Cytosolic and Nuclear Calcium Imaging by Confocal Microscopy

1996 ◽  
pp. 319-328
Author(s):  
Michel Burnier ◽  
Gabriel Centeno ◽  
Hans R. Brunner
Keyword(s):  
Confocal Microscopy ◽  
Calcium Imaging ◽  
Nuclear Calcium

scholarly journals Temporal transcription factors determine circuit membership by permanently altering motor neuron-to-muscle synaptic partnerships

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Julia L Meng ◽  
Yupu Wang ◽  
Robert A Carrillo ◽  
Ellie S Heckscher
Keyword(s):  
Stem Cells ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Confocal Microscopy ◽  
Motor Neuron ◽  
Calcium Imaging ◽  
Motor System ◽  
General Feature ◽  
Partner Choice ◽  
Neuronal Stem Cells

How circuit wiring is specified is a key question in developmental neurobiology. Previously, using the Drosophila motor system as a model, we found the classic temporal transcription factor Hunchback acts in NB7-1 neuronal stem cells to control the number of NB7-1 neuronal progeny form functional synapses on dorsal muscles (Meng et al., 2019). However, it is unknown to what extent control of motor neuron-to-muscle synaptic partnerships is a general feature of temporal transcription factors. Here, we perform additional temporal transcription factor manipulations—prolonging expression of Hunchback in NB3-1, as well as precociously expressing Pdm and Castor in NB7-1. We use confocal microscopy, calcium imaging, and electrophysiology to show that in every manipulation there are permanent alterations in neuromuscular synaptic partnerships. Our data show temporal transcription factors, as a group of molecules, are potent determinants of synaptic partner choice and therefore ultimately control circuit membership.

scholarly journals A Simple Modification of the Stage Insert for Nikon Inverted Microscopes

2000 ◽  
Vol 8 (6) ◽  
pp. 44-45
Author(s):  
Glen MacDonald
Keyword(s):  
Confocal Microscopy ◽  
Calcium Imaging ◽  
Central Portion ◽  
Lateral Motion ◽  
Inverted Position ◽  
Simple Modification ◽  
Sample Compression ◽  
Central Plate ◽  
Wide Range ◽  
Sample Support

This article describes a simple, economical modification of the standard stage insert used on the Nikon TMD, Diaphot and E800 models of inverted microscopes to support slide securely without lateral motion or sample compression. The central portion of the stage on these popular microscopes consists of a removable insert that provides sample support and demarks of the imaging area. The insert may be replaced with holders for a variety of culture chambers and other specialized sample supports. This unique design contributes to the adaptability of these microscopes for a wide range of imaging applications such as confocal microscopy, calcium imaging and electrophysiology.The standard insert is actually constructed of two pieces, an outer ring and a central plate (Figure 1A). The central plate is removed from the modified ring, which is then used in the inverted position (Figures 1B, 3, 4).

High‐Speed Multineuron Calcium Imaging Using Nipkow‐Type Confocal Microscopy

2011 ◽  
Vol 57 (1) ◽  
Author(s):  
Naoya Takahashi ◽  
Shigeyuki Oba ◽  
Naoto Yukinawa ◽  
Sakiko Ujita ◽  
Mika Mizunuma ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Calcium Imaging ◽  
High Speed

Confocal microscopy to analyze cytosolic and nuclear calcium in cultured vascular cells

1994 ◽  
Vol 266 (4) ◽  
pp. C1118-C1127 ◽  
Author(s):  
M. Burnier ◽  
G. Centeno ◽  
E. Burki ◽  
H. R. Brunner
Keyword(s):  
Confocal Microscopy ◽  
Calcium Concentration ◽  
Fluorescent Dyes ◽  
Cytosolic Calcium ◽  
Initial Increase ◽  
Vascular Cells ◽  
Nuclear Calcium ◽  
Proliferative Effect ◽  
Perinuclear Area ◽  
Stimulation Of

With the development of calcium-sensitive fluorescent dyes and videomicroscopic imaging, several investigators have located the changes in intracellular calcium in the cytoplasm, in the perinuclear region, and possibly in the nucleus. However, the presence of calcium in the nucleus is often difficult to ascertain because the fluorescence derived from the perinuclear area interferes with that of the nucleus. We have used confocal microscopy together with two calcium-sensitive dyes [acetoxymethyl esters of fluo 3 (fluo 3-AM) and rhod 2 (rhod 2-AM)] to analyze the cytosolic and nuclear calcium distribution in vascular smooth muscle and endothelial cells studied at rest and after stimulation with receptor-dependent (angiotensin, vasopressin) and receptor-independent (KCl) stimuli. With fluo 3-AM, the baseline fluorescence was located in the cytoplasm but was slightly higher in the nucleus. With all stimuli, the fluorescence intensity increased in both compartments but remained more pronounced within the nucleus. Yet, after calibration, the cytosolic calcium concentration was greater than that of the nucleus at rest and was equally high after stimulation, suggesting different properties of fluo 3 in the cytosol and in the nucleus. With rhod 2-AM, baseline fluorescence was low in the nucleus and high in the cytosol. Cell stimulation caused an initial increase in cytosolic calcium with no change in the nucleus followed by a rise in both compartments. Thus the stimulation of vascular cells is associated with marked increases in cytosolic and nuclear calcium. Fluo 3-AM seems to be a better indicator of nuclear calcium than rhod 2-AM. The increases in nuclear calcium induced by angiotensin II and vasopressin may contribute to their cell proliferative effect.

Two-photon excitation microscopy in cellular biophysics

1995 ◽  
Vol 53 ◽  
pp. 62-63
Author(s):  
David W. Piston ◽  
Brian D. Bennett ◽  
Robert G. Summers
Keyword(s):  
Confocal Microscopy ◽  
Laser Beam ◽  
Duty Cycle ◽  
Excited Electronic State ◽  
Input Power ◽  
Two Photon ◽  
Simultaneous Absorption ◽  
Photon Excitation ◽  
Very High ◽  
Two Photon Excitation

Two-photon excitation microscopy (TPEM) provides attractive advantages over confocal microscopy for three-dimensionally resolved fluorescence imaging and photochemistry. Two-photon excitation arises from the simultaneous absorption of two photons in a single quantitized event whose probability is proportional to the square of the instantaneous intensity. For example, two red photons can cause the transition to an excited electronic state normally reached by absorption in the ultraviolet. In practice, two-photon excitation is made possible by the very high local instantaneous intensity provided by a combination of diffraction-limited focusing of a single laser beam in the microscope and the temporal concentration of 100 femtosecond pulses generated by a mode-locked laser. Resultant peak excitation intensities are 106 times greater than the CW intensities used in confocal microscopy, but the pulse duty cycle of 10-5 maintains the average input power on the order of 10 mW, only slightly greater than the power normally used in confocal microscopy.

Measurement of spontaneous neuronal membrane activity by time lapse confocal microscopy

1995 ◽  
Vol 53 ◽  
pp. 972-973
Author(s):  
R H. Selinfreund ◽  
A. H. Cornell-Bell
Keyword(s):  
Membrane Potential ◽  
Confocal Microscopy ◽  
Patch Clamp ◽  
Electrical Activity ◽  
Time Lapse ◽  
Neuronal Firing ◽  
Neuronal Membrane ◽  
Pituitary Cells ◽  
Patch Clamp Recording ◽  
Spontaneous Electrical Activity

Cellular electrophysiological properties are normally monitored by standard patch clamp techniques . The combination of membrane potential dyes with time-lapse laser confocal microscopy provides a more direct, least destructive rapid method for monitoring changes in neuronal electrical activity. Using membrane potential dyes we found that spontaneous action potential firing can be detected using time-lapse confocal microscopy. Initially, patch clamp recording techniques were used to verify spontaneous electrical activity in GH4\C1 pituitary cells. It was found that serum depleted cells had reduced spontaneous electrical activity. Brief exposure to the serum derived growth factor, IGF-1, reconstituted electrical activity. We have examined the possibility of developing a rapid fluorescent assay to measure neuronal activity using membrane potential dyes. This neuronal regeneration assay has been adapted to run on a confocal microscope. Quantitative fluorescence is then used to measure a compounds ability to regenerate neuronal firing.The membrane potential dye di-8-ANEPPS was selected for these experiments. Di-8- ANEPPS is internalized slowly, has a high signal to noise ratio (40:1), has a linear fluorescent response to change in voltage.

Five-Dimensional Microscopy using Widefield-Deconvolution: Practical Considerations and Biological Applications

1996 ◽  
Vol 54 ◽  
pp. 268-269
Author(s):  
W.F. Marshall ◽  
K. Oegema ◽  
J. Nunnari ◽  
A.F. Straight ◽  
D.A. Agard ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
High Speed ◽  
Laser Scanning ◽  
Ccd Camera ◽  
Image Plane ◽  
Time Lapse ◽  
Laser Scanning Confocal Microscopy ◽  
Three Dimensions ◽  
Excitation Light ◽  
Cooled Ccd

The ability to image cells in three dimensions has brought about a revolution in biological microscopy, enabling many questions to be asked which would be inaccessible without this capability. There are currently two major methods of three dimensional microscopy: laser-scanning confocal microscopy and widefield-deconvolution microscopy. The method of widefield-deconvolution uses a cooled CCD to acquire images from a standard widefield microscope, and then computationally removes out of focus blur. Using such a scheme, it is easy to acquire time-lapse 3D images of living cells without killing them, and to do so for multiple wavelengths (using computer-controlled filter wheels). Thus, it is now not only feasible, but routine, to perform five dimensional microscopy (three spatial dimensions, plus time, plus wavelength).Widefield-deconvolution has several advantages over confocal microscopy. The two main advantages are high speed of acquisition (because there is no scanning, a single optical section is acquired at a time by using a cooled CCD camera) and the use of low excitation light levels Excitation intensity can be much lower than in a confocal microscope for three reasons: 1) longer exposures can be taken since the entire 512x512 image plane is acquired in parallel, so that dwell time is not an issue, 2) the higher quantum efficiently of a CCD detect over those typically used in confocal microscopy (although this is expected to change due to advances in confocal detector technology), and 3) because no pinhole is used to reject light, a much larger fraction of the emitted light is collected. Thus we can typically acquire images with thousands of photons per pixel using a mercury lamp, instead of a laser, for illumination. The use of low excitation light is critical for living samples, and also reduces bleaching. The high speed of widefield microscopy is also essential for time-lapse 3D microscopy, since one must acquire images quickly enough to resolve interesting events.

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