocus and
auto brightness.
At any time, the Operator
can click “Stop”.
Once the progress bar
disappears, the map
image acquisition will
begin. It is at this point
that the Operator can
manually select the boxes
for the machine to raster
if “Manual” was selected.
This can be used if the
Operator finds the
desired feature and
wants to move on, or see
if mistakes have been
made and wants to move
back. “Stop” can be an
excellent time saver,
allowing the Operator to
move forward or
backward in the imaging
process.
Adapted from Graupp, P. & Wrona, R. (2006) The TWI Workbook: Essential Skills for Supervisors. New York, NY. Productivity
Press.
SHINE ATE Regional Center – NSF DUE# 1204279
Before moving on to
using the Observation
Window, it should be
noted:
Look to the window at
the right indicated by the
green square. In this area
the Operator can select
the cannels that the
image is being displayed
on. By turning these
channels on and off, very
different images and
features can be observed.
When the Operator is
ready to move on, click
“NEXT”.
The image directly above
is the image map created
during the previous
steps. In this window the
Operator can navigate
Try turning off the phase
contrast first. If most of
the image is in phase
contrast, a new sett of
dyes needs to be
selected that will cause
the sample to fluoresce.
DO NOT CLICK “Start” or
the acquisition process
start all over.
Observation Window Layout
This is done by double
clicking anywhere on the
image to focus the lasers
at that point.
Adapted from Graupp, P. & Wrona, R. (2006) The TWI Workbook: Essential Skills for Supervisors. New York, NY. Productivity
Press.
SHINE ATE Regional Center – NSF DUE# 1204279
around the sample to find Use the channel buttons
points of interest.
above the image to adjust
which wavelength to be
observed.
Use the “Zoom” bar on
This avoids wasting time
the side to identify
using the laser to look at
important features first. the image; it is a slow
process and it can
damage the sample.
Using the “Repeat”
button.
Select the channels that
the image is being
displayed on by using the
feature illustrated at the
right.
Use the container
selection tool, located at
the very top of the
window, to change the
container.
At 1X the machine rasters
at a standard speed and
yields much clearer
images.
This changes the area of
the slide to be observed.
At 4X the image is
generated much quicker,
but the resolution is poor.
Raster speed does not
affect the final image;
however, it does
influence the rate of
photobleaching of the
sample. A quicker
rastering speed means
more passes by the laser
over the area of interest.
More passes means more
excitation which
inevitably leads to
photobleaching, resulting
in an unimagable section
of the sample.
The Repeat button
determines the rate at
which the microscope
rasters an image.
By turning these channels on and off, very different
images and features can be observed. When multiple
dyes were selected, the Operator can only have two
activated at one time during the image observation.
As the operator switches between lasers, an image is
Adapted from Graupp, P. & Wrona, R. (2006) The TWI Workbook: Essential Skills for Supervisors. New York, NY. Productivity
Press.
SHINE ATE Regional Center – NSF DUE# 1204279
As the Operator moves
around the map image,
the image will change
(ensure that “Repeat” is
ON).
This section of the screen
affects the depth of focus
and the magnification
being used:
generated that combines all the different channels
into a single image.
Each square represents a The bottom right square
different wavelength
combines all other
being recorded.
images into one.
By adding and removing
channels, the individual
squares that are
associated with those
channels will alter the
combined image.
This will not affect the
final image capture.
By clicking and dragging
This allows for precise
within these squares the
feature alignment.
Operator can move the
focus of the laser.
By clicking on the “10X”
button, the Operator will
activate the 60X
magnification. If “60X”
magnification is activated,
click on the button to
change it back to 10X
magnification. Look to
the “60X Lens Oil
Deposition” section
before changing
magnification.
By clicking “Drop Oil” the
Operator prepares the
60X lens for oil
deposition.
By sliding the bar, located
at the very bottom, the
Operator will either zoom
in or out of the lasers
point of focus.
Adapted from Graupp, P. & Wrona, R. (2006) The TWI Workbook: Essential Skills for Supervisors. New York, NY. Productivity
Press.
SHINE ATE Regional Center – NSF DUE# 1204279
Use the “Depth” Tab.
Use the “Other
Parameters” Tab.
“AF” arrow indicates the
point of autofocus. If the
Operator slides this
arrow up and down, the
depth of focus changes
and the top and bottom of
the feature is visible.
“End” and “Start” bars are
used to take stacks of
images. Stacks can be
used to generate 3D
images.
Use “Image Size” menu to
choose the desired size
for saved images.
“Speed & Quality” is
responsible for image
quality.
Use the “Sensitivity” Tab.
Use the “Laser” Tab.
“Confocal Aperture”
affects the size of the
aperture. Smaller
aperture means less light
is being capture. This is
typically observed at
“x1.0” setting.
This tab is used to change
the sensitivity of the
machine to specific lasers
being emitted onto the
sample.
This tab is used to change
the intensity of specific
lasers being emitted onto
the sample.
Higher resolution means
a much larger file size
and a much longer image
acquisition period.
This is typically seen in
“High Quality”.
A higher sensitivity
means the machine will
detect more photons at
that wavelength. This
feature can be useful
when isolating areas of
interest on the sample.
A higher intensity means
more photons of that
wavelength are being
emitted.
Adapted from Graupp, P. & Wrona, R. (2006) The TWI Workbook: Essential Skills for Supervisors. New York, NY. Productivity
Press.
SHINE ATE Regional Center – NSF DUE# 1204279
Be careful not to have
these too high.
High energy lasers can
cause easy
photobleaching effects
and other sample
damage.
60x Lens Selection and Oil Deposition
Look to Area 5 on the
“Observation Window
Layout”.
Click on the “10X” button.
A window will pop up
saying, “Please replenish
objective lens with oil.”
Open the microscope
hood.
Look to the sample stage
and find the oil
deposition site.
Adapted from Graupp, P. & Wrona, R. (2006) The TWI Workbook: Essential Skills for Supervisors. New York, NY. Productivity
Press.
SHINE ATE Regional Center – NSF DUE# 1204279
Obtain the vial of
Olympus brand oil and
apply the oil.
Push the fluid out until
the bubbles in the shaft
are gone and a small drop
is at the tip. Wipe the
drop off with tissue as
shown below:
Push another drop out
onto the tip and gently
place it on the oil
deposition site.
Do NOT touch the lens
with the plastic vial.
Oil on the lens will
damage or even ruin the
lens.
Close the hood, and press
OK; the Operator is now
ready to image at 60X
magnification.
Basic 2D Image Observation and Acquisition
(refer to Image Acquisition Window Layout)
In Area 1, use the “Map”
image to locate an are on
the sample to be imaged.
Use the “Repeat” button
The image will appear in
in Area 2 to start
Area 4.
generating an image.
Use the channel buttons
in Area 3 to isolate
specific wavelengths that
are of interest.
Adapted from Graupp, P. & Wrona, R. (2006) The TWI Workbook: Essential Skills for Supervisors. New York, NY. Productivity
Press.
SHINE ATE Regional Center – NSF DUE# 1204279
Area 4 will contain the
images from all channels.
Click and drag the mouse
around this area to move
around the sample.
Use the focus bar in Area
5 to zoom up on the
feature of interest.
In Area 6, select the
“Other Parameter” tab
and change the image
size to “1024x1024.”
Change “Speed & Quality”
to “High Quality.” Change
the “Confocal Aperture”
to “x1.0.”
In Area 7, adjust the
sensitivity and the laser
intensity.
Use the “10x” button to
obtain a deeper zoom.
Reference “60x Lens
Selection and Oil
Deposition” section
before changing the
magnification.
Use the depth tab if you
would like to change the
depth of the focus.
There is no formula for
Selecting 1 will give the
this. Uses simple rail and Operator a good idea of
error to find the best
what the final saved
possible image quality.
image will look like.
Before selecting “Save”,
go back to Area 2 and
turn the repeat to “1x.”
Area 8 is relatively
“Series Start” is only used
straightforward. There
during 3D image
are two lines. The first
acquisition. “One Shot” is
asks the Operator to
used when only taking a
select a save location.
single image. When ready,
The second line asks for
select “One Shot” and
the file name.
move away from the
computer to avoid
vibrations. It may take a
few minutes depending
on some of the settings.
Basic 3D Image Observation and Acquisition
(refer to Image Acquisition Window Layout)
In Area 1, use the “Map”
image to locate an are on
the sample to be imaged.
Adapted from Graupp, P. & Wrona, R. (2006) The TWI Workbook: Essential Skills for Supervisors. New York, NY. Productivity
Press.
SHINE ATE Regional Center – NSF DUE# 1204279
Use the “Repeat” button
in Area 2 to start
generating an image.
Use the channel buttons
in Area 3 to isolate
specific wavelengths that
are of interest.
Area 4 will contain the
images from all channels.
Click and drag the mouse
around this area to move
around the sample.
Use the focus bar in Area
5 to zoom up on the
feature of interest.
The image will appear in
Area 4.
Use the “10x” button to
obtain a deeper zoom.
Reference “60x Lens
Selection and Oil
Deposition” section
before changing the
magnification.
In Area 6, select the
“Other Parameter” tab
and change the image
size to “1024x1024.”
Change “Speed & Quality”
to “High Quality.” Change
the “Confocal Aperture”
to “x1.0.”
Adjust the “End” and
These are the z-stack
“Start” bars located under markers. For a complete
the “Depth tab in Area 6. 3D image, move these
markers to the highest
and lowest points of the
observed feature. Once
the Operator finds a place
where the feature is
barely visible, one of the
extremes has been
reached; click the “Set”
button to anchor that
marker in place.
Adjust the “Step” section.
More steps mean a more
complete image, but a
longer imaging period.
Adapted from Graupp, P. & Wrona, R. (2006) The TWI Workbook: Essential Skills for Supervisors. New York, NY. Productivity
Press.
SHINE ATE Regional Center – NSF DUE# 1204279
Fewer steps is quicker,
but will only image a
limited number of slices
between the two
specified heights and
thus the resolution will
not be as good.
Look to the “Time &
Number” section located
below Area 7. Adjust the
max/mn points and
change the number of
steps observed in the
timeframe change
feature.
In Area 7, adjust the
sensitivity and the laser
intensity.
Area 8 is relatively
straightforward. There
are two lines. The first
asks the Operator to
select a save location.
The second line asks for
the file name.
Often the image quality is
limited to the amount of
time available to take
images.
There is no formula for
this. Uses simple rail and
error to find the best
possible image quality.
Before selecting “Save”,
go back to Area 2 and
turn the repeat to “1x.”
“Series Start” is only used
during 3D image
acquisition. When ready
to image, click “Series
Start” and move away
from the computer to
avoid vibrations. The
machine will begin taking
images between two
specified points. The
Operator will know how
long it takes by looking at
the “Time & Number”
section.
Image Editing
Adapted from Graupp, P. & Wrona, R. (2006) The TWI Workbook: Essential Skills for Supervisors. New York, NY. Productivity
Press.
SHINE ATE Regional Center – NSF DUE# 1204279
Open Editing Program
FV10-ASW 3.1
Sign in to program:
Navigate the “Explore”
window and locate a
desired image. Double
click on it to open it.
User ID: Administrator
Password: Administrator
The below window will be visible:
A window like this
appears:
Adapted from Graupp, P. & Wrona, R. (2006) The TWI Workbook: Essential Skills for Supervisors. New York, NY. Productivity
Press.
SHINE ATE Regional Center – NSF DUE# 1204279
The next four steps use
the toolbar to the right:
The orange highlighted
area (a) is the channel
select bar. Turning these
channels on and off has
the same effect as it does
when acquisitioning an
image. Only the
wavelengths selected will
be displayed.
The red highlighted
arrow button (b) opens
the 2D control panel.
Features in this window
have not been thoroughly
explored, so as far as
image editing goes, it will
entail a lot of trial and
error.
The green highlighted c)
rainbow button with the
scale on it opens the LUT
window. In this window,
the Operator has the
freedom to color your
image in any way. Each
channel has its own set of
RGB settings, and each of
those has their own subsettings. Image editing is
Adapted from Graupp, P. & Wrona, R. (2006) The TWI Workbook: Essential Skills for Supervisors. New York, NY. Productivity
Press.
SHINE ATE Regional Center – NSF DUE# 1204279
preferential, and this
window allows the
Operator to cater images
with unique style. There
eis no defined method for
maximizing image
quality, so trial and error
will be the primary tool
to maximize quality.
The pink highlighted
button (d) with the pencil
on it is the basic shapes
tool bar. Here the
Operator can draw a
number of different
shapes. The primary
concern in this toolbar is
the “Scale” feature and
the “Text” feature.
Save the image.
Click on the giant “A” near
the top of the toolbar. It
will ask to specify the
location of the text. Click
on the screen to write at
that location.
Click on the ruler at the
bottom of the toolbar.
Once selected, click and
drag across the area
where the scale bar is to
go.
Right click on the screen
to pull up the available
saving options.
Clicking “Save” and Save
As” will save only the
image. Anything added to
it will NOT BE SAVED.
Clicking on either of the
“Export” buttons will
allow the operator to save
Adapted from Graupp, P. & Wrona, R. (2006) The TWI Workbook: Essential Skills for Supervisors. New York, NY. Productivity
Press.
SHINE ATE Regional Center – NSF DUE# 1204279
the image AS IT HAS
BEEN EDITED in various
formats.
Image editing in 3D (All
the previous editing
techniques can also be
applied to 3D images).
The box to the left opens
“3D Animation.”
The box to the right
combines all the images
into a 3D rendering for
further editing. After
clicking the right box, the
windo will look like the
one to the right.In this
view, the Operator can
click and drag around the
image to rotate it 360
degrees in any direction.
Right click on the image
to open more settings.
Observe the scale bar.
Clicking “Save Display”
will capture whatever is
on the image screen at
that time.
When the Operator opens
a 3D image, a slightly
different window
appears. The two boxes
in the upper right corner
will be highlighted and
explained in the following
two steps.
This takes the entire
stack of images and
creates a slideshow that
makes them appear as on
rotating image.
In this view the Operator
also has access to the 3D
control panel and the 3D
image settings and can
access these panels the
same way as in the 2D
view.
In the “3D Control Panel”
switch between the two
rendering modes; both
will give a unique image.
In the “3D Image Settings”
window, go to the
“Image” tab and adjust
the resolution with the
slide bar; this usually
helps increase the image
quality.
Write down the
dimensions of the feature.
When converting back to
a 2D image the scale does
Adapted from Graupp, P. & Wrona, R. (2006) The TWI Workbook: Essential Skills for Supervisors. New York, NY. Productivity
Press.
SHINE ATE Regional Center – NSF DUE# 1204279
not carry over, so the
Operator must manually
create a scale bar.
Once the Operator has
oriented and edited the
image, go to the top left of
the 3D window.
A button that shows a box
turning into a square is
the 3D to 2D button.
Once an image is
converted to 2D, it
behaves the same was as
all other 2D images, with
one small difference. If
the Operator tries to add
a scale bar now, it will
only give the dimension
in pixels.
There is a pixel to
distance conversion, but
all aspects of the image
quality must be known.
Therefore, the scale bar
must be manually created
using the scale bar
function and the text
function.
Adapted from Graupp, P. & Wrona, R. (2006) The TWI Workbook: Essential Skills for Supervisors. New York, NY. Productivity
Press.