Field of View for the IRIS Digital Fluorescence Microscope as it Relates to Zebrafish Embryo Observations
Please Note: As of 5/1/2017, the iRiS™ Digital Imaging System has been renamed as the CELENA® S Digital Imaging System.
When providing demonstrations of the innovative CELENA® S Digital Fluorescent Cell Imaging System, we are often asked what would be the best objectives to include with the CELENA® S as it relates to a particular model system. Essentially, the question comes down to what objective has the most appropriate Field of View (FOV).
For traditional microscopes, the view field is circular and the FOV is the diameter of the light circle measured in millimeters (i.e., what you see when you look through the scope). The lower the magnification, the larger the FOV. Field numbers usually are included on the objective's side. For digital imagers such as the CELENA® S, the same general concepts apply, but the field view is rectangular and not circular, and the FOV is provided as a dimension. Recently, we've performed a few demonstrations for Zebrafish researchers working on early embryo development. Below, we provide a table (Table 1) that provides the FOV for the CELENA® S objectives and how it relates to what may be observed in early Zebrafish embryonic development (Based on the growth chart found at Zfin.Org Table 2). The Zebrafish egg is about 700 - 800 um or 0.7 -0.8 mm in diameter. During the development, the embryo size grows to 14 mm long over 45 days.
Table 1: CELENA® S FOV as it relates to embryonic Zebrafish observations
Objective | X (mm) | Y (mm) | Area (mm²) | Development Stage |
---|---|---|---|---|
1.25X | 5.9 | 4.72 | 27.95 | Midway thru larval stage (≤ 2wks) |
4X | 1.84 | 1.48 | 2.7 | Up to pharyngula stage (≤ 24 h) |
10X | 0.74 | 0.59 | 0.44 | |
20X | 0.37 | 0.3 | 0.11 | |
40X | 0.18 | 0.15 | 0.03 | |
100X | 0.07 | 0.06 | 0.004 |
For Zebrafish observation, the 4x objective maybe used up to 24 h development or late in the phayngula stage -- when the embryo is under 1.9 mm. Beyond that the 24 h stage, the 1.25x objective may be used up to ~ a little under 2 weeks or about mid-way through the larval stage
The CELENA® S digital fluorescence microscope has both phase contrast optics for live cell imaging and fluorescence modules that can be configured for various fluorescence dyes. The system also has time-lapse and Z-stacking imaging capabilities that may be relevant for early Zebrafish observations.
For more information or to inquire about pricing for the CELENA® S Digital Imaging System: Click here >>
Plus, for better flurorescent imaging results try the new refractive index matching X-Clarity™ Mounting Solution: Click here >>
Table 2: Zebrafish Developmental Stages (Source: Zfin.Org)
Period | Stage | Begins | Developmental Landmarks |
---|---|---|---|
Zygote (0 - 0.75 h) | 1-cell | 0.00 h | Cytoplasm streams toward animal pole to form blastodisc |
Cleavage | 2-cell | 0.74 h | Partial Cleavages |
(0.75 - 2.25 h) | 4-cell | 1.0 h | 2 x 2 array of alastomeres |
8-Cell | 1.25 h | 2 X 4 array of alastomers | |
16-cell | 1.5 h | 4 x 4 array of blastomeres | |
32-cell | 1.75 h | 8 x 4 array of blastomers | |
32-cell | 1.75 h | 8 x 4 array of blastomeres | |
64-cell | 2.0 h | 3 regular tiers of blastomerse | |
Blastula | 128-cell | 2.25 h | 5 blastomeres, cleavage plane irregular |
(2.25 - 5.25 h) | 256-cell | 2.50 h | 7 blastomere tiers |
512-cell | 2.75 h | 9 blastomere tiers; YSL forms | |
1K-cell | 3.00 h | 11 blastomere tiers; single row of YSL nuclei; asynchronous cell cycle | |
High | 3.33 h | >11 blastomere tiers; blastodisc flattening begins; YSL nuclei in two rows | |
Oblong | 3.66 h | Blastodisc flattening; multiple rows of YSL nuclei | |
Sphere | 4.00 h | Spherical shape; flat border between blastodisc and yolk | |
Dome | 4.33 h | Yolk cell bulging toward animal pole as epiboly begins | |
30%-epiboly | 4.66 h | Blastoderm an inverted cup of uniform thickness | |
Gastrula | 50%-epiboly | 5.25 h | Blastoderm remains of uniform thickness |
(5.25 - 10.33 h) | Germ Ring | 5.66 h | Germ ring visible from animal pole; 50%-epiboly |
Shield | 6.00 h | Embryonic shield visible from animal pole; 50%-epiboly | |
75%-epiboly | 8.00 h | Dorsal side distinctly thicker; epiblast, hypoblast, evacuation zone visible | |
90%-epiboly | 9.00 h | Axis and neural plate; brain and notochord rudiments | |
Bud | 10.00 h | Tail bud prominent; early polster; 100%-epiboly | |
Segmentation | 1-4 somites | 10.33 h | First somite furrow |
5-9 somites | 11.66 h | Polster prominent; optic vesicle, Kupffer's vesicle, neural keel | |
10-13 somites | 14 h | Pronephros forms | |
14-19 somites | 16.00 h | EL (embryo length) = 0.9 mm; otic placode, brain neuromeres | |
20-25 somites | 19 h | EL = 1.4 mm; lens, otic vesicle, hindbrain neuromeres | |
26+ somites | 22.00 h | EL = 1.6 mm; blood islands, otoliths, midbrain-hindbrain boundary | |
Pharyngula | Prim-5 | 24 h | EL = 1.9 mm; early pigmentation, heartbeat |
(24 - 48 h) | Prim-15 | 30.00 h | EL = 2.5 mm; early touch reflex, retina pigmented |
Prim-25 | 26 h | EL = 2.9 mm; rudiments of pectoral fins | |
High-pec | 42.00 h | EL = 2.9 mm; rudiments of pectoral fins | |
Hatching | Long-pec | 48 h | EL = 3.1 mm; elongated pectoral fin buds |
(48 - 72 h) | Pec-fin | 460.00 h | EL = 3.3 mm; pectoral fin blades |
Larval | Protruding-mouth | 72 h | 3.5 mm total body length |
Day 4 | 96.00 h | 3.7 mm total body length | |
Day 5 | 120 h | 3.9 mm total body length; 6 teeth | |
Day 6 | 144.00 h | 4.2 mm total body length | |
Day 7-13 | 168 h | 4.5 mm total body length; 8 teeth | |
Day 14-20 | 14 d | 6.2 mm total body length;10 teeth | |
Day 21-29 | 21 d | 7.8 mm total body length | |
Juvenile | Day 30-44 | 30 d | 10 mm total body length; adult fins/pigment |
Day 45-89 | 45 d | 14 mm total body length; 12 teeth | |
Adult (90 d -2 y) | 90 d | Breeding Adult |
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