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. |
|
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 |
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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. |