TILL Photonics iMic TIRF System

This total internal fluorescence (TIRF) microscope has a unique design, allowing for a great deal of customization of the sample space and experimental design. The system is equipped with two EMCCD cameras and a customized dual emission beam splitter (dichrotome) allowing for up to four channels simultaneously imaged and makes it ideal for fluorescence resonance energy transfer (FRET) on camera based system. Its scan head (Yanus) allows for flexibility in applications such as photoactivation (PA) and fluorescence recovery after photobleaching (FRAP) experiments, all at high frame rates. Scan head allows for fast 2, 4, 100 and 360 points TIRF illumination, which makes the excitation profile more uniform than standard single point TIRF illumination. An environmental chamber allows for these techniques to be extended to live cell samples. Protocol editor makes the microscope versatile for more complex experiments.
Microscope body:
Inverted
Motroised Stage:
Yes
Temp Control:
Full Enclosure
CO2:
No
Software:
Live Acquisition

Common Applications

FRAP: Fluorescence Recovery After Photobleaching is used to gain insight into the dynamics or both diffusion and binding rates within a sample. Photobleaching is an irreversible process by which a fluorophore loses the ability to absorb and emit light. By deliberately inducing photobleaching within a restricted region of the sample observing the recovery within this region over time is affected by the rate at which unbleached molecules entre this region and bleached molecules are moved. 
FRET:

FRET (Fluorescence Resonance Energy Transfer) is a technique used largely to investigate molecular interactions between two or more molecules.  It works by using two carefully selected fluorophores that when in close enough proximity (10–100 Å), and suitable orientation, energy is transferred nonradiatively from the excited donor fluorophore to another acceptor fluorophore.

Photo-activation within live cells:

Photoactivation is a technique of photomanipulation used to investigate intracellular protein movement. A laser is used to cause a structural change to a population of molecules like fluorophores. These photoactivated molecules can then be tracked to assess their movements.

 

 

Objective Lenses

 
MAG N.A CORRECTIONS IMMERSION W.D MISC.
10x 0.3 EC Plan-Neuofluar Dry    
100x 1.46 Alpha-Plan apochromat Oil 0.17mm (UV) Vis-IR

 

Light Sources

Lasers Wavelengths Description
Diode 405 405nm Used for TIRF imaging
Diode 488 488nm Used for TIRF imaging
Diode 561 561nm Used for TIRF imaging
Diode 633 633nm Used for TIRF imaging
Polychrome V monochomator 320nm to 680nm Provides continueous selection of non-coherent light wavelength (5nm)

 

Transmitted light: Green LED

Filters

Name Description
491/561 For illumination with either/both 488 and 561nm lasers
Quad filter Used for any/all of the four lasers
T740 FCS filter for IR transmttance LED and any of the for lasers for FCS experiments

Before each camera there is a filter wheel containing a range of emission band pass filters that can be swapped depending of the users need and spectra of dyes used. The beam splitter consists of dichroic at 561 nm, but this unit can be swapped upon request of user. Note this system is capable of 2 camera simultaneous imaging

Detector

Detector Sensor Type Pixel Format Pixel Size Description
2x Andor iXon3 897 EM-CCD 512x512 16um x 16um Sensitiviey and speed at 56 fps or 595 fps with 128 x 128 crop

 

Miscellaneous

The Dichrotome is an extension of TILL Photonics‘Digital Microscope, the iMIC. It is integrated into the microscope and uses a single beam splitter design to separate the emission light from the sample into two independent colour channels. Each channel is simultaneously projected onto half of the CCD-chip. Open system with no defined laser safety interlock, therefore take extreme caution and follow SWP when using.