Estimation of temperature rise at the focus of objective lens at the 1700 nm window

Optical microscopy of biological tissues at the 1700[Formula: see text]nm window has enabled deeper penetration, due to the combined advantage of relatively small water absorption and tissue scattering at this wavelength. Compared with excitation at other wavelengths, such as the commonly used 800[Formula: see text]nm window for two-photon microscopy, water absorption at the 1700[Formula: see text]nm window is more than one order of magnitude higher. As a result, more temperature rise can be expected and can be potentially detrimental to biological tissues. Here, we present theoretical estimation of temperature rise at the focus of objective lens at the 1700[Formula: see text]nm window, purely due to water absorption. Our calculated result shows that under realistic experimental conditions, temperature rise due to water absorption is still below 1[Formula: see text]K and may not cause tissue damage during imaging.

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Functional studies of the kidney of living animals using multicolor two-photon microscopy

AJP Cell Physiology ◽

10.1152/ajpcell.00159.2002 ◽

2002 ◽

Vol 283 (3) ◽

pp. C905-C916 ◽

Cited By ~ 213

Author(s):

Kenneth W. Dunn ◽

Ruben M. Sandoval ◽

Katherine J. Kelly ◽

Pierre C. Dagher ◽

George A. Tanner ◽

...

Keyword(s):

Intravital Microscopy ◽

Cell Biology ◽

Fluid Transport ◽

Biological Tissues ◽

Two Photon Microscopy ◽

Functional Studies ◽

Two Photon ◽

Cell Vitality ◽

The Way

Optical microscopy, when applied to living animals, provides a powerful means of studying cell biology in the most physiologically relevant setting. The ability of two-photon microscopy to collect optical sections deep into biological tissues has opened up the field of intravital microscopy to high-resolution studies of the brain, lens, skin, and tumors. Here we present examples of the way in which two-photon microscopy can be applied to intravital studies of kidney physiology. Because the kidney is easily externalized without compromising its function, microscopy can be used to evaluate various aspects of renal function in vivo. These include cell vitality and apoptosis, fluid transport, receptor-mediated endocytosis, blood flow, and leukocyte trafficking. Efficient two-photon excitation of multiple fluorophores permits comparison of multiple probes and simultaneous characterization of multiple parameters and yields spectral information that is crucial to the interpretation of images containing uncharacterized autofluorescence. The studies described here demonstrate the way in which two-photon microscopy can provide a level of resolution previously unattainable in intravital microscopy, enabling kinetic analyses and physiological studies of the organs of living animals with subcellular resolution.

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Multiplex assessment of the positions of odorant receptor-specific glomeruli in the mouse olfactory bulb by serial two-photon tomography

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1512135112 ◽

2015 ◽

Vol 112 (43) ◽

pp. E5873-E5882 ◽

Cited By ~ 20

Author(s):

Bolek Zapiec ◽

Peter Mombaerts

Keyword(s):

Olfactory Bulb ◽

Surface Area ◽

Odorant Receptor ◽

Three Dimensional ◽

Two Photon Microscopy ◽

Two Photon ◽

Automated Method ◽

Order Of Magnitude ◽

Or Gene ◽

Or Genes

In the mouse, axons of olfactory sensory neurons (OSNs) that express the same odorant receptor (OR) gene coalesce into one or a few glomeruli in the olfactory bulb. The positions of OR-specific glomeruli are traditionally described as stereotyped. Here, we have assessed quantitatively the positions of OR-specific glomeruli using serial two-photon tomography, an automated method for whole-organ fluorescence imaging that integrates two-photon microscopy with serial microtome sectioning. Our strategy is multiplexed. By repeated crossing, we generated two strains of mice with gene-targeted mutations at four or five OR loci for a total of six ORs: MOR23 (Olfr16), mOR37A (Olfr155), M72 (Olfr160), P2 (Olfr17), MOR256-17 (Olfr15), and MOR28 (Olfr1507). Glomerular imaging relied on intrinsic fluorescence of GFP or DsRed, or on whole-mount immunofluorescence with antibodies against GFP, DsRed, or β-gal using the method of immunolabeling-enabled three-dimensional imaging of solvent-cleared organs (iDISCO). The high-resolution 3D-reconstructed datasets were segmented to identify the labeled glomeruli and to assess glomerular positional variability between the bulbs of one mouse (intraindividual) and among the bulbs of different mice (interindividual). In 26 mice aged 21 or 50 d or 10 wk, we made measurements of the positions of 352 glomeruli. We find that positional variability of glomeruli correlates with the OR: For instance, the medial MOR28 glomerular domain occupies a surface area that is an order of magnitude larger than the surface area of the medial MOR23 glomerular domain. Our results quantify the level of precision that is delivered by the mechanisms of OSN axon wiring, differentially for the various OSN populations expressing distinct OR genes.

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