Articles de revista
http://hdl.handle.net/2117/23849
2024-03-29T06:58:42Z
-
DNA Origami offers a versatile method for quantifying protein copy‐number in superresolution
http://hdl.handle.net/2117/109680
DNA Origami offers a versatile method for quantifying protein copy‐number in superresolution
Zanacchi, Francesca Cella; Manzo, Carlo; Sandoval Alvarez, Angel; Derr, Nathan D.; Garcia-Parajo, Maria F.; Lakadamyali, Melike
Single-molecule-based super-resolution microscopy offers researchers a unique opportunity to quantify protein copy number with nanoscale resolution. However, while fluorescent proteins have been characterized for quantitative imaging using calibration standards, similar calibration tools for immunofluorescence with small organic fluorophores are lacking. Here we show that DNA origami, in combination with GFP antibodies, is a versatile platform for calibrating fluorophore and antibody labeling efficiency to quantify protein copy number in cellular contexts using super-resolution microscopy.
2017-11-02T16:16:03Z
Zanacchi, Francesca Cella
Manzo, Carlo
Sandoval Alvarez, Angel
Derr, Nathan D.
Garcia-Parajo, Maria F.
Lakadamyali, Melike
Single-molecule-based super-resolution microscopy offers researchers a unique opportunity to quantify protein copy number with nanoscale resolution. However, while fluorescent proteins have been characterized for quantitative imaging using calibration standards, similar calibration tools for immunofluorescence with small organic fluorophores are lacking. Here we show that DNA origami, in combination with GFP antibodies, is a versatile platform for calibrating fluorophore and antibody labeling efficiency to quantify protein copy number in cellular contexts using super-resolution microscopy.
-
Super-resolution microscopy: going live and going fast
http://hdl.handle.net/2117/109676
Super-resolution microscopy: going live and going fast
Lakadamyali, Melike
Super-resolution microscopy is increasingly becoming an important
tool for biological research, providing valuable information at the
nanometer length scales inside cells and tissues. In the past decade
numerous technological advancements have transformed superresolution
microscopes into powerful tools of discovery. While the first
super-resolution images took several hours to acquire, recent
progress has led to tremendous improvement in acquisition speed,
enabling researchers to probe dynamic processes in living cells with
unprecedented spatiotemporal resolution. This mini-review focuses
on the recent developments in live-cell super-resolution microscopy
and its biological applications.
2017-11-02T16:00:02Z
Lakadamyali, Melike
Super-resolution microscopy is increasingly becoming an important
tool for biological research, providing valuable information at the
nanometer length scales inside cells and tissues. In the past decade
numerous technological advancements have transformed superresolution
microscopes into powerful tools of discovery. While the first
super-resolution images took several hours to acquire, recent
progress has led to tremendous improvement in acquisition speed,
enabling researchers to probe dynamic processes in living cells with
unprecedented spatiotemporal resolution. This mini-review focuses
on the recent developments in live-cell super-resolution microscopy
and its biological applications.
-
Human N-Methyl-D-aspartate receptor antibodies alter memory and behavior in mice
http://hdl.handle.net/2117/88911
Human N-Methyl-D-aspartate receptor antibodies alter memory and behavior in mice
Planagumà, Jesús; Leypoldt, Frank; Mannara, Francesco; Gutiérrez-Cuesta, Javier; Martín-García, Elena; Aguilar, Esther; Titulaer, Maarten J.; Jain, Ankit; Balice-Gordon, Rita; Lakadamyali, Melike; Graus, Francesc; Maldonado, Rafael; Dalmau, Josep
Anti-N-methyl D-aspartate receptor (NMDAR) encephalitis is a severe neuropsychiatric disorder that associates with prominent memory and behavioural deficits. Patients’ antibodies react with the N-terminal domain of the GluN1 (previously known as NR1) subunit of NMDAR causing in cultured neurons a selective and reversible internalization of cell-surface receptors. These effects and the frequent response to immunotherapy have suggested an antibody-mediated pathogenesis, but to date there is no animal model showing that patients’ antibodies cause memory and behavioural deficits. To develop such a model, C57BL6/J mice underwent placement of ventricular catheters connected to osmotic pumps that delivered a continuous infusion of patients’ or control cerebrospinal fluid (flow rate 0.25 µl/h, 14 days). During and after the infusion period standardized tests were applied, including tasks to assess memory (novel object recognition in open field and V-maze paradigms), anhedonic behaviours (sucrose preference test), depressive-like behaviours (tail suspension, forced swimming tests), anxiety (black and white, elevated plus maze tests), aggressiveness (resident-intruder test), and locomotor activity (horizontal and vertical). Animals sacrificed at Days 5, 13, 18, 26 and 46 were examined for brain-bound antibodies and the antibody effects on total and synaptic NMDAR clusters and protein concentration using confocal microscopy and immunoblot analysis. These experiments showed that animals infused with patients’ cerebrospinal fluid, but not control cerebrospinal fluid, developed progressive memory deficits, and anhedonic and depressive-like behaviours, without affecting other behavioural or locomotor tasks. Memory deficits gradually worsened until Day 18 (4 days after the infusion stopped) and all symptoms resolved over the next week. Accompanying brain tissue studies showed progressive increase of brain-bound human antibodies, predominantly in the hippocampus (maximal on Days 13–18), that after acid extraction and characterization with GluN1-expressing human embryonic kidney cells were confirmed to be against the NMDAR. Confocal microscopy and immunoblot analysis of the hippocampus showed progressive decrease of the density of total and synaptic NMDAR clusters and total NMDAR protein concentration (maximal on Day 18), without affecting the post-synaptic density protein 95 (PSD95) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. These effects occurred in parallel with memory and other behavioural deficits and gradually improved after Day 18, with reversibility of symptoms accompanied by a decrease of brain-bound antibodies and restoration of NMDAR levels. Overall, these findings establish a link between memory and behavioural deficits and antibody-mediated reduction of NMDAR, provide the biological basis by which removal of antibodies and antibody-producing cells improve neurological function, and offer a model for testing experimental therapies in this and similar disorders.
2016-07-20T08:03:45Z
Planagumà, Jesús
Leypoldt, Frank
Mannara, Francesco
Gutiérrez-Cuesta, Javier
Martín-García, Elena
Aguilar, Esther
Titulaer, Maarten J.
Jain, Ankit
Balice-Gordon, Rita
Lakadamyali, Melike
Graus, Francesc
Maldonado, Rafael
Dalmau, Josep
Anti-N-methyl D-aspartate receptor (NMDAR) encephalitis is a severe neuropsychiatric disorder that associates with prominent memory and behavioural deficits. Patients’ antibodies react with the N-terminal domain of the GluN1 (previously known as NR1) subunit of NMDAR causing in cultured neurons a selective and reversible internalization of cell-surface receptors. These effects and the frequent response to immunotherapy have suggested an antibody-mediated pathogenesis, but to date there is no animal model showing that patients’ antibodies cause memory and behavioural deficits. To develop such a model, C57BL6/J mice underwent placement of ventricular catheters connected to osmotic pumps that delivered a continuous infusion of patients’ or control cerebrospinal fluid (flow rate 0.25 µl/h, 14 days). During and after the infusion period standardized tests were applied, including tasks to assess memory (novel object recognition in open field and V-maze paradigms), anhedonic behaviours (sucrose preference test), depressive-like behaviours (tail suspension, forced swimming tests), anxiety (black and white, elevated plus maze tests), aggressiveness (resident-intruder test), and locomotor activity (horizontal and vertical). Animals sacrificed at Days 5, 13, 18, 26 and 46 were examined for brain-bound antibodies and the antibody effects on total and synaptic NMDAR clusters and protein concentration using confocal microscopy and immunoblot analysis. These experiments showed that animals infused with patients’ cerebrospinal fluid, but not control cerebrospinal fluid, developed progressive memory deficits, and anhedonic and depressive-like behaviours, without affecting other behavioural or locomotor tasks. Memory deficits gradually worsened until Day 18 (4 days after the infusion stopped) and all symptoms resolved over the next week. Accompanying brain tissue studies showed progressive increase of brain-bound human antibodies, predominantly in the hippocampus (maximal on Days 13–18), that after acid extraction and characterization with GluN1-expressing human embryonic kidney cells were confirmed to be against the NMDAR. Confocal microscopy and immunoblot analysis of the hippocampus showed progressive decrease of the density of total and synaptic NMDAR clusters and total NMDAR protein concentration (maximal on Day 18), without affecting the post-synaptic density protein 95 (PSD95) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. These effects occurred in parallel with memory and other behavioural deficits and gradually improved after Day 18, with reversibility of symptoms accompanied by a decrease of brain-bound antibodies and restoration of NMDAR levels. Overall, these findings establish a link between memory and behavioural deficits and antibody-mediated reduction of NMDAR, provide the biological basis by which removal of antibodies and antibody-producing cells improve neurological function, and offer a model for testing experimental therapies in this and similar disorders.
-
Super-resolution imaging with stochastic single molecule localization: concepts, technical developments, and biological applications
http://hdl.handle.net/2117/88880
Super-resolution imaging with stochastic single molecule localization: concepts, technical developments, and biological applications
Oddone, Anna; Verdeny Vilanova, Ione; Tam, Johnny; Lakadamyali, Melike
Light microscopy has undergone a revolution with the advent of super-resolution microscopy methods that can surpass the diffraction limit. These methods have generated much enthusiasm, in particular with regards to the new possibilities they offer for biological imaging. The recent years have seen a great advancement both in terms of new technological developments and exciting biological applications. Here, we review some of the important milestones in the field and highlight some recent biological applications. Microsc. Res. Tech. 77:502–509, 2014. © 2014 Wiley Periodicals, Inc.
2016-07-19T08:30:19Z
Oddone, Anna
Verdeny Vilanova, Ione
Tam, Johnny
Lakadamyali, Melike
Light microscopy has undergone a revolution with the advent of super-resolution microscopy methods that can surpass the diffraction limit. These methods have generated much enthusiasm, in particular with regards to the new possibilities they offer for biological imaging. The recent years have seen a great advancement both in terms of new technological developments and exciting biological applications. Here, we review some of the important milestones in the field and highlight some recent biological applications. Microsc. Res. Tech. 77:502–509, 2014. © 2014 Wiley Periodicals, Inc.
-
Quantitative super-resolution microscopy: pitfalls and strategies for image analysis
http://hdl.handle.net/2117/88879
Quantitative super-resolution microscopy: pitfalls and strategies for image analysis
Durisic, Nela; Laparra Cuervo, Lara; Lakadamyali, Melike
Super-resolution microscopy is an enabling technology that allows biologists to visualize cellular structures at nanometer length scales using far-field optics. To break the diffraction barrier, it is necessary to leverage the distinct molecular states of fluorescent probes. At the same time, the existence of these different molecular states and the photophysical properties of the fluorescent probes can complicate data quantification and interpretation. Here, we review the pitfalls in super-resolution data analysis that must be avoided for proper interpretation of images.
2016-07-19T08:16:58Z
Durisic, Nela
Laparra Cuervo, Lara
Lakadamyali, Melike
Super-resolution microscopy is an enabling technology that allows biologists to visualize cellular structures at nanometer length scales using far-field optics. To break the diffraction barrier, it is necessary to leverage the distinct molecular states of fluorescent probes. At the same time, the existence of these different molecular states and the photophysical properties of the fluorescent probes can complicate data quantification and interpretation. Here, we review the pitfalls in super-resolution data analysis that must be avoided for proper interpretation of images.
-
Single molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate
http://hdl.handle.net/2117/88877
Single molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate
Durisic, Nela; Laparra-Cuervo, Lara; Sandoval-Álvarez, Ángel; Borbely, Joseph Steven; Lakadamyali, Melike
Photoswitchable fluorescent probes are central to localization-based super-resolution microscopy. Among these probes, fluorescent proteins are appealing because they are genetically encoded. Moreover, the ability to achieve a 1:1 labeling ratio between the fluorescent protein and the protein of interest makes these probes attractive for quantitative single-molecule counting. The percentage of fluorescent protein that is photoactivated into a fluorescently detectable form (i.e., the photoactivation efficiency) plays a crucial part in properly interpreting the quantitative information. It is important to characterize the photoactivation efficiency at the single-molecule level under the conditions used in super-resolution imaging. Here, we used the human glycine receptor expressed in Xenopus oocytes and stepwise photobleaching or single-molecule counting photoactivated localization microcopy (PALM) to determine the photoactivation efficiency of fluorescent proteins mEos2, mEos3.1, mEos3.2, Dendra2, mClavGR2, mMaple, PA-GFP and PA-mCherry. This analysis provides important information that must be considered when using these fluorescent proteins in quantitative super-resolution microscopy.
2016-07-19T08:07:05Z
Durisic, Nela
Laparra-Cuervo, Lara
Sandoval-Álvarez, Ángel
Borbely, Joseph Steven
Lakadamyali, Melike
Photoswitchable fluorescent probes are central to localization-based super-resolution microscopy. Among these probes, fluorescent proteins are appealing because they are genetically encoded. Moreover, the ability to achieve a 1:1 labeling ratio between the fluorescent protein and the protein of interest makes these probes attractive for quantitative single-molecule counting. The percentage of fluorescent protein that is photoactivated into a fluorescently detectable form (i.e., the photoactivation efficiency) plays a crucial part in properly interpreting the quantitative information. It is important to characterize the photoactivation efficiency at the single-molecule level under the conditions used in super-resolution imaging. Here, we used the human glycine receptor expressed in Xenopus oocytes and stepwise photobleaching or single-molecule counting photoactivated localization microcopy (PALM) to determine the photoactivation efficiency of fluorescent proteins mEos2, mEos3.1, mEos3.2, Dendra2, mClavGR2, mMaple, PA-GFP and PA-mCherry. This analysis provides important information that must be considered when using these fluorescent proteins in quantitative super-resolution microscopy.
-
Human N-methyl D-aspartate receptor antibodies alter memory and behaviour in mice
http://hdl.handle.net/2117/78937
Human N-methyl D-aspartate receptor antibodies alter memory and behaviour in mice
Planagumà, Jesús; Leypoldt, Frank; Mannara, Francesco; Gutiérrez-Cuesta, Javier; Martín-García, Elena; Aguilar, Esther; Titulaer, Maarten J.; Petit-Pedrol, Mar; Jain, Ankit; Balice-Gordon, Rita; Lakadamyali, Melike; Graus, Francesc; Maldonado, Rafael; Dalmau, Josep
Anti-N-methyl D-aspartate receptor (NMDAR) encephalitis is a severe neuropsychiatric disorder that associates with prominent memory and behavioural deficits. Patients’ antibodies react with the N-terminal domain of the GluN1 (previously known as NR1) subunit of NMDAR causing in cultured neurons a selective and reversible internalization of cell-surface receptors. These effects and the frequent response to immunotherapy have suggested an antibody-mediated pathogenesis, but to date there is no animal model showing that patients’ antibodies cause memory and behavioural deficits. To develop such a model, C57BL6/J mice underwent placement of ventricular catheters connected to osmotic pumps that delivered a continuous infusion of patients’ or control cerebrospinal fluid (flow rate 0.25 µl/h, 14 days). During and after the infusion period standardized tests were applied, including tasks to assess memory (novel object recognition in open field and V-maze paradigms), anhedonic behaviours (sucrose preference test), depressive-like behaviours (tail suspension, forced swimming tests), anxiety (black and white, elevated plus maze tests), aggressiveness (resident-intruder test), and locomotor activity (horizontal and vertical). Animals sacrificed at Days 5, 13, 18, 26 and 46 were examined for brain-bound antibodies and the antibody effects on total and synaptic NMDAR clusters and protein concentration using confocal microscopy and immunoblot analysis. These experiments showed that animals infused with patients’ cerebrospinal fluid, but not control cerebrospinal fluid, developed progressive memory deficits, and anhedonic and depressive-like behaviours, without affecting other behavioural or locomotor tasks. Memory deficits gradually worsened until Day 18 (4 days after the infusion stopped) and all symptoms resolved over the next week. Accompanying brain tissue studies showed progressive increase of brain-bound human antibodies, predominantly in the hippocampus (maximal on Days 13–18), that after acid extraction and characterization with GluN1-expressing human embryonic kidney cells were confirmed to be against the NMDAR. Confocal microscopy and immunoblot analysis of the hippocampus showed progressive decrease of the density of total and synaptic NMDAR clusters and total NMDAR protein concentration (maximal on Day 18), without affecting the post-synaptic density protein 95 (PSD95) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. These effects occurred in parallel with memory and other behavioural deficits and gradually improved after Day 18, with reversibility of symptoms accompanied by a decrease of brain-bound antibodies and restoration of NMDAR levels. Overall, these findings establish a link between memory and behavioural deficits and antibody-mediated reduction of NMDAR, provide the biological basis by which removal of antibodies and antibody-producing cells improve neurological function, and offer a model for testing experimental therapies in this and similar disorders.
2015-11-09T13:12:34Z
Planagumà, Jesús
Leypoldt, Frank
Mannara, Francesco
Gutiérrez-Cuesta, Javier
Martín-García, Elena
Aguilar, Esther
Titulaer, Maarten J.
Petit-Pedrol, Mar
Jain, Ankit
Balice-Gordon, Rita
Lakadamyali, Melike
Graus, Francesc
Maldonado, Rafael
Dalmau, Josep
Anti-N-methyl D-aspartate receptor (NMDAR) encephalitis is a severe neuropsychiatric disorder that associates with prominent memory and behavioural deficits. Patients’ antibodies react with the N-terminal domain of the GluN1 (previously known as NR1) subunit of NMDAR causing in cultured neurons a selective and reversible internalization of cell-surface receptors. These effects and the frequent response to immunotherapy have suggested an antibody-mediated pathogenesis, but to date there is no animal model showing that patients’ antibodies cause memory and behavioural deficits. To develop such a model, C57BL6/J mice underwent placement of ventricular catheters connected to osmotic pumps that delivered a continuous infusion of patients’ or control cerebrospinal fluid (flow rate 0.25 µl/h, 14 days). During and after the infusion period standardized tests were applied, including tasks to assess memory (novel object recognition in open field and V-maze paradigms), anhedonic behaviours (sucrose preference test), depressive-like behaviours (tail suspension, forced swimming tests), anxiety (black and white, elevated plus maze tests), aggressiveness (resident-intruder test), and locomotor activity (horizontal and vertical). Animals sacrificed at Days 5, 13, 18, 26 and 46 were examined for brain-bound antibodies and the antibody effects on total and synaptic NMDAR clusters and protein concentration using confocal microscopy and immunoblot analysis. These experiments showed that animals infused with patients’ cerebrospinal fluid, but not control cerebrospinal fluid, developed progressive memory deficits, and anhedonic and depressive-like behaviours, without affecting other behavioural or locomotor tasks. Memory deficits gradually worsened until Day 18 (4 days after the infusion stopped) and all symptoms resolved over the next week. Accompanying brain tissue studies showed progressive increase of brain-bound human antibodies, predominantly in the hippocampus (maximal on Days 13–18), that after acid extraction and characterization with GluN1-expressing human embryonic kidney cells were confirmed to be against the NMDAR. Confocal microscopy and immunoblot analysis of the hippocampus showed progressive decrease of the density of total and synaptic NMDAR clusters and total NMDAR protein concentration (maximal on Day 18), without affecting the post-synaptic density protein 95 (PSD95) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. These effects occurred in parallel with memory and other behavioural deficits and gradually improved after Day 18, with reversibility of symptoms accompanied by a decrease of brain-bound antibodies and restoration of NMDAR levels. Overall, these findings establish a link between memory and behavioural deficits and antibody-mediated reduction of NMDAR, provide the biological basis by which removal of antibodies and antibody-producing cells improve neurological function, and offer a model for testing experimental therapies in this and similar disorders.
-
Cross-Talk-Free Multi-Color STORM Imaging Using a Single Fluorophore
http://hdl.handle.net/2117/24928
Cross-Talk-Free Multi-Color STORM Imaging Using a Single Fluorophore
Tam, Johnny; Cordier, Guillaume Alan; Borbely, Joseph Steven; Sandoval Alvarez, Angel; Lakadamyali, Melike
Multi-color stochastic optical reconstruction microscopy (STORM) is routinely performed; however, the various approaches for achieving multiple colors have important caveats. Color cross-talk, limited availability of spectrally distinct fluorophores with optimal brightness and duty cycle, incompatibility of imaging buffers for different fluorophores, and chromatic aberrations impact the spatial resolution and ultimately the number of colors that can be achieved. We overcome these complexities and develop a simple approach for multi-color STORM imaging using a single fluorophore and sequential labelling. In addition, we present a simple and versatile method to locate the same region of interest on different days and even on different microscopes. In combination, these approaches enable cross-talk-free multi-color imaging of sub-cellular structures.
2014-12-04T15:46:00Z
Tam, Johnny
Cordier, Guillaume Alan
Borbely, Joseph Steven
Sandoval Alvarez, Angel
Lakadamyali, Melike
Multi-color stochastic optical reconstruction microscopy (STORM) is routinely performed; however, the various approaches for achieving multiple colors have important caveats. Color cross-talk, limited availability of spectrally distinct fluorophores with optimal brightness and duty cycle, incompatibility of imaging buffers for different fluorophores, and chromatic aberrations impact the spatial resolution and ultimately the number of colors that can be achieved. We overcome these complexities and develop a simple approach for multi-color STORM imaging using a single fluorophore and sequential labelling. In addition, we present a simple and versatile method to locate the same region of interest on different days and even on different microscopes. In combination, these approaches enable cross-talk-free multi-color imaging of sub-cellular structures.