Biological Microscopes

Assured capture of reactions immediately following laser light stimulation.

The compact design incorporates two laser scanners, one for confocal imaging and the other for simultaneous stimulation. They can be illuminated separately and independently, making it possible to stimulate the specimen during observation. As a result, the rapid cell reactions that occur right after laser stimulation can be accurately and reliably captured, making the FV1000 ideal for such applications as FRAP, FLIP, photoactivation and photoconversion.

Stimulation area can be changed during imaging.

Two laser beams, one for imaging and laser light stimulation, can be controlled independently and separately. The stimulation area can be moved to a different position on the cell during imaging, so the system is powerful for a variety of experiments.

Wide choice of different breaching modes.

Various scan modes can be used for both the observation area and stimulation area. This enables free-form bleaching of designated points, lines, free-lines, rectangles and circles.

Multi-laser combiner enables 2-channel laser output.

Laser light is branched in the laser combiner, and each laser wavelength provided in the system can be selected and used for imaging and stimulation.

Tornado scanning for highly efficient bleaching.

Conventional raster scanning cannot always complete photobleaching quickly. Tornado scanning makes the procedure much more efficient by significantly reducing unnecessary scanning.

Stimulus setting This enables selection of the laser wavelength, intensity, scanning mode and other parameters for the stimulation laser. When using the SIM scanner, control can be done in linkage with imaging in µs units.

Positive fluorescence imaging of live cells by laser light stimulation while imaging is in progress.

In contrast to bleaching, light stimulation enables many changes inside and outside cells, such as intensifying fluorescence or changing colors, and can be performed with minimal light. Live cell imaging is dramatically enhanced by capturing a diffusion or transfer of fluorescence-labeled molecules, or by marking a specific live cell with an appropriate color stain. The FV1000’s SIM system can provide excitation scanning separately from imaging scanning; this allows the user to illuminate the specimen as required, without the restrictions of image settings.
The illumination area is not limited to the imaging field of view, but covers areas outside as well. The imaging area and laser light stimulation areas can be freely set up on the reference image.

Kaede

This fluorescent protein is made through cloning from Trachyphyllia geoffroyi. It emits a strong green light after synthesis, but when stimulated by ultraviolet laser illumination, changes its color from green to red, like a maple tree in autumn. Its name is derived from this characteristic ("kaede" means "maple tree" in Japanese). When violet (or ultraviolet) laser illumination is directed onto a Kaede-expressing cell, diffusion of the reddish Kaede can be monitored throughout the whole area of the cell, providing an easy and accurate method for capturing the whole cell labeling. The FV1000 allows this to be done while observation is in progress.

When Kaede is expressed in the cytoplasm of a live cell, it shows a high-speed diffusion coefficient value (about 30µm2/s) in spite of its tetramic structure. Taking advantage of this property, the movements of Kaede molecules in the cell can be observed more easily than molecules in general GFP/FRAP experiments. Since Kaede photoconversion requires only slight laser light, less intense than that used in ordinary photobleaching, labeling can be completed in a very short time.

Kaede-expressing astroglia cells are stacked on the Kaede-expressing neurons. By illuminating two colonies with a 405nm laser, the Kaede color can be photoconverted from green to red. The glial cells in contact with the neurons are observed while they are forming colonies and extending their processes, and the nuclei of these colonies can also be observed. The FV1000 SIM scanner makes it easy to change cell colors from green to red while conducting an observation, and to control neutral colors between red and green.
Data courtesy of : Dr. Hiroshi Hama, Ms. Ryoko Ando and Dr.Atsushi Miyawaki
RIKEN Brain Science Institute Laboratory for Cell Function Dynamics

FRAP (Fluorescence Recovery After Photobleaching)

Conventional FRAP applications are performed using a laser controlled via the AOTF (Acousto-Optical Tuneable Filters). Introduction of the SIM scanner further improves FRAP performance. For instance, while conducting an observation with the main scanner laser, the user can use a second laser to carry out photobleaching on a particular targeted area. As a result, the rapid movements of fluorescence molecules that come from outside the targeted area immediately after photobleaching are not overlooked.
FRAP: Fluorescence recovery after photobleaching is a method for analyzing molecular movements. It measures diffusion rates, tethering to other structural components and the separation speed of molecules.

FLIP (Fluorescence Loss In Photobleaching)

The FV1000 SIM scanner system optimizes FLIP operation. Instead of the conventional method of alternating between photobleaching and imaging, this system conducts both procedures at the same time, enabling reliable capture of even rapid molecular movements.

PA-GFP (Photo Activatable-Green Fluorescent Protein)

Fluorescent protein PA-GFP can be used to mark targeted cells, organelles and proteins. The SIM scanner allows illumination of any designated area at any time. In the following photos, 405nm laser excitation is conducted intermittently on part of a PA-GFP expressing cell, and time-lapse changes of fluorescent signals on different points of the cell are monitored. This provides information about protein diffusion within the cell.

Dronpa

Dronpa is a new fluorescent protein with photochromic properties. Using photoactivation like PA-GFP, this method is suitable for observation of molecular movement, but has unique additional functions.

1.

Since fluorescence emission is extremely faint before activation of PA-GFP, it is difficult to confirm expression and cell fixation before imaging. By contrast, Dronpa provides very bright fluorescence signals before activation, so that expression and cell fixation are easy to confirm.

2.

Photochromism appears broadly similar to photobleaching, with fluorescence intensity reduced by irradiation with a strong 488nm laser. However, fluorescence intensity reduced by photochromism is readily restored with illumination from a faint 405nm laser. As a result, user-selected control of fluorescence intensity using 405nm (for excitation) and 488nm (for imaging) enables repeated photoactivation experiments on the same cell.

3.

The fluorescence intensity before fading and previous photoactivation results are used as control values, which makes experiments much more quick to perform.

Uncaging

With a 405nm laser or ultraviolet laser attached, the SIM scanner system can be used for uncaging. Caged compounds can be uncaged point-by-point or as an ROI operation, while the FV1000’s main scanner captures images of the uncaging without any time lag.

Caged-Glutamate