This article discusses the basic design concepts of a UV-visible-NIR range microscope spectrometer in several different configurations. These include configurations to acquire absorbance, reflectance, fluorescence and Raman spectra of microscopic samples. A brief summary of some of the uses of the microscope-spectrometer is also included.
The microscope-spectrometer is a hyphenated instrument that combines the magnifying power of a microscope with the analytical capabilities of different types of spectrometers. As such, these instruments are used to acquire spectra, color spaces and even thin film thickness of micron-scale samples areas.
With the flexibility inherent in their design, the microscope-spectrometer can be configured in many different ways and used to measure absorbance, reflectance, Raman and even emission spectra, such as fluorescence and photoluminescence, of sub-micron-sized sample areas. With the addition of specialized algorithms, the microscope-spectrometer can also be used to measure the thickness of thin films or to act as a colorimeter for microscopic samples.
There are many reasons to use tools that can acquire spectra of microscopic sample areas. These instruments require only small amounts of samples in either solid or liquid form. Another advantage is that very little or no preparation is required for many samples. Also, color comparisons done by spectroscopy tend to be more accurate because these instruments have a broader spectral range, can correct for lighting variations, and can measure the intensity of each wavelength band of light.
Before the advent of microspectroscopy, the only way to analyze many types of microscopic samples was to use micro-chemical testing and then some sort of visual examination. Unfortunately, this testing tends to be destructive, requires a considerable volume of sample, and suffers from the inaccuracies of the human visual system. The microscope-spectrometer avoids these issues yielding improved accuracy and speed of analysis. Microscope-spectrometers can also “see” beyond the range of the human eye and thus distinguish variations that would not be apparent visually.
The Components of a Microscope Spectrometer
The microscope-spectrometer integrates UV-visible-NIR range spectrometers with an optical microscope designed for both spectroscopy and imaging (see Figure 1). The microscope must feature an operational spectral range from the deep ultraviolet to the near infrared while maintaining good spectral and image quality. Standard microscopes cannot be used as they only cover a portion of the visible spectrum due to their optical designs and light sources used in such devices.
A UV-visible-NIR range microscopespectrometer, on the other hand, utilizes a custom-built microscope with an optics and light sources optimized for the deep UV through the NIR. The microscope consists of air-mounted lenses of fused silica and other materials, as well as ultraviolet enhanced mirrors, mounted in a configuration designed to evenly illuminate a sample while producing a sharp image in all regions of the designated spectral range of the instrument. Depending upon the type of spectroscopy to be done, the illumination is provided either by mixing the output of a deuterium lamp with that of a halogen lamp, use of arc lamps or even lasers. The advantages of building this type of UV-visible-NIR microscope is that both the image quality, throughout the UV, visible and NIR regions, as well as the spectral quality can be optimized when the microscope is integrated with both its imaging and spectroscopy components.
The spectrometer itself must also be designed for microspectroscopy in order to obtain good spectral results. This means that the spectrometer must be highly sensitive while still maintaining an acceptable spectral resolution and signal-to-noise ratio. Stability is also an issue since the microscope-spectrometer is a single beam instrument and reference spectra must be obtained prior to measuring the sample. The instrument must also have a high dynamic range since one frequently switches from transmission or reflectance microspectroscopy to fluorescence microspectroscopy. This allows the user to obtain different types of spectral information from exactly the same location on the microscopic sample.
Integration of the spectrophotometer with the microscope is critically important. While the microscope and spectrophotometer must both be optimized for microspectroscopy, the key to a microscope spectrophotometer’s operation is the hardware that enables them to work together. This interface has several basic requirements. Most importantly, it must channel the electromagnetic energy collected by the microscope from the sample into the spectrophotometer.
However, the user must be able to visualize the sample measurement area as well as the surroundings. This is done by having the entrance aperture of the spectrophotometer at the same focal plane as the sample image. The sample may then be moved with the microscope stage, as one would normally do with a microscope, until the image of the entrance aperture is over the area to be measured. The black square in the center of the image is the entrance aperture of the spectrophotometer. All this is done in real time so that the spectroscopy of microscope samples is quick and easy.
Configuration of the Microscope Spectrometer
The job of the UV-Visible-NIR microscope is to illuminate the sample and then to funnel the electromagnetic energy collected from the sample into the spectrometer. In order to do this, the user must be able to visualize the area to be measured as well as see the surrounding sample. This is done by having the entrance aperture of the spectrometer at the same focal plane as the sample image. Thus, a video image of the two shows the aperture in sharp focus over the sample in addition to the surrounding field of view of the sample. In operation, the sample stage is moved until the image of the entrance aperture is over the area of the sample to be measured. When the aperture is placed over the sample area of interest, the spectrum is then measured. Of course, images of the measured sample with the aperture in place can also be captured. For many research applications, this sample-aperture alignment operation is done manually. For industrial operations, this procedure is often automated.
The optical path of a microscope-spectrometer is more complex than the operation. See Figure 2. The microscope’s optics focus light onto the sample. Photons interact with molecules in the sample and the electromagnetic energy from the sample is collected by the microscope objective and focused onto the entrance aperture of the spectrometer. Much of the light is imaged by a digital camera allowing the user to see the both the sample and the entrance aperture of the spectrophotometer superimposed thus allowing the user to see exactly what they are measuring. The electromagnetic energy that passes through the entrance aperture passes into the spectrometer and a spectrum is collected. Depending on the type of experiment to be performed, the microscope can be configured with different illumination schemes: white light allows for reflectance microspectroscopy from the deep UV to the NIR. Incident illumination can also be used for fluorescence or photoluminescence microspectroscopy by using a filter-based monochromator with an arc lamp source or one of a series of lasers.
Transmission microspectroscopy is done with a white light focused onto the sample through the microscope’s condenser. As stated earlier, the optical materials and the light sources used to build the microscope mean that the spectral range for both imaging and spectroscopy goes as low as 200 nm and into the near infrared to 2500 nm. Comparably, a modern optical microscope will have a very limited spectral range of only 450 to 700 nm depending upon its configuration. Thus, the need for a custom microscope if the user requires ultraviolet or near infrared spectra.
Raman microspectroscopy can also be done with the microscope spectrometer. The Raman module integrates a laser, a Raman spectrometer and optics for illumination of the sample and collection of the Raman scattered light. See Figure 3. Again, this module is also designed for simplicity of operation while giving the user confocal Raman spectroscopic capabilities. When used, the laser from the Raman module illuminates the sample. The Raman scattered light is collected from the sample by the microscope objectives and again imaged onto the Raman spectrometer entrance aperture. A Raman spectrum is then collected from a specific sample area.
How the Microscope Spectrometer is Used
The microscope spectrometer is used in many different fields and applications. In materials sciences, these instruments are used to develop and characterize novel materials. The display industry uses them to characterize individual pixels for color consistency (See Figure 4).
In semiconductors, they are used to measure the thickness of thin films (see Figure 5). The biologist uses microscope spectrometers to study vision and the forensic scientist uses them to characterize trace evidence such as textile fibers and paint chips.
The microscope-spectrometer is a device that integrates an optical microscope with a spectrometer to acquire spectra of microscopic sample areas. Such instruments are capable of absorbance and reflectance spectra from the deep ultraviolet through the visible and in to the near infrared regions. The microscope spectrometer can also measure fluorescence, photoluminescence and Raman spectra. These devices are used in many fields including, but not limited to, forensic science, semiconductor and optical thin film thickness measurements, biotechnology and cutting-edge materials science research.