The Science of Microspectroscopy
Uses of Microspectrophotometers
Units of UV-Visible-NIR Microspectroscopy
Algorithms of UV-Visible-NIR Microspectroscopy
20/30 PV™ Microspectrophotometer
General diagram of a transmission microspectrophotometer
The UV-visible-NIR microspectrophotometer is designed to measure the spectrum of microscopic areas or microscopic samples. It can be configured to measure the transmittance, absorbance, reflectance, polarization and fluorescence of sample areas as smaller than a micron.
The microspectrophotometer is sometimes called a microscope spectrophotometer as it combines an optical microscope and a highly sensitive spectrophotometer. In actuality, the microspectrophotometer is a fully integrated, purpose built instrument while the microscope spectrophotometer is an add-on component for a standard microscope. As such, add-on components have certain performance limitations due to the microscope itself. An integrated instrument avoids those limitations as it is designed specifically for microspectroscopy.
Individually, the microscope is an optical instrument that uses lenses and mirrors to produce magnified images of microscopic objects or microscopic areas of larger objects. The sample is illuminated in one several ways:
- Transmission illumination where light is focused onto the sample by the condenser. The portion collected by the microscope objective has been transmitted through the sample
- Incident or reflectance illumination where light is again focused onto the sample but through the objective. The reflected light from the sample is collected by the same objective. Reflectance can be further subdivided into specular and diffuse incident illumination.
- Emission: where the sample emits light after the sample has been stimulated in some manner. One example is fluorescence where a fluorophore is excited with one wavelength of light and emits light of a different wavelength. The emitted light is collected by the microscope objective.
The objective of the microscope is used to collect the light from the sample and focus it on the spectrophotometer aperture. The digital imaging system images both the aperture and the sample simultaneously so that it can present an image that shows what the spectrophotometer aperture is measuring..
The spectrophotometer portion of the microspectrophotometer is an optical instrument for measuring the intensity of light relative to its wavelength. The electromagnetic energy, collected from the sample, enters the device through the aperture and is separated into its component wavelengths by a diffraction grating. The separated light is then focused onto a CCD array detector where the intensity of each wavelength is then measured by a pixel of the array. The CCD is then read to a computer and the result is a spectrum which displays the intensity of each wavelength of light.
In a microspectrophotometer, such as those made by CRAIC Technologies, the spectrophotometer is integrated with a specially designed microscope. The microscope optics and light sources are of the highest quality and able to operate in the ultraviolet, visible and near infrared regions (normal microscopes only operate in the visible range). The spectrophotometer is built into the microscope, along with a digital imaging system, so that the maximum amount of light can be collected from the smallest samples. As such, microspectrophotometers are very flexible instruments able to measure absorbance, transmittance, reflectance, and emission (such as fluorescence) spectra of even sub-micron sized samples.