Field of View for the IRIS Digital Fluorescence Microscope as it Relates to Zebrafish Embryo Observations

Dec-09-2015 0 comments Cube Biosystems

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).

FOV with Celena S Digital Microscope as it relates to embryonic Zebrafish observations.

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