Laser Scanning Multiphoton Microscopes
Figure 6: A) Nose-to-Brain (N2B) drug delivery WSI image of a sagittal mouse head cryosection. Cell nuclei (blue), neurofilament NF200 (green), mouse IgG (red). Scale bar = 1 mm. B–D) Multiphoton close-up images of the neuronal bundle shows a time series of drug exposure for 2h (B), 4h (C), and 20 h (D). Cell nuclei (blue), NF200 (green), mouse IgG (red), and SHG-signal (magenta). Scale bar = 50 μm. E–F) Epifluorescence images show the transport scheme with intracellular uptake at the apical mucosa, distribution to the lamina propria, and transport along neuronal bundles from the lamina propria to the olfactory bulb 0–48 h after nasal application of the drug. Cell nuclei (blue), NF200 (green), mouse IgG (red). Scale bars = 100 μm. See Reference 9.
New methods of intravenous drug delivery techniques to
the CNS, for example, refined catheter-based administration at the olfactory or respiratory area with an adjusted smaller administered volume, are promising. Multimodal microscopy proved very beneficial for better visualizing the full transport scheme with time-dependent intracellular uptake and antibody distribution and for observing immunoreactivity along neu- ronal bundles. Tis method was successful in increasing the uptake via an olfactory mechanism and for decreasing the intake in peripheral tissues, such as muscles and surrounding areas.
Multimodal Microscopy in Tissue Engineering Te field of tissue engineering focuses on tissue regenera-
tion and aims to find approaches to replace injured or patho- genic tissue with ex vivo engineered tissue. Tissue engineered constructs mainly consist of scaffolds that serve as biodegrad- able carrier systems for the cells. Tus, multiphoton microscopy is a requirement to image the constructs mimicking native 3D tissue not only at the surface, but also deep in the tissue. A com- mon problem is the limited deep migration of cells and hypoxia due to insufficient vasculature with increasing size of the scaf- folds. For imaging deep into these 3D constructs, multiphoton imaging is far superior to classical confocal microscopy. Figure 7 shows different approaches of scaffold design and
materials in the field of tissue engineering. Popular scaffold materials are collagen types 1 or 2, which show self-assembly capacity of fiber-based networks. Due to native integrin recog- nition sequences such as the “RGD-motif,” which is frequently found in collagens, the collagen scaffold provides multiple adhe- sion points for cell attachment. Most cells are highly adaptable
22
to their surrounding environment and can remodel the collagen depending on their needs. Multiple tissue types can be devel- oped through varying the molecular composition, the selection of cells, and the chemical and mechanical parameters during the cultivation of these constructs. Another approach is to predefine the exact shape of the cell growth area by nano 3D printing, as shown in Figure 7A. Here, native decellularized tissue is used as a template, which can then be adapted or optimized by a CAD program. For 3D printing, cell-compatible non-toxic biomateri- als such as GM10 are used. Aſter printing the scaffold, cells are seeded and grown on the scaffold according to the template.
Conclusion Te use of multiphoton imaging has rapidly spread
throughout biomedical research. Experiments are becoming more complex, that is, using awake animals, and differ sig- nificantly from standard setups. Te future requires an all- in-one microscope that is agile and adaptable, with features that lower the technical barrier, expand on diagnostics with multiple modes, and save resources and time while improv- ing results. Systems also need to be industry-ready, portable, and easily operated in any indoor environment, even the home office. Te MPX satisfies these requirements, advancing LSMM microscopy research and translating it to a wide range of med- ical applications. Te 3D scan head is tightly integrated and includes the Prospective Instruments tuneable ultrafast fiber laser, perfectly matched for multiphoton microscopy, allowing robust and 24/7 reliable operation. Te need for a highly flexible microscope with easily and quickly switchable modes is a premium feature for advanced
www.microscopy-today.com • 2022 May
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68 |
Page 69 |
Page 70 |
Page 71 |
Page 72
