Scattering TIRF

[Claire Brown]

I have a user who wants to use our objective based TIRF to measure particle absorption to the coverslip using scattering. They are currently using a 633 nm laser and looking at scatter with a 10x lens and a PMT on a prism based TIRF.

 

My system has a 633 laser and we have typical filters to look at fluorescence but I’m not sure if there is something special I would need to do to look at the scattering. I’m not sure if the bandpass filter we have for fluorescence will pick it up or not.

 

Any suggestions would be appreciated.

 

Sincerely,

 

Claire

 

Claire M. Brown, PhD

Life Sciences Complex Imaging Facility Director

 

[John Oreopoulos]

Hi Claire,

Hope all is well up in Montreal.
The simplest thing to do to image the scattered light of TIR  
illumination would be to just remove the emission filter from your  
filter cube and perhaps replace the dichroic with a half-silvered  
mirror like you find in reflected brightfield filter cubes, but this  
second part isn't totally necessary. Couple of things to keep in mind:

1. Be very careful looking at the sample through the eyepieces (if  
any are present) since you are now viewing laser light directly.  
You'll want to have a way to adjust and turn down the laser power  
that is incident on the sample either with a gradient neutral density  
filter wheel or electronically if your laser has this ability. The  
same applies for your detector or imaging device. Don't expose these  
to too much light or they could be damaged.

2. Scattered TIR images don't look great - usually there is a lot of  
background and laser interference fringes in the image. Nevertheless,  
the intensity of the scattering object that is being imaged can be  
used to do some very interesting quantitative work. For example, see  
these publications:

TOTAL INTERNAL-REFLECTION MICROSCOPY - A SURFACE INSPECTION TECHNIQUE
Author(s): TEMPLE PA Source: APPLIED OPTICS    Volume: 20    Issue:  
15    Pages: 2656-2664    Published: 1981

Measurement of colloidal forces with TIRM.
Author(s): Prieve DC Source: ADVANCES IN COLLOID AND INTERFACE  
SCIENCE    Volume: 82    Issue: 1-3    Pages: 93-125    Published:  
OCT 10 1999

Measuring colloidal forces using evanescent wave scattering
Author(s): Bike SG Source: CURRENT OPINION IN COLLOID & INTERFACE  
SCIENCE    Volume: 5    Issue: 1-2    Pages: 144-150    Published:  
MAR 2000

A new approach for analyzing particle motion near an interface using  
total internal reflection microscopy
Author(s): Oetama RJ, Walz JY Source: JOURNAL OF COLLOID AND  
INTERFACE SCIENCE    Volume: 284    Issue: 1    Pages: 323-331    
Published: APR 1 2005

Surface Plasmon Resonance Imaging Using a High Numerical Aperture  
Microscope Objective
Author(s): Huang B (Huang, Bo), Yu F (Yu, Fang), Zare RN (Zare,  
Richard N.) Source: ANALYTICAL CHEMISTRY    Volume: 79    Issue: 7    
Pages: 2979-2983    Published: APR 1 2007

These publications also contain instrumental diagrams which are (with  
the exception of the last paper) essentially prism-based TIRF  
microscopes without an emission filter to block scattered laser  
light. It is interesting to note that "TIR" microscopy ("TIRF"  
microscopy with the F-fluorescence dropped from the name) seems to  
have developed in the materials science and analytical chemistry  
fields independent of the the advances made in the biological  
sciences with TIRF microscopy.

3. The first and the last papers also show that additional contrast  
and information about the orientation of scattering particles can be  
gained by varying the polarization of the incident illumination beam,  
but you probably won't have the ability to easily change that on a  
commercial system. Try rotating the optical fiber connector that  
delivers the laser light into the microscope system. If it's a  
"polarization maintaining" fiber, you may see some variation in the  
scatting particle's intensity.


John Oreopoulos, BSc,
PhD Candidate
University of Toronto
Institute For Biomaterials and Biomedical Engineering
Centre For Studies in Molecular Imaging


On 11-Jan-10, at 12:22 PM, Claire Brown, Dr. wrote:

> I have a user who wants to use our objective based TIRF to measure  
> particle absorption to the coverslip using scattering. They are  
> currently using a 633 nm laser and looking at scatter with a 10x  
> lens and a PMT on a prism based TIRF.
>
> My system has a 633 laser and we have typical filters to look at  
> fluorescence but I’m not sure if there is something special I would  
> need to do to look at the scattering. I’m not sure if the bandpass  
> filter we have for fluorescence will pick it up or not.
>
> Any suggestions would be appreciated.
>
> Sincerely,
>
> Claire
>
> Claire M. Brown, PhD
> Life Sciences Complex Imaging Facility Director
>