Leica DLS Light Film Imaging Module Upgrade Confocal Microscopy: Faster Imaging and Less Phototoxicity

Tracking the growth of fungi and the formation of biofilms is of great importance in the food and pharmaceutical industries: understanding the patterns and mechanisms of fungal growth can help us use beneficial fungi more effectively and contain harmful bacteria. The growth of fungi at different developmental stages depends on intracellular activity and the biochemical environment in which it is located. However, confocal microscopy and electron microscopy techniques that are currently used are not perfect for this task.

Published in ChemPhysChem's "Exploring Morphological and Biochemical Linkages in Fungal Growth with Label-Free Light Sheet Microscopy and Raman Spectroscopy" using Leica light sheet technology DLS, directly using the autofluorescence of the fungus itself: due to different metabolic states, this autofluorescence "specificity The mark "is on the spores, while the hyphae are essentially absent. It can be seen from the figure that the distribution of spores is clearly seen by using DLS: toward the top, a gradient distribution, and the closer to the top, the greater the density of spores. The imaging depth is 1.1mm, which fully satisfies the study of intact fungi. This gradient distribution indicates that there is an internal growth regulation mechanism in the fungus. Combined with Surface-Enhanced Raman Spectroscopy (SERS), the complex physicochemical interactions are directly related to the morphological characteristics of fungal growth, providing a study of the relationship between secondary metabolites and fungal growth. Very good tool.

Let's first take a look at what is the light microscopy technology [2]:

Fluorescent samples are excited from the side of the sample using a beam of light, detected using CCD or sCMOS, and the illumination path and the fluorescence detection path are perpendicular to each other. Since the plane in which the sample is excited is the imaging plane, there is no defocus excitation, and optical slicing can be automatically obtained, thereby minimizing photobleaching and optical damage. The light film microscopy system uses CCD or sCMOS imaging, and the speed is usually tens of frames per second or even hundreds of frames. Therefore, by moving the sample under the light sheet to inject the incident light beam into different planes, the whole can be easily and very quickly obtained. 3D map of the organization.

Then we will also find that traditional light microscopy microscopes usually require the installation of dedicated optics in a separate system, where the illumination and detection objectives are arranged perpendicular to each other. However, in practical applications, many times we need both light and confocal functions.

The Leica TCS SP8 DLS creatively combines optical and confocal imaging : the light is placed directly on the confocal platform, and the unique TwinFlect mirror unit allows the excitation beam to be incident on the sample from both left and right, with uniform illumination. and eliminate the natural double-sided lighting dark areas to ensure that the interference levels of resolution cells. The fast imaging speed , high resolution and low phototoxicity allow the sample to retain its biological activity in the system, completing long-lived biological culture and imaging for hours or even days.

Light sheet microscope has never been easy, but at the same time without sacrificing the confocal functionality, the joint can also be achieved using a focused light sheet microscope and common, complete activation light, the light conversion operation and follow-up experiments.