SOW.pdf

The Keyence BZ-X800 fluorescent microscope is the only one that fulfills our research requirements as we need the microscope to:

(1). Include, as standard components, an electronic shutter, XY and Z motorized stages (tilling), an electronic revolver, an electronic filter turret, electronic color filters, electronic phase-contrast slit switching mechanism, electronic dimmer filters, and an electronic aperture control; it is necessary that all of these components can be operated with the mouse.

=> Manual adjustment takes much more labor and time before optimum settings are reached and will require several training sessions before the user acquires the level of skill necessary. An electronic control system that can be operated easily with the mouse is required to make the tasks efficient and shorten overall evaluation time by ensuring that even novices can easily perform operation.

(2) include a specimen enclosure to provide an observation environment that, even without a darkroom, will not impair the background contrast required for fluorescence observation.

=> To ensure that researchers can observe in a bright open space without suffering from darkroom-related restrictions because the microscope is installed in a place best suited to the researchers.

(3) Must have a small footprint because of limited laboratory space.

(4) The product must have a built-in vibration dampening system with the ability to use up to 100x objectives in a typical lab environment.

=> To ensure that additional funds are not necessary to purchase an air table or granite anti-vibration table.

(5) The microscope must be able to recall all capture settings which include the shutter, aperture, light intensity, XYZ stage position, magnification, filter, imaging mode (bright field/fluorescence/phase). In addition, there must be no manual/mechanical adjustments related to the microscope structure or light path (aperture, focus, stage, condenser, lighting). Everything must be controlled electronically.

=> This requirement is to ensure absolute reproducibility of the imaging technique to obtain consistent results without errors due to manual mechanical adjustments.

(6) The stroke of the motorized XY stage must be at least 114 mm x 80 mm and the minimum travel pitch must be 1 um or smaller. Also, the stroke of the motorized Z axis stage must be at least 8 mm and the minimum travel pitch must be 0.1 μm or smaller.

=> To accurately determine signal localization, it is necessary to perform positioning at pitches of 0.1 um or smaller so that the focus can be adjusted with steps finer than 0.1 um. In addition, considering different types of experiments planned for the future, a travel range of 114 mm x 80 mm is required when observing various organs, including the developing gonads.

7) Fluorescence filter placement must be possible at four locations.

=> This requirement is associated with the need to select a filter most suitable for the reagents being used in order to make multiple fluorescent wave lengths capturing possible.

(8) Up to six objective lenses with magnifications ranging from 2x to 100x must be available.

=> To use a wide range of objective lenses for research purposes, from high magnification lenses for localized observation in high resolution to lower magnification lenses for macro observation.

(9) The microscope must be equipped with a monochrome CCD that is cooled to a temperature that is lower than the ambient temperature by at least 25°C. The microscope must also be furnished with a mechanism that can capture full-color images using color filters inserted under electronic control.

=> This requirement is associated with the research-related need to achieve high sensitivity, high color gradation, and quick capturing. To have a single camera achieve both color and monochrome mode is essential for user friendliness, repeatability and reproducibility. Systems with 2 camera systems have higher reliability issues as well as optical path misalignments.

(10) The microscope must be equipped a “low photobleach mode” which physically blocks the light to the sample when it is just stationary. Light is only let through when we move the sample or change the capture settings.

=> This requirement protects the sample from over-exposure and prolongs the life of delicate samples and fluorescence signal.

(11) The microscope must be equipped with a navigation function that enables the user the following: to make observations at a high magnification while checking the position over the entire region using a wide-area map image linked with information on the XY stage coordinates and to move the observation position by clicking the desired point on the wide-area map image. It must also be possible to create the wide-area map image using any of the mountable lenses.

=> This requirement is associated with the need to shorten capture time by ensuring that it is possible to perform positioning while using a high-magnification lens without having to frequently switch between high-magnification and low-magnification lenses.

(12) The microscope must include a function that enables the user, when using any of the mountable lenses, to set a maximum range of 50,000 x 50,000 pixels automatically by clicking the edges of the desired capture range, capture images in this range, and stitch them seamlessly.

=> This requirement is associated with the need to increase the visibility of the stitched image by enabling the user to view an image showing the entire observation range. This function enables the user to capture wide-area images easily and accurately; this means that the user can observe both the entirety and details accurately at the same time. This feature is needed when evaluating a transportation range or the like.

(13) The number of pixels that can be recorded in a single image (not stitched) must be at least 12,500,000.

=> This requirement is associated with the capability of observing even fine parts clearly and proceeding to more detailed analysis.

(14) It must be possible to report the progress status and completion of capturing by email.

=> This requirement is associated with the need to perform other tasks efficiently in parallel to capturing.

(15) The analysis software must include some basic image processing software such as Haze reduction (no-neighbor deconvolution), image overlays, scale bar, cut/paste, zoom, lookup table, black balance.

=> This requirement is associated with the ability to do simple image analysis that is pre-calibrated to ensure time savings.

(16) The microscope must include a function to record a video at a frame rate of 95 fps or higher.

=> To accurately record and evaluate events that change rapidly.

(17) It must be possible to equip the microscope with a temperature/CO2 regulation chamber to capture time lapse images. It must also be possible to set a multi-well plate on the mounted chamber.

=> The mounting of a temperature/CO2 chamber is indispensable to keep cultured cells active and recordable for a long time. Since cells may be cultured not only in dishes, but also in multi-well plates, it must be possible to set a multi-well plate on the chamber.

(18) The microscope must include a function that automatically selects cells based on the contour, color contrast, etc. and automatically counts/calculates the number of cells, area, perimeter, Feret diameter, major axis, minor axis, and/or integrated brightness value. This software must be compatible with brightfield, phase and fluorescence imaging. The microscope must also include a function that, using selected image information as a target area, allows the user to set conditions again for images in each of such target areas and automatically counts various items. This function must also enable the user to measure area ratios or the like easily.

=> This function enables the user to evaluate the efficiency of accumulation into the nucleus or the like quantitatively. Manual measurement results may be arbitrary or vary among measurers. For objective and reproducible evaluation, an automatic counting function is required.

(19) It must be possible to output a conditions file that indicates the measurement conditions that apply when the automatic counting function is used. A macro processing function must also be available that performs automatic counting on multiple image files in a batch under the same condition based on the conditions file.

=> This function is required when performing measurement under the same condition in a short time on a large number of specimens for the purpose of comparison.

(20) Optical sectioning image mode must be available. Optical sectioning’s operating principal is a Structured Illumination Microscopy (SIM) based design with a built in spatial light modulator using a programmable liquid filter (LC filter), projecting structured light patterns that can be adjusted to accommodate a variety of samples, vessel types and fluorescence intensities. The built-in software will utilize a user defined structured projection and raster the light pattern across each focal plane to be captured. The software then utilizes an algorithm to perform a scaled subtraction of out of focus fluorescence light, isolating in-focused fluorescence signal which will allow construction of high-resolution 3D models. Both 1D slits and 2D pinhole options must be available for maximum flexibility.

=> If the observation target section has a thickness, its image may include a fluorescence blurring component caused by scattered light from positions other than the focus. The optical sectioning image function is required to realize clear observation and determine accurate signal localization.