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Hi Kathy,

First of all, did you do the measurement of the halide lamp with the proper
excitation filter in place?

In terms of fluorescence, you cannot directly compare both numbers. The
metal halide lamp has a broadband spectrum (see e.g.
http://www.jhtechnologies.com/pdfs/xcite120.pdf), while lasers have one or
more lines at discrete wavelengths. And as you know, the energy of a photon
depends on its wavelength.

Moreover, the type of illumination in widefield epi-fluorescence is
completely opposite to what is used in a laser scanning confocal microscope.
In widefield fluorescence the exciatation light is spread out over a large
area of the sample, while in a CLSM the laser beam is focused to a highly
intense and very small diffraction limited spot (this is why you don't need
so much laser power in confocal microscopy). So, in any case, you should be
comparing the irradiance (energy per second and per unit area) at the focal
plane, rather than the power of both light sources at the nosepiece.

In short, the extra power of the Halide lamp is necessary:
1. because of the widefield type of illumination;
2. because the power is distributed over a range of wavelengths which do not
all excite the fluorophores equally efficiently (see the fluorophore
absorption spectrum). In confocal microscopy one tries to match the laser
wavelength as well as possible to the absorption maximum so that the
available excitation photons are all used in an efficient manner.


Best regards,

Kevin



Kevin Braeckmans, Ph.D.
Lab. General Biochemistry and Physical Pharmacy
Ghent University
Harelbekestraat 72
9000 Ghent
Belgium
Tel: +32 (0)9 264.80.78
Fax: +32 (0)9 264.81.89
> -----Oorspronkelijk bericht-----
> Van: Confocal Microscopy List [mailto:[log in to unmask]]
> Namens Kathryn Spencer
> Verzonden: dinsdag 24 juli 2007 21:22
> Aan: [log in to unmask]
> Onderwerp: Comparing light output of metal halide vs. laser
> 
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> 
> Hello all;
> 	Question of comparison between metal halide illumination and ion
> lasers. Can you compare numbers obtained with a power meter at the
> objective nosepiece (objective removed)? We took readings from a Prior
> Lumen 200 vs. ion and diode lasers. The metal halide was an order of
> magnitude brighter (same scope system). The exposure times for each
> illuminator were milliseconds. Is there an advantage to the higher
> output of the metal halide? Are these photons extra (i.e., is the fluor
> saturated and these photons are only contributing to phototoxicity)?
> Canyou really compare these two by the numbers?
> 	Thanks in advance.
> 	Kathy