Fluorescent light source [X-cite 120]
An added benefit of X-Cite 120 is it's broader spectrum excites more fluorophores, see the table below:
| Fluorophore | Peak Excitation Wavelenth (nm) |
X-Cite 120 output (mW) |
HBO output (mW) |
|---|---|---|---|
| GFP/Ds Red (FRET) | 471-488 | 8.0 | 2.8 |
| FITC | 490 | 8.1 | 3.0 |
| Texas Red | 595 | 7.0 | 3.3 |
| TRITC | 555 | 17.9 | 6.5 |
| Cy3 | 552 | 27.6 | 12.1 |
| Rhodamine | 550 | 33.9 | 23.3 |
| Lucifer Yellow | 428 | 15.0 | 6.7 |
Camera Spectral Response [QImaging Retiga EXi digital camera]
GFP - Green fluorescent proteins
Green fluorescent proteins are being used for more and more applications in molecular and cellular biology. As a result of the variety of applications several variants form the original wild type green fluorescent protein (wtGFP) have been developed. Several of these variants have different excitation and emission spectra than wtGFP.
The red-shifted mutants have been developed for use with the standard "fluorescein" filter set and as such utilize the 485/20 excitation and 530/25 emission filter set. Note that although the 508/20 emission filter is closer to the emission peak of the sample, overlap with the 485/20 filter precludes the use of that filter, necessitating the use of the 530/25 nm filter. The blue emitting variants generally require the use of the 360/40 excitation filter in conjunction with the 460/40 emission filter.
Excitation and Emission Data of GFP Variants
| GFP Variant | Excitation max (nm) | Emission max (nm) |
|---|---|---|
| wtGFP | 395 | 509 |
| GFPuV | 395 | 509 |
| Stemmer | 395 | 509 |
| EGFP | 488 | 509 |
| GFP-S65T | 489 | 509 |
| Y66H | 382 | 459 |
| EBFP | 380 | 460 |
CFSE [CellTrace™ CFSE Cell Proliferation Kit, InVitrogen]
Fluorescein goat anti mouse IgG antibody/pH 8.0
Absorption and fluorescence emission spectra of fluorescein goat anti–mouse IgG antibody in pH 8.0 buffer.
RGB New Spectral Curves
The following curves were taken using a NIST-traceable visible-wavelength spectrometer and CRI-manufactured off-the-shelf liquid crystal tunable RGB filters commonly used in the Micro*Color™ and Macro*Color™ product families.
Second-generation Micro*Color filters, which succeeded the VariSpec™ RGB filters in 1997, were phased out in late 2002 and early 2003. The filters featured a “Color-Shaping Glass” (CSG) component that helped to correct the overly red-sensitive spectral response of most CCD-equipped cameras, produced at the time. Some OEM customers who manufactured their own cameras with a similar CSG component purchased a “bare” Micro*Color filter with no CSG component, but with an AR-coated cover slip.
Third-generation Micro*Color 2 filters, introduced in December of 2002, feature a near-infrared hot mirror component, instead of the CSG. Since most camera manufacturers now include a CSG component in their visible wavelength models, it was decided to eliminate the CRI-supplied CSG. The hot mirror was judged to be necessary, since many end-users utilize illumination systems, such as tungsten-halogen lamps, that output significant amounts of energy in the near-infrared that may not be completely eliminated by a significant number of current camera configurations.