Okolab H301-OLYMPUS-IX3-SVR User manual

H301-OLYMPUS-IX3-SVR User Manual
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H301-OLYMPUS-IX3-SVR
Compatible with the following XY stage
Compatible with the following Okolab Controllers
•Olympus IX3-SVR
•H301-T-BL-PLUS
•UNO-T
•UNO-T-H-PREMIXED
•UNO-T-H-CO2

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Index
1. Components and dimensions................................................................................................... 3
2. Available Sample Holders....................................................................................................... 3
3. Available Lids ..................................................................................................................... 5
3.1 Sliding lid .......................................................................................................................... 5
3.2 Koehler Lid........................................................................................................................ 6
3.3 Injection Lid ...................................................................................................................... 6
3.4 Laser Interlock Lid............................................................................................................... 7
4. Insertion of the Sample Feedback Temperature Sensor.................................................................. 7
5. Insertion of Sample Holder into Chamber ................................................................................... 8
6. Working with 35 or 60 mm Petri Dish –Spacing Rings and Magnetic Locks ........................................... 9
7. Working with 1x3’’ and 1x2’’ chamber slides - magnetic locks........................................................11
8. Working with MW Plates - Magnetic locks and Chamber riser ..........................................................12
9. Connection of the Gas Supply ................................................................................................12
10. Working with Perfusion ........................................................................................................13
10.1 MICROFLUIDIC-C-RISER (Optional)............................................................................................15
11. Connecting the Chamber to the adapter ...................................................................................16
12. Connection of the Chamber with XY stage .................................................................................17
13. Cleaning ..........................................................................................................................18

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1. Components and dimensions
H301-OLYMPUS-IX3-SVR includes the following components:
•Chamber main body with embedded temperature sensor. It connects on the stage with screws.
•Sliding heated glass lid with Indium Tin Oxide (ITO) conductive coating and embedded temperature sensor.
•Chamber riser. It is a removable frame increasing the height of the chamber from 27 to 33 mm, often re-
quired when using multi-well (MW) plates.
Distance between focal plane
and upper side of the glass
lid without the chamber riser
Distance between focal plane
and upper side of the glass
lid with the chamber riser
Chamber top view
Sliding Lid
Chamber Riser
Sample Holder-Order Separately
Chamber Base
Adapter
Figure 1. H301-OLYMPUS-IX3-SVR - Components and Dimensions.
2. Available Sample Holders
The following sample holders are available.
NOTE: Please contact [email protected] if you cannot find the sample holder you are looking for. We are constantly
adding new inserts to the list.
1x35-M
#1 35mm Petri-dish
1xGS-M
#1 1x3in. chamber slide
1xLABTEK-M
#1 Lab-Tek 1in.x2in. chambered cover glass
1xLABTEK-II-M
#1 Lab-Tek II 1in.x2in. chambered cover glass
1x60-M
#1 60mm Petri-dish
1xT25-M
#1 Nunc and Greiner T25 flask
2x35-M
#2 35mm Petri-dish
2xGS-M
#2 1x3in. chamber slides
2xLABTEK-M
#2 Lab-Tek 1in.x2in. chambered cover glass
2xLABTEK-II-M
#2 Lab-Tek II 1in.x2in. chambered cover glass
2x60-M
#2 60mm Petri-dish
4x35-M
#4 35mm Petri-dish

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GS35-M
#1 1x3in. chamber slide and #2 35mm Petri-dish
LABTEK-35-M
#1 Lab-Tek 1in.x2in. chambered cover glass and #2 35mm Petri-dish
LABTEK-II-35-M
#1 Lab-Tek II 1in.x2in. chambered cover glass and #2 35mm Petri-dish
LABTEK-60-M
#1 Lab-Tek 1in.x2in. chambered cover glass and #1 60mm Petri-dish
LABTEK-II-60-M
#1 Lab-Tek II 1in.x2in. chambered cover glass and #1 60mm Petri-dish
GS60-M
#1 1x3in. chamber slide and #1 60mm Petri-dish
6035-M
#1 60mm Petri-dish and #1 35mm Petri-dish
6MW
6-well plates
12MW
12-well plates
24MW
24-well plates
24MW-NUNC
24-well NUNC/GREINER plates
48MW
48-well plates
96MW
96-well plates. *Includes MW-OIL
MW-OIL
Open frame to image multi well plates with oil immersion objectives
Figure 2.Available sample holders.

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3. Available Lids
The following heated glass Lids are available for H301-OLYMPUS-IX3-SVR:
•H301-SLIDING-LID: For easy sample loading and pipetting –Included in code H301-OLYMPUS-IX3-SVR
•H301-KOEHLER-LID*: Reduces chamber height to 22 mm and allows imaging under Koehler illumination
•H301-INJECTION-LID*: Heated glass lid with two small openings (sealed with flexible plastic) allowing injec-
tion or permanent access to the sample. Compatible only with sample holder 1x35
•H301-LASER-INTERLOCK-LID*: Heated glass lid with safety switch. Connects to laser controller and automati-
cally turns laser off when lid is lifted. Compatible only with sample holder 1x35
* OPTIONAL –not included with H301-OLYMPUS-IX3-SVR
3.1 Sliding lid
The Sliding Lid is a heated glass lid allowing for easy sample loading and pipetting. The Sliding Lid is screwed onto the
chamber.
Figure 3 shows chamber dimensions with the Sliding Lid (with and without chamber riser). Figure 4 illustrates how to
remove Sliding Lid, when a different Lid is necessary.
NOTE: Sliding Lid MUST BE REMOVED when using any other Lid. The Sliding Lid is fixed onto the chamber with 4
screws. Screws location is indicated by letter A in Figure 4 (image 2 and 3). Keep Sliding Lid fully closed to access
screws labeled A in image 2. Open Sliding Lid to access screws labeled A in image 3.
Distance between focal plane
and upper side of the glass
lid without the chamber riser
Distance between focal plane
and upper side of the glass
lid with the chamber riser
Chamber top view
Sliding Lid
Figure 3. H301-OLYMPUS-IX3-SVR(Sliding Lid comes as standard)

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Sliding Lid
A
A
Screws M2x4
Figure 4. Assembly of the sliding lid
3.2 Koehler Lid
The Koehler Lid is a heated glass lid reducing the chamber’s height to 22 mm and allowing imaging under Koehler il-
lumination. The Koehler Lid is placed onto the chamber (NO SCREWS or TOOLS are necessary).
Figure 5 shows chamber dimensions with Koehler lid (with and without chamber riser).
Distance between focal plane
and upper side of the glass
lid without the chamber riser
Distance between focal plane
and upper side of the glass
lid with the chamber riser
Chamber top view
H301-KOEHLER LID
Figure 5.H301-OLYMPUS-IX3-SVR + H301-KOEHLER-LID
3.3 Injection Lid
The Injection Lid is a heated glass lid with two small openings sealed with flexible plastic allowing injection or perma-
nent access to the sample. NOTE: Compatible with sample holder 1x35 ONLY. The Injection Lid is placed onto the
chamber (NO SCREWS or TOOLS are necessary).
Figure 6 shows chamber dimensions with the Injection Lid (with and without chamber riser).

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Distance between focal plane
and upper side of the glass
lid without the chamber riser
Distance between focal plane
and upper side of the glass
lid with the chamber riser
Chamber top view
H301-INJECTION LID
Figure 6. H301-OLYMPUS-IX3-SVR+ H301-INJECTION-LID
3.4 Laser Interlock Lid
The Laser Interlock Lid is a heated glass lid with a safety switch connecting to the laser controller. It automatically
turns the laser off when the lid is lifted. NOTE: Compatible with sample holder 1x35 ONLY. The Laser Interlock Lid is
placed onto the chamber (NO SCREWS or TOOLS are necessary).
Figure 7 shows chamber dimensions with the Laser Interlock Lid (with and without chamber riser).
Distance between focal plane
and upper side of the glass
lid without the chamber riser
Distance between focal plane
and upper side of the glass
lid with the chamber riser
Chamber top view
H301-LASER-INTERLOCK LID
Figure 7. H301-OLYMPUS-IX3-SVR + H301-LASER-INTERLOCK-LID
4. Insertion of the Sample Feedback Temperature Sensor
Insert the Sample Feedback Temperature Sensor through the dedicated opening located in the H301-OLYMPUS-IX3-SVR
(see Figure 8, Front and 3D views).

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1.Frontal view 2.3D view
Temperature Sensor dedicated opening
Temperature Sensor dedicated opening
Figure 8. Insertion of the temperature sensor inside the chamber.
5. Insertion of Sample Holder into Chamber
Sample holders fit into the chamber base and are held in place by magnets embedded within both chamber and hold-
er. To introduce a sample holder with the proper orientation, match the red dot on the holder to the one on the
chamber base, as illustrated in Figure 9.
Insert the adapter by
superimposing the red points
Figure 9. Introduction of the Specimen Holder inside the Chamber Base.

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6. Working with 35 or 60 mm Petri Dish –Spacing Rings and
Magnetic Locks
Magnetic locks prevent movement of 35 and 60 mm dishes inside the sample holder.
Figure 10 illustrates the available magnetic locks for 35 and 60 mm dishes. Threaded magnetic posts allow adjusting
holder’s height.
Magnetic Locks for 35 and 60 mm petri dishes.
Included in the corresponding sample holder
Figure 10.Magnetic locks for 35 and 60 mm dish
NOTE: Magnetic locks are included with sample holder.
Spacing rings accounting for variability in dish diameter from one manufacturer to another are also included. Figure
11 shows how to position the ring. Ring selection guide diagrams: rings for 35 and 60 mm dish -Figure 12 and Figure
13, respectively.
1
3
2
Figure 11. 1) Insertion of the ring, 2) insertion of the 35 mm dish, 3) insertion of the magnetic lock.
NOTE: Spacing rings are included with sample holder.
NOTE: Spacing rings do not block the petri inside its accommodation, but they are designed to center the petri with
respect to the observation circular window.

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BD Falcon-35x10 Willco-35x10
Grainer-Petri35x10 Ibidi μ-Dish 35 mm-low
MatTek-P35G-X14-XCorning-35x10 mm
Figure 12. Ring selection scheme for 35 mm dish.
Corning-60x15 mm
BD Falcon-60x10
Ibidi μ-Dish 50 mm-low
Grainer - Petri 60x15
MatTek-P50G-X30-X
Willco 60x15
Figure 13. Ring selection scheme for 60 mm dish.

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7. Working with 1x3’’ and 1x2’’ chamber slides - magnetic
locks
Magnetic locks prevent movement of 1’’x 3’’ and 1’’x 2’’ chamber slides inside of the sample holder.
NOTE: Magnetic locks are included with sample holder.
1
2
Figure 14. Magnetic lock for 1'' x 3'' chamber slide.
1
2
3
Figure 15. Magnetic lock for 1'' x 2'' chamber slide. To lock the slide, push simultaneously the buttons indicated with (3).

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8. Working with MW Plates - Magnetic locks and Chamber ris-
er
Magnetic locks hold MW plates in place into sample holder illustrated in Figure 16
Figure 16. Magnetic locks for MW plates. Ships with 6, 12, 24, 48 MW plates.
Chamber Riser increases chamber’s height and is REQUIRED with 6, 12, 24, 48 MW plates, regardless of the Lid em-
ployed.
Figure 17 shows how to install and fasten the chamber riser. Screws location is indicated by letter A.
A
Chamber Riser
A
Chamber Base
Figure 17. Riser Assembly.
9. Connection of the Gas Supply
A single silicon tubing carries output gas from the Okolab Gas Controller to the H301-OLYMPUS-IX3-SVR. Silicon tubing
connects to a gas input - brass opening - located on a corner of the H301-OLYMPUS-IX3-SVR. See Figure 18. Connect by
gently pushing silicon tubing onto brass opening.

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Gas Input
Figure 18.Connection with gas supply.
10. Working with Perfusion
The Chamber Riser included with H301-OLYMPUS-IX3-SVR features 12 perfusion holes for the insertion of perfusion
tubing up to 2.5 mm in outer diameter. To lock the Chamber Riser into the base use a 1.5mm Allen key to secure the
4 GUTB2-2,5-6 screws as shown in Figure 19.
Figure 19. How to lock the Chamber Riser.
Small screws plug the perfusion holes when not in use. (Grub screws M3x6). Remove small screws with a 1.5 mm Allen
key as necessary before introducing perfusion tubing.
Figure 20 shows location of perfusion holes.

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1.Frontal view 2.3D view
Perfusion dedicated opening
Figure 20. Perfusion.
Figure 21. How to access the perfusion holes.

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10.1 MICROFLUIDIC-C-RISER (Optional)
The MICROFLUIDIC-C-RISER is a chamber riser with 19x3 mm windows (on the two short sides) to allow insertion of
microfluidic tubings. Windows are magnetically closed. Compatible with most of H201 and H301 Okolab chambers.
Thickness of this chamber riser is 8mm (2mm more than default chamber riser). Requires Koehler lid and single ac-
commodation specimen holders.
Figure 22. MICROFLUIDIC-C-RISER.

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11. Connecting the Chamber to the adapter
Follow the steps shown in the images of Figure 23 and listed below in order to correctly connect the chamber with its
adapter:
1. Follow the arrows shown in Figure 23 Images 1 and 2 for placing the captive screws in the proper housings
2. Place the chamber on the adapter as shown in, Figure 23 Image 3
3. Use a 1.5mm metric Allen Wrench to tighten two of the four captive screws in place (A in Image 4) while keeping
the sliding lid closed. (See Figure 23 Image 4)
4. Tighten the remaining two captive screws (A in Image 5) while keeping the chamber sliding lid open.
5. Image 6 of Figure 23 shows the chamber fixed on the adapter
1 2
34
56
Captive Screw
A
A
Figure 23. How to assemble the chamber to the adapter.

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12. Connection of the Chamber with XY stage
Once you have assembled the chamber with adapter you can place it on the XY stage, following the steps shown in
Figure 24.
1. Place the adapter on the stage (See Image 1 of Figure 24).
2. Follow the instructions shown in Image 2 of Figure 24 for placing socket head cap screws; use the provided Allen
wrench for tightening the socket head cap screws and fastening the adapter on the stage. Screws housings are in-
dicated with letter A in Image 2 of Figure 24.
1 2
Screw M3x6
A
A
Figure 24. How to fix the H301-OLYMPUS-IX3-SVR to the XY stage.

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13. Cleaning
•Turn the system off and pull the mains plug out the socket
•Remove the lid from the chamber main body, and keep it separate from the chamber main body while the
chamber cools down.
•To clean the body and the glass lid of the chamber, wipe with a soft micro-fiber cloth. For stubborn smudges,
you can damp the soft micro-fiber cloth with ethyl alcohol (product code UN1170). Do not put any liquid di-
rectly on the chamber. While cleaning the glass lid, do not apply strong force to the surface of the glass lid
because it can be damaged.
H301-OLYMPUS-IX3-SVR_User Manual Rev 03_Published in January 2020