The base microscope for "The A1" is an inverted Nikon Eclipse Ti-E equipped for standard widefield fluorescence, 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:
|Plan Apo VC 20X||1.00||0.75||Air|
|Plan Fluor 40X*||0.20||1.30||Oil|
|Plan Apo VC 60X||0.21||1.40||Oil|
|Plan Apo VC 60X||0.27||1.20||Water|
|Plan Apo 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, argon gas, DPSS, and diode lasers, interface control unit, and an HP xw8600 workstation running NIS-Elements C imaging software. The system produces fluorescent excitation through the use of six laser lines: 408 nm for blue fluorophores, 457 nm for cyan fluorophores, 488 nm for green fluorophores, 514 for yellow fluorophores, 561 nm for red fluorophores, and 638 nm for far-red fluorophores. The fluorescent emission is directed to four highly sensitive photomultiplier tubes (PMTs) to collect emitted blue, green, red, and far-red light or to a specialized 32-channel "spectral detector" for spectral analysis and unmixing. 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:
|Laser Line||Fluorophore Type||Examples||PMT||Filter Options|
|408 nm||Blue||Alexa Fluor 405, Cascade Blue, Coumarin 30, DAPI, Hoechst, Pacific Blue, most quantum dots||1||BP 425-475 nm|
|488 nm||Green||Alexa Fluor 488, ATTO 488, Calcein, Cy2, eGFP, FITC, Oregon Green, YO-PRO-1||2||BP 500-550 nm|
|561 nm||Red||Alexa Fluor 546, 555, 568, and 594, Cy3, DiI, DsRed, mCherry, Phycoerythrin (PE), Propidium Iodine (PI), RFP, TAMRA, tdTomato, TRITC||3||BP 570-620 nm|
|638 nm||Far Red||Alexa Fluor 633 and 647, Allophycocyanin (APC), Cy5, TO-PRO-3||4||BP 663-738 nm|
A unique feature of the system is the ability to collect spectral information from a sample. Using a specialized multichannel PMT detector, 32 images can be collected simultaneously in about half a second. Each image covers a discrete spectral band with a width of 2.5, 5, or 10 nm. Spectral imaging can be used to computationally unmix the signals of fluorophores with similar emission spectra. For example, for most imaging modalities, separating GFP from YFP is difficult, but can be done relatively easily using a spectral detector. Even probes with closely spaced emission spectra, such as GFP and Alexa Fluor 488, can potentially be separated. Autofluorescence from endogenous fluorophores in cells and tissues can also be separated and subsequently removed from images. In addition to the separation of overlapping fluorescence emissions, the spectral detector can also be used to generate emission spectra from an unknown fluorophore present in samples.
The AR1 is equipped with two sets of galvanometer-driven mirrors for scanning. The conventional pair is utilized for slow and moderate-speed collection of high-resolution images. The other pair, which comprises the "resonant scanner" (the "R" in A1R), has a set of galvanometers with a resonance frequency of 7.8 kHz, which allows for imaging of up to 30 frames per second (for a standard box size of 512x512 pixels). Faster frame rates can be achieved using smaller box sizes or limiting the scanning to a single line ("line scan").
FRAP, FLIP, and Photoactivation
For experiments involving fluorescence recovery after photobleaching (FRAP), fluorescence loss in photobleaching (FLIP), or the photoactivation of fluorophores, an ultraviolet or violet laser is typically used to perform the photobleaching/photoactivation, which is preceded or followed by standard image collection. The A1R can utilize both sets of galvanometer-driven mirrors to perform photobleaching/photoactivation simultaneously with image collection. However, for this approach, the 408 nm violet laser must be used for photobleaching/photoactivation. Additionally, traditional sequential experiments can be performed using any combination of lasers for photobleaching/photoactivation and image collection.
A product brochure for the Nikon A1R Confocal Microscope can be downloaded here.