Base Microscope
The base microscope for "The Radiance" is an inverted Nikon Eclipse TE2000 equipped for standard widefield fluorescent, brightfield, and differential interference contrast (DIC) microscopy (all used mainly to locate objects of interest). The system is fitted with the following set of objectives:

Objective Working
Distance (mm)
Numerical
Aperture
Immersion
Medium
PlanFluor 10X* 16.0 0.30 Air
PlanApo 20X 1.00 0.75 Air
PlanFluor 40X 0.20 1.30 Oil
PlanApo VC 60X* 0.27 1.20 Water
PlanApo 60X 0.21 1.40 Oil
PlanApo 100X* 0.13 1.40 Oil
*These objectives are not normally present on the microscope turret, but can be attached upon request.
*These objectives are not normally present on the microscope turret, but can be attached upon request.


Confocal Hardware
The confocal components include a side-mounted scanhead, argon gas, helium/neon mixed-gas, and red diode lasers, interface control unit, and Dell OptiPlex computer with dual displays running LaserSharp 6.0. The system produces fluorescent excitation through the use of seven laser lines: 457 nm for cyan fluorophores, 476 nm and 488 nm for green fluorophores, 514 for yellow fluorophores, 543 nm for red fluorophores, and 637 for far-red fluorophores. The fluorescent emission is directed to three highly sensitive photomultiplier tubes (PMTs) to collect emitted green, red, and far-red light. Unlike film or CCD cameras, the PMTs collect a single pixel at a time as the laser scans across the sample. Before the emitted light reaches the PMTs, however, it passes through a confocal pinhole to remove out-of-focus light and either a bandpass (BP) or longpass (LP) filter to remove unwanted wavelengths. See the table below for a summary of the excitation/emission schema:

Excitation Emission
Laser Line Fluorophore Type Examples PMT Filter Options
457 nm Cyan Alexa Fluor 430, BO-PRO-1, CFP 1 Numerous
476 nm Green DiO, FM 1-43, NBD-X 1 Numerous
488 nm Green Alexa Fluor 488, Calcein, Cy2, eGFP, FITC, Oregon Green, YO-PRO-1 1 Numerous
514 nm Yellow Alexa Fluor 514, EthD-1, YFP 1 Numerous
543 nm Red Alexa Fluor 546, 555, and 568, Cy3, DiI, DsRed, Phycoerythrin (PE), Propidium Iodine (PI), Rhodamine, Texas Red, TRITC 2 Numerous
637 nm Far Red Alexa Fluor 633 and 647, Allophycocyanin (APC), Cy5, TO-PRO-3 3 Numerous


Multiphoton Hardware
The multiphoton components include a high-power laser, beam-conditioning unit including a Pockel's cell, and two non-descanned direct PMTs, all of which are integrated into the normal excitation and emission paths. The key component is a diode-pumped Ti:Sapphire tunable red-to-infrared (715-955 nm) femtosecond laser. This "multiphoton laser" emits short pulses of long wavelength light at high intensities. At the focal plane--but not below or above--these photons reach a high enough intensity to produce the multiphoton effect. In this situation, fluorescent excitation is achieved by two lower energy photons arriving nearly simultaneously, which is sufficient to excite fluorophores normally requiring a single higher energy photon. For example, FITC can be excited by a multiphoton laser at ~950 nm, whereas its normal excitation peak is ~490 nm. The emission curves of fluorophores excited in this manner are essentially identical to those for single-photon excitation.