Spectrophotometry is a powerful analytical tool used to measure the luminous intensity of various samples across a measurement area of around 1 x 1 centimetres (cm). But what if you want to observe the spectra of microscopic samples? Standard microscopes image samples using visible light with a maximum optical resolving power of two micrometres (μm). However, it is possible to add spectroscopy to UV-visible-NIR optical microscopy to observe spectra and images at high resolutions (1 x 1 μm).
The microspectrophotometer is a hybrid instrument with a few different designations. Some manufacturers use the terms microscope spectrophotometer or microscope spectrometer. We prefer the contracted term microspectrophotometer for the sake of accuracy and simplicity. After all, users cannot simply combine a standard microscope and spectrophotometer in the hopes of obtaining accurate spectral fingerprints from microscopic sample areas. That requires a purpose-built system.
Understanding the Design of a Microspectrophotometer
Microspectrophotometry has enormous advantages, but the breadth of its functionality depends on the configuration of the instrument. Generally, a UV-VIS-NIR microspectrophotometer can measure absorbance, emission spectra, and fluorescence across microscale sample areas. Understanding how this works requires a basic overview of a microspectrophotometer’s anatomy.
A microspectrophotometer’s optical path will vary by design, but generally, a UV-VIS-NIR light source (i.e. a xenon lamp) illuminates a sample via a condenser and a compatible objective acquires the photons emitted, transmitted, or reflected. Naturally, the microscope column must be optimized for both imaging and spectroscopy, meaning it requires an operational spectral range from the deep ultraviolet through to the near-infrared regions of the electromagnetic spectrum. This demands uniform sample illumination and optimal visualization of the whole measurement area plus the surrounding field of view. Positioning the objective’s aperture directly over the measurement area can be a manual or automated process, with the latter requiring high-precision stages.
The obtained spectrum is funnelled through the microscope column, using a series of high-performance lenses and mirrors, and is focused onto the spectrophotometer’s entrance aperture. A diffraction grating is typically used to separate incident light into selected wavelengths across the full measurement range. These separated wavelengths impinge on the multi-channel array detector, typically a CCD (charge-coupled device), enabling different spectra to be measured rapidly and precisely.
Optimal performance hinges on the spectrophotometer's dynamic range, high sensitivity, spectral resolution, and stability. High-performance instruments will allow users to switch between different spectral modes while measuring a single area, to allow for full spectral data acquisition from a single location.
Looking for a Microspectrophotometers?
CRAIC Technologies is a leading supplier of cutting-edge UV-visible-NIR microspectrophotometers built to custom requirements. Our flagship solution is the 2030PV PRO, a fully-integrated micro-analysis instrument with an extremely wide spectral range (200–2500 nm) and a complete suite of spectroscopy capabilities. The 2030PV PRO allows for simultaneous digital imaging of sample areas with micro spectral acquisition, providing unmatched spectral data and superior ease of use. Contact us today for more information about how our microspectrophotometers work.