Multiphoton Non-linear Optical Microscope (NLOM)

This custom-built NLOM utilizes a Zeiss AxioImager inverted microscope and a Femtolasers Synergy TiSa pulsed laser with extremely short (~ 10 fs) pulses  to simultaneously excite a broad range of fluorochromes. Another imaging modality is Second Harmonics generation (SHG), useful for instance for imaging of collagen. 

The NLOM has three non-descanned detectors with interchangeable filters, as well as a 16-channel spectral detector, based on an aberration-corrected concave grating and a linear PMT array.  All detectors are equipped with a constant fraction discriminators and work in a photon counting mode.

The hardware is controlled through National Instruments acquisition cards and custom software written in Labview.

 Objectives:

  • 10x/0.25 dry; 20x/0.75  dry; Long working distance Plan Apochromat 25x/0.8 multi-immersion objective (water, glycerol, oil)

Stage:

Ludl MAC5000 motorized XYZ stage with a z-axis linear encoder.

 

Figure 1. Pulse width characterization using an autocorrelator. The number of fringes permits calculation of the pulse width, here it was measured as 9.9 fs.

NLOM-autocorrelation

The ultra-short pulses (10-100x shorter than most commercial systems) provide two advantages: higher peak power without excessive photodamage, and broad-band excitation (spectrum width over 120 nm, see Fig. 2) that permits excitation of many fluorochromes without the need for laser tuning.

Figure 2. Emission spectrum of the femtosecond laser in mode-locked operation

NLOM-spectrum

 

Figure 3: Second Harmonics signal from collagen fibres form rat tail tendon. 20x/0.7 dry objective. 65 mW laser power.

 

The NLOM was built in collaboration with Dr. Alvin Yeh (Biomedical Engineering) and is housed in the Microscopy and Imaging Center.  For more inforamtion, contact Stan Vitha (mailto:vitha@tamu.edu).