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Greetings Orin,

I've spent a substantial amount of time working with scientists from the 
physical/engineering disciplines in the context of laser scanning 
microscopy.  A favorite platform for researchers investigating colloidal 
crystal formation and doing particle tracking was the old Noran 
systems.  At least one group I know runs one of the newer multipoint 
scanners from VisiTech. 

    One problem you are likely to run into is the fact that the sample 
is part of the optical system for laser scanning microscopy.  The second 
problem that is common is the fact that lenses which provide the high 
numerical aperture and index matching which is pre-requisite for 
accurate position determination w/ respect to the z axis have very small 
working distances (usually less than 100um).  If lower numerical 
aperture dry lenses (eg. 10x) are used to facilitate deeper penetration 
into the sample, you most certainly will have severe spherical 
abberations and axial scaling.  The problem confounds accurate 
positional determination w/ respect to the z-axis.

    Many of the microchannels I've seen are composed of materials such 
as PDMS or PMMA and have a high refractive index, the channel usually 
runs some sort of water-based (low RI) solvent, and then the beads are a 
high refractive index such as silica or polystyrene.  This is an optical 
nightmare: there are many highly reflective interfaces and if the beads 
are large enough they will act as ball lenses.  You may be able to get 
the information you need, but you should be careful to take the optics 
of your sample into account when deriving conclusions about bead 
velocities (if they are permitted to travel with 3 degrees of freedom) 
or conclusions about microscope performance.  They are all designed for 
biological specimens and the refractive indeces for which corrected 
optics are available are 1.51 (fixed, dehydrated protein; borosilicate 
glass), 1.42 (glycerol), and 1.33 (water).  A lens for optical 
sectioning must be an immersion lens for quantitative performance at any 
depth beyond the coverslip/sample interface.

    Some pertinent references include:

Carlsson, K. (1991). The influence of specimen refractive index, 
detector signal integration, and non-uniform scan speed on the imaging 
properties in confocal microscopy. J. Microsc. 163,2: 167-178.

Hell, S., Reiner, G. Cremer, C. and Stelzer, EHK. (1993). Aberrations in 
confocal fluorescence microscopy induced by mismatches in refractive 
index. J. Microsc. 169, 3: 391-405.

Visser, T.D. Oud, J.L. and Brakenhoff, G.J. (1992). Refractive index and 
axial distance measurments in 3-D microscopy. Optik, 87, 1: 17-19.

There are many others as well.  Hope this helps.

Best Regards,

Karl Garsha
Applications Specialist
Photometrics and Optical Insights
Office: 520-547-2704
www.photomet.com
www.optical-insights.com



ORIN HEMMINGER wrote:

>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>I'm trying to find a system for getting quantitative (velocity profiles) information for microfluidic devices, microchannels with a fluid and a solid tracer particle driven by electo-osmosis.  This is a new field to me so I'm struggling to find the best system.  Essentially we are looking at a confocal system because of the high spatial resolution and z plane resolution and because confocal systems keep getting faster and faster I think we can get the required temporal resolution to track flowing particles.  So my main requirement is speed, at least 20-30 frames per second, beyond that I'm wondering what is the best system avaliable for tracking particles flowing in microchannels using fluorescent dyes like FITC (~490 nm).  I'd like to do 4-D particle tracking and generate velocity profiles (I hope that the software that comes with a confocal system will have that capability).  I'm assuming that I should go with a spinning disk, I'm trying to set up demo's to see the PerkinEl
>mer Ultraview EMCCD, the Zeiss LSM 5 LIVE, and the Olympus DSU.  I've never used confocal microscopy and I'm not sure what my hurdels and complications are going to be when trying to use a spinning disk(or slit or point scanning) confocal microscopy unit for microfluidic applications.  Most literature I find is for Bio applications so any advice or input would be greatly appreciated.  Thanks in advance.
>
>Orin Hemminger
>Graduate Student
>The Ohio State University
>Chemical and Biomolecular Engineering
>USA
>1-614-292-4935
>  
>