Biological Microscopes

Multiphoton simultaneous imaging and laser stimulation.

Laser light stimulation can be adjusted as desired without the user being limited by imaging settings. The FV1000's SIM scanner for laser light stimulation, which is independent of the scanner for observation, is available. A multiphoton laser provides simultaneous excitation of the same focal plane as that used in imaging.

Calcium signal of a single dendritic spine examined by multiphoton uncaging and fluorescence

a) Stacked fluorescent image of dendritic spines in the hippocampus (excitation of 830 nm). Whole-cell recording was performed, and Alexa 594 and the calcium indicator OGB-5N were injected. At the head of the single spine (red), multiphoton uncaging of caged glutamate was performed and glutamate was administered (excitation of 720 nm). A line scan was performed on the line (the line linking the 2 triangles) from the head of this single spine toward the dendritic trunk.
b), c) Simultaneous line scanning for Alexa 594 and OGB-5N.
d) Calcium concentration determined from the fluorescence emission ratios of OGB-5N and Alexa 594
e) Changes in calcium concentration at the head of the spine (H, black), changes in calcium concentration at the dendritic trunk (D, red), current from whole-cell recorded NMDA receptors (INMDA). Calcium flow into the trunk via NMDA receptors at the head of the spine is apparent from these observations.
Reprinted from Noguchi et al. Neuron 46(2005)609-622.
Jun Noguchi, Haruo Kasai
Center for Disease Biology and Integrative Medicine, Faculty of Medicine,
University of Tokyo

Synchronization of laser light stimulation and patch clamp signals.

The FV1000MPE's analog unit enables voltages to be converted into images and handled just like fluorescence images. For example, electrical signals measured by a patch clamping during laser light stimulation can be synchronized with the image acquisition and displayed with pseudo color.

Laser light mapping

• Laser light mapping

The observation field is divided into a grid and separate fields are discretely irradiated with a laser, allowing laser light stimulation while excluding the effects of light from adjacent spaces. You can observe only a cell reacting to stimulation.

Laser light mapping

• Multipoint stimulation

Light stimulation only to the appointation cell is possible. FV1000MPE give the point that you can appoint to 256 points at the maximum light stimulation in turn and, in what take imaging and an electrical signal in at the same time, can analyze reaction to the cell.

Multipoint stimulation

Functional mapping of glutamate receptors at the single spine level via multiphoton excitation of caged glutamate.

Left: Stacked multiphoton fluorescence images (excitation of 830 nm, Alexa594 as fluorochrome) of hippocampal CA1 pyramidal cells.
Top right: An enlargement of the mapping field.
Bottom right: Electrical signals from glutamate receptor current, indicated by whole-cell recording when separate points in the top right figure are irradiated with the laser, are captured and then mapped with color-coding to peak values in cell response. At that point, caged glutamate (CDNIglutamate) is then administered to specimen slices.

Masaki Matsuzaki, Haruo Kasai
Center for Disease Biology and Integrative Medicine, Faculty of Medicine,
University of Tokyo