7 replies to this topic

[cardosopedro]

Can someone explain me what is exactly the difference between a fluorometer and a spectrophotometer?

Edited by cardosopedro, 10 November 2009 - 10:40 AM.

[bob1]

a fluorometer measures fluorescence, a spectrophotometer measures absorbance/transmittance.

[Prep!]

why did u get this doubt!!! is there more to it or only this??!!! if you lookin for some application wud like if more details are givenm.. if its just a question tat popped up.. bob is absolutely bang on target!!!!

[cardosopedro]

Thanks for both answers. Yeah, I know spectro is for absorbance and fluoro is for fluorescence. But I would like to know the difference in terms of the mechanism of the machines, i.e., how they detect the signals... How color (Abs) is different from fluorescence (Fluor)?

When I was discussing this with my colleagues several doubts came out. For instance, if I want to detect ROS production with a probe that as a certain excitation/emission profile I should use a fluoremeter, right? However, I see in several articles that the authors use "microplate readers", which I've always associated as being a spectrophotometer...

So this generated some confusion.

[bob1]

Microplate readers can be either fluorometer or spectrometer or both, as is more common today. If you have something with emission/excitation values then, yes it is a fluorescent marker, so use a fluorometer.

Fluorometers work by shining a light of shorter frequency (higher energy) onto the marker, which then emits light at a longer frequency (lower energy)... e.g. blue excitation will give green fluorescence for an alexafluor 488 dye (488=excitation frequency). The machine uses a filter between light source and the sample to provide the excitation light and between the sensor and the sample to block out light from the excitation, so that only emission light gets through.

Absorbance kind of works in reverse to this. A sample of colour yellow, is not yellow itself, it is reflecting light that is yellow and absorbing the rest, so you could shine a light at it and measure in any colour other than yellow, how much light is getting through the sample on the other side. In this case you are looking for a loss of light or absorbance of the sample. This can be done for the whole spectrum or only at a particular wavelength(s) (e.g. DNA and RNA are measured based on absorbance at 260 and 280 nm). The light for specific wavelengths can be filtered before or after the sample, but usually before.
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It is often simpler to think of it like this: absorbance passes through but fluorescence bounces back. See attached images for illustration.

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[cardosopedro]

Thanks Bob for the detailed explanation.

[NWU99]

People also get "spectrophotometer" confused with "spectrometer." A spectrophotometer is an instrument that measures absorbance and it might use either filters, a monochromator or a spectrometer for absorbance wavelength selection. A "spectrometer" typically collects data from an entire wavelength range simultaneously using a CCD, so it's faster. This is how the Nanodrop and the Omega readers from BMG collect full spectrum data for UV/Vis DNA and protein analysis.

[mdfenko]

NWU99, on Dec 3 2009, 02:12 PM, said:

People also get "spectrophotometer" confused with "spectrometer." A spectrophotometer is an instrument that measures absorbance and it might use either filters, a monochromator or a spectrometer for absorbance wavelength selection. A "spectrometer" typically collects data from an entire wavelength range simultaneously using a CCD, so it's faster. This is how the Nanodrop and the Omega readers from BMG collect full spectrum data for UV/Vis DNA and protein analysis.

a spectrometer is an instrument that measures properties of light. a spectrophotometer is a spectrometer that measures light intensity (key term-photometer) at some wavelength(s).

a wavelength scan, whether performed all at once (using a multi-element ccd) or sequentially (by moving a prism or diffraction grating), is generally made with a scanning spectrophotometer (measuring absorbance profile).

Edited by mdfenko, 04 December 2009 - 08:51 AM.