1. Introduction

The International Iberian Nanotechnology Laboratory (INL) in Braga, Portugal, is seeking to expand the capabilities of its Life Science microscopy platform, acquired in 2010, with total internal reflectance microscopy (TIRFM). The technical requirements of which are detailed below.

2. Technical Requirements

— TIRFM illumination:

— Monolithic laser module providing illumination at 405 nm, 488 nm, 561 nm and 647 nm with power output levels of 20 mW, 50 mW, 50 mW and 125 mW respectively;

— Integration with Nikon Ti-e microscope:

— Laser safety module;

— Laser coupling module for Nikon Ti-e microscope.

— 60x oil immersion TIRFM compatible objective with a numerical aperture (NA)>1.48.

— Electron-multiplication Charge Coupled Device (emCCD) with:

— 512 x 512 pixels;

— Cooling down to —100 ºC;

— Full frame read-out > 50Hz;

— Read noise < 1e- (with electron multiplication gain);

— Pixel well depth > 150.000e-.

— Software:

— Off-line software license for TIRFM data analysis with co-localization and FRET analysis capabilities.

— Software integration of TIRFM illumination module.

— TIRFM filter sets:

— 4-band filter set compatible with the TIRFM laser illumination wavelengths;

— Single band filter sets for TIRFM operation at 488, 561 and 647 nm respectively;

— Cy3 and CFP dichroic filters accompanied by empty filter cubes to migrate existing Cy3/Cy5 and CFP/YFP FRET imaging capabilities to the TIRFM upgrade.

— Migration of the INL Life Science microscope's fluorescence lamp with excitation and emission filter wheel (Lambda LS, Sutter Instrument) to INL's microfluidics microscope (MA200, Nikon)

— Lamp and filter wheel adapter components;

— Software upgrade:

— Computer controlled operation of lamp and filter wheels;

— Image based spectral unmixing module.

— 5-band dichroic filter set for DAPI, FITC, TRITC, Cy5 and Cy7 imaging.