Fluorescence Microscopy System

Introduction | Equipment | Operating Hours | Booking | Rules & Regulations | Charges | Contact |

Introduction

Axio Observer is an inverted research microscope from Carl Zeiss. It has been developed for maximum flexibility in the live cell techniques of today and tomorrow and realized as a fully integrated research platform for cell observation, cell manipulation and cell analysis.

Equipment

Hardware

• Axio Observer.Z1 inverted microscope (fully motorized version, including motorized Z-drive)
• Objectives: EC Plan-Neofluar 10x/0.3 Ph1, EC Plan-Neofluar 10x/0.3, LD Plan-NeoFluar 20x/0.4 Corr, LD Plan NeoFluar 40x/0.6 Corr, Plan-Apochromat 63x/1.40 Oil DIC
• DIC Slider LD PN 20x/0.40 II EP 20x, DIC Slider LD PN 40x/0.60 II Corr, DIC Slider PA 63x/1.4 III HR
• Hamamatsu ORCA R2 C10600-10B Cooled Digital CCD Camera
• TFT touchscreen
• Motorized scanning stage with joystick for XY control
• HAL 100 Illuminator for transmitted light
• HXP 120 Illuminator (fluorescence light source)
• FL filter set for DAPI, FITC, HE mRFP, HE BFP+GFP+HcRed
• Colibri Illumination System (high-end LED light source for fluorescence applications where rapid excitation changes are required; LED module 365 nm, 470 nm, 590 nm)
• Environmental chamber with temperature and CO2 control (Incubator XL S1, Heating Unit XL S1, TempModule S1, CO2 module S1, CO2 cover PM S1)
• Heating insert for 35 or 60 mm dishes and 6-well dishes

Software

• AxioVision Release 4.8.

Applications

• Epifluorescence/immunofluorescence, observation of intracellular processes in living cell cultures, cell/cell interactions, motility, growth, time-lapse studies with automated processes, and z-sectioning.

Operating Hours

Operating Hours: Monday – Sunday, 8.30 am – 8.30 pm daily.

For night usage after 8.30 pm, please kindly inform A/P Tang Bor Luen at bchtbl@nus.edu.sg, Dr. Lim Cheh Peng at bchlimcp@nus.edu.sg and Miss Gan Bin Qi at bchgbq@nus.edu.sg, at least 3 days prior to use.

Booking

Booking (Users at Department of Biochemistry)
1. Operating hours: Monday to Sunday, 8.30 am to 8.30 pm.
2. Priority booking on Fridays, 8.30 am to 5.30 pm, is granted to other NUHS users.
3. Minimum booking per slot is 30 minutes. For booking of >3 hours or after 8.30 pm, please inform A/P Tang Bor Luen, Dr. Lim Cheh Peng and Ms. Gan Bin Qi, at least 3 days prior to use.
4. Please book online prior to use through the Microsoft Outlook online calendar named “Gan Bin Qi - Fluorescence Microscope” and indicate the following in the booking calendar:
Full name / lab / contact no. / trained by Zeiss or others (name)
5. Access to “Gan Bin Qi - Fluorescence Microscope” calendar is by invitation only, and authorized to trained users only.
6. Please delete your online booking if you wish to cancel your slot.
7. Booking for the next month starts on the last week of the current month.

Booking (Other NUHS Users)
1. Operating hours: Fridays, 8.30 am to 5.30 pm.
2. Maximum booking per slot is 3 hours, except for live cell imaging. Exceptions may be granted for booking of >3 hours or after 5.30 pm, on a case-by-case basis.
3. Please fill in the booking form and e-mail it to Ms. Gan Bin Qi at least 3 days prior to use.

Rules & Regulations for the usage of Zeiss Fluorescence Microscopy System

Use of machine
1. Only primary-trained and secondary-trained users are allowed to use the microscope.

• a. Definition of primary-trained users: those who are trained by Zeiss and hold the Zeiss certificate.
• b. Definition of secondary-trained users: those who are trained by a Zeiss-certificate holder. Adequate training should be provided by a primary-trained user from the same lab. Trainees can only start using the microscope independently when the trainer decides that they are competent to safely operate the machine.

2. Please log in your details in the keys logbook upon collection of the microscope room key from MD7 Level 1. Please do not leave the key inside the microscope room unattended when you leave the room temporarily, nor take the key out of MD7. For a prolonged period of absence (e.g. lunch), please lock the door and either return the key to Level 1 or leave your phone number on the door where you can be contactable.
3. Please log in your Start time in the user logbook in the microscope room.
4. Please do not use gloves at the “No Gloves” areas, i.e. the computer, keyboard, mouse etc.
5. Procedures for switching on the system:

• a. Switch on the main switches #1, #2 and #3 on the wall to power up the computer, microscope and main accessories.
• b. If you are doing live cell imaging, switch on main switch #4 on the wall to power up CO₂ Module, Temperature Module, and Heating Unit. Please follow through step 5.g. below. Please ignore this step 5.b. if you are not doing live cell imaging.
• c. Switch on #5 for HXP 120 lamp, #6a for Colibri and #6b for SVB1 trigger box for Colibri and camera control.
• d. Switch on #7 for Hamamatsu camera, #8 for stage control, #9 for Power Supply 232, and #10 for microscope (on/off button on left-hand side of the microscope).
• e. Switch on #11 for the computer. Username: User.
• f. Click on the software, AxioVision Rel. 4.8.
• g. For live cell imaging, open the valve of the CO₂ tank gently (anti-clockwise) and check the CO₂ level remaining in the tank (right gauge). Do not adjust the regulator knob connected to the CO₂ tank which is pre-set to 1 bar (left gauge), nor adjust the Open/Close knob. To switch on the heating insert, heating chamber unit and CO₂, go to “Work Area” and “Incubation” in the AxioVision Rel. 4.8, and select “On” for “H Insert P” (60 mm dish heating insert) or “H Mount Fr” (6-well heating insert) and “H Unit XL” (heating chamber unit) in Temperature tab, and “CO2 Small V” (CO₂) in Atmosphere tab. The default settings are 37^oC, 37^oC, 37^oC and 5%, respectively. Alternatively, you may use the TFT touchscreen by choosing “Microscope” and “Incubation” to switch on the heating for 60 mm dish heating insert (labeled “H Insert P”) or 6-well heating insert (labeled “H Mount Fr”), heating chamber unit (labeled “H Unit XL”), and release of CO₂ (labeled “CO₂ Small V”). Please ensure that the level of sterile water in the bottle connected to the CO₂ tubing in the chamber is between the minimum and maximum level. Please ignore this step 5.g. if you are not doing live cell imaging.
• h. If you are doing multichannel fluorescence acquisition or normal time lapse acquisition, please use the "6D Acquisition" from the toolbar. Please refer to Zeiss manual for multichannel fluorescence acquisition.
• i. If you are doing very fast acquisition for 1 color, e.g. > 3 fps, please use the "Fast Acquisition" function from toolbar. Please refer to Zeiss manual for Fast Acquisition.

6. Please lower the objectives (click “Load Position” on the TFT touchscreen) before removing the slide to prevent scratching of lens.
7. Please clean the immersion oil off the 63x objective using 100% ethanol and lens paper immediately before switching to another objective to prevent dripping of oil and soiling the sides of the objectives.
8. Please do not adjust any buttons or knobs other than those taught during the training.
9. Please save all your data in D drive only. Any files saved on the desktop or other drives will be removed without notice. Please note that regular clearing up of the disk space will also be performed at the end of every month, and any data more than 3 months old will be deleted without further notice.
10. Procedures for standby/shutdown:

• a. Please select the 10x objective.
• b. If you are doing live cell imaging, switch off the heating insert, heating chamber unit, and CO₂ by selecting “Off” for “H Insert P” or “H Mount Fr”, “H Unit XL”, and “CO₂ Small V” in AxioVision Rel. 4.8, or select “Off” using TFT touchscreen. Close the valve of the CO₂ tank gently (clockwise).
• c. If the next user is using within the next 30 minutes, please do not turn any switches off.
• d. If the next user is using more than 30 minutes later, please turn off all switches in reverse order for a complete shutdown.
• e. Please clean the immersion oil off the 63x objective using 100% ethanol and lens paper.

11. Special instruction for HXP 120 lamp: when it is switched on, leave it on for a minimum of 15 minutes before switching it off and when it is off, leave it off for a minimum of 15 minutes before switching it on.
12. Please log in the End time and CO2 level (for live cell imaging) in the user logbook at the end of your usage. Please alert Miss Gan Bin Qi when the CO2 level drops to 20 bar (red mark on the right gauge of the regulator).
13. Please ensure that the microscope room is securely locked upon exit. Please return the microscope room key immediately to MD7 Level 1 and log in the return time in the keys logbook.

Charges

1. The usage of the microscope will be charged quarterly according to the following rates:

Users	Rate/hour
Department of Biochemistry	S$15.00
Other NUHS users	S$20.00

2. For long-term time-lapse experiments, a package price of S$5.00 per hour for a minimum booking and usage of 12 hours, are applicable for both Department of Biochemistry and other NUHS users.

No. of hours	Package Price
12 hrs	S$60.00
24 hrs	S$120.00
36 hrs	S$180.00
48 hrs	S$240.00
60 hrs	S$300.00
72 hrs	S$360.00

3. Training is compulsory for first-time users, and can be arranged upon request. Training charges may apply.
4. Please refer to SOP for cost-sharing (Department of Biochemistry users) for cost-sharing of Fluorescence Microscopy System.

Contact

**For enquiries, please contact A/P Tang Bor Luen, Dr. Lim Cheh Peng or Ms. Gan Bin Qi.**

PI-in-charge A/P Tang Bor Luen E-mail: bchtbl@nus.edu.sg Tel: 65161040	Core Facilities Manager Dr. Lim Cheh Peng E-mail: bchlimcp@nus.edu.sg Tel: 65168891	Laboratory Executive Miss Gan Bin Qi E-mail: bchgbq@nus.edu.sg Tel: 65161040
Address Department of Biochemistry Yong Loo Lin School of Medicine National University of Singapore 8 Medical Drive Singapore, 117597