The search for novel bioimaging techniques is essentially driven by the need to increase our knowledge about diseases, with the objective of investigating their critical molecular basis in their environment. This usually occurs at the cellular or sub-cellular level, making spatial resolution and time–resolved observation critical issues. Many techniques have failed because of their inability to reach targets revealing the development or occurrence of a disease and/or because they damage or alter the samples. The development of modes of imaging for biological research requires the merging of specific achievements in imaging technology and related methods for reliable, safe, and reproducible analysis of biosamples. UV-fluorescence-based microscopes have become routine tools for biological research because of their exceptional analytical performance. First, their main strength is to remove any doubt about the result. The number of molecular probes is growing every day, giving the sensation that almost everything can be probed. They can also be coupled to morphological (3D confocal) or topographic (3D non-confocal) techniques with satisfactory resolution, well below 1 μm, thus enabling determination of the location of probes down to the sub-cellular level. Another important advantage is their capability of time-resolved and kinetic measurements, thus making live imaging possible. However, these approaches are rather limited by the number of probes that can be used at the same time, usually no more than 2– 4. Another major limit is the predetermined way of investigating a disease: probes usually target given molecules, which can be relevant for diagnosis, but this remains largely questionable for exploratory studies. Other limits are also reducing interest in these techniques, for example photobleaching of fluorescent probes over time, and the possibility of false-positive results. For these reasons, probe-free techniques able to provide global information about the sample are now being developed as non-oriented means of analysis in the biological sciences. Spectroscopic techniques are among the best candidates for such coupling to 3D imaging; some have also been developed for non-destructive analysis of biosamples. Fourier-transform infrared (FTIR), X-ray fluorescence (XRF), and Raman techniques enable laterally or spatially resolved chemical mapping of biosamples. The recent commercial release of multimodal systems combining AFM with FTIR or Raman microscopy shows the trend toward utilization of morpho-spectral imaging in the biosciences. Other initiatives include the use of ellipsometry, another non-destructive technique, for rapid 3D analysis of thin samples; this technique can be also coupled to IR microscopy for safe multimodal analysis of biosamples. Ellipsometry, and X-ray microscopy with a CCD camera, can also be regarded as valuable tools for timeresolved studies, at least down to the scale of seconds. Coupling of X-ray fluorescence microscopy to X-ray phase-contrast (XR-PC) imaging in synchrotron radiation facilities has also been tentatively proposed; this would enable elemental characterization of samples with 3D rendering. Therefore, several hybrid imaging arrangements are—or can be—proposed for characterization of biosamples with time-resolved, high-resolution, and in-vivo applications, potentially all combined. Biosafety issues have also become a major issue in routine in-vivo imaging and now concern all applications, from fundamental biological research to clinical routines. Morpho-spectral imaging approaches can result in great achievements in biological research only if biological Published in the special paper collection Imaging Techniques with Synchrotron Radiation with guest editor Cyril Petibois.