Base Microscope
The base microscope for "The 10-24" is an inverted Nikon Diaphot 200 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
Plan 4X* 17.1 0.13 Air
Plan 10X 16.0 0.30 Air
Plan 20X 2.10 0.50 Air
Fluor 40X* 0.30 0.85 Air
Fluor 40X 0.20 1.30 Oil
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.


Confocal Hardware
The confocal components include a side-mounted scanhead, krypton/argon mixed-gas laser, interface box, and Dell PowerEdge 1800 computer with dual displays running LaserSharp 5.2. The system produces fluorescent excitation through the use of three laser lines: 488 nm for green fluorophores, 568 nm for red fluorophores, and 647 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
488 nm Green Alexa Fluor 488, Calcein, Cy2, eGFP, FITC, Oregon Green 2 BP 505-539
568 nm Red Alexa Fluor 555 and 568, Cy3, DsRed,
EthD-1, Phycoerythrin (PE), Propidium Iodine (PI), Rhodamine, Texas Red, TRITC
1 BP 589-621
LP 585-
647 nm Far-Red Alexa Fluor 633 and 647, Allophycocyanin (APC), Cy5, TO-PRO-3 3 BP 664-696


Capabilities
Fixed tissue sections and cells as well as live cell cultures are routinely imaged using the 1024. Up to three fluorophores may be imaged simultaneous on this system. In addition, DIC-like images (often termed "transmitted light images") can be produced using the laser light that is not absorbed by the sample, which is collected by the condenser above the sample and directed to a photodiode detector.