CMI STANDARD OPERATING
PROCEDURE
Optigrid Structured Light Microscope
Room
CMI documentID:SOP002
CONTACT INFORMATION:
Peter Owens: 091 494036 (office) [email protected]
Kerry Thompson: 091 495704(office)
[email protected]
REQUIREMENTS FOR EQUIPMENT USAGE:
1. CMI user
2. Completion and signing of Microscope Safety Checklist
3. Certification by Peter Owens or Kerry Thompson
REVISION LOG
Revision
Date
Author
Changes
1.0
5/3/13
P.Owens
Initial draft
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1. Purpose
1.1.
This document specifies the work instructions for the CMI Optigrid structured light microscope located in
room 102 of Block F in Anatomy. If you see an area where more clarification is needed, if additional
information is needed, or if you have suggestions on how to make this guide more useful in the lab, please
contact the CMI.
1.2.
Note that this document is not a detailed instrument manual and does not intend to be one. For
detailed questions, please refer to the manuals present in the lab, or ask CMI personnel for help.
2.
Scope
2.1.
These work instructions are applicable to all work that is carried out using the Optigrid Structured Light
Microscope.
3. Applicable Documents
PLEASE LEAVE ALL HARDCOPIES IN THE
LAB
3.1.
CMI Safety Manual
3.2.
MSDS Sheets
3.2.1.
3.2.2.
Immersion Oil
Isopropanol
3.3.
Olympus IX81 manual (hardcopy in the lab, soft copy on the instrument PC)
3.4.
Equipment Folder (hardcopy in the lab, soft copy on the instrument PC)
3.5
The CMI access policy , available online at
http://imaging.nuigalway.ie/access%20policy/cmi_access_policy_1.5.pdf
3.6.
Before starting, please read the following carefully:
3.6.1.
3.6.2.
This manual was developed to assist in the training process of users. Be aware that only the basic
operation details will be presented. Please contact the CMI staff for more assistance if required.
Changes may occur when a new software version or patch is installed. Please contact the CMI
staff if you are not sure about new features and functions.
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4. Structured Light (‘Optigrid’) Microscopy Primer
The OptiGrid is a device that when coupled to a standard research fluorescence microscope, will allow you to
acquire confocal quality images without using laser scanning technology.
To do this, two additional pieces of hardware are necessary:
The optigrid paddle, which is inserted into the microscope’s fieldstop location and is connected to the optigrid
controller box
The optigrid imaging system is based on a technique called “Structured Light” (Neil et al 97).
This technique modifies the illumination system of a microscope to project a grid pattern onto the specimen. The
grid is moved into three different positions, and an image is acquired at each position. An algorithm is then used to
produce a single, derivative, confocal image from which the grid pattern has been removed. The algorithm takes
advantage of the fact that in-focus information changes as the grid moves, whilst out-of-focus information remains
constant. Therefore, if information does not change between images, it is out of focus and can be removed from the
final image.
Structured light illumination systems, project a sinusoidal pattern onto specimens by placing a one-dimensional
grating in the microscope’s illumination path. The grid is moved in the Y plane into three different positions and an
image is acquired at each position. The following algorithm is then used to produce a single, derivative, confocal
image from
which the grid pattern has been removed.
Confocal Image= �((𝐼1 − 𝐼2 ) + (𝐼1 − 𝐼3 ) + (𝐼2 − 𝐼3 ))
where
I1 = Image with the grid in position 1
I2 = Image with the grid in position 2
I3 = Image with the grid in position 3
The optigrid paddle consists of a one-dimensional optical grid mounted on a piezo-electronically driven actuator.
Once inserted into the light path of the illuminator, amplified voltage is applied to the piezo crystal to change its
length. The piezo crystal provides accurate repositioning of the grid pattern.
The grid pattern is systematically projected onto the specimen and is moved perpendicularly to the grid lines across
the sample. One structured-light image actually consists of three split-second captures of the grid. The first image is
taken at any position of the grid, the grid is then moved linearly by 1/3 of the grid period length to capture the
second image, and another 1/3 to capture the third image. All this typically occurs in under one second, generating
one structured-light image or optical section.
Optically speaking, the grid returns a strong signal wherever focus is sharp and a weak signal where focus is soft.
The optigrid algorithm then eliminates the weaker signals from above or below the primary image plane as defined
by the grid. The resulting image is free of any stray light or soft focus data, and can be viewed live on your computer
monitor at near real time.
The two-point discrimination of the OptiGrid is the same as the resolving power of your selected microscope
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objective. Objective lens with a high degree of spherical aberration correction and UV-transmission capability are
recommended to use with the grid.(i.e., the OptiGrid resolution using a 60X, 1.4NA oil objective would be 0.19μm
with 550nm wavelength). Because OptiGrid utilizes the illumination source of the host wide-field microscope, all
fluorescence capabilities of that microscope are maintained. When using fluorescence with the OptiGrid, the
excitation and emission filters and dichloric mirrors should be optimized to the specific fluorophore being used.
1 The grid is projected onto the specimen and moved
in 1/3 steps of the grid period length. Elapsed image
capture time = <1 second.
2
Algorithm extrapolates only strong image signals
from grid.
3
Three grid movements yield one optical section
containing only image data that is exactly within
the focal plane.
4
Collapse incrementally imaged optical section
stacks.
5
The resulting structured-light composite image
features haze-free, sharp focus. Also 3-D
reconstructions using Volocity.
Figure 1: Theory of the optigrid image production (Medusa form of Obelia jellyfish (200μm section)
fluorescence. 10X/0.40NA objective.)
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5. Responsibilities
5.1.
Operators of this equipment are responsible for the following:
5.1.1
5.1.2
5.1.3
5.1.4
5.1.5
5.1.6
Complying with all safety regulations.
Compliance with procedures and specifications contained in this document.
Reporting misuse of the instruments, or in a manner inconsistent with this specification, by
any personnel, to the supervising CMI staff.
Maintaining a clean workspace. Food and drink are NOT allowed!
Last-minute bioprep work should be done in the cell culture room XXX (observe sign-up
rules for that room) and not in the optigrid room.
Reporting any and all maintenance issues/concerns to the supervising CMI staff member
immediately.
6. System Overview
6.1 The Optigrid structured light microscope consists of the following devices:
6.1.1
6.1.2
6.1.3
6.1.4
6.1.5
6.1.6
6.1.7
6.1.8
Instrument Control PC, 64 bit running Volocity software (version 6.2)
Olympus power unit IX2-UCB.
Epifluorescence illumination lamp (Mercury Halide lamp XCite 120)
Olympus IX81 Inverted Microscope with 0.55 NA condenser
Hammamtsu Orca ER camera and controller
Optigrid controller and paddle containing grid.
Improvision Acquisition Hub.
Several Olympus microscope objectives are available:
1.
UPlanSAPO 10x/0.4 N.A.
2.
UPlanSAPO 20x/0.75 N.A.
3.
UPlanSAPO 40x/0.90 N.A.
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Control boxes
Transmitted light
Camera
Light source
Inverted Microscope
IX81
Fluorescence
turret Control Box
Figure 2 Optigrid System
6.2. Familiarise yourself with the system folder, located on the shelf above the equipment.
6.3. This microscope can be used in both ordinary widefield mode , obtaining fluorescence images without the use
of the optigrid system; and in optigrid confocal mode.
For grid confocal mode the grid paddle must be placed in the light path , otherwise it should NOT be placed in the
light path.
6.3 A metal halide light source directs white light to the microscope via a liquid light guide. This light is directed
via the grid (if used) into the back of the microscope, up through the filter wheel and into the objective and sample is
illuminated. The emission returns through the objective and is directed to the camera on the left hand port of the
microscope.
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7.0 Start Up Procedure
7.1
7.2
Book equipment on CMI web booking system.
Turn on metal halide light source.
Intensity wheel
Figure 3: Light source
Allow for 5 minutes to stabilize. Do not turn on/off unless finishing up for the day. Each time you turn
off/on the light source you reduce the lifespan of the lamp by 1 hour.
Make sure the lamp intensity wheel is set to the lowest setting above 0.
7.3. Turn on the control boxes
Turn on camera control
Turn on microscope control
If using the grid feature:
7.4. Turn on the grid controls 1 & 2.
Otherwise there is no need to turn on these components.
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Grid control 1
Microscope control
Camera control
Grid control 2
Figure 4: Control boxes
7.5.
Turn on PC, Login as CMI user (no password) and start Volocity software.
This brings up the following login window:
Password for username ‘optigridanatomyuser’ is CMI10.
7.6.
There are a number of configuration options when starting volocity
depending on what you want to do.
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These are vis_aquire: visualization and acquisition only, allows you to acquire and do image processing.
Vis_rest: visualization and restoration only, allows yout to view , process images and do deconvolution.
Vis_quant_rest: visualization , quantitation and restoration, includes quantition optin for counting, measuring etc.
It is expected that users will choose the vis_aquire option first , record images and then login again under a
configuration in order to free up the acquisition license. There are only 2 acquisition licenses shared between 2 sites
so the usage of this needs to be monitored carefully. If these two licenses are used up then acquisition will NOT be
possible on this machine.
7.7. Press connect and you should see the following splash window :
If you get an error , check the configuration option that you have chosen and try again. If error still persists contact a
CMI staff member.
7.8. When Volocity has started Select either “Create a new library” or “Open an existing library”. The following
screen will appear:
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Create a new library if needed in the appropriate area on the PC.
Library name mvd2 file extension
Select ‘video preview’
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The following screen appears:
7.9.
Load your sample on the microscope. CAUTION: Avoid any hard contact of the objective lens with
your sample at any time.
7.10.
Check that the light path selector button on the front of the microscope body is in the eye position.
Check that the filter wheel below the nose piece is in position 1, 2 or 3 (for DAPI, FITC , TRITC epi
fluorescence observation. Read the number from the filter turret.
7.11.
Check that manual shutter on right side of the filter turret is open. Make sure the light source
intensity
is at the lowest setting to start off with.
7.12.
Start with a low power objective and find the region of interest on your sample. The manual XY
stage
control is to the right of the microscope body.
7.13.
Use the focus knob which is also on the right side of the microscope body to bring your sample into
focus being careful not to crash the objective into the sample.
7.14.
Rotate the objective turret to switch to a higher power objective if desired. If applicable, put ONE
SMALL drop of immersion medium on the microscope objective lens. Remember: Water ONLY on
water immersion lenses, oil ONLY on oil immersion lenses. Immersion oil is provided close to the
microscope.
7.15.
When the image is in focus, and correct location is observed, push the light path selector button to
the
camera mode.
CAUTION: Avoid wrong immersion contact (oil on water lens, water on oil lens). Do NOT REMOVE objective
lenses from the microscope unless approved by CMI personnel. If you accidently place water on an oil objective
lens or vice versa , wipe it off with lens tissue / isopropanol (no kimwipes, THEY ARE TOO HARSH ON THE
GLASS!) and notify CMI staff.
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7.16. You should now see an image in the camera window, but the channels will have to be optimized.
8.0 Optimising the Image on screen through Volocity control:
1. First you must select the
light path you are using in
theVolocity software:
3. Double click to get to acquisition
window.
DAPI
FITC
TRITC
2. Set the gain for the camera.
This alters the sensitivity of the
camera. Hold the cursor directly
over the slider to display its
current value. A gain setting of
about 100 is generally fine –
4. Set the exposure time.
This determines the length of time
over which the camera will acquire an
image. To adjust the time click in one
of the number fields and edit the
number –you must press return for it
to take effect. You can also drag the
exposure slider to make quick
changes – the time will automatically
update.
If you click the Auto exposure button,
AE, exposure time will be
automatically optimised.
5. Adjust top and bottom settings to
create z series through sample if
required.
5. Set binning if required.
Increasing binning increases signal,
however it decreases resolution.
1x binning -image size 1344x1024
pixels
2x binning – image size 672x512
pixels
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8.1
Double click the icon in section 8 to open the acquisition window:
8.2.
Assign channels as appropriate. Order of operation is best as ‘Channels first then z’.
8.3.
Grid selection: Note you must get proper training in order to use the grid correctly.
If using grid confocal mode, ensure grid paddle is in the light path.
Click on the grid ‘ON’ button and the Improvision Acquisition Hub part of the side bar becomes
expanded as above.
Grid controls :
Off : grid movement stopped
On : grid movement on
Widefield grid : grid on but shows
widefield signal.
Grid gain, usually set to 40%
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9.
9.1.
9.2.
9.3
9.4.
9.5.
9.6.
Shut Down Procedure
Discard cover slips and glass sides in the red sharps container. This includes BioHazard material.
CLEAN UP the workplace, and leave it better than you found it.
Ensure you have saved all your images and shut down volocity.
Turn off the light source and all the instrument control boxes (no particular order).
Shut down the PC.
Sign out on the log-book.
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10. APPENDIX A - CERTIFICATION: DEMONSTRATION OF SKILLS
Name:
Operation:
Date:
Tested by:
DEMONSTRATION of SKILLS
Demonstrate system/ software start up and configuration
Pass Fail
Demonstrate loading a sample on the tool
Demonstrate finding the sample, optimizing channels and taking images
Demonstrate changing the lens during the measurement session
Demonstrate finishing and cleaning up
SAFETY
Where are cover slips, glass slides discarded?
Where are biological samples discarded?
Which chemicals used require careful handling?
VALIDATION
Certification:
Pass:
Trainee
Fail:
Certified by:
Date:
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