Cell damage in two-photon microscopes

Koenig, Karsten; So, Peter T. C.; Mantulin, William W.; Gratton, Enrico

doi:10.1117/12.260794

Cited by 5 publications

(4 citation statements)

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“…The average power densities (0.2mW/mm 2 ) and pulse energy densities (0.04nJ/mm 2 ) used in TeFo imaging are $100x and $5x lower than the corresponding values for a DL PSF (20mW/mm 2 and 0.2nJ/mm 2 ). Reported safe power densities are in the range of 2-5mW/mm 2 and safe pulse energy densities are in the range of 0.025-0.06nJ/mm 2 (Ko ¨nig et al, 1997;Hopt and Neher, 2001). Thus, our conditions are more than an order of magnitude below both the established linear heat-induced damage threshold and the threshold for non-linear damage while at the same time being about 100x (average power density) and 5x (pulse energy density) lower than typical values for standard DL 2p scanning microscopy.…”

Section: Star+methodsmentioning

confidence: 99%

Volumetric Ca2+ Imaging in the Mouse Brain Using Hybrid Multiplexed Sculpted Light Microscopy

Weisenburger

Tejera

Demas

et al. 2019

Cell

178

148

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Calcium imaging using two-photon scanning microscopy has become an essential tool in neuroscience. However, in its typical implementation, the tradeoffs between fields of view, acquisition speeds, and depth restrictions in scattering brain tissue pose severe limitations. Here, using an integrated systems-wide optimization approach combined with multiple technical innovations, we introduce a new design paradigm for optical microscopy based on maximizing biological information while maintaining the fidelity of obtained neuron signals. Our modular design utilizes hybrid multi-photon acquisition and allows volumetric recording of neuroactivity at single-cell resolution within up to 1 3 1 3 1.22 mm volumes at up to 17 Hz in awake behaving mice. We establish the capabilities and potential of the different configurations of our imaging system at depth and across brain regions by applying it to in vivo recording of up to 12,000 neurons in mouse auditory cortex, posterior parietal cortex, and hippocampus.

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Section: Star+methodsmentioning

confidence: 99%

Volumetric Ca2+ Imaging in the Mouse Brain Using Hybrid Multiplexed Sculpted Light Microscopy

Weisenburger

Tejera

Demas

et al. 2019

Cell

178

148

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“…This imaging modality allowed research groups to pursue bioimaging at larger depths and excite samples in ultraviolet range with lesser phototoxicity (19,20). Instead of the traditional NADH excitation at 340-390nm, multiphoton excitation at 740nm-780nm was employed, which presented no toxicity (unlike the UV radiation) at moderate laser powers(<30mW)(21). Another advantage was that most multiphoton microscopes used a mode-locked laser which had excellent temporal instrument response function that helped FLIM to separate fluorescence signal from excitation laser.…”

Section: Introductionmentioning

confidence: 99%

Autofluorescence lifetime imaging of cellular metabolism: Sensitivity toward cell density, pH, intracellular, and intercellular heterogeneity

Chacko

Eliceiri

2018

Cytometry Pt A

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Autofluorescence imaging (AFI) has greatly accelerated in the last decade, way past its origins in detecting endogenous signals in biological tissues to identify differences between samples. There are many endogenous fluorescence sources of contrast but the most robust and widely utilized have been those associated with metabolism. The intrinsically fluorescent metabolic cofactors Nicotinamide adenine dinucleotide (NAD+/NADH) and Flavin adenine dinucleotide (FAD/FADH2) have been utilized in a number of AFI applications including basic research, clinical and pharmaceutical studies. Fluorescence lifetime imaging microscopy (FLIM) has emerged as one of the more powerful AFI tools for NADH and FAD characterization due to its unique ability to non-invasively detect metabolite bound and free states and quantitate cellular redox ratio. However, despite this widespread biological use, many standardization methods are still needed to extend FLIM based AFI into a fully robust research and clinical diagnostic tools. FLIM is sensitive to a wide range of factors in the fluorophore microenvironment and there a number of analysis variables as well. To this end there has been an emphasis on developing imaging standards and ways to make the image acquisition and analysis more consistent. However biological conditions during FLIM based AFI imaging are rarely considered as key sources of FLIM variability. Here we present several experimental factors with supporting data of the cellular microenvironment such as confluency, pH, inter/intra cellular heterogeneity, and choice of cell line that need to be considered for accurate quantitative FLIM based AFI measurement of cellular metabolism.

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“…This technique also allows deep penetration into tissues compared to other microscopic systems by using infrared wavelengths and minimizes photobleaching and damage compared to white light in confocal microscopy. Two-photon microscopy enables 3D fluorescence imaging of live cells of tissues or organs and allows for timelapse imaging of photosensitive samples with minimal phototoxicity over a long period 3,4,5 . It has been used to study the mechanism of glioblastoma cells in the tumor microenvironment, cellular interactions that contribute to biliary membrane integrity, and fast spatiotemporal cellular dynamics within neural networks.…”

Section: Introductionmentioning

confidence: 99%

Stabilized two-photon intravital imaging using dexmedetomidine

Kim¹,

Cho²,

Paulson³

et al. 2023

Advanced Biomedical and Clinical Diagnostic and Surgical Guidance Systems XXI

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Two-photon microscopy provides subcellular-resolution imaging deep into animal tissues, and is frequently used for the study of biological and disease processes. However, its applicability in vivo is complicated by physiological movement. We demonstrate treatment with dexmedetomidine (DEX), an already FDA-approved alpha-2 adrenergic receptor agonist, to reduce tissue oscillation frequency and amplitude in the livers of anesthetized mice. Fluorescence intensity and focal quality fluctuations were found to improve for 30 minutes after administration, indicating that dexmedetomidine may be used to improve imaging quality in two-photon intravital microscopy studies. These results will likely generalize to other imaging modalities, target tissues, and animal models.

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Cell damage in two-photon microscopes

Cited by 5 publications

References 0 publications

Volumetric Ca2+ Imaging in the Mouse Brain Using Hybrid Multiplexed Sculpted Light Microscopy

Volumetric Ca2+ Imaging in the Mouse Brain Using Hybrid Multiplexed Sculpted Light Microscopy

Autofluorescence lifetime imaging of cellular metabolism: Sensitivity toward cell density, pH, intracellular, and intercellular heterogeneity

Stabilized two-photon intravital imaging using dexmedetomidine

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