A hyphenated instrument is one that blends the capabilities of two different technologies to form a new analytical technique with novel abilities. The microscope spectrophotometer is one such hyphenated instrument; it is a hybrid that combines the magnifying power of an optical microscope with analytical proficiencies of a UV-visible-NIR range spectrophotometer. As such, microscope spectrophotometers can be used to measure the molecular spectra of microscopic sample areas from the deep ultraviolet to the near infrared region. They can be configured for many different types of spectroscopy and, as such, are used to measure absorbance, reflectance, and even emission spectra, such as fluorescence and photoluminescence, of micron-sized samples. With the addition of specialized algorithms, the microscope spectrophotometer can also be used to measure the thickness of thin films or act as a colorimeter for microscopic samples.
There are many reasons to use the microscope spectrophotometer. The most obvious is that spectra can be acquired from a sample area smaller than a micron. Additionally, 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. And color comparisons by spectroscopy tend to be more accurate with spectrophotometers 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, these methods tend to be destructive, require a lot of samples, and suffer from the inaccuracies of the human visual system. The microscope spectrophotometer avoids these issues and can “see” beyond the range of the human eye and detect variations that would not otherwise be apparent.
Microscope Spectrophotometer Design
The microscope spectrophotometer integrates an optical or light microscope with a UV-visible-NIR range spectrophotometer (Figure 1). The microscope is a device designed to enlarge an image of small objects to allow them to be studied. The spectrophotometer is an instrument that measures the intensity of each wavelength of light from the ultraviolet through the visible and near infrared regions. With a properly configured microscope spectrophotometer, one is able to acquire absorbance, reflectance, and emission spectra with sampling areas on the submicron scale.
In order to cover such a broad spectral range with good image and spectral quality, a custom designed microscope is built and integrated with the spectrophotometer. Standard optical microscopes have a limited spectral range covering only a portion of the visible region because of the materials used for the optics as well as the light sources themselves. The modern microscope spectrophotometer utilizes a custom-built microscope with an optical design and light sources optimized for the deep UV through the NIR.
The spectrophotometer itself must also be designed for microspectroscopy in order to obtain good spectral results. This means that the spectrophotometer must be highly sensitive while still maintaining an acceptable spectral resolution. Stability is also an issue since the microscope spectrophotometer 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 spectroscopy when measuring the same sample. This allows you 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, but also see the surrounding sample. 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. In Figure 2, 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.
As shown in Figure 3, the microscope optics focus light onto the sample. The electromagnetic energy is then collected from the sample by the microscope objective. The light from the objective is focused onto the mirrored entrance aperture of the spectrophotometer. The majority of the light is reflected from the entrance aperture surface onto the camera. The spectrophotometer aperture is also imaged by the camera so that it appears as a black square on the sample (Figure 2). This allows for easy and quick alignment of the microscope spectrophotometer. The light that passes through the entrance aperture then passes into the spectrophotometer where a spectrum is measured.
The microscope can be configured with different illumination schemes depending upon the type of experiment to be performed. Incident illumination with white light allows for reflectance microspectroscopy from the deep UV to the near IR. Incident illumination can also be used for fluorescence or photoluminescence microspectroscopy. In addition, transmission microspectroscopy is possible with white light focused onto the sample through the microscope condenser.
Applications of Microspectroscopy
The first microscope spectrophotometers were developed in the 1940s and since then, a host of different applications have been developed. With the ability to acquire spectra of microscopic sample areas, microscope spectrophotometers are used everywhere from university laboratories to production lines for quality control and failure analysis.
Forensic Science. The analysis of forensic evidence has been one of the most important applications for microscope spectrophotometers since the early 1980s. The greatest effort was in the analysis of trace evidence, specifically textile fibers and paint chips1, 2. As their names suggest, these types of samples are usually microscopic and, being evidence, should not be damaged or destroyed by testing. With fibers, microscope spectrophotometers are used to measure the UV-visible-NIR absorbance and fluorescence spectra of individual fibers. Paint chips are usually cross-sectioned and then the absorbance spectrum of each layer is measured so that known and questioned samples may be compared with a high degree of discrimination.
Flat Panel Displays. Modern flat panel displays consist of millions of multi-colored pixels. As the technology has progressed, the pixels become ever smaller and more tightly packed across ever larger surfaces. The most modern displays use different technologies, such as quantum dots and organic light emit diodes, to create pixels of different colors on the microscopic scale. The microscope spectrophotometer is used to help develop these materials as viable light sources and ultimately as displays 3,4. The microscope spectrophotometer is also used in the production process to ensure that both the color and the intensity of the pixels are consistent across the entire display, thereby ensuring bright and evenly illuminated images across the display.
Energy. Coal and petroleum source rock contain vitrinite and other macerals. Microscope spectrophotometers are used to grade the thermal maturity5, and therefore the energy content, of coal, coke and petroleum source rock. This is done by measuring the absolute reflectivity of vitrinite on a polished sample. Depending upon the reflectivity, the thermal maturity of the sample may be determined.
Nanotechnology. The microscope spectrophotometer is also advancing nanotechnology and materials science based on their ability to measure transmission, reflectance, and emission spectra microscopic sample areas. One rapidly growing area of applications is in the development and use of surface plasmon resonance (SPR)6,7,8.
Surface plasmons are excited by illuminating a planar metallic surface or nanoscale metallic particles with light (Figure 4). Changes in the optical characteristics of these materials occur when these nanoparticles or surfaces interact with other materials. As such, much work is being done to develop new materials that exhibit some form of plasmon resonance, but also to build devices that feature these phenomena. The latter includes biosensors and microfluidic device sensors of various types. The microscope spectrophotometer measures how the spectra of the SPR materials change under different conditions giving the researcher the ability to characterize a new material, then to “tune” that material for specific optical effects.
The microscope spectrophotometer is a hyphenated technique that combines the optical microscope with a spectrophotometer so that one can acquire spectra of microscopic sample areas. Such instruments are capable of absorbance and reflectance spectra from the deep UV through the visible and into the near infrared regions. The microscope spectrophotometer can also measure fluorescence and other types of emission spectra. These devices have found uses in many fields including forensic science, semiconductor and optical film thickness measurement, biotechnology, and the latest in materials science.
- S. Walbridge-Jones, Microspectrophotometry for textile fiber color measurement, Identification of Textile Fibers, Woodhead Publishing, 2009, Pages 165-180,
- Standard Guide for Microspectrophotometry in Forensic Paint Analysis, American Society of Testing and Materials.
- Buchnev, O., Podoliak, N., & Fedotov, V. A. (2018). Liquid crystal-filled meta-pixel with switchable asymmetric reflectance and transmittance. J. Molecular Liquids, 267, 411-414.
- Rezaei, S. D., Hong Ng , R. J., Dong, Z., Ho, J., Koay, E. H., Ramakrishna, S., & Yang, J. K. (2019). Wide-gamut plasmonic color palettes with constant subwavelength resolution. ACS nano, 13 (3), 3580-3588.
- “Methods for the petrographic analysis of coals – Part 5: Method of determining microscopically the reflectance of vitrinite”, ISO 7404-5, International Organization for Standardization, 2009.
- Ng, R.J.H., Krishnan, R.V., Dong, Z., Ho, J., Liu, H., Ruan, Q., Pey, K.L. and Yang, J.K (2019). Micro-tags for art: covert visible and infrared images using gap plasmons in native aluminum oxide. Optical Materials Express, 9 (2), 788-801.
- Alali, M., Yu, Y., Xu, K., Ng, R.J., Dong, Z., Wang, L., Dinachali, S.S., Hong, M. and Yang, J.K. (2016). Stacking of colors in exfoliable plasmonic superlattices. Nanoscale, 8 (42), 18228-18234.
- Jiang, M., Siew, S.Y., Chan, J.Y., Deng, J., Wu, Q.Y.S., Jin, L., Yang, J.K., Teng, J., Danner, A. and Qiu, C.W., (2020). Patterned resist on flat silver achieving saturated plasmonic colors with sub-20-nm spectral linewidth. Materials Today, 35, 99-105.