Multimodal microscopy and spectroscopy


Optical microscopy methods are routinely used for spatially-resolved localization and visualization of optical contrast generating probes in various media. Nevertheless, these methods are of limited utility in their ability to analyze the dynamics, interactions, and physical environment of molecules when unassisted by spectroscopy. Imaging spectroscopy methods enable the extension of simple spatial analyses to demonstrate function, co-localization, and molecular interaction.

We are interested in combining different modes of microscopy with spectroscopic tools to better study molecular interactions. We focus on exploiting the sensitivity and specificity of fluorescent probes in an imaging mode to yield spatial, spectral, and temporally resolved information about molecular systems of interest.

Schematic of the multimode microscope. The setup is designed for maximum flexibility with different illumination (widefield transmission or epi-illumination by halogen or mercury lamp; pulsed laser for confocal illumination) and detection (true color intensity, spectra, lifetimes) possibilities.

We have designed and realized a multimode microscope capable of widefield transmission, reflectivity, and emission imaging. The instrument also incorporates confocal spectral and lifetime imaging, enabling convenient high-content imaging of complex samples, allowing the direct correlation of the data obtained from the different modes.

We have used multimodal fluorescence microscopy to research to detect and visualization of J-aggregate coupling of small molecule dyes intercalated into nanochannels in zeolites.

Correlating intensity images with lifetime images plus emission spectra evidenced J-aggregate coupling of small molecule dyes intercalated into nanochannels in zeolites

Currently we are using multimodal fluorescence detection to research the modulation of fluorescent protein emission by nanophotonic structures like inverse opal photonic crystals, and for the visualization of fluorescent proteins micro- and nanopatterned onto surfaces.

In all cases, the combination of different microspectroscopic modes is essential for the resolution of specific photophysical details of the complex systems in question.

Fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) is an increasingly used technique for the study of the dynamic properties of molecules. FCS is a correlation analysis of the fluctuating fluorescence intensity that results from changes in the number of fluorophores in a small focal volume. If the number of fluorophores within the focal volume is low enough, these fluctuations can actually be observed. By correlating the intensity time signals, it is possible to extract the parameters that give rise to the fluctuations, such as the diffusion of molecules, changes in the local concentration of particles, and even chemical reactions.

FCS is a useful and versatile technique that can be used, for example, to study conformational changes, the binding of molecules to membranes, or protein aggregation.

Selected publications


Blum, C., Y. Cesa, M. Escalante, and V. Subramaniam, Multimode Microscopy: Spectral and Lifetime Imaging Journal of the Royal Society Interface, 2009. 6: p. 35-43, doi:10.1098/rsif.2008.0356.focus.


Busby, M., C. Blum, M. Tibben, S. Fibikar, G. Calzaferri, V. Subramaniam, and L. De Cola, Time, Space and Spectrally Resolved Studies on J-Aggregate Interactions in Zeolite-L Nanochannels. Journal of the American Chemical Society, 2008. 130: p. 10970-10976,


Escalante, M., C. Blum, Y. Cesa, C. Otto, and V. Subramaniam, FRET Pair Printing of Fluorescent Proteins. Langmuir, 2009. 25(12): p. 7019-7024, doi:10.1021/la900223f.

PhD student: Niels Zijlstra, Martijn Stopel
Project leader: Christian Blum