FluoView 1200 Confocal Microscope

Optimized for Imaging Live Cells and Tissues, FV1200 brings sensitivity and power to your research through a range of innovative technology and sensitivity improvements, allowing you to capture the most critical elements of your biological samples with speed, precision and reliability.

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Applications

FV-OSR (Olympus Super Resolution) Technology

Olympus'''' widely applicable super-resolution method requires no special fluorophores, and can work for a wide range of samples in combination with a large selection of superior optics and high sensitivity detectors. Ideal for colocalization analysis, sequential or simultaneous acquisition of 2 fluorescent signals can lead to resolution of approximately 120nm, nearly doubling the resolution of typical confocal microscopy. Operation is simple, with minimal training requirements, and can be added to any FV1000 or FV1200 confocal system, making FV-OSR a truly accessible method for achieving super-resolution.

*Subject to objective magnification, numerical aperture, excitation and emission wavelength and experiential conditions.


Trachea multi-ciliated epithelial cells (Culture)

Immunofluorescence microscopy:
Odf2 staining (Alexa Fluor 488, green)
ZO-1 staining (Alexa Fluor 568, magenta)
Staining for Odf2 encircled the base of cilia at the upper part of the basal body (green). Staining for ZO-1 revealed the tight junctions (magenta).
Objective: UPLSAPO60XS

Courtesy of Hatsuho Kanoh, Elisa Herawati, Sachiko Tsukita,Ph.D.
Graduate School of Frontier Biosciences and Graduate School of Medicine, Osaka University.

Cultured epithelial HeLa (EpH) cells.

Immunofluorescence microscopy:
a-tubulin staining (Alexa Fluor 488, green)
ZO-1 staining (Alexa Fluor 568, magenta)

Staining for ZO-1 revealed the tight junctions (TJs) (magenta).
Staining for a-tubulin showed an apical network of microtubules. This network associates with the TJ to form the "TJ-apical complex" (green).
Objective: UPLSAO100SXS

Courtesy of Hatsuho Kanoh, Tomoki Yano, Sachiko Tsukita,Ph.D.
Graduate School of Frontier Biosciences and Graduate School of Medicine, Osaka University.

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 to label the entire cell.

Whole cell labeling with Kaede

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 FV1200 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

In these images the red fluorescence of photoconverted Kaede has been pseudocolored magenta for easy discrimination by red/green color blind individuals.

Zebra fish
Embryo where Huc:Kaede has been injected into a fertilized egg (5th day after insemination) Image observed after 3 days via 405nm stimulation of sensory spinal nerve (Rohon-Beard) cells. Objective: LUMPlan FL20 x W (N.A.0.8) Dr. Tomomi Sato, Dr. Hitoshi Okamoto RIKEN Brain Science Institute Laboratory for Developmental Gene Regulation Reference: Sato T, Takahoko M, Okamoto H. (2006) HuC:Kaede, a useful tool to label neural morphologies in networks in vivo. Genesis. 44:136-42.

Zebra fish
Embryo where Huc:Kaede has been injected into a fertilized egg (5th day after insemination) Image observed approximately 1 hour after 405nm stimulation of the trigeminal ganglion Objective: LMPlanFL60 x W (N.A.0.9) Dr. Tomomi Sato, Dr. Hitoshi Okamoto RIKEN Brain Science Institute Laboratory for Developmental Gene Regulation Reference: Sato T, Takahoko M, Okamoto H. (2006) HuC:Kaede, a useful tool to label neural morphologies in networks in vivo. Genesis. 44:136-42.

When Kaede is expressed in the cytoplasm of a live cell, it shows a high-speed diffusion coefficient value (about 30m2/s) in spite of its tetramic structure. Taking advantage of this property, the movements of Kaede-labeled molecules in the cell can be observed. Since Kaede photoconversion requires only minimal laser light, less intense than that used in ordinary photobleaching, protein trafficking can be observed with less perturbation to the cell or organism.

Diffusion Measurement Techniques:

The FV1200 Diffusion Measurement Package uses confocal images to analyze molecular interactions and movements within cells. There are 4 main techniques embodied in the software package.

Point FCS

Point Fluorescence Correlation Spectroscopy (FCS) analyzes the fluctuations in signal as fluorescently labeled particles move in and out of a confocal volume. Diffusion coefficients, rates of transport or conformational changes can be measured. Molecular concentration can be determined by quantifying the number of fluorescent molecules in the observation volume.

  • High time resolution and spatial resolution.
  • Good for fast particle movement or small molecule diffusion in solution.
  • Data from single location through Point Scanning.
  • Able to analyze cross correlations which reduce the background contribution.
  • Outputs or results are given as diffusion constant and number of molecules.

Raster Image Correlation Spectroscopy (RICS)

Raster Image Correlation Spectroscopy is a powerful new technique covering a range of different diffusion speeds. The basis of this technique is that the time difference between pixels in the X direction is microseconds but pixels of lines in the Y direction differ by milliseconds, and frame to frame by seconds. Correlating over these 3 different time scales can identify different mobilities. Because the diffusion is calculated from images, the results indicate the spatial distribution of the diffusion constant across the image, that is, in different regions of the cell.

Measurement in regions.
  • A spatial mapping of diffusion time constant and local concentration.
  • Based on both time and spatial correlation.
  • Can handle multiple diffusion time scales.
Measures a wide range of molecular processes.
  • From protein in solution (medium diffusion time scale) to protein on cell membrane (slow diffusion time scale).

Outputs or results are given as Diffusion Constant and Number of Molecules as a function of location.

Cross-Correlation Cross-correlation analysis such as point FCCS or ccRICS measures protein interactions.
  • A tool to assess the interaction (binding) of two groups of fluorescently labeled molecules or proteins.
  • Determine how many molecules are binding.
  • Data acquisition from two simultaneous channels.
  • Able to measure wide range of applications.
  • Cross correlation signal exists only if observed in both channels.

FRAP

Fluorescence recovery after photobleaching (FRAP) is a method for analyzing molecular movements. It measures diffusion rates, tethering of molecules to cellular structural components and the separation speed of molecular complexes.

The FV1200 SIM scanner further improves FRAP performance. 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. 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.

Example of FRAP:



FLIP

Fluorescence loss in photobleaching (FLIP) is another method for analyzing molecular movements. The FV1200 SIM scanner system optimizes FLIP operation, allowing constant measurement of fluorescence intensities outside the photobleached area.

Example of FLIP:



PA-GFP

Photo Activatable-Green 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.

Example of PA-GFP:



Dronpa

Dronpa is a new fluorescent protein with photochromic properties that enable it to be reversibly switched on and off. Using photo activation 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 photo bleaching, 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 photo activation experiments on the same cell.
  3. The fluorescence intensity before fading and previous photo activation results are used as control values, which makes experiments more accurate and reliable.

FRET

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, a wizard dialog allows easy and reproducible experimental setup.

Acceptor bleach can be set easily using wizard setting
Acceptor bleach can be set easily using wizard setting.
FRET
FRET chart

Calcium concentration measurement by FRET using YC3.60 (Yellow Cameleon 3.60) Cameleon reporter molecules function by FRET occurring between two fluorescent moieties linked by a calcium binding site. When calcium is bound, a conformational change increases the FRET efficiency and this change can be used as a measure of calcium concentration. 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 high precision, high-speed calcium concentration imaging, which is difficult by conventional methods. 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

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Software Capabilities:

Uncaging

With a 405nm 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 FV1200''''s main scanner captures images of the uncaging without any time lag.

Example of Uncaging:

Multi-point imaging and Montage Imaging

Tiling image acquisition by equipping the system with a motorized XY stage, repeated image acquisition of multiple points located over a large specimen can be automated.

Image made by acquiring images at multiple points of a mouse brain section (YFP) and integrated.

Objective: UPlanSApo10x
Acquired visual fields: 25 fields
XYZ acquisition conditions for each visual field: 512 x 512, 15 slices
Sample provided by:
Ms. Mikako Sakurai, Mr. Masayuki, Sekiguchi (Section Chief) Department of Degenerative Neurological Diseases, National Institute of Neuroscience, National Center of Neurology and Psychiatry.


VIDEOS

The Use of Wide-Scale Imaging to Study Retinal Cells in Wholemounts

The Use of Wide-Scale Imaging to Study Retinal Cells in Wholemounts

Dr. Steven Fisher, Research Professor, Neuroscience Institute; Professor Emeritus, Cellular Molecular & Developmental Biology, University of California, Santa Barbara
Delivered at the Imaging Symposium sponsored by Olympus at the Society of Neuroscience annual meeting on November 15th, 2010.

PLAY VIDEO

Long-term, efficient time-lapse of multiple live cells

In addition to tiled images covering a large area, time-lapse observation of multiple regions can be scheduled. The software allows easy setup of imaging multiple wells in a microtiter well plate containing cells under different conditions. These functions dramatically improve throughput of experiments requiring long-term observation.

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Scanning Modes:

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 scanning modes

Observation of complex time-lapse combined with Z or λ dimensions is possible. Line scanning for a straight line, slanted line or freely drawn curve enables easy analysis of rapid changes on the order of milliseconds.

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 use an external trigger for each image frame acquisition.

Laser monitoring function

Feedback is applied to the laser output so that the sample is always exposed to a fixed level of excitation light and the amount of fluorescence can be measured accurately.

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Time Course:

Complex protocols can be constructed using the Time Controller. Ramp laser power and detection parameters, create nested loops and other special protocols.

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:

1. Image acquisition while storing data on the hard disk.
2. Changing time-lapse intervals during the course of an experiment.
3. Image acquisition while changing the excitation laser in mid-procedure.
4. Data output from a specified point, using an external trigger.
5. 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.

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Stimulation Applications

Photostimulation, optogenetics, photobleaching, photoconversion, photoactivation and uncaging can be accomplished using the laser light stimulation function (Stimulus Setting). Laser light stimulation experiments can be done 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. Systems that only have the main scanner can perform laser light stimulation during a time course by automatically 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. 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.
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3D Rendering

Selectable rendering modes

Maximum intensity Projection methods

Interactive volume rendering

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 moving the mouse. A variety of display functions are available, including extended focus images and the ability to display a cross section at an arbitrary location.

interactive volume rendering

4D animation creation function

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



Brightness compensation function for the depth direction

When acquiring 3D images, the images become dimmer with depth. The system is equipped with a function for increasing laser intensity or PMT sensitivity with depth so that images of deeper regions can be acquired with equal brightness.

Brightness compensation
Image without compensation
Image without compensation
Image with compensation
Image with compensation
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SPECTRAL UNMIXING

Easy fluorescence separation

Fluorescence can easily be separated through two modes (Normal and Blind). In Normal mode, separation is performed using stored fluorochrome spectra, or data derived from acquired images. In Blind mode, the separation uses an iterative process to derive the best fit of a chosen 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.
  • The use of a diffraction grating for linear dispersion ensures accurate spectral analysis.

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

MitoTracker (mitochondria) POPO-3 (nucleus)XYλ acquisition conditionsWavelength detection range: 550nm~640nm in 2nm

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COLOCALIZATION

Analysis of emission intensity overlap

Threshold lines can be set interactively. Setting the ROI on the scatter plot creates a colocalization image highlighting structures with colocalization. Values can also be obtained for Pearson correlation, Manders overlap coefficient and colocalization index.

This method is also applicable for analysis of an image series.

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Hardware additions/applications:

FV1200/IX83ZDC (Zero Drift)

Corrects for thermal drift during confocal time-lapse imaging

During long time-lapse observations, temperature changes around the microscope and drug administration during the observation cause thermal drift, resulting in loss of focus. For confocal laser scanning biological microscopes with high resolution in the Z-direction, even slight focal drift can impair image acquisition to the point that images are no longer useful to researchers. Olympus is the world leader in equipping a confocal laser scanning biological microscope with zero drift compensation; it corrects automatically for thermal drift during confocal time-lapse imaging.

  • In time-lapse imaging, focus is automatically corrected immediately prior to imaging.
  • Compensation is performed in reference to the bottom surface of the dish, allowing target Z-slice images to be obtained regardless of sample conditions.
  • Without thermal drift compensation, several Z-slice images must be taken to ensure acquisition of target image plane. Thermal drift compensation eliminates this need and minimizes sample exposure to irradiation.

TIRF (Total Internal Reflection Fluorescence Microscopy)

High S/N images near the cell surface

This special TIRF unit employs the FV1200''''s laser for TIRF illumination. The incident angle of the excitation laser toward the specimen is controlled through FV1200 software FV10-ASW, to set up the necessary laser filtering light volume. The optimum light path length is provided automatically through the selection of excitation wavelength and the objective. Since TIRFM observation can be done by exchanging confocal observation, protein localization on the cell surface and cross section images of the cell interior may be acquired simultaneously. A CCD is required for TIRFM image acquisition and image-capturing software is required. Note that time-lapse imaging by interchanging CCD and confocal images and then overlapping them is not possible.

TIRFM LSM
Features Acquisition of high S/N images at 50-200nm depths from the specimen surface (surface observation only). Allows observations from the surface to the intracellular structure.
Capable of 3D observation for each Z-axis position.
Acquisition of high S/N images with minimal influence from the background. Capable of composing 3D images from slice images.
Method Imaging through CCD camera.
Frame rate depends on camera performance.
Point scanning.
Detector incorporates photomultiplier.
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