Biological Microscopes

Live plot function Changes in fluorescent intensity in a region designated as ROI are plotted in real-time during image acquisition.

Live tile mode, Look back function.

This allows the user to check the acquired images during time lapse experiment. Adjustment of focus and/or image brightness can be done during rest time.

High-speed image acquisition (4KHz/line).

High-speed scanning mode can capture confocal images at 16 frames per second with 256 x 256 pixel resolution. In combination with clip scanning, images can be acquired even faster than video rate.

Wide variety of line scanning modes.

Line scanning for a straight line, slanted line or free curve enables easy
analysis of changes over time at the msec order. Observation of complex time lapse combined with Z or λ element is also possible.

Trigger function.

The system also has a trigger function for synchronizing scanning with external devices. Scanning can be started/stopped with a trigger signal, and it is also possible to gather frames for each external trigger.

Laser monitoring function.

Feedback is applied to the laser output so that the sample is always exposed to a fixed level of excitation light. There is no need to pay attention to variation in laser output, and the amount of fluorescence can be measured accurately.

Using the Time Controller to schedule experiment flows and protocols.

The Time Controller allows image acquisition conditions to be changed easily while observation is in progress. In addition to experiments such as FRAP and FLIP, the following protocols are supported:

• Image acquisition while storing data on the hard disk.
• Changing time-lapse intervals during the course of an experiment.
• Image acquisition while changing the excitation laser in mid-procedure.
• Data output from a specified point, using an external trigger.
• After acquisition of reference images, laser intensity and excitation area can be changed.

The experiment protocol can be entered from the taskbar in the protocol schedule area. Settings can be entered freely by clicking and dragging the mouse on the column of items to be scheduled.

Acceptor bleach can be set easily using wizard setting.

FRET is the phenomenon by which the excitation energy of fluorescent molecules transfers to other fluorescent molecules, with the degree of transfer efficiency depending on the relative positions of two molecules. FRET allows observation of the interaction between two protein molecules, analysis of structural changes, and imaging of the calcium concentration in cells.

• Equipped with FRET analysis functions using the Ratio Imaging method, Acceptor Photobleach method and Sensitized Emission method.
• For the Acceptor Photobleach method, the system is also equipped with an experiment protocol setting function using the Wizard format, and this facilitates setting of experiment procedures.

YC3.60 (Yellow Cameleon 3.60) Due to structural improvement using circular permutation, YC3.60 offers superior performance and achieves a relatively high rate of change. Among other advantages, it allows highprecision, high-speed calcium concentration imaging, which is difficult by conventional methods. In LSM observation, calcium dynamic movements are observed with simple ratio imaging: this has contributed to making FRET imaging more popular. In the above ratio image photos, YC3.60 is expressed on HeLa cells, with calcium concentration changes captured when they are stimulated by histamine. Laser for observation: LD440, 0.3% output Objectives: 40x, zoom 1.3x References: Takeharu Nagai, Shuichi Yamada, Takashi Tominaga, Michinori Ichikawa, and Atsushi Miyawaki 10554-10559, PNAS, July 20, 2004, vol. 101, no.29

Using the laser light stimulation setting function (Stimulus Setting), laser light stimulation experiments can be done with either the main scanner (image scanner) or SIM scanner. Even with the main scanner only, it is possible to perform laser light stimulation during a time course by instantaneously switching the laser scanning mode (laser wavelength, laser irradiation range). However, images cannot be acquired during laser light stimulation.

• Tornado scanning can also be used for laser light stimulation (with SIM scanner only). This is suitable for FRAP, Kaede protein and other photoconversion experiments.
• In intensity analysis, the timing of laser light stimulation is displayed simultaneously with the changes in fluorescent light intensity.

Selectable rendering modes.

Using the laser light stimulation setting function (Stimulus Setting), laser light stimulation experiments can be done with either the main scanner (image scanner) or SIM scanner. Even with the main scanner only, it is possible to perform laser light stimulation during a time course by instantaneously switching the laser scanning mode (laser wavelength, laser irradiation range). However, images cannot be acquired during laser light stimulation.

Alpha Blend method (Cultured nerve cells derived from mouse hippocampus) Dr. Koji Ikegami, Dr. Mitsutoshi Setou Molecular Geriatric Medicine, Mitsubishi Kagaku Institute of Life Sciences		Maximum Intensity Projection method (Ptk2 cells)

Using the interactive volume rendering method with the 3D display function, the angle of a 3D rendered image can be freely changed to the direction you wish to see by operating the mouse. A variety of display functions are available, including the ability to display a cross section at an arbitrary location, and extend focus images.

		Wild-type embryo in stage 17 of drosophila Dr. Tetsuya Kojima Laboratory of Innovational Biology, Department of Integrated Biosciences Graduate School of Frontier Sciences, University of Tokyo
Lily pollen

4D animation creation function.

3D structures which change with the passage of time can be animated for images acquired with XYZT.

Brightness compensation function for the depth direction.

	Image without compensation	Image with compensation

When acquiring 3D images, the images darken in the depth direction. The system is equipped with a function for increasing laser intensity or PMT sensitivity in the depth direction, so that images of more distant parts can be acquired while retaining a fixed brightness.

Easy fluorescence separation.

Fluorescence can easily be separated through two modes (Normal and Blind). In Normal mode, separation is performed based on a designated ROI fluorescence spectrum using already-known fluorochrome wavelength data, or data derived from acquired images. In Blind mode, the separation uses an iterative process to derive the best fit of a given number of fluorescence spectra.

• 2nm spectral resolution allows two fluorochromes with similar emission peaks to be clearly separated
• Spectral unmixing is successful even when there are emission intensity differences in each fluorochrome
• A fine diffraction grating is used to gain precise separation for unmixing.

EGFP (dendrite) — EYFP (synapse) XYλ acquisition conditions Wavelength detection range: 495nm~561nm in 2nm steps Excitation wavelength: 488nm		Rhodamine-Phalloidin (actin) — PI (nucleus) XYλ acquisition conditions Wavelength detection range: 560nm~630nm in 2nm steps Excitation wavelength: 543nm		MitoTracker (mitochondria) — POPO-3 (nucleus) XYλ acquisition conditions Wavelength detection range: 550nm~640nm in 2nm steps Excitation wavelength: 543nm