Location: U3202 MRB3
*Reservation calendar online at VU C.O.R.E.S.
A reservation is not required for this instrument. However, if you do not have a reservation and someone comes to use the instrument and they do have a reservation, then you must remove your sample and give them immediate access.
The Synergy HT can be used for fluorescence, luminescence and absorbance readings (see user manual at bottom).
- Absorbance can read at wavelengths 340, 405, 562, 590, 600 and 620 nm.
- Luminescence readings use a Photomultiplier Tube (PMT). The readings are detected from the top of the plate. It should be noted we do not have injectors on the Synergy. Please be sure to use plates suitable for luminescence assays. An example of these plates are Costar white PS microtiter plates (Cat# 3917). These are more expensive because they are sterile which may not be necessary for your application. There are many other manufacturers of these plates and so you should look for the best product to suit your needs.
- Fluorescence readings use filter wheels with excitation and emission filters at specific wavelengths. Information about white vs. black plates is included below from the PerkinElmer website. These are the filter sets we have:
- Center wavelength/bandpass (range)
- 360/40 Excitation (340-380 nm)
- 460/40 Emission (440-480 nm)
- 485/20 Excitation (475-495 nm)
- 528/20 Emission (518-538 nm)
- 530/25 Excitation(517.5-542.5 nm)
- 645/40 Emission (625-665 nm)
The VBI Equipment Resource has one additional platereader in the building. The links to this instrument is below:
- POLARstar Omega Platereader (BMG Labtech)
Black vs. white plates (Information taken from PerkinElmer)
Autofluorescence is fluorescence resulting from substances other than the fluorophore-of-interest, and can negatively affect an assay by increasing background signal. Many components of assays buffers and biological samples can autofluoresce. Autofluorescence is triggered by the same excitation light used to excite the fluorophore in the fluorescence assay. The severity of background autofluorescence can vary based on the excitation wavelength being used in a particular assay. For example, higher excitation wavelengths (above 650 nm) usually cause less autofluorescence than wavelengths in the UV/Vis range.
Because white plates reflect light and black plates tend to quench light, background fluorescence will be higher in white plates as opposed to black plates. For this reason, black plates are typically recommended for fluorescence assays that use short half-life fluorophores. Time-resolved fluorescence assays, which use longer half-life fluorophores, can use either white or black plates (read below for more information).
General fluorescence assays (fluorescence intensity, fluorescence polarization, FRET)
General fluorescence assays include traditional fluorophores such as fluorescein, cyanine 3, cyanine 5, green fluorescent protein (GFP), rhodamine, Texas Red, coumarin, and other fluorophores. These fluorophores have relatively-short half-lives (< µsec, for microplate-based assays). We recommend running assays utilizing short half-life fluorophores in black plates to reduce background autofluorescence.
Time-resolved fluorescence assays (TRF, TR-FRET)
Time-resolved fluorescence assays involve fluorophores that have longer half-lives (µsec – msec, for microplate-based assays). Examples of such fluorophores include Europium chelates and cryptates, Samarium chelates, and Terbium chelates and cryptates. Because of the longer half-life of the fluorescent signal, you can set up your instrument to incorporate a "lag time" or "delay time" between the time the fluorophore is excited, and the time you begin reading the emission signal (time-resolved mode). This allows background autofluorescence to fade before you begin collecting emission signal from assays involving a long half-life fluorophore. Because of this, time-resolved fluorescence assays can be run in either black or white plates. The use of white plates will result in higher raw signals, because the light is reflected maximally by the white color of the plate. The use of black plates will result in lower raw signals, because the black color of the plate can quench the light. However, black plates may help in situations where cross-talk is an issue, resulting in better sensitivity. In general, we recommend white plates for higher-density time-resolved fluorescence assays (for example, assays run in 384-well and 1536-well plates) and other assays where a lower signal might be expected.