Light-emitting diode (LED) illumination for fluorescence microscopy systems has come a long way since the turn of the century, when the LED sources available had low optical power and a limited range of wavelengths. While the benefits of LED technology were attractive, their limitations prevented large-scale adoption. Twenty years later, advances in the semi-conductor industry have eliminated many of those fundamental hurdles. Today, LED technology is rapidly replacing the lamps used in microscopy and fluorescence imaging, even for applications on the extreme ends of the light microscopy spectrum. This article describes the latest advances in illumination for fluorescence imaging, from near-UV to near-IR fluorophores. Case studies demonstrate the new X-Cite® NOVEM™ as successful replacements for traditional arc lamps in calcium imaging applications, producing equivalent results with the convenience of LEDs. For its usefulness to the microscopy community, the X-Cite NOVEM system was awarded the prestigious 2021 Microscopy Today Innovation Award.

Combining the biological specificity of fluorescence microscopy with topographical features revealed by atomic force microscopy (AFM) provides new insights into cell biology. However, the lack of systematic alignment capabilities especially in scanning-tip AFM has limited the combined application approach as AFM drift leads to increasing image mismatch over time. We present an alignment correction method using the cantilever tip as a reference landmark. Since the precise tip position is known in both the fluorescence and AFM images, exact re-alignment becomes possible. We used beads to demonstrate the validity of the method in a complex artificial sample. We then extended this method to biological samples to depict membrane structures in fixed and living human fibroblasts. We were able to map nanoscale membrane structures, such as clathrin-coated pits, to their respective fluorescent spots. Reliable alignment between fluorescence signals and topographic structures opens possibilities to assess key biological processes at the cell surface such as endocytosis and exocytosis.

The painting materials and drawing techniques of a pair of two-panel folding screens entitled Red and White Plum Blossoms by Ogata Korin, a national treasure in Japan, were investigated directly and nondestructively by XRF, X-ray radiography, and high resolution digital imaging. Several assumptions were made about the materials used in the red and white plum trees drawn on the right and left screens, respectively, and the river drawn at the center. By the present investigation, the materials used for the paintings were revealed, and some of them contradicted what have been previously believed.

Cellular autofluorescence, though ubiquitous when imaging cells and tissues, is often assumed to be small in comparison to the signal of interest. Uniform estimates of autofluorescence intensity obtained from separate control specimens are commonly employed to correct for autofluorescence. While these may be sufficient for high signal-to-background applications, improvements in detector and probe technologies and introduction of spectral imaging microscopes have increased the sensitivity of fluorescence imaging methods, exposing the possibility of effectively probing the low signal-to-background regime. With spectral imaging, reliable monitoring of signals near or even below the noise levels of the microscope is possible if compensation for autofluorescence and background signals can be performed accurately. We demonstrate the importance of accurate autofluorescence modeling and the utility of spectral imaging and multivariate analysis methods using a case study focusing on fluorescence confocal spectral imaging of host-pathogen interactions. In this application fluorescent proteins are produced when Francisella novicida invade host macrophage cells. The resulting analyte signal is spectrally overlapped and typically weaker than the cellular autofluorescence. In addition to discussing the advantages of spectral imaging for following pathogen invasion, we present the spectral properties and cellular origin of macrophage autofluorescence.

Light-emitting diodes (LEDs) can be easily and inexpensively integrated into modern light microscopes. There are numerous advantages of LEDs as illumination sources; most notably, they provide brightness and spectral control. We demonstrate that for transmitted light imaging, an LED can replace the traditional tungsten filament bulb while offering longer life; no color temperature change with intensity change; reduced emission in the infrared region, which is important for live cell imaging; and reduced cost of ownership. We show a direct substitution of the typical tungsten bulb with a commercially available LED and demonstrated the color stability by imaging a histology section over a wide range of light intensities. For fluorescent imaging, where the typical illumination sources are mercury or xenon lamps, we demonstrate that LEDs offer advantages of providing a longer lifespan, having a more constant intensity output over time, more homogeneous illumination, and significantly lower photon dose. Our LED equipped system was used to image and deconvolve dual fluorescently labeled cells, as well as image cells undergoing mitosis expressing green fluorescent protein–histone 2B complex. The timing of the stages of mitosis is well established as an indicator of cell viability.