TIRF Microscope
TIRF (Total Internal Reflection Fluorescence) microscopy is the ideal technique to study cellular membranes and their local environment close to the cover slip. TIRF uses the evanescent field generated by the total reflection at the coverslip to excite fluorophores. Thereby the evanescent field illuminates the specimen to a depth of up to 200 nm. Fluorochromes at deeper levels of the specimen are not excited and only those within this excitation zone (<200nm) from the cover glass. The system uses a CoolSNAP HQ2 CCD camera providing fast acquisition speed and excellent sensitivity. In addition the system is equipped with a 2D-VisiFRAP Real-time Scanner to preform FRAP and Photo-activation experiments. It also has an on stage control chamber to allow precise control of temperature and CO2.
The TIRF microscope is the system to use if you would like to study processes close to the plasma membrane. However you could also use this system to study plant material if you would use it in a semi TIRF mode which allows to excite fluorochromes at deeper levels of the sample.
- TIRF microscopy
- Life cell imaging
- Z sectioning
- Time series capture
- Co –localisation studies
- FRAP / Photo activation
Advantage
- Outstanding signal-to-noise ratio. Due to the low penetration depth of the evanescent field, the out-of-focus fluorescence is minimized and therefore hardly any background fluorescence occurs.
- High imaging speed
- Only a portion of the cell is exposed to the energy of the electromagnetic evanescent wave (~200nm), the production of harmful oxygen and therefore the phototoxic stress for cells is greatly reduced
Disadvantage
- TIRF is a surface interface phenomenon, and as such is restricted to studying sample that is within 100nm or so of the glass interface of the slide.
Motorised inverted microscope (Olympus IX81)
Objectives
- PlanApo 100x/1.45 TIRF Oil
Laser lines
| Laser | Excitiation Lines (nm) | Colour of fluorophores | Examples of fluorophores |
|---|---|---|---|
| Blue diode | 405 | Blue | DAPI / Alexa 350 |
| 488 Solid State Lasers | 488 | Green | GFP / Alexa 488 / FITC |
| 561 Solid State Lasers | 561 | Red | mCherry / TagRFR / Alexa 568 |
- VisiTIRF Dual port condenser VS T1 (Visitron Systems)
- 2D-VisiFRAP Realtime Scanner (Visitron Systems)
- Dual Beam Splitter for simultaneous observation of green and red
- CoolSNAP HQ2 CCD camera
- Objective Piezo of 100µm range for high precession and fast Z sectioning
Olympus TIRF: Actin polymerisation in Arabidopsis thaliana
