Confocal Microscopy

Introduction

The Carl Zeiss on the inverted Axio Observer microscope is ideal for research in cell and molecular biology. The LSM 710 laser-scanning microscopy has excellent sensitivity combined with outstanding noise and excitation laser light suppression to deliver the best results. Our confocal microscope is equipped with 6 laser lines and is also installed for live cell imaging.

Equipment

Hardware
  • Axio Observer.Z1 inverted microscope (fully motorized version with apochromatic beampath, including motorized Z-drive)
  • Objectives: EC “Plan-Neofluar” 5x/0.16 M27, 10x/0.3 M27, 20x/0.5 M27, 40x/1.3 Oil DIC M27, 63x/1.4 Oil DIC M27, and 100x/1.4 Oil DIC M27
  • LD condenser 0.55 H, Ph1, Ph2, Ph3, DIC
  • DIC slider EC PN 10x/0.3 I, 20x/0.5 II, PA 20x/0.8 II, DIC slider EC PN and Fluar 40x/1.3 III, PA 40x/0.95 III, DIC slider PA 63x/1.4 III, DIC slider PA 100x/1.4 III
  • Transmitted Light Detector T-PMT
  • TFT touchscreen
  • Motorized scanning stage with joystick for XY control
  • HAL 100 Illuminator for transmitted light
  • X-Cite Illuminator for reflected light
  • Epifluorescence filter sets for DAPI, Cy3 and GFP
  • LSM 710 scanning module with 3 spectral reflected/fluorescence detection channels and 6 laser lines (405/458/488/514/543/633 nm)
  • Environmental chamber with temperature and CO2 control (Incubator XL S1 LSM 710, Heating Unit XL S1, TempModule S1, CO2 module S1, CO2 cover PM S1)
  • Heating insert for 35 or 60 mm dishes
Software
  • ZEN 2010. It is a Zeiss navigation software that offers a logical, easy-to-understand user interface and an improved color scheme for work that involves microscopy.
Applications
  • Confocal microscopy with spectral imaging acquisition, immunofluorescence, 2D imaging, co-localization, 3D optical sectioning/z-sectioning, observation of intracellular processes in living cell cultures, cell/cell interactions, motility, growth, time-lapse studies with automated processes, Ca2+ fluxes, photomanipulation (photoactivation or photobleaching e.g. fluorescence recovery after photobleaching (FRAP) and fluorescence resonance energy transfer (FRET) analyses), tile scan (stitch 2D or 3D images with motorized xy stage), mulitple location (2D or 3D) imaging (mark and find with motorized xy stage), High Dynamic Range (HDR) imaging (can be combined with z-sectioning, tile scan, multiple location, etc.), Physiology analysis (with Physiology software module to analyze intensity vs time), and 6D analysis (xyz, time, different colours and different locations).

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 and Dr Yap Lai Lai at , 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 and , at least 3 days prior to use.
  4. Please book online prior to use through Department Core Facilities Booking System booking system http://hippo/cfbs/ (Must be on NUS Network) and indicate the following in the booking calendar:
  5. Full name / lab / contact no. / trained by Zeiss or others (name)
  6. Access to “Confocal Microscopy” calendar is by invitation only, and authorized to trained users only.
  7. Please delete your online booking if you wish to cancel your slot.
  8. 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 at least 3 days prior to use.
Download Booking Form

Rules & Regulations for the usage of Zeiss Confocal Microscopy System

Use of machine

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

  • Definition of primary-trained users: those who are trained by Zeiss and hold the Zeiss certificate.
  • 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. Primary- trained user should email Dr Yap Lai Lai with the name of secondary-trained user to request for access to booking calendar.
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:
  • Switch on sequentially from switches #2 to #9.
  • Select Biochem User in the computer screen.
  • Click on the software, ZEN 2010. Choose “Start System” for new image acquisition.
  • 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 “Ocular” and “Incubation” in the ZEN 2010, click to check “Channel 1” (heating insert) and “Channel 2” (heating chamber unit) under “Temperature”, and “CO2” under “Atmosphere”. The default settings are 37oC, 37oC and 5%, respectively. Alternatively, you may use the TFT touchscreen by choosing “Microscope” and “Incubation” to switch on the heating for heating insert (labeled “H Insert P”) and 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.d. if you are not doing live cell imaging.

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 40x/63x/100x objectives 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:
  • Please select the 5x objective.
  • If you are doing live cell imaging, switch off the heating insert, heating chamber unit, and CO2 by unchecking Channel 1, Channel 2 and CO2 in ZEN2010, or select “Off” using TFT touchscreen. Close the valve of the CO2 tank gently (clockwise).
  • If the next user is using within the next 30 minutes, please do not turn any switches off.
  • If the next user is using within the next 30 minutes to 1 hour, please switch off #9 (X-Cite lamp) and #8 (idle-run switch) to idle for standby mode. The next user can switch #8 to run and switch on #9 when ready to use.
  • If the next user is using more than 1 hour later, please turn off all switches in reverse order from #9 (X-Cite lamp) to #2 (safety lock) for a complete shutdown. After switching off #7 (key on Ar-ML laser), please wait for the fan to stop (~10 min) before switching off the rest. To turn off #6 (computer), please shut down the PC.
  • Please clean the immersion oil off the 40x/63x/100x objectives using 100% ethanol and lens paper.

11. Special instruction for X-Cite 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 Dr Yap Lai Lai 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.

Click Here to download PDF version

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 hoursPackage 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 Confocal Microscopy System.

Contacts

PI-in-charge Core Facilities Manager Address
A/P Tang Bor Luen

+65 6516 1040
Dr. Yap Lai Lai

+65 6516 8891
Department of Biochemistry
Yong Loo Lin School of Medicine
National University of Singapore
8 Medical Drive
Singapore, 117597

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