Method and apparatus for analyzing the nature of a sample

A method and apparatus for analyzing the absorption spectra of a minute sample. The sample is placed in a hollow enclosure which is sealed after a suitable working fluid is introduced into the enclosure. When a vapor equilibrium is reached within the enclosure creating a droplet condensate of working fluid about the sample, the sample is first irradiated with dark-field visible illumination with an image thereof stored, and is then irradiated with infrared radiation and a second dark-field image stored. The stored images are analyzed and the procedure is repeated for a plurality of different frequencies spanning the range of infrared absorption of the sample.

BACKGROUND OF THE INVENTION 
This invention relates to determining the nature of a minute particle or 
sample through infrared detection techniques, and in particular to a 
method and apparatus for determining the nature of the sample by heating 
the sample over a range of infrared frequencies and measuring the 
resultant infrared absorption of the sample. 
There are known methods for determining the molecular characterization, and 
therefore nature, of small samples of matter. Standard infrared 
spectrometric methods have been well know for quite some time, but are not 
readily useable for very small particles, and therefore not available for 
determining the molecular nature, as opposed to the elemental nature, of a 
particle. Other methods of spectrometry, such as the Raman method, are 
known and used to determine the characteristics of particles as small as 
10 microns, with difficulty at that small size. The Raman method suffers 
the deficiency that the number of reference spectra for the Raman method 
is not nearly as large as the number of reference spectra available when 
using standard infrared spectrometric methods. In addition, when using the 
Raman method, large amounts of energy are employed. When organic samples 
are studied, the Raman method cannot be employed since live cells would be 
destroyed. 
SUMMARY OF THE INVENTION 
The invention pertains to a method and apparatus for analyzing the 
absorption spectra of a minute sample which avoids the detracting features 
of the prior art, the spectra obtained from the invention can be compared 
with the large number of available infrared reference spectra in order to 
determine the molecular characterization of the sample studied. 
The apparatus includes a container for retaining the sample, with the 
container comprising a carrier, a sealed, hollow sample enclosure formed 
in the carrier, means forming a transparent path through the enclosure 
with the path being transparent to both infrared and visible radiation, 
and a working fluid disposed within the enclosure, the working fluid also 
being transparent to infrared radiation. A source of tunable infrared 
radiation is oriented to pass infrared radiation through the enclosure 
along the transparent path. A visible light source is positioned to pass 
visible radiation through the enclosure. An image of the sample within the 
enclosure is then captured and stored. 
The stored image is also analyzed. The image is converted in a digitizer to 
a computer-readable format and a computer is then used for examining the 
converted image. In accordance with the preferred embodiment of the 
invention, the computer is used to analyze an image of the sample when 
first irradiated only by visible radiation, and then when subsequently 
irradiated also by infrared radiation. 
In accordance with the illustrated embodiment of the invention, the 
container includes a raised sample platform formed in the enclosure and a 
well formed about the platform, with the working fluid being located in 
the well. Preferably, the well is annular. 
For capturing and storing an image of the sample, preferably a dark-field 
light microscope and a camera are employed. A dark-field condenser is used 
to produce dark-field illumination. 
The transparent path is comprised of first and second windows on opposite 
sides of the enclosure of the sample container. The windows, in 
combination with the carrier, form a hollow cell for containing the 
sample. One of the windows also serves as the sample platform for the 
sample. 
The working fluid comprises an infrared transparent liquid. Suitable 
liquids have been determined to be pentane, liquid argon and water, the 
latter when analyzing organic matters. 
To analyze the sample, the sample is first located in the hollow enclosure. 
The working fluid is then introduced into the enclosure without directly 
contacting the sample, and the enclosure is then sealed to create a vapor 
equilibrium within the enclosure. When the equilibrium has been reached, a 
droplet condensate of the working fluid is created about the sample. The 
sample is then irradiated with radiation, the size of the droplet is 
detected and stored, and the procedure of irradiating, detecting and 
storing is repeated for a plurality of frequencies spanning the range of 
infrared absoption of the sample.

DESCRIPTION OF EXAMPLES EMBODYING THE BEST MODE OF THE INVENTION 
The apparatus of the invention is depicted schematically in the single 
drawing FIGURE. The depiction of the apparatus is not intended to be to 
scale and parts are exaggerated in size and shown schematically for the 
purposes of description. 
For retaining the sample, a container 10 is employed. The container 10 
includes a carrier 12 which is shaped for mounting in a stationary 
orientation. Thus, the carrier 12 is shown in the rectangular form of a 
common slide for microscopic examination. 
The carrier 12 includes a sealed, hollow sample enclosure 14 formed 
centrally therein. A transparent path is formed through the enclosure 14 
by means of top and bottom windows 16 and 18, respectively, which are 
formed of a material which is transparent to both infrared and visible 
radiation. A sample 20 to be analyzed is situated within the enclosure 14. 
Since the invention is intended for analysis of particles of very small 
size, the sample 20 will normally be a particle of 10 microns or less in 
diameter. 
The window 18 is shaped to form a raised sample platform 22 for the sample 
20. A well 24, preferably annular, is formed in the window 18 about the 
platform 22 and a suitable working fluid 26 is disposed within the well 24 
about the sample 20. 
A visible light source 28 is oriented beneath the container 10. In order to 
permit conversion to dark-field illumination, a dark-field condenser 30 is 
interposed between the light source 28 and the container 10. 
Infrared radiation is provided by an infrared radiator or source 32, 
emitting an infrared beam 34 as illustrated which is reflected by a beam 
splitter 36 to pass through the sample 20. As is conventional, the beam 
splitter 36 is transparent to dark-field light from the combination of the 
light source 28 and dark-field condensor 30. 
A dark-field microscope 38 is employed to capture an image of the 
irradiated sample 20. The image is recorded by a camera 40. For analysis 
purposes, in accordance with the preferred procedure of the invention, the 
recorded image from the camera 40 is digitized and stored in a downstream 
combination of a digitizer and computer 42. In the computer, the image may 
then be studied further. 
The working fluid 26 must be compatible with the sample 20 to be analyzed. 
The fluid 26 must not dissolve or react with the sample, must have a 
suitable working temperature range, and must be transparent to infrared 
radiation in the region of spectral interest. The applicants have found 
that, for non-soluble substances, pentane is an appropriate working fluid 
for working at room temperature, and has wide ranges of infrared 
transparency. Also, by proper design to accomodate the temperatures 
involved, liquid argon may also be used. Liquid argon has almost complete 
infrared transparency, and is entirely non-reactive. Finally, for analysis 
of biological materials such as single cells, water must be used as the 
working fluid. While water has extensive infrared absorption bands, these 
absorption bands duplicate those present in a cell due to the water it 
contains, and therefore do not interfere with observation. Because water 
must be used at biologically compatible temperatures, an appropriate 
desired vapor/liquid equilibrium state within the sample enclosure 14 must 
be established by increasing the pressure within the enclosure 14 by 
various pumping methods. 
In operation, the sample to be anlyzed is placed in the enclosure 14 on the 
sample platform 22. The working fluid 26 is introduced into the annular 
well 24 and the enclosure 14 is closed and sealed by the window 16. An 
equilibrium vapor pressure is established within the enclosure 14, and 
working fluid 26 condenses about and forms a droplet around the sample 20. 
The droplet, which is not illustrated in the drawing, is tightly bonded to 
the sample 20 by surface tension, and the size of the droplet depends upon 
establishment of a vapor/liquid equilibrium within the enclosure 14, and 
therefore upon the temperature within the enclosure 14. It has been found 
that the size of the droplet ranges from about twice the size of the 
sample 29 for particles 10 microns in diameter to many times the particle 
diameter for smaller particles. Under the dark-field illumination method 
of the invention, the brightness of the droplets makes their observation 
quite simple. 
As is well known, the total light scattered by a droplet is a function of 
the diameter of the droplet. Therefore, an increase in the temperature of 
the particle due to infrared heating will result in a decrease in the size 
of the droplet. Consequently, the light scattered by the droplet will 
decrease as the droplet assumes a new, smaller equilibrium size. Thus, the 
temperature of the particle can be accurately monitored by monitoring the 
total light scattered by the droplet surrounding the sample 20. 
The infrared beam 34 striking the sample 20 heats the sample at those 
wavelengths where the sample absorbs the infrared radiation. By tuning the 
infrared beam 34 and source 32 through the region of spectral interest, 
the infrared absorption spectrum of the sample 20 may be determined and 
stored by the computer 42. 
It therefore is practical to determine the absorption spectrum of any 
particle that will serve as a nucleus for a condensation droplet visible 
in the dark-field microscope 38. It has been determined that particles as 
small a 2 or 3 microns can easily be analyzed by the method and apparatus 
of the invention using an infrared source 32 which can deliver intensities 
of about 1 watt per square centimeter. Such intensities are available from 
commercial monochromators of from Fourier transform infrared illuminators. 
For test purposes any other appropriate source can be substituted for the 
infrared source 32, so long as the source can simulate an infrared source. 
A low-power helium-neonlaser has been utilized with success. 
Two images of the sample 20 are taken for each different frequency of the 
infrared source 32. The first is of the sample 20 when illuminated by 
dark-field light but without being irradiated by the infrared beam 34. 
After that image is captured, the source 32 is activated and, after 
equilibrium within the enclosure 14 is again established, a second 
dark-field image is captured in the camera 40. The process is repeated for 
each desired frequency from the infrared source 32. 
In the digitizer and computer 42, the two images for each infrared 
frequency are first digitized, and then the image obtained after heating 
by the infrared beam 34 is subtracted from the image without heating. The 
resultant data is stored in the computer for each frequency, and following 
accumulation of data throughout the desired spectral range, the nature of 
the sample 20 can then readily be determined using the spectral 
information obtained. 
Various changes to the invention can be made without departing from the 
spirit thereof or scope of the following claims.