Patent Application
20080278719
This invention relates generally to Raman and fluorescence microscopy and, in particular, to apparatus and method for reducing undesirable substrate-induced scattering in systems of this type.
It is common to use a glass slide to hold a material of interest (sample) when performing Raman or fluorescence microscopy. Referring to
This invention solves the above-referenced problem through the use of a material which is generally transparent in the visible region of the spectrum but reflective at the laser wavelength. In a typical configuration, a substrate provides a surface for supporting the sample, with the material being disposed between the surface of the substrate and the sample. The material is substantially transparent in the visible region of the spectrum but reflective at the laser wavelength, thereby minimizing unwanted Raman or fluorescence scattering that would be produced by the substrate if the material were not present.
In preferred embodiments the substrate is a microscope slide, dimpled sample holder or multi-cell well plate. Although other materials such as polymer may be used, such substrates are ordinarily glass. Although other types of optical filters such as holographic filters may be used, in the preferred embodiment the material is a multilayer dielectric filter acting as a “hot mirror” that reflects near-infrared energy. For laser wavelengths in manufacturing environments, a useful material becomes reflective above 700 nm or thereabouts.
Both surfaces of the substrate may include the optical filter material so that either side of the substrate may be used to support the sample. Although a full-spectrum mirror coating would work to protect the substrate from laser energy, an advantage of visible transmission is that it allows back illumination from behind/underneath the slide or well plate, thereby being visible to a microscope's eyepiece or video camera. In addition, polarization microscopy may still be employed to locate regions of interest in the sample.
A method of investigating a sample according to the invention comprises the steps of placing the sample on the surface of a substrate having an optical filter with a transmissive wavelength region and a reflective wavelength region; illuminating the sample with a laser having a wavelength in the reflective wavelength region; and analyzing the Raman or fluorescence scattering from the sample resulting from the laser illumination.
As discussed in the Summary, this invention uses a layer of laser-reflective material on a glass substrate such that when material of interest is present on the coating, the glass is essentially hidden from the laser illumination and thus little if any unwanted Raman or fluorescence scattering is produced.
In the preferred embodiment, a “hot minor” dielectric coating is applied to one or both glass surfaces. A useable hot mirror coating includes that sold by Edward Scientific, e.g. item #M43-843 on page 79 of its 2007 catalog #N071A. Such a coating is generally transparent in the visible and at least slightly reflective in the mean IR region above 700 nm or thereabouts.
Referring to
An added advantage of the coating is that the desired scattering that can be collected from the material of interest will be increased by as much as a factor of four due to 1) backscatter from direct illumination; 2) forward scatter from the direct illumination that is reflected back by coating; 3) forward scatter from laser illumination that is reflected back by the coating; and 4) backscatter from the laser illumination (as in 3, above) which is then reflected back by the dielectric.
Another advantage is that any Raman or fluorescence scattering produced by laser illumination that leaks through the filter to the substrate material is also reflected back by the filter and thus does not interfere with scattering from the sample.
Another advantage is that the material of interest can be observed in the visible spectrum through the back of the substrate, e.g. with an inverted microscope, or can be illuminated in the visible from the back for transmission imaging.
Another advantage is that polarization microscopy may still be employed to locate regions of interest in the sample since polarized illumination in the visible spectrum coming from beneath the substrate will pass through the filter largely unaffected.
An extension of the invention is with well plates. Glass (quartz) well plates are often used with inverted microscopes at high magnification looking through the bottom of the well plate, since the high magnification objectives generally have short working distances. Long working distance objectives can be used with Raman through the open end of the well, however, silica Raman from the well plate bottom can overwhelm the Raman from the material of interest which is often just a few small crystals at the bottom of the well. The solution is to add a dielectric “hot minor” coating to the bottom of the wells.
