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Hello;
	Should have supplied more information. Both illumination sources
were tested on spinning disk systems (one with microlenses, one
without). Appropriate filters were in place for the metal halide source,
and were compared to specific laser lines (diode or ion). We took the
objective out of the system, just to eliminate differences between NAs.
I guess my basic question; is more always better? If you have short
exposure times (200msec) with the lower intensity illumination source,
what does 10 times that really get you? Once the fluor is saturated, it
cannot absorb any more photons. Am I at that saturation point?
	The power meter is a broadband power/energy meter that does not
need tuning to specific wavelengths. It has a thermopile sensor.
	We're demoing two systems, and my colleagues are excited (pun
intended) about the one system, because it is "10 times better". Is it
really?
	Kathy




-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On
Behalf Of Kevin Braeckmans
Sent: Tuesday, July 24, 2007 11:23 PM
To: [log in to unmask]
Subject: Re: Comparing light output of metal halide vs. laser

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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